key: cord-331045-i33nr27j authors: addie, diane d. title: feline coronavirus – that enigmatic little critter date: 2003-11-13 journal: vet j doi: 10.1016/s1090-0233(03)00083-2 sha: doc_id: 331045 cord_uid: i33nr27j nan feline coronavirus -that enigmatic little critter at the recent second international feline coronavirus/feline infectious peritonitis symposium (siffs scotland 2002), dr. jim richards aptly described feline coronavirus (fcov) as an ''enigmatic little critter!'' feline infectious peritonitis (fip) was first described in 1964 (holzworth, 1963) and nearly 40 years on, very little is known about this complicated disease, there is no single diagnostic test, no treatment and only one vaccine (which is not at present available in the uk). in fact, the pathogenesis of fip is hardly understood at all and every advance of science seems to make it harder, rather than easier, to understand. for diagnosis, clinicians use a panel of tests including fcov serology, albumin to globulin ratio, haematology, cytology of effusion and measurement of acute phase proteins, especially a1-acid glycoprotein (agp). there are many publications about the virtues and limitations of these tests in cats with fip, but, as far as i know, dr. gi-ordanoõs paper, published in this issue of the veterinary journal, is the first time that workers have looked at the relevant differences in these parameters in the cats who get fip and their in-contact cats who remain healthy (giordano et al., 2004) . physically (if not emotionally) it is easy to take laboratory grown viruses and inoculate them into groups of specific pathogen free cats and then publish the results. what professor paltrinieriõs group does is far more difficult -following naturally infected cats -but their results are much more trustworthy and more likely to represent what really happens in the field. dr. giordano and her colleagues address the down-to-earth questions: ''how do i diagnose this disease in the living cat?'' and ''how do i distinguish the fcov infected cat from the cat with fip?'' like duthie et al. (1997) , they found that cats with fip were likely to have higher agp concentrations, and that haptoglobin levels were not predictive. but, unlike duthie, they examined a group of fcovexposed cats and asked what was the difference between them and the cats with fip? this is a question which constantly arises in real life, and dr. giordano is the first to present an answer: she found that there was a massive increase in serum amyloid a (saa) compared with fcov exposed cats. it would appear that saa should be added to the panel of tests performed on the suspect fip case. what is very interesting and unique about this study is the following of four fcov exposed cats over 83 days. it was extraordinary that when fip occurred in one cat, the in-contact catsõ acute phase proteins fluctuated. the significance is that these fluctuations did not appear with fcov infection, but with the development of fip in one of the cats. if this is truly the case, it would imply that the mutated, pathogenic form, fipv, had spread to the other cats. present belief is that for cats to develop fip, a mutation (more accurately -a deletion) must occur in the viral genome of non-pathogenic fcovs (so called enteric coronaviruses) which allows the virus to replicate in macrophages (vennema et al., 1998) . the current theory is that the mutated virus cannot transmit to other cats, although this theory was challenged at siffs as delegates had experienced households where many cats had developed fip, implying that virulent virus had spread (addie et al., in press) . the mechanism by which cats do not get fip is not understood at present. in giordanoõs paper, four surviving cats had a transient rise in saa and the authors ask the interesting question as to whether this increase and the decrease in agp seen in these cats had some protective role? although the biological function of agp is not completely known, it is a natural anti-inflammatory and immunomodulatory agent. its effects in relation to fip development could be protective or damaging. examples of agpõs protective properties are: (1) it has anti-complement activity (fournier et al., 2000) , and fip is an immune-mediated disease such that cats which are decomplemented do not develop fip and (2) agpõs immunomodulatory and anti-neutrophil activity: in fip chemokines are released which attract neutrophils -one of the cell types in fip pyogranulomas. on the other hand, agp may exacerbate the effects of fip by maintaining capillary permeability in animals with shock (fournier et al., 2000) and clearly cats with effusive fip have very permeable capillaries. moreover, agp from humans with cancer suppressed the augmentation of natural killer (nk) cell activity by interferon a or c (aso et al., 1999) ; suppression of nk cell activity could allow the virus to replicate more. the immunomodulatory function of agp is affected by its carbohydrate composition (aso et al., 1999; fournier et al., 2000) . the sialyl lewis x form of agp the veterinary journal 167 (2004) 5-6 the veterinary journal www.elsevier.com/locate/tvjl is induced during inflammation and ameliorates both complement and neutrophil-mediated injuries while the non-sialyl lewis x form does not (fournier et al., 2000) . moreover, sialyl lewis x is the ligand for the cell adhesion molecules involved in adhesion of monocytes to endothelial cells (fournier et al., 2000) , and one of the earliest stages of the pathogenesis of fip is the adhesion of fcov-infected monocytes to the endothelium in fip vasculitis. is it possible that development of fip has got little to do with the virus after all and everything to do with the agp response of the cat? sequential testing of symptomatic cats is a large gap in the area of fip diagnosis and treatment and needs to be filled. i have followed one cat with fip over the time of treatment until death and i found that agp and globulin levels correlated well with response to treatment and improving or worsening clinical signs, whereas repeatedly measuring fcov antibody titre was unhelpful. agp and globulin levels fell when the cat responded well to treatment and rose when the cat relapsed, but the fcov antibody titre remained high. i hope that professor paltrinieriõs group will expand this particular area of research in future as it would be especially good to be able to correlate clinical pathology results with clinical response to treatment. establishment of objective markers for clinical improvement would also make evaluation of potential treatments easier. dr. giordanoõs paper is interesting, and it points the way to future research. nevertheless, her results must be confirmed on larger numbers of cats, saa levels should be studied in the many differential diagnoses of fip and, of course, we need to understand whether and how acute phase proteins enable fcov exposed cats to recover from the infection in order to ascertain if therein lies a potential for fip treatment -and control of that critter. diane d. addie recommendations from workshops of the second international feline coronavirus/feline infectious peritonitis symposium effect of a1-acidic glycoprotein in the ascitic fluid of cancer patients on human nk cells: selective suppression of interferon-induced nk activation value of a1-acid glycoprotein in the diagnosis of feline infectious peritonitis a-1-acid glycoprotein changes in some acute phase protein and immunoglobulin concentrations in cats affected by feline infectious peritonitis (fip) or exposed to feline coronavirus infection some important disorders of cats feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses key: cord-323932-l14sjufm authors: ishida, t; shibanai, a; tanaka, s; uchida, k; mochizuki, m title: use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis date: 2004-02-25 journal: j feline med surg doi: 10.1016/j.jfms.2003.08.011 sha: doc_id: 323932 cord_uid: l14sjufm a total of 12 clinically ill cats previously diagnosed as feline infectious peritonitis (fip) were treated with a combination of recombinant feline interferon and glucocorticoid. a complete remission (over 2 years) and a partial remission (2 to 5 months) were observed in four (33.3%) and four (33.3%) cases, respectively. those that survived for more than 2 years were all older cats (6 to 16 years old) with the effusive form of fip. summary a total of 12 clinically ill cats previously diagnosed as feline infectious peritonitis (fip) were treated with a combination of recombinant feline interferon and glucocorticoid. a complete remission (over 2 years) and a partial remission (2 to 5 months) were observed in four (33.3%) and four (33.3%) cases, respectively. those that survived for more than 2 years were all older cats (6 to 16 years old) with the effusive form of fip. © 2004 esfm and aafp. published by elsevier ltd. all rights reserved. feline infectious peritonitis (fip), a feline coronavirus (fcov) disease, has been recognized worldwide, and is considered to be a fatal disease of this species (rohrbach et al., 2001) . generally accepted therapies include glucocorticoid with or without other immunosuppressive agents such as cyclophosphamide (mcreynolds and macy, 1997) . other documented therapies include use of human interferon and a propionibacterium acnes preparation (weiss et al., 1990) , and ozagrel hydrochloride (watari et al., 1998) , but complete resolution of the disease was not reported. clinical diagnosis of fip in the practice setting is a challenge to practitioners. to date, there is no single ante-mortem test both sensitive and specific for fip, and sparkes et al. (1991) state that the diagnosis of fip should depend on the presence of multiple abnormalities which are compatible with this disease to increase the specificity. recently, a recombinant feline interferon (rfeifn) has been marketed in japan, the uk and ec countries with some feline (japanese market) and canine (european market) viral infections as the label indications (martin et al., 2002; sakurai et al., 1992; ueda et al., 1993) . although significant antiviral effect of rfeifn against feline coronaviruses has not been demonstrated in vitro (mochizuki et al., 1994) , the immunomodulatory effects of interferon are generally accepted (tompkins, 1999) and might be of some use in the treatment of immunologic disease such as fip. clinical diagnosis of fip was established for 12 cats presented at the hospitals. the cases included six males and six females with ages varying from 3 months to 16 years. the criteria of the diagnosis included: antibiotics non-responsive chronic fever, low normal pcv values or mild non-regenerative anemia (pcv <32%; normal range 29-48%), hyperglobulinemia with electrophoretic evidence of polyclonal gammopathy, non-septic inflammatory ascites/pleural effusion (effusive) with characteristic findings, cytologic or pathologic evidence of pyogranuloma (dry-type), and fcov serum antibody titer by an immunoperoxidase method using infected cell antigen. the mandatory findings for effusion were: no evidence of bacterial infection, high specific gravity (sg>1.017), high protein content (tp>3.0 g/dl), low albumin/globulin ratio (a/g<0.8), low cellularity (<10,000 cells/ microliter), mixed cell reaction with nondegenerate neutrophils, lymphocytes and macrophages (sparkes et al., 1991) . the diagnosis of fip was established when the cat had either the characteristic effusion or needle biopsy-confirmed granuloma and all the other findings above. after the patients were stabilized by general supportive therapies including thoracocenthesis, oxygen administration and fluid therapy, the medical treatment was initiated. for the treatment regimen, rfeifn 1 was initially administered subcutaneously at 1 mu/kg every other day until remission, followed by weekly subcutaneous injections with the same dosage. glucocorticoid was used either as intrathoracic injection of dexamethasone at 1 mg/kg once, in case of respiratory emergency, followed by oral doses of prednisolone at 2 mg/kg daily gradually tapering to 0.5 mg/kg every other day, or oral prednisolone only from the start. table 1 shows the summary of cases studied and the outcome. case 5 was a 6-year-old spayed female domestic shorthair (dsh) cat presented with difficulty breathing and fever, and diagnosed as the thoracic effusive form of fip. the fcov antibody titer was 1:400 with a marginal low normal pcv of 32% and an elevated serum total protein of 10.8 g/dl with a marked polyclonal gammopathy. with rfeifn at 1 mu/kg every other day and prednisolone at 1 mg/kg po bid, the pleural effusion disappeared in 1 week. the maintenance therapy with the weekly doses of rfeifn and prednisolone at 1 mg/kg po every other day, the cat was healthy at 14 months from the diagnosis, when the treatment was terminated and the fcov antibody was <1:100. the cat was still healthy at the end of the 2-year observation period. case 6 was a female persian cat, 11 years and 4 months of age, presented with fever and abdominal distension. the cat had a fcov antibody titer of 1:3,200, elevated total serum protein of 9.1 g/dl with polyclonal gammopathy, a marginal low pcv and proteinuria. with the induction therapy with rfeifn at 1 mu/kg sc every other day and prednisolone at 1 mg/kg po bid, the ascites decreased in 1 month and completely disappeared in 2 months. for the maintenance therapy, the weekly doses of rfeifn at 1 mu/kg were continued until 2 years from diagnosis and prednisolone was not given. at 2 years, the fcov antibody titer was 1:800 and the cat was healthy. case 7 was a male dsh cat, 12 years and 3 months of age, presented with difficulty breathing, anemia and fever. the thoracocenthesis revealed a characteristic non-septic effusion (tp=4.1 g/dl, a/g=0.47). the serum fcov antibody titer was 1:200 and total protein was elevated (tp=8.6 g/dl) with polyclonal gammopathy. the induction therapy with rfeifn at 1 mu/kg sc every other day alone was effective in completely removing the effusion and fever in 17 days, and the treatment was terminated on day 22 with no further maintenance therapy because of the cat owner's financial concern. the cat was reportedly healthy at 2 years from diagnosis. case 11 was a 16-year-old castrated male dsh cat with fever, pleural effusion (tp=3.4 g/dl, sg1.027, a/g=0.6) and anemia (pcv=27%). although the serum total protein was not significantly elevated (tp=6.2 g/dl), a marked polyclonal gammopathy was noted and the serum fcov antibody titer was 1:12,800. after thoracocenthesis, an intrathoracic injection of 1 mg/kg dexamethasone was given, and rfeifn was started at 1 mu/kg sc every other day with daily oral prednisolone at 2 mg/kg. the pleural effusion disappeared in 1 week, when the maintenance therapy with weekly rfeifn and daily oral prednisolone at 1 mg/kg was started. prednisolone was then tapered to 0.5 mg/kg po every other day, and the treatment was stopped at 12 months, when the serum fcov antibody titer was 1:1,600. the cat was healthy at 2 years from diagnosis. those with partial remission included one drytype and three effusive form fip cases in relatively young ages. their survival times were from 2 months to 5 months. the other four cases showed no response to the therapy and all died within 1 month. the overall efficacy in this study was 33.3% complete response (remission >2 years) and 33.3% partial response (remission 2 to 5 months). since those that survived for 2 years were all older cats (6 years or older), the disease progression in older cats may be slower or the immunologic pathogenesis may be different in those cats, so that the rfeifn therapy may have a possible impact on the disease. since the glucocorticoid therapy has not been proven effective in inducing complete remission of fip (mcreynolds and macy, 1997) , the present results may demonstrate apparent therapeutic effect of rfeifn in some cases. a possible criticism for the present study includes the experimental design with no matched case controls. further controlled studies with confirmed fip cases are warranted to prove the efficacy of rfeifn therapy. although the accuracy of the clinical diagnosis in the present study was in part confirmed by necropsy of the dead cases, there was no way to confirm the diagnosis in those that survived. therefore, in future studies, immunofluorescence detection of fcov antigen in the mononuclear cells in the effusion by using a monoclonal antibody may be a good alternative for invasive biopsy. also, specificity and sensitivity of pcr detection of fcov genome in the effusion should be established. the concurrent use of glucocorticoid did not seem to interfere with the effect of interferon in the cases that survived. if the immunomodulatory effects of interferon involves enhancement of the anti-viral immunologic events, it is not compatible with the immunosuppressive effects, against both th1 and th2 responses, of glucocorticoid. if interferon exerts its effect through modulation of immunologic inflammatory process, it will be interesting to study optimal dosing schedule with glucocorticoid. treatment of canine parvoviral enteritis with interferon-omega in a placebo-controlled challenge trial feline infectious peritonitis. part ii. treatment and prevention inhibitory effects of recombinant feline interferon on the replication of feline enteropathogenic viruses in vitro epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals feline interferon production in silkworm by recombinant baculovirus feline infectious peritonitis: a review of clinicopathological changes in 65 cases, and a critical assessment of their diagnostic value immunomodulation and therapeutic effects of the oral use of interferon-alpha: mechanism of action homogeneous production of feline interferon in silkworm by replacing single amino acid code in signal peptide region in recombinant baculovirus and characterization of the product effect of thromboxane synthetase inhibitor on feline infectious peritonitis in cats effect of interferon or propionibacterium acnes on the course of experimentally induced feline infectious peritonitis in specific-pathogen-free and random-source cats key: cord-335434-lgvoethn authors: cannon, martha .j.; silkstone, malcolm a.; kipar, anja m. title: cutaneous lesions associated with coronavirus-induced vasculitis in a cat with feline infectious peritonitis and concurrent feline immunodeficiency virus infection date: 2005-02-12 journal: j feline med surg doi: 10.1016/j.jfms.2004.12.001 sha: doc_id: 335434 cord_uid: lgvoethn this report describes a clinical case of feline infectious peritonitis (fip) with multisystemic involvement, including multiple nodular cutaneous lesions, in a cat that was co-infected with feline coronavirus and feline immunodeficiency virus. the skin lesions were caused by a pyogranulomatous-necrotising dermal phlebitis and periphlebitis. immunohistology demonstrated the presence of coronavirus antigen in macrophages within these lesions. the pathogenesis of fip involves a viral associated, disseminated phlebitis and periphlebitis which can arise at many sites. target organs frequently include the eyes, abdominal organs, pleural and peritoneal membranes, and central nervous tissues, but cutaneous lesions have not previously been reported. a 1-year-old domestic shorthair cat was presented with a 2-week history of pyrexia, lethargy, inappetence and weight loss, sneezing, bilateral nasal and ocular discharge and conjunctivitis. these signs had not responded to treatment with amoxycillin/clavulanic acid (synulox; pfizer) or doxycycline (ronaxan; merial animal health). some improvement was seen following treatment with dexamethasone (azium; schering plough animal health). four days before presentation the cat had suffered sudden onset blindness and had developed a number of small skin nodules over the neck and forelimbs. clinical examination revealed bilateral mydriasis with severe iritis and extensive areas of retinal detachment. there were also bilateral serous ocular and nasal discharges. on abdominal palpation the right kidney was painful and irregular in outline. the skin over the ventral and lateral aspects of the cat's neck and the proximal forelimbs exhibited multiple well-circumscribed slightly raised, red nodules of approximately 2 mm diameter, which were associated with partial alopecia, but were non-painful and nonpruritic. the major differential diagnoses were diseases that are expected to have multisystemic involvement and which may involve both the eye and the kidney. the most likely differential diagnoses were considered to be feline infectious peritonitis (fip), multifocal lymphosarcoma, feline immunodeficiency virus-associated disease, feline leukaemia virus-associated disease or toxoplasmosis. a concurrent upper respiratory tract virus infection was suspected as the most likely cause of the nasal and ocular discharges and the sneezing. the cat was treated with oral clindamycin (antirobe; pharmacia and upjohn, 50 mg twice daily) and with prednisolone eye drops (pred forte; allergan, one drop to each eye three times daily), pending the results of further diagnostic tests. serum biochemistry revealed mild hyperbilirubinaemia (total bilirubin 12.0 mmol/l, reference range 0e8.6 mmol/l) and an elevation in a1-acid glycoprotein (3.26 g/l, reference range 0.1e0.48 g/l). the levels of total protein (76 g/l, reference range 57e89 g/l), albumin (26 g/l, reference range 26e39 g/l) and globulin (50 g/l, reference range 28e51 g/l) were within reference range and the albumin:globulin ratio was 0.52. routine haematology indicated mild normocytic, normochromic anaemia (red blood cell count 4.7 ! 10 12 /l, mchc 35.2 g/l, mcv 45.7 fl) and profound lymphopenia (0.1 ! 10 9 /l, reference range 1.5e7.0 ! 10 9 /l). urinalysis was unremarkable and indicated normal renal concentrating ability (specific gravity o 1.050). culture from ocular and oropharyngeal swabs was negative for feline herpesvirus, feline calicivirus and chlamydophila felis (clinical pathology diagnostic service, university of bristol), but rapid immunomigration tests (witness, rhone-merieux) revealed that the cat was feline immunodeficiency virus (fiv) antibody positive and feline leukaemia virus antigen negative. the coronavirus antibody titre was zero (companion animal diagnostic laboratory, university of glasgow) and the toxoplasma gondii igg titre was negative (!8 iu/ml; scottish toxoplasma reference laboratory, inverness). the cat remained non-pyrexic but over the next 2 days right-sided renomegaly became apparent. ultrasound examination of the right kidney revealed multiple hypoechoic areas throughout the renal cortex and foci of increased echogenicity within the medulla. the cat was anaesthetised and biopsies were taken from skin lesions and right kidney. biopsies were fixed in 10% neutralised formalin and submitted for histopathological examination. histopathology revealed a severe extensive pyogranulomatous nephritis. in the skin, a multifocal pyogranulomatous perivascular infiltration and phlebitis was seen in the mid and deep dermis, centred around mid dermal and deep dermal vascular plexuses (figs 1, 2a) . there was intense degeneration and necrosis of infiltrating cells. additionally, moderate atrophy of adnexae and epidermis was seen. immunohistology for feline coronavirus (fcov) antigen, using a mouse monoclonal antibody (fcv3-70, custom monoclonals international, west sacramento, usa), was performed on renal and skin biopsies as previously described (kipar et al 1998, in press ). scattered macrophages expressing low amounts of fcov antigen were identified in the renal infiltrates. in the skin lesions, numerous fcov antigen-positive cells were found (fig 2b) . the histological and immunohistological findings together confirmed the diagnosis of fip. palliative treatment was instituted using immunosuppressive doses of methyl-prednisolone (medrone v; pharmacia and upjohn; 8 mg twice daily). the cat's respiratory signs improved and the uveitis was less severe, but 6 days later a neuropathy developed, with flaccid paralysis of the tail and proprioceptive deficits of the hindlimbs. treatment was maintained at the owner's request until the cat died 3 days later. a post-mortem examination was not performed. this is the first reported case of fip in which skin lesions have been recognised as a feature of the disease. the nodular erythematous skin lesions were associated with pyogranulomatous phlebitis and periphlebitis in the dermis. the presence of fcov antigen within a significant proportion of macrophages in these infiltrates demonstrated their association with fip. this case also illustrates the important role of histopathology and immunohistology for the premortem diagnosis of fip. in this case the clinical signs were consistent with a number of possible diagnoses, the most likely of which were considered to be fip, lymphosarcoma, feline leukaemia virus-associated disease, feline immunodeficiency virus-associated disease and toxoplasmosis. fip was suspected because of the presence of clinical, clinicopathological and histopathological findings that were consistent with the disease. the diagnosis was confirmed by the presence of numerous fcov antigen-positive macrophages within the granulomatous lesions, a finding only seen in, and therefore pathognomonic for, fip (kipar et al 1998, in press) . clinicopathological findings that were consistent with fip included the presence of elevated a 1 -glycoprotein concentration, an albumin:globulin ratio of less than 0.6, mild non-regenerative anaemia, lymphopenia and mild hyperbilirubinaemia. an elevated a 1 -glycoprotein concentration is frequently seen in cats with fip, but is a non-specific feature of inflammatory diseases in general, and is also found in terminal stages of fiv (duthie et al 1997) . an albumin:globulin ratio of less than 0.6, mild non-regenerative anaemia, lymphopenia and mild hyperbilirubinaemia are also common findings with fip but are not specific to the disease (sparkes et al 1994) . in this cat the cov antibody titre was zero and this was an unusual feature of the case. a positive cov antibody titre is not a universal finding in cats with fip (sparkes et al 1994 , gaskell and dawson 2000 , lappin 2003 ) and negative cov antibody titres may occur in cats affected by either 'effusive' or 'non-effusive' forms of the disease (sparkes et al 1991 , harvey et al 1996 , paltrinieri et al 1998 . a number of explanations have been postulated, including lack of circulating cov antibodies due to immune-complexing with cov antigen and loss of ability to produce antibodies in the terminal stages of disease (hoskins 2001) . taken together, the clinical signs, clinical pathology, histological changes and immunohistological findings in this case confirm that the cat had a 'non-effusive form' of fip, with involvement of the kidneys, skin and most likely brain and eyes. this cat was found to be fiv positive using an 'in-house' test kit (witness rim; rhone-merieux), but in view of the cat's clinical condition and the histopathological findings, confirmation of this finding by western blot was not pursued. the apparent presence of concurrent fiv infection in this case might have predisposed the cat to the development of fip, as it has been shown that immunosuppression, eg, due to fiv infection, is associated with a higher risk of mutation of nonpathogenic fcov to pathogenic, fip-inducing fcov mutants (poland et al 1996) . other possible differential diagnoses were not identified by the initial clinicopathological and routine histopathological findings in this case. further detailed investigations would have been required to rule out their presence as concurrent diseases in addition to the presence of fip in this cat, but these tests were not undertaken. fip is a systemic, fcov-induced disease which has long been regarded as an immune complexmediated, type iii hypersensitivity disease (hayashi et al 1977 , pedersen and boyle 1980 , weiss et al 1980 . one of the morphological hallmarks of fip is a granulomatous to necrotising phlebitis and periphlebitis (hayashi et al 1977 , weiss et al 1980 , kipar et al 1998 which was recently shown to be triggered by activated monocytes which attach to venous endothelial cells, migrate out of the veins, thereby destroying the basal lamina, and then accumulate perivascularly (kipar et al in press) . the process appears to be cytokine-mediated (kipar et al in press ). the morphological features (predominance of macrophages) and distribution (only veins are affected) of vascular lesions in fip do not support a primarily immune complex-mediated development of vasculitis, although fcov immune complexes may well participate in the disease (jennette and falk 1995, kipar et al in press) . animals with fip exhibit generalised activation of venous and, to a lesser extent, arterial endothelial cells, likely mediating selective adhesiveness of monocytes (kipar et al in press) . a selective endothelial cell reactivity to systemic cytokines could explain why fip-associated phlebitis is not seen in all organs, but predominantly occurs in leptomeninges, renal cortex and eyes (hayashi et al 1977 , weiss et al 1980 , male et al 1990 , kipar et al 1998 ). however, phlebitis in, for example, lungs and liver has been shown (hayashi et al 1977 , weiss et al 1980 and the present report demonstrates that dermal veins can also be affected. this report indicates that a differential diagnosis of fip should be considered in cats showing nodular erythematous skin lesions, especially where these occur in conjunction with signs of systemic disease that are consistent with the diagnosis. value of a1-acid glycoprotein in the diagnosis of feline infectious peritonitis fip-related disease an uncommon intestinal manifestation of feline infectious peritonitis: 26 cases (1986e1993) systemic vascular lesions in feline infectious peritonitis update on feline coronavirus disease update on the pathobiology of vasculitis cellular composition, coronavirus antigen expression and production of specific antibodies in lesions of feline infectious peritonitis morphological features and development of granulomatous vasculitis in feline infectious peritonitis polysystemic viral diseases: feline coronavirus some aspects of humoral and cellular immunity in naturally occurring feline infectious peritonitis immunologic phenomena in the effusive form of feline infectious peritonitis two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus feline infectious peritonitis: a review of clinicopathological changes in 65 cases, and a critical assessment of their diagnostic value an appraisal of the value of laboratory tests in the diagnosis of feline infectious peritonitis disseminated intravascular coagulation in experimentally induced feline infectious peritonitis. american journal of veterinary research 41 the authors wish to thank dr p mcneill for initial immunohistological examination of the skin biopsies, and mr r e thomas for his help and advice in the initial management of this case. key: cord-308557-mvu97jsu authors: pesteanu-somogyi, loretta d.; radzai, christina; pressler, barrak m. title: prevalence of feline infectious peritonitis in specific cat breeds() date: 2005-07-01 journal: j feline med surg doi: 10.1016/j.jfms.2005.04.003 sha: doc_id: 308557 cord_uid: mvu97jsu although known that purebreed cats are more likely to develop feline infectious peritonitis (fip), previous studies have not examined the prevalence of disease in individual breeds. all cats diagnosed with fip at a veterinary teaching hospital over a 16-year period were identified. breed, sex and reproductive status of affected cats were compared to the general cat population and to mixed breed cats evaluated during the same period. as with previous studies sexually intact cats and purebreed cats were significantly more likely to be diagnosed with fip; males and young cats also had a higher prevalence of disease. abyssinians, bengals, birmans, himalayans, ragdolls and rexes had a significantly higher risk, whereas burmese, exotic shorthairs, manxes, persians, russian blues and siamese cats were not at increased risk for development of fip. although additional factors doubtlessly influence the relative prevalence of fip, this study provides additional guidance when prioritizing differentials in ill purebreed cats. f eline infectious peritonitis (fip) is a progressive systemic disease with a wide spectrum of clinical signs and high mortality (hartmann 2005) . it is caused by a mutation in the feline enteric coronavirus, a common pathogen of cats that may cause no clinical signs or transient diarrhea , mcreynolds and macy 1997 , hartmann 2005 . the mutated fip virus disseminates via the monocyte phagocytic system, and variations in an individual cat's immune response produce one of two recognized forms of disease , mcreynolds and macy 1997 , hartmann 2005 . the 'wet' form of fip, seen in approximately 75% of cases, is caused by complement-mediated vasculitis initiated by immune complex deposition in vessel walls, and typically results in body cavity effusions , mcreynolds and macy 1997 , hartmann 2005 . the 'dry' form of fip, found in the remainder of cases, results when a cell-mediated immune response dominates and granulomas form in various organs , mcreynolds and macy 1997 , hartmann 2005 . epidemiologic studies of cats with fip have identified several risk factors for development of disease. the highest prevalence is in young cats (3 months to 3 years of age) with the majority of cases (75%) in multi-cat environments (kass and dent 1995 , foley et al 1997a , mcreynolds and macy 1997 , rohrbach et al 2001 . males and sexually intact cats are also at increased risk for development of fip (robison et al 1971 , rohrbach et al 2001 . other factors that have been less commonly reported to be associated with an increased disease prevalence include season (more cases are typically diagnosed in winter), felv infection, an increase in factors associated with 'stress', high coronavirus antibody titer, regular introduction of new cats to a cattery, and increased frequency of coronavirus shedding (kass and dent 1995 , mcreynolds and macy 1997 , foley et al 1997a , rohrbach et al 2001 . two studies have reported that fip is more common in purebreed cats (robison et al 1971 , rohrbach et al 2001 . although the relative prevalence of fip in different cat breeds has been reported in at least one study, statistical differences were not calculated (scott 1991) . therefore, to the authors' knowledge, whether a specific breed predisposition exists has never been thoroughly investigated. the purpose of this study was to determine whether such a breed predilection exists in cats. sex and age of affected cats were also examined in order to allow some comparison between the current study population and those in previous studies. the final diagnosis was reviewed for all cats entered in the computerized patient database of the north carolina state university college of veterinary medicine (ncsu-cvm) between december 22, 1986 and december 22, 2002 . cats with fip were identified using the coding terms 'feline infectious peritonitis' or 'fip'. final diagnosis in all cases had been determined by the attending clinician; criteria used for diagnosis and results of antemortem or post-mortem diagnostic test results were not reviewed. breed, sex, and reproductive status of all cats evaluated at the ncsu-cvm during the 16-year study period were reviewed; all cats of unknown breed were excluded. mixed breed cats of all hair lengths (domestic shorthair, mediumhair and longhair) were considered a single breed (termed 'mixed breed') for data analysis purposes. descriptive statistics were calculated for each variable studied for the fip population and for the total cat population. descriptive statistics for cat age at time of evaluation were calculated only for fip-affected cats. breed, sex, and reproductive status differences were compared using the fisher's exact test; values of p less than or equal to 0.05 were considered significant. odds ratios (or) and 95% confidence intervals (ci) were also calculated for each variable. during the 16-year study period, 11,535 cats of known breed were examined at the ncsu-cvm. cats examined included mixed breed cats (9511 cats) and 36 different purebreed varieties (2024 cats). sixty cats (0.52%) had a final diagnosis of fip; breed was known for all affected cats. sex and reproductive status information was avail-able for 57 of the 60 fip cats and 11,303 of the 11,475 non-fip cats. age information was available for 58 of the 60 fip cats. cats diagnosed with fip included mixed breed cats (33 cats) and 13 different purebreeds (27 cats). prevalence of fip in the mixed breed cat population was 0.35% versus 1.3% in the purebreed cat population (fig 1) . purebreed cats were significantly more likely to be diagnosed with fip than were mixed breed cats (or 4.5, ci 2.7e7.5; p ! 0.001). breeds with a prevalence of fip significantly greater than mixed breed cats included the abyssinian, bengal, birman, himalayan, ragdoll, and rex (including cornish and devon varieties) breeds (table 1 , fig 2) . the prevalence of fip in burmese, exotic shorthair, manx, persian, russian blue, and siamese cats was not significantly different from mixed breed cats. the two havana brown cats evaluated at the ncsu-vth during the study period were both diagnosed with fip, but this small number precluded statistical analysis. twenty-three cat breeds had an fip prevalence of zero. these included the angora (11 cats evaluated during study period), balinese (25 cats), belgian (two cats), bombay (four cats), british blue (two cats), british shorthair (three cats), chartreux (four cats), colorpoint shorthair (one cat), egyptian mau (one cat), japanese bobtail (six cats), korat (five cats), maine coon (151 cats), maltese (two cats), norwegian forest cat (five cats), ocicat (16 cats), ragamuffin (one cat), scottish fold (15 cats), siberian (one cat), snowshoe (two cats), somali (three cats), sphinx (one cat), tonkinese (18 cats), and turkish van (two cats) breeds. unfortunately, the low prevalence of fip in the mixed breed cat population prevented determination of significance or relative risk in these purebreed cat varieties. cats with fip were significantly more likely to be sexually intact when compared to the general cat population, regardless of whether the cats were male or female (intact male versus castrated male, p ! 0.001; intact female versus spayed female, p z 0.002; all intact cats versus all altered cats, p ! 0.001; prevalence of intact cats in the general population was 15.8%, versus 45.6% in the fip population). although more cats with fip were male than female, the difference in prevalence was not statistically significant (p z 0.425; 53.6% of the total cat population was male, versus 59.6% of the fip population). at the time of last evaluation the median age of cats with fip was 0.96 years (25th percentile 0.5 years, 75th percentile 2.0 years). sixty-seven percent of cats with fip were less than 2 years of age. although the increased prevalence of fip in purebreed cats has been previously reported, this is the first time that a predisposition of specific breeds to the development of disease has been examined (robison et al 1971 , rohrbach et al 2001 . our results show that certain breeds may in fact be more likely to develop fip, particularly the birman, ragdoll, bengal, rex, abyssinian, and himalayan breeds. other breeds of cats, the burmese, exotic shorthair, manx, persian, russian blue, and siamese, did not appear to be at increased risk as compared to mixed breed cats. our results on the effects of sex, reproductive status, and age on the relative prevalence of fip are similar although not identical to previous studies (robison et al 1971 , horzinek and osterhaus 1979 , kass and dent 1995 , rohrbach et al 2001 . previous evidence supports an influence of host genetics on mutation of the feline enteric coronavirus or on susceptibility to fip. cheetahs, whose genome has become more homozygous with minimal allelic diversity due to an evolutionary bottleneck, have a very high prevalence of fip (o'brien et al 1985) . similarly, the increased prevalence we found in some purebreed varieties could be due to a concentration of inherited factors through in-breeding or small founder populations. given a common environment and viral strain, foley and pedersen (1995) calculated that slightly greater than 50% of fip susceptibility in purebreed cats from six catteries could be attributed to inherited differences between individuals. interestingly, in this study one of the catteries with numerous closely related fip-affected cats was a birman cattery (foley and pedersen 1995) . birmans were by far the most commonly affected in the study reported here, and therefore we may not be the first to provide evidence of an increase in susceptibility to fip in this breed. other investigators have questioned whether the increased prevalence of fip in purebreed cats may actually be due to confounding factors. purebreed cats are more likely to be raised in catteries, which may be inherently more stressful because of the multi-cat environment, regular introduction of new cats, and frequent breeding (kass and dent 1995, pedersen 1995) . additionally, cattery cats presumptively have greater exposure to feline enteric coronavirus (a requirement for development of fip) due to increased population density (foley et al 1997a , 1997b , mcreynolds and macy 1997 . finally, the possible increased willingness of owners of expensive purebreed cats to pursue advanced diagnostics and supportive treatment at a referral veterinary facility such as the ncsu-vth may skew the apparent prevalence of disease. however, these factors would be expected to falsely increase the prevalence of fip in all purebreed cats and not just those breeds we report to be at increased risk of disease development. in this report we chose to include cases based on final diagnosis entered into our computerized medical database rather than by review of records and histopathology reports. as a result, we must acknowledge that future investigations that are limited to cases with confirmed diagnoses could yield different results. however, because the ante-mortem diagnosis of fip at our tertiary care treatment hospital is expected to be similar to diagnostic algorithms proposed by other authors, we feel that our results, particularly in breeds with larger numbers or particularly strong associations with disease, are unlikely to conflict with future studies (sparkes et al 1991 , rohrer et al 1993 , addie and jarrett 1998 . multivariate analysis of the variables studied here would further define specific breed susceptibility to fip. for example, it is unknown if the breeds with an increased prevalence of fip actually had larger numbers of intact cats evaluated, thus influencing our calculations. furthermore some breeds had very few individuals examined, and the large ci reflect the lack of precision in determining risk. we doubt that the absence of cats diagnosed with fip in 23 breeds indicates an absolute resistance to disease, although it is possible that some of these breeds (such as maine coon cats, which were seen at the ncsu-vth in relatively larger numbers) possess unrecognized protective factors that influence susceptibility. unfortunately, because of the low prevalence of fip in all cats a much larger population would need to be examined to determine if lack of disease in these breeds is statistically significant. the predisposition of certain breeds to the development of fip demonstrated here warrants further research. our results suggest that the index of suspicion for fip should possibly be increased in some ill purebreed cats. a multicenter study that includes cases from primary as well as referral facilities with multivariate analysis is likely necessary to definitively answer the question of individual breed susceptibilities to fip. infectious diseases of the dog and cat the inheritance of susceptibility to feline infectious peritonitis in purebred catteries risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus patterns of feline coronavirus infection and fecal shedding from cats in multiple-cat environments feline infectious peritonitis. veterinary clinics of north america, small animal practice 35, 39e79. horzinek mc, osterhaus adme (1979) feline infectious peritonitis: a worldwide serosurvey the epidemiology of feline infectious peritonitis in catteries feline infectious peritonitis. part i. etiology and diagnosis. compendium of continuing education for the practicing veterinarian genetic basis for species vulnerability in the cheetah epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals the diagnosis of feline infectious peritonitis (fip): a retrospective and prospective study feline infectious peritonitis: transmission and epidemiology feline infectious peritonitis: a review of clinicopathological changes in 65 cases, and a critical assessment of their diagnostic value the authors thank cavell brownie, phd (department of statistics, north carolina state university) for performing the statistical analyses and malcolm roberts, bvsc, phd, mph, frcvs, facvsc (department of clinical sciences, north carolina state university) for manuscript review. key: cord-023034-j8zwcfys authors: osterhaus, albert d. m. e.; horzinek, marian c.; wirahadiredja, r. m. s. title: feline infectious peritonitis virus: ii. propagation in suckling mouse brain date: 2010-05-13 journal: j vet med b infect dis vet public health doi: 10.1111/j.1439-0450.1978.tb01683.x sha: doc_id: 23034 cord_uid: j8zwcfys summary: feline infectious peritonitis (fip) virus multiplication was demonstrated in the brains of one‐day‐old laboratory mice using direct immunofluorescence tests. specificity was assessed by virus reisolation, indirect immunofluorescence and reproduction of fip after inoculation of spf kittens using brain material from the 6th mouse passage. zusammenfassung: virus der felinen infektiösen peritonitis ii. vermehrung im gehirn von säuglingsmäusen mit hilfe der direkten immunofluoreszenz wurde die vermehrung des virus der felinen infektiösen peritonitis (fip) im gehirn eintägiger laboratoriumsmäuse nachgewiesen. die spezifität wurde durch reisolierung des virus und indirekte immunofluoreszenz belegt, sowie durch die auslösung der fip nach inokulation von gehirnmaterial der sechsten mäusepassage in spf katzenwelpen. résumé: virus de la péritonite infectieuse du chat ii. multiplication dans le cerveau de la souris nouveau‐née a l'aide de l'immunofluorescence directe la multiplication du virus de la péritonite infectieuze féline a été démontrée dans le cerveau de la souris nouveau‐née. epreuves de spécificité étaient le réisolement du virus, l'immunofluorescence indirecte et la reproduction de la maladie par inoculation de chats spf utilisant du material cerveau provenant du 6ième passage en souris. resumen: virus de la peritonitis infecciosa del gato ii. multiplicación en cerebro de ratoncitos recién‐nacidos utilizando la inmunofluorescencia directa se mostró la multiplicación en cerebro de ratoncitos recién‐nacidos del virus de la peritonitis infecciosa del gato. se pudo evidenciar la especificidad por medio de re‐aislamiento del virus y de la inmunofluorescencia indirecta; además, se logró reproducir la enfermedad en gatos spf, inoculándoles material del 6° pasaje en cerebros de ratoncitos. feline infectious peritonitis (fip) virus was shown to possess many of che physical properties of coronaviridae family members (2, 5, 10). support for this tentative classification has been obtained recently from neutralization and immunofluorescence studies showing an antigenic relationship between fip virus and the coronavirus causing transmissible gastroenteritis (tge) of swine (6, 8, 13) . numerous attempts to isolate fip virus in chicken embryos (11, 14) , in primary feline cells (i, 3, 7, 12) and continuous lines (7) have failed; in vitro virus growth could be demonstrated so far only in cultures of cells derived from the peritoneal exudates of kittens after experimental infection with fip virus (7) . the present report describes the successful propagation of fip virus in the brain of one-day-old mice. this system was selected since coronaviruses of such diverse species as man, mouse, rat and chicken have been shown to grow in suckling mouse tissues (for review see 4). for mouse inoculation experiments, material from the third cat passage of the dahlberg strain (2) was used as a 10 o/o (w/v) homogenate of infectious liver tissue in phosphate buffered saline (pbs). the material was stored at for virus identification using the indirect immunofluorescence test (ift), the following sera were employed: paired pre-and postinoculation sera of an experimental fip case (dahlberg strain) from our laboratory (utrecht). paired pre-and postinoculation sera from an spf kitten, kindly provided by dr. m. c. pedersen, davis. cal. -20 field sera from randomly selected cats in the netherlands; their antibody titers had been determined in heterologous indirect immunofluorescence tests published previously ( 6 ) : 13 of them had high (anti-tge-virus) titers and 7 were negative (< lo). -20 sera from spf cats from a barrier-contained colony (centraal proefdierenbedrijf tno, zeist, the netherlands) in which no antibodies to tge virus had been found (6). specified pathogen-free mice (strain cpb se) were obtained from the breeding colony mentioned above (cpb zeist) and kept in a laminar flow hood throughout the experiments. litters containing 7 to 14 animals not older than 24 hours were infected by the intracerebral route, inoculating 5 pullanimal of a 10 o / o (w/v) cat liver suspension (starting material) or of a 40 o / o mouse brain homogenate (passage material), using a hamilton syringe. a control passage series was done using a normal cat liver homogenate as starting material. the animals were examined daily for clinical signs. after 7 days the mice were decapitated and the brains removed and divided into two portions each, one serving for demonstrating viral antigen using the direct ift, the other (after pooling and homogenizing) for further passaging. a group of three kittens nine weeks of age was obtained as a gift from the aforementioned spf cat breeding colony (courtesy of j. c. j. van vliet, cpb, zeist) which had been shown seronegative for tge virus ( 6 ) . the animals were housed together in a pressurized sterile stainless steel glove box; it formed part of an isolator unit belonging to an institute (gezondheidsdienst voor pluimvee, doorn, the netherlands) where cats had no access. two of the animals were inoculated with passage material by injection of 1.0 mi.-quantities of a clarified (2.5 min at 10.000 x g.) 40 o / o (w/v) mouse brain homogenate via the intraperitoneal route. the third kitten served as a contact control. the animals were checked daily for rise in body temperature and overt clinical signs. from the ascitic fluid of an fip-field case y-globulin was prepared by repeated precipitation (3 times) with ammonium sulphate at 50 o/o saturation. the preparation was labelled with fitc using standard techniques (9). cryostat sections from mouse brains and cat organs were acetone-fixed (10' at ~--20 "c), dried, washed with pbs and with distilled water and dried again. a working dilution of the conjugate in pbs was applied to the sections and the preparations were incubated for 30' at 37 oc in a moist chamber. after three rinses in pbs and one in distilled water the slides were dried and mounted in uvak (searle, high wycombe bucks. eng.). the specificity of the conjugate was demonstrated in a blocking test using tge virus (strain purdue) infected porcine thyroid cells; significant quenching of fluorescence was observed when the antigen preparations had been preincubated with unlabelled anti-fip serum prior to the application of the fitc conjugated y-globulin. indirect immunofluorescence tests were performed essentially as described previously ( 6 ) , using infected porcine kidney cells as tge antigen source and an fitc labelled rabbit anti cat y-globulin as second antibody. demonstration of virus multiplication in the brains of suckling mice three litters of one day old mice were inoculated intracerebrally with infectious cat liver material and one litter with normal cat liver material; passages were performed at weekly intervals as described above. no clinical signs were observed in the experimental animals during the short observation period. the results of direct ift applied to brain sections at different passage levels are shown in table 1 . using the labelled anti fip y-globulin, clearcut fluorescence was observed in one of the three fip virus-infected mouse series (a), from the second passage onward; the two other fip-series and the control series remained negative through four subsequent passages. to avoid any personal bias, coded samples were examined by one of the authors (r. m. s. w.) in a different institute. predominantly cytoplasmic fluorescence was observed in neurons and glia cells (fig. 1 ) in focal accumulations throughout the brain. their number and size increased during the first positive passages. in order to confirm the results, reisolation was attempted from the same cat liver material. virus multiplication was detected in two of the four passage series (table i , e and f) from the third passage on. table 1 passage history of fip virus (strain dahlberg) in suckling mouse brain in order to determine the specificity of the observed fluorescence for fip virus, indirect ift were carried out in parallel on poslitive mouse brain sections (homologous reaction) and on porcine kidney cells infected with tge virus (heterologous reaction). the preinoculation sera of experimentally fip virus-infected cats (sera utrecht and davis) showed no fluorescence whereas the postinoculation sera were unequivocally positive. twenty sera from the dutch open cat population (6) with varying titers in the heterologous reaction were assayed on mouse-brain sections; complete agreement was observed between the results of both tests: the 13 sera positive in the heterologous reaction fig. 1 . immunofluorescence in a positive mouse brain section (table i a were also positive in the homologous reaction on infected mouse brain sections. fluorescence using the indirect test was localized in foci throughout the brain (fig. 2) as was the case with the direct method. none of the 20 sera from the spf cat colony showed a reaction with fip mouse brain sections, as they were also negative in the heterologous test ( 6 ) . the conclusive experiment for establishing the fip virus specificity of the immunofluorescence in mouse brain was performed by inoculating spf kittens with fluorescence-positive material of the 6th mouse passage (isolation series a, table 1 ). employing the heterologous indirect ift, no antibodies reacting with tge virus were found in the preinoculation sera of the animals. the temperature curves of two inoculated kittens and one control animal are given in figure 3 . one inoculated cat showed a distinct rise in body temperature on day 13 p. i. and gradually developed classical fip symptoms: anorexia, depression, enlarged abdomen; death occurred on day 20 p. i. upon post mortem examination the clinical diagnosis was confirmed. granulomatous foci were present in the liver, spleen, lung, mediastinal lymph nodes, kidneys, bladder wall and the small intestine; all these tissues showed specific fluorescence. the experiments described in this paper have established that fip virus multiplies in brain tissue of one-day-old laboratory mice. the presence in the material from several subsequent mouse passages of an antigen reacting with fip antibodies of naturally and experimentally infected cats is one line of evidence; the reproduction of the clinical and anatomo-pathological picture of the feline disease by inoculation of brain material of the 6th mouse passage into the natural host constitutes the final proof. it must be emphasized that all experiments were done under conditions of careful isolation using spf animals. one reason for selecting the mouse as an experimental animal has been given in the introduction: the multiplication of coronaviruses of several species in this host. ecologic reasons supported this choice. as pointed out in an earlier zbl. vet. med., reihe b, bd. 25, heft 4 306 osterhaus, horzinek and wirahadiredja publication (5) the sporadic occurrence of clinical fip could reflect virus spillover from a non-feline reservoir. although serology has shown a rather high percentage of healthy cats to possess antibodies (indicating cat-to-cattransmission of the virus), the mouse as predominant prey mammal could still play a role in fip epidemiology. since clinical symptoms have never been observed even in older fip virus infected mice (osterhaus and horzinek, unpublished observations), they might be inapparent carriers; this possibility is being investigated. the authors feel that their findings are significant in the virology of fip. although in vitro cell systems will certainly follow, important questions of virus characterization, demonstration of (cross-)neutralizing antibodies, virus attenuation etc. can be answered now, using an easily available small laboratory animal. feline infectious peritonitis (fip) virus multiplication was demonstrated in the brains of one-day-old laboratory mice using direct immunofluorescence tests. specificity was assessed by virus reisolation, indirect immunofluorescence and reproduction of fip after inoculation of spf kittens using brain material from the 6th mouse passage. virus der felinen infektiosen peritonitis 11. vermehrung im gehirn von sauglingsmausen mit hilfe der direkten immunofluoreszenz wurde die vermehrung des virus der felinen infektiosen peritonitis (fip) im gehirn eintagiger laboratoriumsmause nachgewiesen. die spezifitat wurde durch reisolierung des virus und indirekte immunofluoreszenz belegt, sowie durch die auslosung der fip nach inokulation von gehirnmaterial der sechsten mausepassage in spf katzenwelpen. virus de la pkritonite infectieuse du chat 11. multiplication dans le cerveau de la souris nouveau-nee a l'aide de l'immunofluorescence d,irecte la multiplication du virus de la pkritonite infectieuze fkline a ktk dkmontrke dans le cerveau de la souris nouveau-nke. epreuves de spkcificitk ktaient le rkisolement du virus, l'immunofluorescence indirecte et la reproduction de la maladie par inoculation de chats spf utilisant du material cerveau provenant du 62me passage en souris. virus de la peritonitis infecciosa del gato 11. multiplicacih en cerebro de ratoncitos recih-nacidos utilizando la inmunofluorescencia directa se mostr6 la multiplicaci6n en cerebro de ratoncitos recikn-nacidos del virus de la peritonitis infecciosa del gato. se pudo evidenciar la especificidad por medio de re-aislamiento del virus y de la inmunofluorescencia indirecta; ademis, se logr6 reproducir la enfermedad en gatos spf, inoculdndoles material del 6' pasaje en cerebros de ratoncitos. feline infectious peritonitis: experimental studies feline infectious peritonitis virus report of the isolation of an agent from cell cultures of kidneys from domestic cats coronaviruses: a comparative review untersuchungen zur atiologie der felinen infektiosen peritonitis (vorlaufige mitteilung) seroepidemiology of feline infectious peritonitis virus infections using transmissible gastroenteritis virus as antigen morphologic and physical characteristics of feline infectious peritonitis virus and its growth in autochthonous peritoneal cell cultures detection of transmissible gastroenteritis virus neutralizing antibody in cats immunofluorescent detection of viral antigens morphogenesis of a virus in cats with experimental feline infectious 11 feline infectious peritonitis: experimental evidence for its multiphasic nature untersuchungen iiber die antigenverwandtschaft der viren der felinen infektiosen peritonitis (fip) und der transmissiblen gastroenteritis (tge) des schweines the skilful technical assistance of miss ali kroon and mr. julius ana-kotta is highly appreciated. post mortem examination was kindly performed by j. koeman authors' address: instituut voor virologie, faculteit der dier eneeskunde, rijksuniversiteit utrecht, yalelaan 1 , practicumgebouw, utrecht, the netherlanfs. key: cord-329866-io9fvy58 authors: lorusso, eleonora; mari, viviana; losurdo, michele; lanave, gianvito; trotta, adriana; dowgier, giulia; colaianni, maria loredana; zatelli, andrea; elia, gabriella; buonavoglia, domenico; decaro, nicola title: discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis date: 2019-08-31 journal: research in veterinary science doi: 10.1016/j.rvsc.2017.10.004 sha: doc_id: 329866 cord_uid: io9fvy58 abstract intra-vitam diagnosis of feline infectious peritonitis (fip) is a challenge for veterinary diagnosticians, since there are no highly specific and sensitive assays currently available. with the aim to contribute to fill this diagnostic gap, a total of 61 effusions from cats with suspected effusive fip were collected intra-vitam for detection of feline coronavirus (fcov) antibodies and rna by means of indirect immunofluorescence (iif) assay and real-time rt-pcr (qrt-pcr), respectively. in 5 effusions there was no evidence for either fcov rna or antibodies, 51 and 52 specimens tested positive by iif and qrt-pcr, respectively, although antibody titres≥1:1600, which are considered highly suggestive of fip, were detected only in 37 effusions. three samples with high antibody levels tested negative by qrt-pcr, whereas 18 qrt-pcr positive effusions contained no or low-titre antibodies. qrt-pcr positive samples with low antibody titres mostly contained low fcov rna loads, although the highest antibody titres were detected in effusions with c t values>30. in conclusion, combining the two methods, i.e., antibody and rna detection would help improving the intra-vitam diagnosis of effusive fip. feline infectious peritonitis (fip) is a lethal disease of cats caused by a hypervirulent variant of feline coronavirus (fcov), an alphacoronavirus that usually causes self-limiting infections of the intestinal epithelium, leading to mild or no gastroenteric signs (addie et al., 2009) . two different fcov genotypes are currently known, fcov type i (fcov-i) and type ii (fcov-ii), both involved in the occurrence of mild gastroenteritis or fatal fip (decaro and buonavoglia, 2011) . fip is a perivascular pyogranulomatosis that may occur in two clinical forms, effusive and non-effusive fip, which are characterized by prevalence of effusions in the body cavities and of pyogranulomatous lesion in organs, respectively. fip diagnosis is challenging since the 'gold standard' is the post-mortem demonstration of fcov antigens in tissues by immunohistochemistry. therefore, alternative tools are commonly used for the intra-vitam diagnosis. haematological and biochemical analyses can support a presumptive diagnosis of fip, but they usually require further investigations, such as assessment of the fcov antibody titres and molecular detection of fcov rna in the effusions (effusive form) or bioptic samples (non-effusive fip) from ill cats. unfortunately, both methods lack specificity and sensitivity, thus often leading to an inconclusive diagnosis (addie et al., 2009) . recently, a comparison between the intra-vitam detection of fcov antibodies and that of fcov rna in the effusions of cats with confirmed fip has been carried out, showing a trend toward negative or low antibody levels in cats with high viral rna titres (meli et al., 2013) . however, these findings have not been confirmed by other studies. in the present paper, a total of 61 effusions from cats with confirmed fip have been screened for fcov antibodies and rna, suggesting that intra-vitam diagnosis of effusive fip needs to be assessed by means of combined antibody-and virus-detection methods. t reported (meli et al., 2013) . all samples were sent to our lab for fip confirmation by diagnostic labs that had carried out some preliminary analyses on the effusions, including rivalta's test, total proteins, albumin/globulin ratio, total leukocyte counts and identity of cells (table 1) . collected samples included 58 ascitic fluids and 3 pleuric effusions. for fcov antibody detection and titration, an indirect immunofluorescent (iif) assay was used (campolo et al., 2005) , with minor modifications. briefly, fcov-ii strain 25/92 (buonavoglia et al., 1995) was cultivated on crandell feline kidney (crfk) cells grown on coverslips. infected cells were fixed in acetone 100% and twofold dilutions of the effusion (starting from dilution 1:100 to 1:51,200) were tested. goat anti-cat igg conjugated with fluorescein isothiocyanate was used as secondary antibody solution (sigma aldrich srl). the assay was proven to detect both fcov-i and fcov-ii antibodies (addie and jarrett, 1992; campolo et al., 2005) . effusion with qrt-pcr positive and iif-negative results were treated with ammonium thiocyanate to dissociate immune complexes, as previously described (pullen et al., 1986; macdonald et al., 1988) . for fcov rna detection, 140 μl of the effusions were used for rna extraction by means of qiaamp® viral rna mini kit (qiagen s.p.a., milan, italy), following the manufacturer's protocol and the rna templates were stored at −70°c until their use. fcov reverse-transcriptase quantitative pcr (fcov qrt-pcr) was performed as previously described (gut et al., 1999) , with minor modifications. in brief, a one-step method was adopted using platinum® quantitative pcr supermix-udg (invitrogen srl, milan, italy) and the following 50-μl mixture: 25 μl of master mix, 300 nm of primers fcov1128f (gatttgatttggcaatg-ctagattt) and fcov1229r (aacaatcactagatccagacgttagct), 200 nm of probe fcov1200p (fam-tccattgttggctcgtcatagcg-ga-bhq1) and 10 μl of template rna. the employed oligonucleotides bind to the 3′ untranslated region (gut et al., 1999) . the thermal profile consisted of incubation with udg at 50°c for 2 min and activation of platinum taq dna polymerase at 95°c for 2 min, followed by 45 cycles of denaturation at 95°c for 15 s, annealing at 48°c for 30 s and extension at 60°c for 30 s. threshold cycle (c t ) number was used as the measure of viral load. the lower the c t , the more virus present in the sample. spearman's rank correlation coefficient was calculated to evaluate the possible correlation between viral rna loads and antibody titres by the use of the online tool social science statistics (http://www. socscistatistics.com/tests/spearman). fifty-one (48 ascitic and 3 pleuric fluids) of the 61 tested samples had fcov antibody (table 2 and fig. 1 ), although only 37 positive effusions contained antibody levels ≥ 1:1600, which are considered highly suggestive of fip diagnosis (hartmann et al., 2003) . additional 13 samples presented fcov antibody titres between 1:200 and 1:800, which are quite high for an enteric infection but cannot be considered enough high for a systemic infection. only one effusion had an antibody titre of 1:100 and two samples displayed an antibody titre of 1:51,200. by means of qrt-pcr, fcov rna was detected in a total of 52 samples (49 ascitic and 3 pleuric fluids). c t values were generally above 30 (mean c t value of 32.87), accounting for low viral titres, with higher viral rna loads (c t values < 30) being detected in only 11 effusions. by comparing the results qrt-pcr with those of iif assay using an antibody titre ≥ 1:1600 as cut-off (fig. 1b) , 6 samples tested negative by both assays (no viral rna and no fcov antibodies), possibly accounting for diseases other than fip, and 3 samples tested negative only by qrt-pcr, although they contained fcov antibody titres between 1:3200 and 1:12,800, which were highly suggestive of fip. eighteen effusions were found to contain fcov rna in the absence of specific antibodies (or at least in the presence of antibody titres < 1:1600); 5 of these qrt-pcr positive specimens had no fcov antibodies (or at least antibody titres < 1:100), while additional 13 effusions contained antibody titres ranging from 1:100 to 1:800, which are not considered as suggestive of fip. therefore, based only on antibody detection, a total of 18 cats whose effusions contained viral rna were predicted not to be affected by fip, while taking advantage on molecular detection of fcov rna, 3 animals with high antibody titres would have been considered fip negative. unfortunately, samples with fcov rna tested negative by iif even after treatment with the chaotropic thiocyanate ion, which had been proven to dissociate immune complexes pullen et al., 1986. most effusions displaying the highest viral loads (c t values < 30) contained antibody titres ≥ 1:1600; only one sample with a low c t value displayed an antibody titre (1:200) not suggestive of fip (table 2) . therefore, qrt-pcr positive samples with low antibody titres mostly contained low fcov rna loads, although the highest antibody titres were detected in effusions with c t values > 30. overall, no statistically significant correlation (r = 0.1178; twotailed p-value = 0.36576) was found between viral rna loads and antibody titres. intra-vitam fip diagnosis still represents a challenge for veterinarians and diagnosticians, since there is no available tool to unambiguously diagnose the disease. fip cannot be differentiated from an fcov enteric infection based on serology because the antibodies are directed against the same pathogen and there are no relevant antigenic differences between the enteric and hypervirulent strains. it is recognised that fip-ill cats have very high antibody titres in their serum and effusions due to the systemic spreading of the virus through the infected monocytes/macrophages (addie et al., 2009 ). however, detection of high antibody titres alone is not a confirmatory test. in addition, the absence of specific antibodies or the presence of very low antibody titres has been recently demonstrated in the effusions of cats with confirmed fip, likely due to antibody sequestration by the high number of viral particles in the same sample of some cats (meli et al., 2013) . hartmann et al. (2003) demonstrated that about 10% of cats with fip tested seronegative for fcov. however, in that study a transmissible gastroenteritis virus strain was used as antigen, which could affect the sensitivity of fcov-antibody testing (giori et al., 2011) . accordingly, fcov antibody titres were found to dramatically drop in terminal cases of fip (pedersen, 1995) . this phenomenon is not restricted to fip, but it has been also demonstrated for other viral infections characterized by high-level virus replication (quirós-roldán et al., 2000; guihot et al., 2014) . overall, detection of fcov antibodies in the effusions is affected by poor specificity and sensitivity. molecular methods have been used for detection of fcov rna in the effusions of cats with suspected fip (gut et al., 1999; simons et al., 2004; hornyák et al., 2012; soma et al., 2013; doenges et al., 2017; felten et al., 2017; longstaff et al., 2017) . however, these methods display similar issues related to the diagnostic performances (lack of sensitivity and specificity). in fact, they are not able to distinguish between enteric and virulent fcovs, since no specific genetic markers have been identified for the latter strains. in addition, the enteric fcovs have been proven to cause transient viremia and even have a low replication in the blood (can-sahna et al., 2007; kipar et al., 2010; fish et al., 2017) , thus potentially being able to passively spread to the effusions associated to other diseases. a recent paper (meli et al., 2013) has investigated the agreement between fcov antibody titres and rna detection in the effusions of 13 cats with confirmed fip, showing a correlation between high amounts of virus and lower signals in iif assay, likely due to the fact that antibodies bound to viral antigens of the effusions are not able to bind to the antigens of the fcov-infected cells used in serological tests. here, we have analysed by the same methods the effusions of 61 cats with suspected fip, thus including also potential samples from animals with non-fip related diseases. accordingly, using an iif antibody titre of 1:1600 as a cut-off, 5 samples tested negative by both iif and qrt-pcr assays, possibly accounting for diseases other than fip, while 21 effusions gave contrasting results (low-titre or no antibodies in the presence of fcov rna or viceversa). these 21 samples with conflicting results are likely to be true positive since an iif-negative result could be related to antibody sequestration by high viral loads (meli et al., 2013) . in addition, addie et al. (2015) demonstrated that up to 43% antibodypositive effusions from fip cases were negative for fcov rna, likely as a consequence of pcr inhibition by interfering substances or rna degradation during sample transportation and storage. however, in the absence of alternative diagnosis, even those 5 cats with neither fcov antibodies nor rna in their effusions could not be definitively considered as non-fip animals (addie et al., 2015) . unfortunately, clinical cases were mostly untraceable and confirmatory necropsy was not done in any case, so that the lack of confirmatory testing represents the main limitation of the present study. in contrast with what observed by meli et al. (2013) , there was no statistically significant correlation between high viral loads and lowtitre or negative antibody results. in fact, most effusions with low or no fcov antibody titres displayed low amounts of virus, although samples with very high levels of fcov rna contained slightly lower antibody titres (generally < 1:3200) in comparison with effusions with the lowest amounts of virus, which reached iif antibody titres of 1:26,600-1:51, 200 ( table 2) . the present study confirms that, when performed singularly, neither the detection of fcov nucleic acid nor that of specific antibodies in the effusions of cats with suspected fip is able to warrant an affordable diagnosis of the disease. therefore, in order to increase the diagnostic performances, we suggest combining the two methods (antibody and rna detection) for an intra-vitam diagnosis of effusive fip. using this diagnostic approach, only 6 out of 61 cats whose effusions were analysed would be considered fip negative, even if also in these cases fip could not be completely ruled out (meli et al., 2013) . thus, the combined serological and molecular protocol should improve the ability of fig. 1 . comparison between indirect immunofluorescence (iif) assay and real-time rt-pcr (qrt-pcr) carried out on 61 effusions from cats with suspected feline infectious peritonitis (fip) . numbers indicate the samples positive (+) or negative (−) for fcov antibodies or rna. results according to both techniques are shown in bold. for iif assay, the cut-off was set to 1:100 (a) or 1:1600 (b), the latter being considered highly suggestive of fip. laboratories to diagnose effusive fip, especially if the test results are supported by clinical and haematological findings. however, intravitam diagnosis of non-effusive fip still remains highly inconclusive, even if recent studies tried to address this issue (doenges et al., 2016) . therefore, future studies are needed to develop and validate tools for the intra-vitam diagnosis of non-effusive fip, which still represents a challenge for veterinary diagnosticians. there is no conflict of interest of any authors in relation to the submission. feline coronavirus antibodies in cats feline infectious peritonitis. abcd guidelines on prevention and management utility of feline coronavirus antibody tests isolamento e caratterizzazione di uno stipite di virus della peritonite infettiva felina identification of a feline coronavirus type i strain from a cat with feline infectious peritonitis by rt-pcr and phylogenetic analysis the detection of feline coronaviruses in blood samples from cats by mrna rt-pcr canine coronavirus: not only an enteric pathogen detection of feline coronavirus in cerebrospinal fluid for diagnosis of feline infectious peritonitis in cats with and without neurological signs comparison of realtime reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis detection of feline coronavirus spike gene mutations as a tool to diagnose feline infectious peritonitis a cross-sectional quantitative rt-pcr study of feline coronavirus viremia and replication in peripheral blood of healthy shelter cats in southern california performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases low titers of serum antibodies inhibiting hemagglutination predict fatal fulminant influenza a(h1n1) 2009 infection one-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses comparison of different tests to diagnose feline infectious peritonitis detection of subgenomic mrna of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (p-sg-qpcr) sites of feline coronavirus persistence in healthy cats feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis the measurement of relative antibody affinity by elisa using thiocyanate elution samples with high virus load cause a trend toward lower signal in feline coronavirus antibody tests the history and interpretation of feline coronavirus serology antibody avidity determination by elisa using thiocyanate elution anti-hepatitic c virus antibodies hidden in circulating antibody/antigen aggregates in hcv-rna positive patients a mrna pcr for the diagnosis of feline infectious peritonitis detection of ascitic feline coronavirus rna from cats with clinically suspected feline infectious peritonitis this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-324530-tac1unnp authors: andré, nicole m; cossic, brieuc; davies, emma; miller, andrew d; whittaker, gary r title: distinct mutation in the feline coronavirus spike protein cleavage activation site in a cat with feline infectious peritonitis-associated meningoencephalomyelitis date: 2019-06-26 journal: jfms open rep doi: 10.1177/2055116919856103 sha: doc_id: 324530 cord_uid: tac1unnp case summary: this report describes a cat with chronic, progressive, non-painful, non-lateralizing multifocal neurologic clinical signs associated with feline infectious peritonitis (fip). the cat initially presented as underweight, despite a good appetite, and a complete blood count showed non-regenerative anemia. three months later the cat was returned having developed ataxia and paraparesis, which then progressed over 2 months to tetraparesis, tail plegia, urinary and fecal incontinence, and titubation. histologic examination of the tissues with subsequent immunohistochemistry confirmed fip-associated meningoencephalomyelitis following necropsy. molecular analysis of the coronavirus spike protein within the tissues identified a specific, functionally relevant amino acid change (r793m), which was only identified in tissues associated with the central nervous system (ie, brain and spinal cord). relevance and novel information: this case report describes an early presentation of a cat with primarily neurologic fip, with molecular characterization of the virus within various tissues. feline infectious peritonitis (fip) is caused by feline coronavirus (fcov) and is widely considered to be one of the most significant infectious diseases to affect the feline population. [1] [2] [3] it is the most common infectious disease of the central nervous system (cns) of cats. 4 fcovs have been reported to exist as two distinct serotypes: type i (more common) and type ii viruses, 5 each with distinct biological properties. 5, 6 both fcov serotypes have distinct 'biotypes'. these are typically classified as either feline enteric coronavirus (fecv) or feline infectious peritonitis virus (fipv), with the biotypes differing based on the severity of infection in cats. [7] [8] [9] infection with fcov is common, especially in highdensity housing situations such as animal shelters and breeding facilities. 10 the fecv biotype transmits readily and causes only a mild infection, with transmission occurring via fecal-oral and possibly other routes. 7, 11 if the viral infection worsens and becomes systemic (typically infecting macrophages), then the virus is classified as the fipv biotype. 8 such viruses are believed to contain an 'internal mutation' that accounts for the altered tropism, although the nature of this mutation is not well understood. 12 clinical signs associated with the fipv biotype can be quite variable and non-specific, and can include fever, lethargy, anorexia, pica, vomiting and diarrhea. 13 these clinical signs can be present in either the 'wet', 'dry' or 'mixed' presentations. 14 the wet form of fip is characterized by an effusion in the abdominal and/or thoracic or pericardial cavities, and the 'dry' form by the presence of pyogranulomatous lesions. the 'mixed' form may present with an array of clinical signs. most commonly, neurologic clinical signs are associated with the 'dry' form but can occur with all presentations and may be the sole clinical sign observed. 12, 13 clinical features of neurologic fip can include, but are not limited to, ataxia, head tremors, seizures and/or paresis. 12, 13, 15 ocular lesions may be present with or without lesions in the cns. fip-associated pathologic changes to the cns include meningitis, encephalitis, ependymitis and choroid plexitis, often with concurrent vasculitis. 12,13 fip presenting with predominantly neurologic clinical signs provides a diagnostic challenge and definitive ante-mortem diagnosis is difficult. mri has been identified as a sensitive method of diagnosis in conjunction with clinical signs and cerebrospinal fluid analysis results such as elevated protein levels and neutrophilic pleocytosis. 16 however, such findings are still not specific to fip and may be financially prohibitive. fip may also be considered a diagnosis of exclusion, following evaluation of clinical signs, history and physical examination findings and biochemical values. 12, 13 this case report describes a cat with neurologic fip that progressed over several months. the observations and findings obtained in this case provide support that fip can present predominantly in the cns. when molecular techniques are applied to the virus, a propensity for certain mutations can be associated with specific clinical presentations or pathological changes. an intact female 8-week-old domestic shorthair cat was taken into a foster/rescue home and cohabited a house with approximately nine other cats. the facility had a periodic history of fip cases, including two deaths in the previous 4 months. the cat was co-housed in a large open sunroom containing seven litterboxes, which were cleaned once daily. the diet consisted of commercially available dry and canned food, which was separately offered in individual dishes. the cat was not rabies vaccinated, but had obtained two feline viral rhinotracheitis, calicivirus and panleukopenia vaccinations. at 14 weeks of age, the cat was presented to a general practitioner for evaluation of poor weight gain, soft stool and upper respiratory tract infection. the cat was underconditioned and weighed 2.5 lb (1.1 kg), with a body condition score (bcs) of 2/5, despite being active, alert and having a good appetite. conjunctivitis and a yellow mucopurulent discharge from the nares were noted, and the cat had a fever of 102.6°f (39.2°c). a fecal flotation was performed owing to the soft but formed stool, and no ova or parasites were detected. a complete blood count (cbc) and chemistry profile were performed (tables 1 and 2 ). the chemistry profile showed marked elevations in alkaline phosphatase, alanine transferase and phosphorus levels. a decrease in creatinine and albumin was also noted (table 1) , along with mild anemia and monocytosis. amoxicillin clavulanic acid (clavamox drops; zoetis) 62.5 mg/ml was dispensed and administered at 15.6 mg (12 mg/kg) po q12h for 10 days. blood parameters were re-evaluated at 20 weeks of age using a less defined panel and values were within the normal range (table 1) . at approximately 6 months of age, the cat returned to the general practitioner for evaluation of pelvic limb gait abnormalities that had progressed over the previous 2 weeks. examination revealed symmetric pulses in both hindlimbs and the presence of a pain response; however, less of a response was noted on the right side. paresis was observed in the right hindlimb. when the forelimbs were lifted, the cat was able to walk minimally on the hindlimbs. the cat had severe non-ambulatory paraparesis, with more severe deficits on the right side. no information about spinal reflexes was available. a cbc and chemistry panel were performed (tables 1 and 2 ). the chemistry panel revealed hypoalbuminemia, a decrease in the albumin:globulin (a:g) ratio, low creatinine values and hyperphosphatemia. the cbc revealed a slight anemia, monocytosis and thrombocytopenia. platelet clumping was noted upon microscopic evaluation. meloxicam (metacam oral suspension) was dispensed and a single 0.2 mg dose was administered orally. at 8 months of age, the cat was returned to the general practitioner due to progression of the paraparesis. the client noted further deterioration of the pelvic limb paresis, and now identified 'stiffness' in the thoracic limbs. there was no information about the pelvic limb reflexes; however, the cat had started to have occasional urinary and fecal incontinence. appetite seemed normal; however, the cat remained thin. physical examination revealed a temperature of 101.5°f (38.6°c), heart rate of 170 beats per min and respiratory rate of 30 breaths per min. bcs was 3/9; however, the weight was not noted. abdominal palpation revealed a large, easily expressible urinary bladder. neurologic examination findings revealed normal mentation with no involuntary movements such as tremors. the cat was still very ataxic and ambulatory but now tetraparetic, which was much worse in the pelvic limbs. there was no information about cranial nerve abnormalities, and the eyes and retinas were within normal limits. from a video provided by the owner (see supplementary material), tail paresis was identified. the lesion was considered to affect the cns and was localized as multifocal. a fecal flotation and direct smear were evaluated, with no ova or parasites seen. cryptococcus and toxoplasma antibody titers were performed and were negative. the tetra-ataxia and paresis significantly worsened over the next few months. additionally, the cat now had titubation, tail plegia (see video in the supplementary material) and consistent urinary and fecal incontinence. owing to the grave prognosis, the client elected humane euthanasia, at which time the cat was 10 months (40 weeks) of age. a necropsy was performed at the animal health diagnostic center, cornell university college of veterinary medicine, and this revealed no significant gross abnormalities outside of mild mesenteric lymphadenomegaly. representative sections of all organs, including the entire brain and spinal cord, were fixed in 10% neutral buffered formalin from which sections were cut, stained with hematoxylin and eosin, and analyzed via light microscopy. immunohistochemistry for fcov was carried out using monoclonal antibody fipv3-70 (1:1000), ap-anti-mouse igg and bond polymer refine red detection (leica microsystems). histologic examination revealed lesions typical of fcov infection within the cns. in the spinal cord, the leptomeninges were diffusely expanded by moderate numbers of predominantly plasma cells, admixed with fewer lymphocytes and macrophages, and surrounded by a moderate amount of edema. the underlying white matter was multifocally vacuolated with numerous dilated myelin sheaths, digestion chambers and rare spheroids (figure 1a) . at the level of the lateral aperture, the choroid plexus was expanded by large numbers of plasma cells, lymphocytes and macrophages (figure 1b) . the ependyma lining the ventricular system was effaced by a similar inflammatory population, admixed with fibrin, edema and was also forming thick perivascular cuffs often disrupting the sub-ependymal parenchyma (figure 1c) . immunohistochemistry revealed strong intracytoplasmic immunoreactivity within macrophages (figure 1d ). no fipassociated lesions were present in other organs. non-fcov comorbid histologic findings were chronic enteritis with mid-mucosal fibrosis and mesenteric lymphoid hyperplasia. molecular analysis of the viral spike protein was performed at several time points during the study. fecal samples were collected at 5 months of age (feces #1) and at 8 months of age (feces #2). following euthanasia (at 10 months of age) tissue samples were collected, along with a fecal sample (feces #3). a central 156 base pair region of the spike protein gene, including the critical s1/s2 activation site of the virus, was pcr amplified and sequenced as described in licitra et al, 17 with the following modifications: 25 μl reverse transcription pcrs were performed with qscript xlt 1-step rt pcr kit (quantbio). pcr conditions were 20 mins at 50°c, 3 mins at 95°c and 40 cycles of 10 s at 95°c, 20 s at 55°c, 40 s at 72°c, then 10 mins at 72°c. pcr products were purified using diffinity rapidtips (diffinity genomics). pcr and sequencing showed the presence of a type i fcov, based on a sequence alignment with reference genomes. the sequence information obtained from this cat is shown in figure 2 . the viral sequences from the cns (brain and spinal cord) contained specific amino acid changes compared with other samples (feces, small intestine, mesenteric lymph node and kidney). the most notable change was an arginine to methionine (r-m) substitution at the critical p1 activation position, 17 corresponding to residue 793. other changes that correlated with viruses present in the cns were present in two other positions: 770 alanine to valine (a-v); and 786 threonine to alanine (t-a). here we report clinicopathologic findings and molecular analysis of a cat with progressive neurologic clinical signs associated with fip. the cat initially presented to the referring veterinarian with respiratory signs and fever, and with abnormal liver enzyme function and anemia. at this time fip was not suspected. these initial signs resolved but were replaced by progressing neurologic signs, which led ultimately to euthanasia and submission to the study for evaluation of fcov involvement. upon euthanasia, fcov was found in various tissues in the cat, including the cns. however, histologic examination revealed fcov-associated pathology only within the cns, where there was meningoencephalomyelitis, ependymitis, choroid plexitis and vasculitis. histo logical lesions were compatible with a recent report describing meningoencephalitis in four cats with fip. 18 molecular analysis of the viral spike protein within the tissues identified a specific, functionally relevant amino acid change (r793m), which was only identified in tissues associated with the cns (ie, brain and spinal cord). the r793m mutation in the spike protein s1/s2 cleavage-activation site is a major chemical change from a basic to a hydrophobic residue, and is consistent with an elimination of furin-mediated proteolytic processing of the s protein, as seen by licitra et al, 17 and a proposed change in the activation properties and entry pathway of the virus. it is interesting to note that the r793m mutation was not present in other tissues tested in this cat at the time of necropsy but was found in our previous study (cat id #08-153990), 17 where samples were of neural origin. while biological confirmation is not available, we consider that the other changes found in the viral spike protein from central nervous tissue of this cat (a770v and t786a) are not related to changes in the activation properties and entry pathway of the virus, as they are not in defined functional regions of the spike protein and are not markedly different in their chemistry. interestingly, all samples tested from this cat contained a distinct leucine residue at position 791 (the p3 position of the furin cleavage site, typically serine or alanine as defined in licitra et al). 17 the relevance of this is currently unclear. overall, our results provide evidence that mutation of the viral spike protein is linked to fip outcome, specifically in the s1/s2 cleavage-activation site (residues 789-794). mutations leading to fip have also been linked to changes in other areas of the spike gene (position 1058) 19 and in the 3c gene. 20, 21 to compare our findings for the s1/s2 region to other proposed fip-linked mutations, we performed additional sequencing, which is summarized in table 3 . all fecal samples, as well as a kidney sample, contained methionine (m) at spike position 1058, indicating that an 'enteric' form of fcov 22 was present in the cat throughout our study. in contrast, samples from the brain and spinal cord contained leucine (l) at position 1058, indicating an fip virus. an intact 3c gene was found in feces, with the 3c gene in neural and other tissues truncated and/or deleted depending on the sample tested. this case report describes a young cat with neurologic fip in which detailed clinical and molecular characterization of the associated fcov infection was performed. while the etiology of fip remains complex and likely involves multiple mutations in the viral genome, our results indicate that a specific mutation of the viral spike protein can be associated with infection of the cns, which may explain the tropism to the cns as opposed to other organ systems. an update on feline infectious peritonitis: diagnostics and therapeutics an update on feline infectious peritonitis: virology and immunopathogenesis cns disease in the cat: current knowledge of infectious causes prevalence of diseases of the spinal cord of cats fenner's veterinary virology improving virus taxonomy by recontextualizing sequence-based classification with biologically relevant data: the case of the alphacoronavirus 1 species infectious diseases of the dog and cat a review of coronavirus infection in the central nervous system of cats and mice immunocytochemical demonstration of feline infectious peritonitis virus within cerebrospinal fluid macrophages practical overview of common infectious disease agents hagan and brunner's microbiology and infectious diseases of domestic animals diagnosis and clinical signs of feline infectious peritonitis in the central nervous system a retrospective study of the neuropathology and diagnosis of naturally occurring feline infectious peritonitis feline infectious peritonitis with neurologic involvement: clinical and pathological findings in 24 cats feline infectious peritonitis with spinal cord involvement in two cats clinicopathologic features and magnetic resonance imaging findings in 24 cats with histopathologically confirmed neurologic feline infectious peritonitis mutation in spike protein cleavage site and pathogenesis of feline coronavirus immunohistochemical studies on meningoencephalitis in feline infectious peritonitis (fip) spike protein fusion peptide and feline coronavirus virulence significance of coronavirus mutants in feces and diseased tissues of cats suffering from feline infectious peritonitis feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis acknowledgements we thank wendy wingate for help with sample collection, all members of the whittaker lab for helpful comments and support, and dr john loftus for clinical consultation and critical reading of the manuscript. video of cat at 8 months and 10 months of age. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. center.ethical approval this study involved the use of clientowned animal(s) only, and followed internationally recognized high standards ('best practice') of individual veterinary clinical patient care. ethical approval from a committee was not therefore needed.informed consent informed consent (either verbal or written) was obtained from the owner or legal guardian of all animal(s) described in this study for the procedure(s) undertaken. for any animals or humans individually identifiable within this publication, informed consent for their use in the publication (verbal or written) was obtained from the people involved.orcid id nicole m andré https://orcid.org/0000-0002-3703-5026 key: cord-287157-6rwevq39 authors: kiss, i.; poland, a.m.; pedersen, n.c. title: disease outcome and cytokine responses in cats immunized with an avirulent feline infectious peritonitis virus (fipv)-ucd1 and challenge-exposed with virulent fipv-ucd8 date: 2004-02-25 journal: j feline med surg doi: 10.1016/j.jfms.2003.08.009 sha: doc_id: 287157 cord_uid: 6rwevq39 eight cats were immunized with an avirulent strain of feline infectious peritonitis virus (fipv)-ucd1, then challenge-exposed to a highly virulent cat passaged strain (fipv-ucd8). th1 and th2 cytokine profiles in the peripheral blood mononuclear cells (pbmcs) were measured throughout in the experiment. no clinical signs of fip were evident in the experimental cats after immunization. after challenge, the immunized cats demonstrated one of four clinical outcomes: (1) classical effusive fip; (2) accelerated fip; (3) non-effusive fip, or (4) resistance to challenge. only minor cytokine changes were observed following immunization, however, several cytokine changes occurred following challenge-exposure. the most noteworthy changes were in tumor necrosis factor-alpha (tnf-α) and interferon gamma (ifn-γ) levels. our preliminary findings suggest that immunity against fip is associated with tnf-α and ifn-γ response imbalance, with high tnf-α/low ifn-γ mrna responses favouring disease and low tnf-α/high ifn-γ mrna responses being indicative of immunity. feline infectious peritonitis (fip) is a highly fatal disease in felidae caused by a coronavirus and usually affects cats between 6 months and 3 years of age (reviewed by pedersen, 1995) . the fipv is a common mutant of the far more ubiquitous and largely non-pathogenic feline enteric coronavirus (fecv) (pedersen, 1987a; vennema et al., 1998) . the enteric virus is highly tropic to the mature epithelium of the small intestine, whereas the mutant fipv gains a tropism for macrophages. this macrophage tropism allows the virus to disseminate throughout the body and is responsible for pathogenicity (stoddart and scott, 1989 ). the disease presents in two major forms: (1) an effusive form associated with peritonitis and/or pleuritis and vasculitis, and (2) a non-effusive form characterized by more classical granulomatous disease of major abdominal organs, uveal tract, and meninges (reviewed by pedersen, 1995) . both forms of the disease are uniformly lethal, with affected cats dying over several days to many months. immunity to fipv is presumed to be cellular (pedersen, 1987b) , as is typical of highly cell-associated pathogens (tizard, 2000) . antibody responses, by themselves, are actually harmful by facilitating virus uptake by macrophages and participating in arthus like reactions (pedersen and boyle, 1980) . therefore, whether a cat develops wet or dry fip is thought to depend on the strength of the cat's cell-mediated immune response. cats producing a negligible cell-mediated response and a strong antibody response rapidly develop acute wet fip. cats producing a partial cell-mediated response develop the chronic form of the disease. there is strong circumstantial evidence that susceptibility to fip has a significant genetic component (foley et al., 1997) . the goal of developing effective fipv vaccines has been elusive. most vaccines either fail to protect or enhance the infection (pedersen, 1989) . partial success has been obtained by using temperature sensitive mutants of a type ii strain of fipv (gerber et al., 1990; hoskins et al., 1995; reeves et al., 1992) . we were fortunate to create another avirulent fipv, but to a preferred type i rather than type ii strain (fipv-ucd1). type ii strains are recombinants with canine coronavirus, while type i strains are uniquely cat. type i strains cause 70-95% or more of disease (hohdatsu et al., 1992) . we decided to test our avirulent strain of fipv-ucd-1 as a vaccine. after primary vaccination cats were then challenge-exposed to a highly virulent type i strain of fipv (fipv-ucd8). in order to see whether there was a relationship between vaccine immunity and challenge-exposure outcome, we also analysed th1 and th2 cytokine profiles during immunization and following challenge-exposure. ten 5-month-old male cats were obtained from the specific pathogen free breeding colony of the feline nutrition laboratory, school of veterinary medicine, uc davis, usa. cats were housed in the facilities of the center for companion animal health, school of veterinary medicine, uc davis, usa under the supervision of the center for laboratory animal sciences. eight cats were inoculated intraperitoneally, first with a non-pathogenic strain of fipv-ucd1, then 32 days later with pathogenic fipv-ucd8. attenuated fipv-ucd1 was passaged on fcwf-4 cells and animals inoculated with 1 ml of infectious tissue culture fluid. fipv-ucd8 was serially passaged in laboratory cats and infectious material was in the form of 1 ml of pooled ascitic fluid. this fluid had been harvested from cats dying of experimentally induced effusive fip. two control animals were mock vaccinated and challenge-exposed with pbs. the clinical status of the cats was monitored throughout the experiment. three millilitres of heparinized blood were collected at the following time points: day −3, day 0 (time of experimental infection with ucd-1), day 4, day 7, day 11, day 14, day 18, day 21, day 28, day 32 (time of experimental infection with ucd-8), day 35, day 39, day 42, day 46, and day 49. all animals were euthanased on day 49. a complete necropsy was performed on each animal, including histologic examination of a range of potential target tissues. necropsies were done to confirm the form of disease and to rule out the presence of subclinical infections in animals that appeared outwardly normal (hoskins et al., 1995) . blood samples were immediately centrifuged in order to separate the buffy coat and plasma for cytokine analysis and serology, respectively. all fractions were stored at −80°c until further processing. three time points (day 21, day 42, and day 49) were selected for the serological investigations of the plasma samples by ifa technique using serum dilutions of 1:25 and 1:100 (pedersen, 1976) . ifa substrate slides were made from fcwf-4 cells infected with fipv-ucd1. cytokine mrna measurements from unstimulated peripheral blood mononuclear cells (pbmc) were performed for the following cytokines: il4, il6, il10, il12 p40, il18, ifn-, and tnf-as described previously (kipar et al., 2001; leutenegger et al., 2000) . relative cytokine mrna levels were determined at the time points indicated above and calibrated against cytokine mrna levels measured 3 days prior to immunization and from the two control animals. experimentally infected cats were euthanased and necropsied when it became obvious that their disease was terminal or at the completion of the study (day 49). the form of fip was determined grossly and histologically. no clinical signs of illness were observed in the eight experimental cats after immunization with fipv strain ucd1, except for slight rise in rectal temperatures lasting 1 to 3 days (data not shown). all but one (cat 622) vaccinated cats seroconverted, but to low titer (table 1) . three of eight vaccinated cats (nos 522, 616, 622) developed effusive fip within 2 weeks of challengeexposure to fipv-ucd8, typical of classical nonenhanced disease (pedersen and boyle, 1980) ( table 1) . one of the eight cats (no. 527) developed fip within 4 days, characteristic of enhanced disease (pedersen and boyle, 1980) . two cats (nos 623, 624) developed non-effusive fip. two of eight cats (nos 524, 625) showed no signs of illness, as did the two control animals. secondary antibody responses appeared to reflect disease outcome (table 1 ). the three cats (nos 522, 616, 622) dying of classical effusive fip had a rise in antibody titer post-challenge-exposure. cats nos 623 and 624, which developed non-effusive fip, had low primary antibody responses at day 21 postimmunization and with the same or decreased antibody titers 10 days post-challenge-exposure (day 42). the antibody titers of the two cats (nos 524 and 625) that resisted challenge-exposure declined to negligible levels even after challengeexposure with fipv-ucd8. no major changes in cytokine mrna levels were detected following the initial non-pathogenic fipv-ucd1 immunization (figs. 1-4 ) when compared to individual pre-infection values and to parallel cytokine responses in the two control cats (data not shown). a moderate elevation of tnf-mrna occurred between the second and third week after vaccination in cat 527. a small post-immunization increase of the ifn-mrna level was seen in cat 524, which was one of the ultimate survivors. various cytokine mrna changes were observed following challenge-exposure with fipv-ucd8 (figs. 1-4) . il-4 and il-6 mrna levels did not change from pre-infection or control cat levels in any of the eight infected animals. slight to low increases in il-10 mrna were seen following fipv-ucd8 infection in all cats, while il-18 mrna levels increases were negligible to low following challengeexposure and bore no relationship to disease outcome (data not shown). changes, or lack thereof, were deemed significant for three cytokines mrnas, ifn-, tnf-and il-12p40. the level of ifn-mrna was strongly elevated in one of the surviving two cats; in the other one it remained unaltered. cats that developed fip had negligible or below normal ifnresponses, save cat 527 that showed slightly elevated levels of this cytokine on the day of challenge-exposure. all of the cats that developed fip, regardless of form, had elevated postchallenge-exposure levels of tnf-mrna. il-12 mrna responses were increased following infection with fipv-ucd8, but did not appear to relate to disease outcome or ifn-/tnf-mrna responses. fipv-ucd1 immunization induced only partial protection at best, as gauged against historical data. animal-passaged fipv-ucd8 usually kills from 90-100% of inoculated cats, almost always from effusive fip (nc pedersen, uc davis, unpublished information). in this study, two of eight vaccinated cats (nos 524 and 625) appeared immune to challenge-exposure with virulent fipv-ucd8 and two (nos 623 and 624) developed non-effusive fip (indicative of partial immunity; pedersen, 1995) . three immunized cats (nos 522, 616, and 622) died of the classical form of effusive fip, indicating a predominance of non-protective humoral immunity. one cat (no. 527) developed a highly accelerated form of effusive fip, indicating that immunization had elicited enhancing type antibodies (pedersen and boyle, 1980) . therefore, immunization induced a spectrum of immune effects, ranging from protection (two cats), partial immunity (two cats), typical humoral immunity (three cats), to antibody enhancement (one cat). this reflects past experiences with attenuated live fipv vaccines (mcardle et al., 1995; pedersen and black, 1983; scott et al., 1995) . although this pilot study of a potential fip vaccine was deemed largely a failure as far as protection was concerned, there were interesting findings in regards to th1 and th2 type cytokine responses and post-challenge exposure disease course. we were fortunate in this study to have cats representing each of the four possible disease outcomes: (1) classical effusive fip occurring about 2 weeks following infection; (2) enhanced fip occurring almost immediately after challenge, (3) non-effusive fip, and (4) resistance. based on the cytokine profile analysis of the different groups, we suggest that disease, regardless of form, is associated with a strong tnf-mrna response in pbmc and a failure to induce ifn-mrna. in contrast, immune cats failed to upregulate tnf-mrna and one manifested strong ifn-mrna responses. the former profile tends to favor th2 (humoral) immunity, while the latter favors th1 (cell-mediated) immun-ity. fipv is an intracellular pathogen of macrophages, and as such, cell mediated immunity would be most important. it was interesting to note the relationship between responses to avirulent or virulent virus and the magnitude and even duration of cytokine mrna responses in pbmcs. cats infected with the avirulent fipv only showed transient fevers (data not shown) and low to negligible changes in cytokine mrnas in pbmcs. the cat that had the most pronounced primary cytokine responses was also the animal that developed the most severe febrile reaction to the vaccine. this indicated that changes in cytokine mrnas within pbmcs were only noticeable when reactions within internal lymphoid organs reached a certain threshold, thus allowing the responses to spill over into the blood. this was also observed after challenge-exposure. cats that became very sick with fip tended to have marked upregulation of cytokine mrnas in their pbmcs. a dichotomy in responses was seen between the two cats that were immune to challenge exposure with virulent fipv-ucd8. cat no. 524 developed weak cytokine reactions during immunization and strong cytokine responses following challenge-exposure, while cat 625 showed very little changes during either immunization or challenge-exposure. this suggested that cat no. 524, while being immune, was none the less infected by the challenge virus and did mount a vigorous secondary immune response (at the cytokine, but not antibody level). in contrast, cat 625 appeared to develop exceptionally strong protective immunity from the onset, precluding the need for a systemic response. therefore, the magnitude and duration of cytokine mrna responses in pbmcs does not always correlate with strength of immunity. the importance of ifn-responses in fipv immunity is strongly supported by what has been described recently for both experimental (kyuwa et al., 1998a,b) and natural (france et al., 1999) mouse hepatitis virus (mhv) infections. mhv, like the feline coronaviruses (horzinek et al., 1995) , exists in two biotypes, a naturally occurring and largely enterotropic biotype and a more laboratoryassociated polytropic biotype (homberger et al., 1998) . the enterotropic biotype of mhv is analogous to fecv, while polytropic biotypes have many parallels with fipv. the similarities between feline and murine coronaviruses and their biotype-associated diseases were quickly noted following the creation of ifn-deficient mice. ifnknockout mice developed severe peritonitis, identical to fip, upon experimental challenge with a polytropic mhv (kyuwa et al., 1998a (kyuwa et al., , 2002 and this disease could be partially inhibited with exogenously administered ifn(kyuwa et al., 2002) . this supports our findings on the importance of ifn-mrna responses in cats exposed to fipv. a granulomatous peritonitis and pleuritis has also been described in a colony of ifn-knockout mice infected naturally with enterotropic mhv (france et al., 1999) . this latter observation parallels what is seen in a group of retrovirus immunocompromised cats exposed to fecv (poland et al., 1996) . other similarities exist between murine and feline coronavirus infections. age and genetic factors have been shown to play a role in naturally occurring mhv infection, with 1 week olds being more susceptible than 3 and 12 weeks, and balb and icr mice more susceptible than sjl mice (barthold, 1987) . age and genetic susceptibility have been shown to be important risk factors for fip in purebred cats (foley et al., 1997; foley and pedersen, 1996) . the central question coming out of this preliminary experiment and previous mhvrelated studies concerns the role of ifn-in fipv immunity. in the presented preliminary experiment, vaccination of cats with an attenuated live strain of fipv-ucd1 appeared to induce a degree of protection, in that two of eight cats were immune and two more developed non-effusive fip post challenge. a more significant finding was the possible relationship between certain cytokine mrna responses and disease outcome. cats developing fip after challenge-exposure failed to demonstrate ifn-mrna responses in pbmcs, but did make high levels of tnf-mrna. conversely, immune cats did not make detectable levels of tnf-mrna, and one made markedly high level of ifn-. although only a pilot study, the findings are supported by parallel observations in mhv disease in ifn-knockout mice, and suggest the need for more in depth studies of the role of ifn-in fipv disease and immunity. host age and genotype effects on enterotropic mouse hepatitis virus infection inheritance of susceptibility to feline infectious peritonitis in purebred catteries risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus granulomatous peritonitis and pleuritis in interferon-gamma gene knockout mice naturally infected with mouse hepatitis virus protection against feline infectious peritonitis by intranasal inoculation of a temperaturesensitive fipv vaccine the prevalence of types i and ii feline coronavirus infections in cats prevalence of enterotropic and polytropic mouse hepatitis virus in enzootically infected mouse colonies perspectives on feline coronavirus evolution independent evaluation of a modified live feline infectious peritonitis virus vaccine under experimental conditions (louisiana experience) cytokine mrna levels isolated feline monocytes acute hepatic failure in ifn-gammadeficient balb/c mice after murine coronavirus infection mhv-induced fatal peritonitis in mice lacking ifn-gamma murine coronavirus-induced subacute fatal peritonitis in c57bl/6 mice deficient in gamma interferon immunization of cats against feline immunodeficiency virus (fiv) infection by using minimalistic immunogenic defined gene expression vector vaccines expressing fiv gp140 alone or with feline interleukin 12 (il-12), il-16, or a cpg motif independent evaluation of a modified live fipv vaccine under experimental conditions (university of liverpool experience) an overview of feline enteric coronavirus and infectious peritonitis virus infection animal virus infections that defy vaccination. equine infectious anemia, caprine arthritis encephalitis, maedi-visna, and feline infectious peritonitis virologic and immunologic aspects of feline infectious peritonitis an overview of feline enteric coronavirus and infectious peritonitis virus infections serologic studies of naturally occurring feline infectious peritonitis attempted immunization of cats against feline infectious peritonitis using either avirulent live virus or sublethal amounts of virulent virus immunologic phenomenon in the effusive form of feline infectious peritonitis two related strains of feline infectious peritonitis isolated from immunocompromised cats infected with feline enteric coronavirus long-term follow-up study of cats vaccinated with a temperaturesensitive feline infectious peritonitis vaccine independent evaluation of a modified live fipv vaccine under experimental conditions (cornell experience) intrinsic resistance of feline peritoneal macrophages to coronaviruses correlates with virulence resistance to viruses feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses funding for i. kiss was provided by a fellowship from the fulbright foundation and laboratory costs by the center for companion animal health center (ccah), school of veterinary medicine, university of california, davis, ca, usa, 95616. additional thanks go to jill mikovich and the animal care staff at the ccah for their care of the animals, and to dr árpád bacsadi, veterinary institute of debrecen, p.o. box 51, h-4002, debrecen, hungary for histopathological studies. we wish to also thank dr christian leutenegger and the taqman ® service, uc davis for invaluable assistance with cytokine assays. key: cord-336639-jaue41mv authors: simons, fermin a.; vennema, harry; rofina, jaime e.; pol, jan m.; horzinek, marian c.; rottier, peter j.m.; egberink, herman f. title: a mrna pcr for the diagnosis of feline infectious peritonitis date: 2004-12-21 journal: j virol methods doi: 10.1016/j.jviromet.2004.11.012 sha: doc_id: 336639 cord_uid: jaue41mv a reverse transcriptase polymerase chain reaction (rt-pcr) for the detection of feline coronavirus (fcov) messenger rna in peripheral blood mononuclear cells (pbmcs) is described. the assay is evaluated as a diagnostic test for feline infectious peritonitis (fip). it is based on a well-documented key event in the development of fip: the replication of virulent fcov mutants in monocytes/macrophages. to detect most feline coronavirus field strains, the test was designed to amplify subgenomic mrna of the highly conserved m gene. the test was applied to 1075 feline blood samples (424 from healthy, 651 from sick cats suspected of fip) and returned 46% of the diseased cats as positive for feline coronavirus mrna in their peripheral blood cells; of the healthy cats, 5% tested positive. of a group of 81 animals in which fip had been confirmed by post-mortem examination, 75 (93%) tested positive, whereas 17 cats with different pathologies (non-fip cases) all tested negative. in view of the low rate of false-positive results (high specificity) the mrna rt-pcr may be a valuable addition to the diagnostic arsenal for fip. coronaviruses are enveloped, positive-stranded ssrna viruses, a genus in the family coronaviridae, order nidovirales. they are ubiquitous in cat populations, with particularly high prevalence in catteries and multiple-cat households. feline coronaviruses (fcovs) show a bimodal pathogenicity distribution, with subclinical or mild enteric infections in young kittens at one extreme and the deadly feline infectious peritonitis (fip) at the other. the low virulence strains are referred to as feline enteric coronaviruses (fecv), the highly virulent ones as fip viruses (fipv) . though occurring only sporadically (i.e. not causing epidemics), fip is an important disease: it is mostly fatal, its biology is still poorly understood and prevention is difficult, to say the least. fip is an immune mediated, progressive polyserositis and pyogranulomatosis. it occurs worldwide, affecting both domestic and wild felids (holzworth, 1973; horzinek and osterhaus, 1979) . antibodies against fcovs are found in 80-90% of the animals living in catteries or multiple-cat households and in up to 50% of solitary cats; however, only some 1-5% of the seropositive cats eventually come down with fip. the reason for this discrepancy became clear when the biological and genetic properties of fecv and fipv isolates had been studied (addie and jarrett, 1992; hohdatsu et al., 1992; horzinek and osterhaus, 1979) : the avirulent fcov strains causing inconspicuous infections are responsible for the high seroprevalence; in cats experiencing some immunosuppressive event, expansion of the quasispecies cloud and mutations in the fecv genome lead to virulent variants that induce fip (vennema et al., 1998) . at present, there are no routine serological and virological assays available for an aetiological diagnosis of fip, and to distinguish avirulent from virulent fcovs. although serology is still used in the diagnosis of fip, it is of very limited value. results can only be interpreted in correlation with clinical symptoms. currently the presumptive diagnosis of fip is based on clinical data and characteristic changes in some blood parameters (cammarata parodi et al., 1993; gouffeux et al., 1975) . a definite diagnosis can only be made on the basis of histological examination of biopsy material or postmortem (sparkes et al., 1991 (sparkes et al., , 1992 . our pcr technique using primers targeted to conserved regions of the viral genome, the 3 -utr (lai and cavanagh, 1997) , and its modifications (using the s gene (gamble et al., 1997) ) became a valuable tool for the detection of fcov in body fluids and tissue samples. unfortunately, the technique detects also avirulent fcovs in healthy cats. although the percentage of pcr-positive healthy animals is much lower when compared to fip cats, a positive pcr result alone does not allow a definite diagnosis gunn-moore et al., 1998 ). an important event in fip pathogenesis is the infection of monocytes and macrophages (stoddart and scott, 1989 ). originally, it was thought that the avirulent fecvs would remain confined to the digestive tract and not spread beyond the intestinal epithelium and regional lymph nodes. virulent fipvs, on the other hand, would leave the gut, enter the bloodstream, generalize and reach different organ parenchymas via infected monocytes. not unexpectedly, however, fcov were detected in blood samples of healthy cats that never developed fip, and also after experimental fcov infection (gamble et al., 1997; gunn-moore et al., 1998; herrewegh et al., 1995; kipar et al., 1999) . there may be a difference between the sheer presence of fcov in peripheral blood mononuclear cells (pbmcs) and their replication in pbmcs, and we hypothesized that the latter may be a correlate of virulence. a rt-pcr that detects messenger rna of the highly conserved m gene of the fcov genome in peripheral blood cell samples (lai and cavanagh, 1997; zhang et al., 1994) would detect the macrophage-tropic variants and bypass non-virulent fcov strains in blood. the present study presents the results of this approach. the fcov reference strains and their sources are listed in table 1 . strains fipv 79-1146, fecv 79-1683, and fipv ucd1 were grown in felis catus whole fetus (fcwf) cells. fipv ucd3 was obtained from tissue cell culture from inkapke and brian (1986) a assignment according to pedersen et al., 1981a. b tentative assignment (hohdatsu et al., 1992) . fected fcwf cells (pedersen et al., 1981b) . fecv ucd was acquired from feline faeces as described by pedersen (1987) and grown to low titers in fcwf cells. fipv dahlberg was obtained from brain of a mouse inoculated with homogenate as described by osterhaus et al. (1978) . fipv wellcome was derived from feline embryonic lung (fel) culture cells as described by o'reilly et al. (1979) . blood samples were collected from diseased cats suspected of having fip based on clinical symptoms (n = 651) as well as from healthy cats (n = 424). the healthy cats were mainly animals living in the same household or cattery as the cats suspected of having fip. these samples were obtained from different veterinary clinics in the netherlands. blood: a maximum of 1 ml of non-coagulated edta blood was centrifuged for 10 min at 2500 rpm. plasma was separated from the cell pellet and stored at −20 • c. one volume of pbs was added to the blood cells and total rna was isolated following the total quick rna blood kit protocol (talent). the oligonucleotide primers were chosen from the highly conserved m gene sequence (primer 212) of the fcov genome combined with a primer aiming at the leader sequence of the fcov-genome (primer 1179). primer sequences are shown in table 2 . as a control to check the efficiency of the rna isolation from all the blood samples and the subsequent reverse transcriptase reaction, a glyceraldehyde-3-phosphate dehydrogenase (gapdh) rt-pcr was performed for every clinical sample (primers 1180 and 1181). for the reverse transcriptase (rt) reactions, 10 l of the rna solution and 2 l of reverse primer 212 or 1181 (each samples were stored at −20 • c before using it in the mrna rt-pcr assay. following reverse transcription, 3 l of the rt reaction mixture was added to 27 l of the pcr reaction mixture. the pcr mix consisted of 3 l pcr buffer 2 (10×; perkin elmer usa, 1 × 10 mm tris-hcl, ph 8.3, 50 mm kcl), 2.5 l magnesium chloride (25 mm; gibcobrl life technologies), 1 l dntps (25 mm each dntp; gibcobrl life technologies), 1 l primer 212 (5 mm), and 1 l primer 1179 (5 mm) (both invitrogen), 0.25 l taq polymerase (5 u/ l; gibcobrl life technologies). for the gapdh rt-pcr reaction the same pcr mix was used but with different primers: 1 l primers 1180 and 1181 (each 5 mm) (both invitrogen). the reaction mixture was placed in a thermal cycler (biozym). the temperature cycling protocol consisted of 10 min incubation at 95 • c followed by 30 cycles of 1 min denaturation at 95 • c, 1 min primer annealing at 62 • c and 1 min primer extension at 72 • c. the 30 cycles were followed by 10 min at 72 • c and finally the reaction mixture was cooled to 4 • c. twenty microliters of each pcr sample was analysed by electrophoresis using a 1.5% tae agarose gel (gibcobrl life technologies) for 45 min at 100 v. a 100 bp molecular weight marker (invitrogen) was used to control the size of the amplified pcr product. amplification products were visualised using ethidium bromide staining and uv radiation. samples revealing a 295 bp fragment for the primers 212 and 1179 and another fragment of 195 bp for the primers 1180 and 1181 were considered positive for coronavirus. amplification products were photographed using the biorad geldoc 1000. twenty-three of the obtained pcr products were sequenced to confirm the rt-pcr product. in order to avoid contamination due to carry-over of amplification products several precautions were taken including physical separation of the pre-and post-pcr procedures, the use of aerosol-resistant filter tips (biozym), and during each step from rna isolation to reverse transcriptase and amplification, negative controls of rnase free water were included to try to rule out any false positives. if possible, necropsy of the cat was performed to confirm or rule out a clinical diagnosis of fip. a total of 98 cats were subjected to post-mortem examination. when macroscopic observations were inconclusive, sections of different organs like liver, kidney, spleen etc. were prepared and examined histopathologically. to determine if a rt-pcr for m gene mrna detects different coronavirus isolates, several laboratory isolates were subjected to this assay (table 1) . rna from fipv serotype i (strains ucd1, ucd3), and serotype ii (strains 79-1146, nor15, wellcome), fecv serotype i (ucd, rm), fecv serotype ii (79-1683), fipv wellcome, ccv-k378 and tgev purdue could all be detected in cell culture and faeces material or tissue homogenates. after amplification, fragments of the expected size of 295 bp were obtained with all isolates, as shown in fig. 1 . in all samples tested, gapdh amplicons were demonstrated. the gapdh gene, which is constitutively expressed at high levels in most tissues, was used for reference as a positive result in the gapdh rt-pcr will rules out any failure of sample rna isolation or reverse transcription. an example of a positive mrna rt-pcr assay and gapdh control is shown in fig. 2 . blood samples from 424 healthy cats were assessed for the presence of fcov in peripheral blood cells. these animals had been living in catteries or multiple cat households, where other cats with fip-related clinical signs were living. twentythree cats out of the 424 cats (5%) indeed tested positive for fcov in the mrna rt-pcr test. two cats from the 23 pcrpositive animals became sick within 2 months, both showing different clinical symptoms, one of them indicative of fip. unfortunately, the cause of death could not be assessed by necropsy. veterinary practitioners had submitted 651 samples from cats they suspected to suffer from fip. the animals had shown one or several of the following symptoms: fever, anorexia, weight loss, diarrhea, poor growth, enlarged abdomen, presence of ascitic or thoracic fluid, uveitis and neurological signs. of these, 301 samples (46%) were positive for fcov degrees of freedom: 1; chi-square = 67.0692431561997; p is less than or equal to 0.001; the distribution is significant. mrna in blood cells. a summary of the pcr results is shown in table 3 . microscopy was performed on 98 cats tested for fcov mrna in the blood. in 81 cases fip was confirmed. of these, 75 cats (93%) were found to have fcov mrna in their peripheral blood cells (table 4 ). in none of 17 animals that were shown to have suffered from other diseases than fip, fcov mrna was detected in peripheral blood cells (e.g. heart failure, neoplastia, and bacterial infections. the obtained results were statistically significant when controlled by a chi-square (table 5) . this report describes an rt-pcr assay to detect fcov mrna in blood samples of cats. our approach was based on the assumption that during the pathogenesis of fip, the mutant virus would replicate in monocytes and macrophages. we postulated that detection of fcov mrna in blood samples would correlate with the development of fip. there are several observations that led to this assumption. infection of monocytes and macrophages is considered as the most important pathogenetic event in fip. the mutant virus has acquired a new tropism and replicates to high titers in monocytes and macrophages (stoddart and scott, 1989; kipar et al., 1999) . in vitro, the virulence of fcov strains correlated with their ability to infect macrophages: avirulent fcovs infected fewer cells and produced lower titers than virulent fcovs. the avirulent fcovs were also inferior in sustaining viral replication and spreading to other macrophages (haijema, personal communication) . in coronavirus-infected cells a nested set of subgenomic mrnas is synthesized, each molecule possessing a "leader sequence". this stretch of 60-98 nucleotides (coronavirus species dependent) has been derived from the 5 -end of the genome through discontinuous transcription and is not translated. making use of primers specific for the m-gene mrna leader sequence (lai and cavanagh, 1997 ) and a conserved part of the m-gene, a molecule of 295 bp will be amplified. using the mrna rt-pcr assay, type ii fcov genomes like fipv 79-1146, fipv wellcome, fipv nor15 and fecv 79-1783 could be detected in cell culture. type i fcovs like fipv ucd1, fipv ucd3, fecv ucd, and fipv dahlberg were detected as well. in view of the fact that also canine (ccv k378) and porcine (tgev purdue) coronaviruses tested positive the assay should detect most, if not all, fcov variants. the high detection rate of fcov from cats suspected of suffering from fip in the field supports this assumption. from its design, the mrna assay would appear to be more specific (only replicating virus detected) and more sensitive (only nucleated blood cells employed) for the diagnosis of fip than previous rt-pcr assays focused on genomic rna in body fluids, feces, and tissues (gamble et al., 1997; gunn-moore et al., 1998; herrewegh et al., 1995) . using this assay, we detected mrna in about 93% of edta blood samples from confirmed fip cases. in the genomic rna pcr, 78-92% of fip cats were found to test positive (gamble et al., 1997; gunn-moore et al., 1998; herrewegh et al., 1995) . more importantly, of the healthy cats living in catteries or multiple cat households with a notoriously large virus burden, only 5% tested positive for fcov. the presence of fcov rna in blood monocytes in healthy cats infected with fcov is an indication that the development of fip is not associated with the capability of an fcov to cause viraemia and systemic infection (meli et al., 2004) previous studies quote figures between 20 and 40% (gamble et al., 1997; gunn-moore et al., 1998; herrewegh et al., 1995) , which can be expected in view of the high sensitivity of the pcr. the specificity of our test format would therefore appear as a significant improvement over previously published methods. the question remains if the mrna positive, healthy cats harbour virulent mutants in an early stage of fip pathogenesis. quantitative analyses of fcov mrna levels would be needed to identify potential differences between healthy and diseased cats. a study of naturally occurring feline coronavirus infections in kittens using direct immunofluorescence to detect coronavirus in peritoneal and pleural effusions sequence analysis of the 3 -end of the feline coronavirus fipv 79-1146 genome: comparison with the genome of porcine coronavirus tgev reveals large insertions fip, easy to diagnose? characterization of a feline infectious peritonitis virus isolate development of a nested pcr assay for detection of feline infectious peritonitis virus in clinical specimens feline infectious peritonitis. proteins of plasma and ascitic fluid detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr elimination of feline coronavirus infection from a large experimental specific pathogen-free catbreeding colony by serologic testing and isolation the prevalence of type i and ii feline coronavirus infections in cats some important disorders of cats the virology and pathogenesis of felineinfectious peritonitis sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene histopathological alterations of lymphatic tissues in cats without feline infectious peritonitis after long-term exposure to fip virus the molecular biology of coronaviruses isolation of feline coronaviruses from two cats with divers disease manifestations high viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (fcov) type i and in naturally fcov-infected cats feline infectious peritonitis: isolation of a coronavirus feline infectious peritonitis (fip) virus; 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without their help this study could not have been completed. this study was supported by a research grant from id-lelystad, the netherlands. key: cord-281179-k7630is6 authors: brown, meredith a. title: genetic determinants of pathogenesis by feline infectious peritonitis virus date: 2011-10-15 journal: vet immunol immunopathol doi: 10.1016/j.vetimm.2011.06.021 sha: doc_id: 281179 cord_uid: k7630is6 feline infectious peritonitis (fip) is a fatal, immune-augmented, and progressive viral disease of cats associated with feline coronavirus (fcov). viral genetic determinants specifically associated with fipv pathogenesis have not yet been discovered. viral gene signatures in the spike, non-structural protein 3c, and membrane of the coronavirus genome have been shown to often correlate with disease manifestation. an “in vivo mutation transition hypothesis” is widely accepted and postulates that de novo virus mutation occurs in vivo giving rise to virulence. the existence of “distinct circulating avirulent and virulent strains” is an alternative hypothesis of viral pathogenesis. it may be possible that viral dynamics from both hypotheses are at play in the occurrence of fip. epidemiologic data suggests that the genetic background of the cat contributes to the manifestation of fip. further studies exploring both viral and host genetic determinants of disease in fip offer specific opportunities for the management of this disease. feline infectious peritonitis (fip) is a fatal, immune-augmented, and progressive viral disease of cats associated with feline coronavirus (fcov). viral genetic determinants specifically associated with fipv pathogenesis have not yet been discovered. viral gene signatures in the spike, non-structural protein 3c, and membrane of the coronavirus genome have been shown to often correlate with disease manifestation. an "in vivo mutation transition hypothesis" is widely accepted and postulates that de novo virus mutation occurs in vivo giving rise to virulence. the existence of "distinct circulating avirulent and virulent strains" is an alternative hypothesis of viral pathogenesis. it may be possible that viral dynamics from both hypotheses are at play in the occurrence of fip. epidemiologic data suggests that the genetic background of the cat contributes to the manifestation of fip. further studies exploring both viral and host genetic determinants of disease in fip offer specific opportunities for the management of this disease. © 2011 elsevier b.v. all rights reserved. feline infectious peritonitis (fip), first described in 1963 (holzworth, 1963) , is a fatal, immune-augmented, and progressive viral disease of cats associated with feline coronavirus (fcov). coronaviruses are enveloped positivestrand rna viruses that infect a wide range of vertebrate species (masters, 2006) . although fcov is common in most domestic, feral and non-domestic cat populations worldwide (seroprevalence from 20 to 100%), less than 10% of fcov seropositive cats develop fip (addie, 2000; addie and jarrett, 1992; kennedy et al., 2002) . fip tends to occur most frequently in cats less than two years of age or, less commonly, in geriatric cats (foley et al., 1997a) . the clinical manifestation of fcov infection can present either as the pathogenic disease manifestation of fip (fipv-cases) or the more common, benign or mild enteric infection (fecvasymptomatic) (de groot, 1995; pedersen et al., 1984b) . specific genetic determinants of these clinical outcomes have yet to be discovered. there is no effective treatment, vaccine, nor a diagnostic protocol that can discriminate the e-mail address: merbrown@nmsu.edu avirulent fecv from the pathogenic fipv. cats infected with fcov that show no evidence of disease are thought to be carriers of fcov and may pose an fip risk to other cats (addie, 2000; foley et al., 1997a,b) . based on serological differences, fcov strains have been separated into a common type 1 form (80-90% prevalent in infected cats) and a less common type 2 form (pedersen et al., 1984b) . fcov types 1 and 2 appear to utilize distinct cell entry receptors and display different growth characteristics in vitro, due to the presence of different spike genes (dye and siddell, 2005; tresnan et al., 1996) . recent in vitro work with chimeric viruses has shown that fcov serotype 2 (strain 79-1146) and a chimera serotype 1 virus (strain black), expressing a serotype 2 spike protein, utilize the feline aminopeptidase n (fapn) cellular receptor. in contrast, serotype 1 fcovs most likely use an alternate main cellular receptor (tekes et al., 2010) . further, a feline homologue to human dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (dc-sign), fdc-sign, has been identified as a coreceptor in both serotype 1 and 2 infection in vitro (regan et al., 2010) . both virulent and avirulent fcov strains are found within types 1 and 2 in vivo. an "in vivo mutation transition hypothesis" also called the "internal mutation hypothesis" postulates that de novo virus mutation occurs in vivo giving rise to viruses that are able to spread systemically and lead to fip pathogenesis (poland et al., 1996; vennema et al., 1998) . this hypothesis has been widely accepted for decades and numerous publications have supported this hypothesis (addie, 2000; addie and jarrett, 1992; foley et al., 1997a,b; pedersen, 2009a; pedersen et al., 1984a pedersen et al., , 2009 poland et al., 1996; vennema et al., 1998) . however, the precise nature of the mutation responsible for pathogenesis has not been identified, although studies have suggested sequence differences in the spike protein (rottier et al., 2005) , membrane protein (brown et al., 2009) , or nsp 3c (pedersen et al., 2009 ) are involved. together with in vitro studies describing the fipv strains affinity for macrophages in contrast to fecv strains (stoddart and scott, 1989) , the hypothesis was extended to propose that the enteric coronavirus (fecv) undergoes a mutational shift in the gastrointestinal system, thus allowing infection of macrophages, systemic dissemination and fatal disease manifestation (poland et al., 1996; vennema et al., 1998) . mutational transition in viral pathogenesis has been shown in hiv infection, where specific amino acid changes in the envelope gene determine which coreceptor (ccr5 or cxcr4) is used and hence virus success in cell entry (hartley et al., 2005) . similarly, key amino acid changes in the spike protein lead to virulence in transmissible gastroenteritis virus (tgev) (ballesteros et al., 1997; sanchez et al., 1999) , although the exact switch to pathogenesis in tgev is still unresolved (paul et al., 1997; saif, 2006) . the existence of distinct "circulating avirulent and virulent strains" is an alternative and less popular hypothesis of viral pathogenesis. because fip occurs sporadically and outbreaks of fip in domestic cat populations are uncommon, there has been little epidemiologic support for this hypothesis (poland et al., 1996) . in this hypothesis, both benign and pathogenic strains of a virus circulate in a population, and those individuals exposed to the virulent strains, with the appropriate predisposition, develop disease sequelae. dengue hemorrhagic fever is an example of this, as four viral strains circulate worldwide and individuals exposed for a second time to a virus of a different strain, mount an inappropriate immune response and exhibit pathology consistent with immune-mediated vasculitis (mongkolsapaya et al., 2003) . the zoonotic equine venezuelan encephalitis virus is another example as virulent and avirulent strains of the alphavirus have both been shown to circulate and ecological and epidemiological factors have been identified that contribute or constrain the frequency of disease sequela in equids and humans (anishchenko et al., 2006) . recent viral sequence analyses of fcovs from 56 shelter cats in maryland suggest that there can be circulating strains of virulent and avirulent fcovs (brown et al., 2009 ). viral dynamics from both the in vivo mutation hypothesis and the circulating virulent and avirulent strain hypotheses may play a role in the complex pathogenesis of fip. certain circulating strains may be predisposed to in vivo mutation in an, as of yet, unidentified locus. alternatively, virulent strains may in some cases be distinctive from avirulent strains in outbreaks of fip, as shown in a maryland phylogenetic study of barn cats (brown et al., 2009) . since the outbreak of the severe acute respiratory syndrome (sars) in 2003 a new highly pathogenic coronavirus, sars-cov, has been identified and many coronavirus genomes have been sequenced. genbank now reports the full-length sequence of over twenty types of coronavirus, including 45 full-length sequences of fcov and 2 annotated full-length sequences siddell, 2005, 2007) . coronaviruses are a large family of enveloped, single strand, positive sense, non-segmented rna viruses. approximately 30 kilobases in length, coronaviruses have the largest viral rna genomes known (rottier, 1995a) . the first 2/3 of the coronavirus genome encodes the replicase genes: open reading frames (orfs) 1a and 1b. proteolytic processing of these polyproteins, mediated by viral cysteine proteinases, produce non-structural proteins (nsps), some of which are responsible for replicating the viral genome and/or generating a nested set of subgenomic mrnas to express all of the other orfs in the genome (thiel et al., 2003; ziebuhr, 2004) . the orfs for the structural proteins, spike, envelope, membrane and nucleocapsid, are encoded in the remaining portion of the genome. coronaviruses may encode different numbers of nsps, and the predicted sequences of these proteins do not share high level of homology (rottier et al., 2005) . viral genetic determinants specifically associated with fipv pathogenesis have not yet been discovered and are currently an area of intense research. viral gene signatures in the spike rottier et al., 2005) , nsp 3c (chang et al., 2010; pedersen, 2009a) , and membrane (brown et al., 2009 ) of the coronavirus genome have been shown to often correlate with disease manifestation. the spike gene encodes a large glycoprotein, which forms spikes on virion surfaces, binds to specific cellular receptors, induces neutralizing antibody, and elicits cell-mediated immunity (rottier et al., 2005) . spike has been implicated as a determinant of virulence in tgev (ballesteros et al., 1997; sanchez et al., 1999) , and neurological murine hepatitis virus (phillips et al., 2002) , but not in fcov, sars, or infectious bronchitis virus (tan et al., 2006) . studies directed at the spike gene have been further complicated because previous work focused on serotype ii isolates (79-1146 and 79-1683) which can be grown in cell culture and encodes a spike gene of canine origin, rather than the more prevalent serotype i (herrewegh et al., 1998; pedersen, 2009a) . amino acid differences were detected in the spike of fipv isolate 79-1146 vs. fecv 79-1683, although additional study of different fcov serotype i isolates did not exhibit similar genetic changes (rottier et al., 2005) . however, more recent in vitro studies of cathepsin b and cathepsin l activity in different isolates of fcov showed that fecv isolates were able to induce a specific cleavage event in the spike protein in contrast to fipv isolates, suggesting that cathepsin activity on the spike gene may play a role in viral pathogenesis at the level of cell entry . nsps are involved with virulence in sars (akerstrom et al., 2007; tan et al., 2006) . recently, the role of nsp 1 has been documented in coronavirus virulence (kamitani et al., 2006) . among the coronavirus family, the sars-cov genome encodes the largest number of nsps (eight) while human hcov-229e, pig (tgev), bird (ibv), mouse (mhv), and cat (fcov) encode two, two, four, two, and five, respectively. in fcov, nsp 3c has been implicated in fip pathogenesis (vennema et al., 1998) . phylogenetic study of the nsp 7b and 3c genes in a small group of cats exposed to fcov, found relatedness a consequence of geographic locale, rather than clinical disease outcome (vennema et al., 1998) . recent work by chang et al. (2010) analyzed the 3c gene in natural isolates from 27 fecv-and 28 fipv-infected cats. they found intact 3c genes in all fecvs, while the majority of fipvs (71.4%) had disrupted 3c genes. further, most cats with fip had no detectable intestinal coronavirus. their findings suggested that 3c-inactivated viruses only rarely replicate in the gut, which possibly explains the rare incidence of fip outbreaks. similarly, pedersen et al. (2009) studied single nucleotide polymorphisms and deletions causing truncation of the 3c gene product in virus isolates from fipv and fecv isolates from two geographic locations. they also found that most, but not all, coronaviruses sequenced from fecal isolates had intact 3c genes while almost all isolates from the diseased tissues of fip cases had disrupted 3c genes. the membrane protein is the most abundant structural protein, with important functions in virus budding (rottier, 1995b) . the membrane protein also interacts with cellmediated host immunity (rottier, 1995b) , and is known to both induce alpha interferon (laude et al., 1992) and induce apoptosis (chan et al., 2007; zhao et al., 2006) . recent studies of the membrane gene (brown et al., 2009) in 56 free-ranging domestic cat isolates from maryland demonstrated distinct viral genotypes highly correlated with disease phenotype. epidemiologic data suggests that the cat's genetic background contributes to the manifestation of fip. indeed, variation in breed susceptibility and/or resistance to fip has been noted by investigators (norris et al., 2005; pesteanu-somogyi and pressler, 2006) , and outbreaks of fip in captive cheetah populations, known for their lack of genetic diversity, is suggestive of a role of host genetics in fip disease pathogenesis (o'brien et al., 1985) . host genetic background has been shown to alter the pathogenesis of viral pathogens relating to inappropriate immune responses (e.g. hiv and dengue virus), susceptibility to infection (ccr5 in hiv aids), and the development of clinical symptoms (ccr5, ccr2, sdf1, rantes in hiv aids) (hutcheson et al., 2008) . studies of cats with fip have shown that cytokine expressions are altered as compared to healthy cats (dean et al., 2003; kiss et al., 2004) . specifically, expression of interleukin (il)-1 beta and interleukin-6 are significantly increased in cats with fip, likely produced by infected macrophages and monocytes (kiss et al., 2004; takano et al., 2007a,b) . it has been shown that tumor necrosis factor (tnf)-alpha is able to induce feline t-cell apoptosis, making it the most likely causative agent of tcell lymphopenia in fipv-infected cats (dean et al., 2003; takano et al., 2007a) . candidate genes such as il-12, il-10, il-6 ( kipar et al., 2006) , interferon (ifn)-gamma, tnf-alpha (de albuquerque et al., 2006) , liver/lymph node specific intracellular adhesion molecules-3 grabbing non-integrin (l-sign) (jeffers et al., 2004) , dc-sign yang et al., 2004) , angiotensin-converting enzyme (ace2) (li et al., 2003) , and fapn (tresnan et al., 1996) have been identified as associated with coronavirus disease findings in cat, mouse, and human cases. further studies exploring both viral and host genetic determinants of disease in fip offer specific opportunities for the management of this disease. to date, these studies have unfortunately been limited in the geographic and temporal diversity of the isolates studied. if additional studies are conducted in additional cat populations, the development of antemortem screening tools for genetic disposition for disease as well as the discrimination of virulent versus avirulent strains of fcov may be possible. the author reports no conflict of interest. clustering of feline coronaviruses in multicat households a study of naturally occurring feline coronavirus infections in kittens inhibition of sars-cov replication cycle by small interference rnas silencing specific sars proteins, 7a/7b, 3a/3b and s venezuelan encephalitis emergence mediated by a phylogenetically predicted viral mutation two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism genetics and pathogenesis of feline infectious peritonitis virus the sars-coronavirus membrane protein induces apoptosis through modulating the akt survival pathway feline infectious peritonitis: insights into feline coronavirus 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spike protein during entry of serotype ii feline coronaviruses utilization of dc-sign for entry of feline coronaviruses into host cells the coronaviridae the coronavirus membrane glycoprotein acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein transmissible gastroenteritis virus and porcine respiratory coronavirus targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence a "possible" involvement of tnf-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis tnfalpha, produced by feline infectious peritonitis virus (fipv)-infected macrophages, upregulates expression of type ii fipv receptor feline aminopeptidase n in feline macrophages understanding the accessory viral proteins unique to the severe acute respiratory syndrome (sars) coronavirus chimeric feline coronaviruses that encode type ii spike protein on type i genetic background display accelerated viral growth and altered receptor usage mechanisms and enzymes involved in sars coronavirus genome expression feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign m and n proteins of sars coronavirus induce apoptosis in hpf cells molecular biology of severe acute respiratory syndrome coronavirus the author would like to acknowledge dr. stephen o'brien, dr. gary whittaker, dr. jennifer troyer, and dr. melody roelke. key: cord-336332-9d1h68mi authors: paltrinieri, saverio; ceciliani, fabrizio; gabanti, elisa; sironi, giuseppe; giordano, alessia; addie, diane title: expression patterns in feline blood and tissues of α(1)-acid glycoprotein (agp) and of an agp-related protein (agprp) date: 2003 journal: comp clin path doi: 10.1007/s00580-003-0489-8 sha: doc_id: 336332 cord_uid: 9d1h68mi α(1)-acid glycoprotein (agp) is an acute-phase protein (app) that modulates immune responses, probably – at least in humans – owing to the modification of its glycosylation pattern. on this perspective, feline agp can be a useful comparative model, as it has different concentrations in cats susceptible or resistant to some disease. as a preliminary approach to the study of feline agp (fagp) we have purified this protein from feline serum by hplc using human agp (hagp) as a model. immunoblotting with a polyclonal antibody against fagp and with a monoclonal antibody against hagp was performed on serum from healthy cats, from cats exposed to feline coronavirus (fcov) infection and from cats with purulent inflammations, such as feline infectious peritonitis (fip), feline immunodeficiency virus (fiv) and feline leukemia virus (felv). immunohistochemistry on tissues from healthy cats and from cats with different diseases (fip, fiv, felv, locally extensive inflammation) was also performed with the same antibodies. both hagp and fagp have been purified to homogenity as determined by sds-page. fagp did not react with the anti-hagp antibody which, in contrast, detected in feline serum a low mw protein that we called fagp-related protein (fagprp). this protein was underexpressed in cats with felv and fip. both fagp and fagprp were immunohistochemically detected in plasma and hepatocytes with a stronger intensity in cats with fip and some inflammatory conditions. moreover, fagprp was detected in the cytoplasm of tissue cells, most likely identifiable with plasma cells. these cells were rarely detectable in cats with fiv and felv, and numerous in cats with fip and with locally extensive inflammation. in conclusion, purified fagp has physicochemical characteristics similar to those of hagp, but does not cross-react with anti-hagp antibodies. in contrast, the anti-hagp detected an agp-related protein whose blood concentration and tissue distribution was not related to that of fagp. moreover, both fagp and fagprp were differently expressed in cats with pathologic conditions compared to controls. further study of these proteins by analysing their structural characteristics is required. introduction a 1 -acid glycoprotein (agp) is a major positive acutephase protein (app) in many species. agp belongs to the lipocalin family, a group of extracellular binding proteins specific for small hydrophobic molecules. together with a 1 -microglobulin and glycodelin, it forms the immunocalin subfamily (logdberg and wester 2000) . human agp (hagp) contains 183 aminoacids and is characterised by low molecular weight (41-43 kda), high solubility, very low ph (2.8-3.8) and a high percentage of carbohydrates (45%). its glycosylation pattern is very variable (12-20 glycoforms in humans), depending on the physiological or pathological conditions, such as pregnancy, inflammation or cancer (biou et al. 1991; kim and varky 1997) . the function of agp has not been completely defined: an immunomodulatory and anti-inflammatory role has been suggested (williams et al. 1997) . it can downregulate neutrophil responsiveness, stimulate il-1r antagonist secretion by macrophages (vasson et al. 1994; bories et al. 1990 ), inhibit platelet aggregation and lymphocyte proliferation and modulate the production of anti-inflammatory cytokines by peripheral blood leucocytes (costello et al. 1979) . these activities are correlated to the carbohydrate moiety of agp (shiyan and bovin 1997) . in particular, the rate of sialylation has been proved to be protective in inflammation and in hiv infection (mackiewicz and mackiewicz 1995; rabehi et al. 1995; williams et al. 1997) . in cats, agp is one of the most examined apps. it has been studied in different viral and neoplastic diseases (duthie et al. 1997; selting et al. 2000) . in particular, the serum levels of agp during feline infectious peritonitis (fip) are very high (duthie et al 97) , but this increase is only transient in fcov-exposed cats (giordano et al. 2003) . this could be due to the protective role of some agp glycoforms similar to humans. although diagnostic kits to quantitate feline agp (fagp) are commercially available, there is currently no information about fagp sequence, structure and tissue distribution, or commercial anti-fagp antibodies. we thus decided to design a research project focused on the structural characteristics of fagp. in this paper we describe the results of the first step of this project, during which fagp was purified from feline blood using hagp purification protocol as a model. fagp distribution in blood and tissues of healthy cats and of cats with different diseases was then assessed using a polyclonal anti-fagp antibody. during the fagp purification a second, different protein was identified. this was named fagprelated protein (fagprp), only because it cross-reacts with a monoclonal antibody directed against hagp. to date, it is not known what this small protein (about 29 kda) is. before studying the fagprp further, however, it was decided to evaluate its possible pathophysiological role by assessing its blood and tissue distribution and its relationship with fagp. hagp and fagp purification hagp and fagp were purified to homogeneity following the same procedure. aliquots (1 ml) of serum from healthy cats and from human donors were brought to 0.01 m citrate-phosphate buffer (cpb), ph 4.0, by ultrafiltration with centricon ym 10 (millipore co., bedford, ma, usa). the solution was centrifuged at 12 000 g and the supernatant directly loaded onto a q-sepharose hitrap column (amersham biosciences, uppsala, sweden) equilibrated in the same buffer. the column was washed with 20 ml of loading buffer, and the proteins were eluted with 0.1 m cpb, ph 4.0. the fraction containing agp was concentrated and loaded on to a second sp-sepharose hitrap column equilibrated in 0.1 m citratephosphate, ph 4.0. agp is not retained on the column but eluted in the void volume. protein concentration was quantified using the bioradtm protein assay (bio-rad, hercules, ca, usa). protein homogeneity was assessed by sds-page (laemmli 1970) . sds-page gels were directly stained with coomassie blue. semiquantitative evaluations of agp concentration were determined by immunoblotting on sera from healthy cats (n ¼ 3), from cats living with fcov shedders (fcov-exposed cats, n ¼ 3), and from cats with fiv (n ¼ 3), felv (n ¼ 2) fip (n ¼ 2), and purulent inflammations (n ¼ 3) such as pyometra, pyothorax and purulent rhinoconjunctivitis. immunoblotting was performed on to nitrocellulose at 250 ma for 120 min. nitrocellulose membranes were immunostained using, as primary antibodies, the following: a) anti fagp polyclonal antibody raised in sheep at the glasgow veterinary school by professor david eckersall using a fagp purified from feline ascites with a procedure similar to the protocol used by us. the specificity of this antibody has been validated by the producers by an elisa against fagp. this antibody is routinely used at the glasgow veterinary school to determine the fagp levels in cats with fip (duthie et al. 2002) . the antibody was used in immunoblotting tests at a final dilution of 1:2000. b) commercial anti-hagp monoclonal antibody (sigma, st louis, mo, usa) recognising an epitope located in the 44 kda subunit of denatured and reduced agp. according to the manufacturers, this antibody should not react with other human acute-phase proteins or with agp of different species other than human and baboon. the anti-hagp antibody was also used at a final dilution of 1:2000. tissue samples were taken from two groups of cats: group a: controls: liver, spleen, pancreas, lymphnodes, kidney, large and small intestine, uterus, adrenal glands, thyroid, lung, muscle, heart, cns and cutis were collected at necropsy from three cats without inflammatory disease. group b: tissue samples were collected at necropsy from cats with fip (n ¼ 6), fiv (n ¼ 2), felv (n ¼ 2), parvoviral enteritis (n ¼ 2) and inhalation pneumonia (n ¼ 2); moreover eight surgical biopsies from fiv-and felv-negative cats with lymphocytic plasmacytic gingivitis (lpg, n ¼ 4), pyometra (n ¼ 2) and lymphoplasmocytic enteritis (lpe, n ¼ 2) were also included in this group. haematoxyilin and eosin and toluidine blue stains were performed on sections prepared from formalin-fixed and paraffinembedded samples. the reactivity and the dilution of the primary antibodies (see below) used in immunohistochemistry were previously assessed on cryostatic sections obtained from frozen samples of control cats. immunohistochemistry was then performed on formalin-fixed and paraffin-embedded samples. monoclonal anti-hagp and polyclonal anti-fagp antibodies (see above) were applied on serial sections at final dilutions of 1:5000 and 1:10 000, respectively. the following monoclonal antibodies were also applied on selected sections of intestine, lymph node and lpg: anti-myeloid cell antigens (mac387 -dako, glostrup, denmark; 1:5000), anti-cd79a (dako, glostrup, denmark 1:1000), anti-feline iga (oxford biomarketing, oxford, uk; 1:40) and anti-feline igm (oxford biomarketing, oxford, uk; 1:20). the avidin-biotin complex (abc) method with a commercially available kit (vectastain elite, vector labs inc., burlingame, ca, usa) was used to detect the positive reaction, as previously described (hsu et al. 1980) , after inhibition of the endogenous peroxidase (h 2 o 2 1% in methanol). antigen unmasking was performed using microwave pretreatment (two cycles of 5 min in citrate-buffered solution, 0.01 m, ph 6.2) (cattoretti et al. 1993) , except for sections stained with the anti-iga and the anti-igm antibodies, which were trypsinised (25 min at 37°c; 0.1% trypsin and calcium chloride 0.1% in distilled water, ph 7.8) (waly et al. 2001) . diaminobenzidine served as the chromogen for the reaction and the slides were counterstained with mayer's haematoxylin. sds-page of homogeneous purified human (lane 1) and feline (lane 2) agp is presented in figure 1 . the different electrophoretical mobility between fagp and hagp is probably due to a different glycosylation pattern, which is one of the characteristics of this family of proteins (hochepied et al. 2003) . the homogeneity of the purification was evaluated after silver staining (data not presented) and coomassie blue staining, which showed a single band in the gel. the identification of the homogeneous band at 51 kda as agp was carried out by western blotting using a monoclonal anti-hagp antibody and a polyclonal anti-fagp. anti-hagp antibody strongly reacts with hagp, both in human serum and in the homogeneous solution containing the purified hagp (fig. 2a , lanes 1 and 2, respectively). it does not react with fagp, neither in feline serum nor with purified fagp (fig. 2a , lanes 3 and 4, respectively). therefore, we concluded that monoclonal anti-hagp does not cross-react with feline agp. unexpectedly, anti-hagp cross-reacted with a low molecular weight (29 kda) human and feline protein: this protein can be observed in serum but is not present in purified fagp solution. we called it 'agp-related protein' (fagprp). we have no further structural information about fagprp: it can be detected in the whole sera of both human and feline, but it is apparently lost during the agp purification procedure. the name of agprp is due to its cross-reactivity with both antihuman and antifeline antibodies. polyclonal anti-fagp does react strongly with fagp (fig. 2b, lanes 3 and 4) , but also cross-reacts with hagp ( fig. 2b, lanes 1 and 2) . it also cross-reacts with a 66 kda protein that exhibits an electrophoretical mobility and shape very similar to that of albumin. the strong binding capability of both proteins and the high glycosylation content of agp may cause an incomplete denaturation of feline agp during preparation of samples before sds-page, and therefore an amount of agp may be delayed by interaction with albumin and comigrates during the electrophoretic run. in order to rule out the possibility that anti-fagp cross-reacts with albumin, feline albumin was purified by means of ionic exchange chromatography, western blotted on to nitrocellulose, and the membrane was probed with anti-fagp polyclonal antibody. the results (data not shown) confirm that anti-fagp does not crossreact with feline albumin. in order to investigate the distribution of fagp and of fagprp in different feline pathological conditions, immunoblotting was repeated on serum from cats with purulent inflammations, fiv, felv and fip, and from fig. 2 immunoblotting of hagp and fagp using as primary antibody the anti hagp monoclonal antibody (a) or the anti fagp polyclonal antibody (b). lane 1: human serum (total protein: 4.5 g); lane 2: purified hagp (total protein: 0.4 g); lane 3: feline serum (total protein: 4.5 g); lane 4: purified fagp (total protein: 0.4 g) fcov-exposed cats. sds-page fractionated proteins were immunoblotted and nitrocellulose membranes were probed with anti-fagp polyclonal antibody. moreover, because the monoclonal antibody anti-hagp crossreacts strongly with fagprp, this antibody was used to evaluate the expression of that protein. results are presented in figure 3 . compared to controls, fagp was slightly underexpressed in cats with fiv and felv, unchanged in fcov-exposed cats, and overexpressed in cats with fip and with purulent inflammations. in contrast, fagprp was underexpressed in cats with fip and absent in one out of the two cats with felv. in control cats, both the anti-fagp and the anti-hagp antibodies strongly stained plasma in small and medium vessels. scattered to abundant positive granules were detected within hepatocytes. the anti-hagp antibody also stained some large round or stellate cells with a small, round, central to eccentric nucleus and abundant strongly positive cytoplasm. although positive cells were occasionally detectable in all the examined tissues, they were particularly abundant in the lamina propria of intestine and uterus and in lymphoid tissues, where they were detectable in sinuses and in the perifollicular areas (fig. 4) . these cells were negative to toluidine blue stain and did not stain with anti-cd79, anti-iga, anti-igm and mac387 antibodies. intrahepatocytic and plasma positivity were also detectable in cats with diseases. the intensity of positivity was particularly high in cats with fip and with lpg. in the same cats, a diffuse weak positivity close to pyogranulomatous and to lymphoplasmocytic foci, respectively, perhaps depending on plasma leakage from vessels, was also detectable. moreover, the endothelium in the inflamed tissues and the alveolar epithelium stained positive with the anti-hagp antibody. the number and the distribution of positive cells were very variable in the different pathological conditions: positive cells in tissues from cats with felv and with parvoviral enteritis were rare or absent. in contrast, a large number of positive cells were found in samples with pneumonia, pyometra and lpg. moreover, in some cats with fip the number of positive cells in perifollicular areas of lymph nodes decreased, whereas in others these cells were particularly abundant and without a clear perifollicular distribution (fig. 5 ). a rapid and straightforward purification procedure is the very first step to investigate the structure characteristics of a protein. to date, we have demonstrated that fagp is a protein similar to hagp but not crossreacting with anti-hagp antibodies. the primary structure of fagp is unknown. so far the primary structure of agp has been determined in human, rat, mouse, pig and chicken (fournier et al. 2000) . the degree of similarity among the agp subfamily is very low. these data clearly indicate that agp is not really well conserved between species; therefore, it is not really surprising that monoclonal anti-hagp does not crossreact with fagp. interestingly, the anti-hagp antibody detected a small protein in human and feline serum. a computational analysis (http://dove.embl-heidelberg.de/blast) of human agp, against which the antibody here used has been raised, did not find protein homologues to agp other than those of the lipocalin family, except for some small proteins of bacterial origin and for a ribosomal protein. from this perspective it is not possible to know whether this protein is structurally related to agp. moreover, the protein disappears when agp is purified to homogeneity, both in human and cat. on the other hand, we may not rule out the possibility that the antibody cross-reacts with a partially deglycosylated form of fagp, which has no equivalent in human serum. because the low isoelectric point of agp is due largely to the glycan moiety of the protein, it is possible that a partial deglycosylated isoform of 29 kda is not copurified with the completely glycosylated agp. based on its cross-reactivity with the anti-hagp antibody, we provisionally called this protein agp-related protein (agprp). agprp purification and sequencing are required to clarify the nature of this protein. these procedures, however, are quite expensive and time-consuming. for these reasons it was decided to evaluate the expression of fagprp and its tissue distribution before engaging in the determination of the primary structure. a semiquantitative analysis of the expression of fagp and fagprp in serum from cats with different pathologies suggests that fagprp concentration is not related to that of fagp: fagprp is underexpressed in cats with fip, in which, by contrast, fagp was overexpressed. the latter finding was in agreement with previous results obtained on the same cats using a commercially available radial immunodiffusion kit (giordano et al. 2003) . the underexpression of fagprp, if confirmed in a larger number of cats, might be a useful support in the diagnosis of fip. furthermore, the expression pattern recorded in blood from cats with fiv, felv and purulent inflammations suggests a possible involvement of both fagp and fagprp in these diseases. the tissue distribution of fagp was consistent with its role of acute-phase protein (app): positivities were detectable within hepatocytes, where app are produced, and in plasma (ceciliani et al. 2002) . both these findings, however, must be carefully interpreted, because of the presence of a 66 kda positive protein, most likely albumin. we ruled out experimentally that feline albumin cross-reacts with polyclonal anti-fagp: it is probable that an aliquot of fagp conjugates with albumin during sds-page of sera, and therefore the crossreaction at 66 kda might be considered as an electrophoretical artefact. as a further support to this hypothesis, fagp resulted in overexpression in fip and in inflammatory conditions usually characterised by decreased albumin (pedersen 1995; kaneko 1997) . the tissue distribution of fagprp does not help us to understand the nature of this protein: the intrahepatocytic localisation and the plasma positivity are typical of app, suggesting that fagprp might be an agp isoform. in contrast, no reports about the presence of agp or of other app on tissue cells are so far available, supporting the hypothesis of a protein different from agp and characterised by the simultaneous cellular and plasma distributions. the morphology and the distribution pattern of positive cells in the intestine were very similar to those described by waly et al.(2001) for igaor igm-producing plasma cells. in the present paper, immunohistochemistry for cd79a, iga and igm was negative but samples were collected from a retrospective analysis of our archives, and this might have altered the preservation of membrane-bound antigens. the nature of fagprp-expressing cells needs to be further verified by the analysis of samples collected with a standardised fixation protocol. in contrast, the fixation procedure does not alter the ability to detect myeloid cells and mast cells by mac387 antibody and toluidine blue stain, respectively, thus allowing us to exclude that positive cells were mast cells and macrophages. the group of cats with pathologies was very heterogeneous: this does not allow for the exact definition of the role of those proteins in each disease. however, the changes in the distribution of positive cells observed in these cats suggest that agprp might play some role in the pathogenesis of the diseases. based on these observations this protein will be further investigated. in conclusion, this study allowed the purification of fagp and the detection of another protein, fagprp, whose concentration in pathological conditions is different from that of fagp. both the proteins have a similar intrahepatocitic and plasmatic distributions, but fagprp is also detectable in tissue cells. all these findings suggest that these two proteins are structurally and functionally different, and that the cross-reactivity is probably due to the conservation of a very small epitope. the different expression pattern of these proteins recorded in cats with pathological conditions compared to controls suggests the need to further investigate the structural characteristics of both fagp and fagprp. alterations of the glycan moiety of human alpha-1-acid glycoprotein in late-term pregnancy prevalence of triand tetraantennary glycans of human alpha-1-acid glycoprotein in release of macrophage inhibitor of interleukin-1 activity antigen unmasking on formalin-fixed, paraffin-embedded tissue sections the systemic reaction during inflammation: the acute phase proteins inhibition of platelet aggregation by native and desialised alpha-1-acid glycoprotein value of alpha-1-acid glycoprotein in the diagnosis of feline infectious peritonitis agp measurement as an aid to the diagnosis of feline infectious peritonitis alpha-1-acid glycoprotein changes in some acute phase proteins and in imunnoglobulin concentrations in cats affected by feline infectious peritonitis or exposed to feline coronavirus infection alpha-1-acid glycoprotein: an acute phase protein with inflammatory and immunomodulating properties use of avidin-biotin-peroxidase complex (abc) in immunoperoxidase techniques: a comparison between abc and unlabeled antibody (pap) procedures serum protein and the dysproteinemias perspectives on the significance of altered glycosylation of glycoproteins in cancer cleavage of structural proteins during assembly of the head of bacteriophage t4 immunocalins: a lipocalin subfamily that modulates immune and inflammatory responses glycoforms of serum alpha-1-acid glycoprotein as markers of inflammation and cancer an overview of feline enteric coronavirus and feline infectious peritonitis virus infection alpha-1-acid glycoprotein binds human immunodeficiency virus type 1 (hiv-1) envelope glycoprotein via n-linked glycans serum alpha-1-acid glycoprotein concentrations in healthy and tumor-bearing cats carbohydrate composition and immunomodulatory activity of different glycoforms of alpha-1-acid glycoprotein effects of alpha-1-acid glycoprotein on human polymorphonuclear neutrophils: influence of glycan microheterogeneity alpha-1-acid glycoprotein reduces local and remote injuries after intestinal ischemia in the rat the distribution of leococytes subsets in the small intestine of healthy cats acknowledgements this work was performed with university grant first 2001. the authors are very grateful to dr paola roccabianca for useful suggestions and to professor david eckersall and dr angela pacitti from the glasgow veterinary school. key: cord-295491-zlah6u5s authors: günther, sonja; felten, sandra; wess, gerhard; hartmann, katrin; weber, karin title: detection of feline coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification date: 2018-03-11 journal: j virol methods doi: 10.1016/j.jviromet.2018.03.003 sha: doc_id: 295491 cord_uid: zlah6u5s feline infectious peritonitis (fip) is a fatal disease in cats worldwide. the aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay to detect feline coronavirus (fcov) in body cavity effusions of cats with and without fip, in order to minimize the time from sampling to obtaining results. rna was extracted from body cavity effusion samples of 71 cats, including 34 samples from cats with a definitive diagnosis of fip, and 37 samples of control cats with similar clinical signs but other confirmed diseases. two reaction mixtures (isothermal mastermix, optigene ltd.and pcrun™ molecular detection mix, biogal) were tested using the same primers, which were designed to bind to a conserved region of the fcov membrane protein gene. both assays were conducted under isothermal conditions (61 °c–62 °c). using the isothermal mastermix of optigene ltd., amplification times ranged from 4 and 39 min with a sensitivity of 35.3% and a specificity of 94.6% for the reported sample group. using the pcrun™ molecular detection mix of biogal, amplification times ranged from 18 to 77 min with a sensitivity of 58.8% and a specificity of 97.3%. although the rt-lamp assay is less sensitive than real time reverse transcription pcr (rt-pcr), it can be performed without the need of expensive equipment and with less hands-on time. further modifications of primers might lead to a suitable in-house test and accelerate the diagnosis of fip. feline coronavirus (fcov), a member of the genus alphacoronavirus of the subfamily coronavirinae, family coronaviridae within the order nidovirales (de groot et al., 2011) , belongs to a group of enveloped, positive-sense rna viruses that cause diseases in several species, such as severe acute respiratory syndrome (sars) in humans or transmissible gastroenteritis (tge) in pigs. despite the high prevalence of fcov infections in the cat population worldwide, only 5-10% of fcov-infected cats develop the fatal disease feline infectious peritonitis (fip) (addie and jarrett, 1992) . this change of virulence of a harmless fcov biotype that usually causes no clinical signs into the pathogenic variant is thought to be caused by mutations in the fcov spike protein gene (chang et al., 2012; vennema et al., 1998) . these mutations cause a change in tropism from enterocytes to macrophages, giving fcov the ability to infect and effectively replicate within cells of the macrophage lineage and cause a lethal systemic disease with multi-organ involvement (pedersen, 2009) . the median survival time of cats with effusive fip is only a few days (ritz et al., 2007) , and the diagnosis of fip commonly leads to euthanasia, since to date, no treatment has been proven to be effective. cats with fip show nonspecific clinical signs such as fever, weight loss and anorexia, often accompanied by body cavity effusions and/or ocular and neurological signs. a definitive diagnosis of fip ante mortem remains challenging, especially when no body cavity effusions can be detected (hartmann et al., 2003) . presently, the gold standard for the diagnosis of fip is considered to be immunostaining of fcov antigen in macrophages within tissue lesions, a technique that requires invasive tissue collection (kipar and meli, 2014) . in cats with fip, fcov can be detected by rt-pcr in cell-free body cavity effusions in more than 80% of the cases, while serum or blood samples often are negative (doenges et al., 2017) . for both immunostaining and rt-pcr, samples have to be sent to specialized laboratories, resulting in the delay of diagnostic results. this leads to further unnecessary testing for other diseases, to withholding necessary therapy of other treatable diseases, or to delayed euthanasia in cats suffering from severe signs of fip. therefore, a fast and simple point of care test would be very beneficial in the diagnostic process. loop-mediated isothermal amplification (lamp) is a simple, rapid, and cost-effective nucleic acid amplification method (notomi et al., 2000) and is already used for the detection of coronaviruses in humans and several animal species (hong et al., 2004; nemoto et al., 2015) . a set of four to six primers is used, that form products with self-hybridizing loop structures. by using a dna polymerase with strand displacement activity, no melting or annealing steps are required, and amplification products of different lengths are formed at a constant temperature of 60-65°c (nagamine et al., 2002) . since lamp reactions only require a simple heat block with constant temperature, and dna amplification can be detected by fluorescence or color change, the method can be applied for point-of-care diagnostics (surabattula et al., 2013) . the aim of this study was to test specificity and sensitivity of two commercially available reaction mixtures in a reverse transcription lamp (rt-lamp) to detect fcov in body cavity effusions of cats with and without fip, and to minimize the time from sampling to obtaining results. this study included 71 cats that were presented to the clinic of small animal internal medicine, lmu munich, germany. all cats included had body cavity effusions. in every cat presenting with body cavity effusions, fip is a potential differential diagnosis. an earlier study showed that fip is responsible for about 40% of effusions, while most of the remaining cases were caused by malignomas, cardiac insufficiency or purulent serositis (hirschberger et al., 1995) . the fip group (n = 34) included cats with a definitive diagnosis of fip by one or more methods: all effusions of cats with fip tested positive for fcov by rt-pcr by a commercial laboratory, and in 26/34 samples putative disease-causing mutations could be detected. the rt-pcr detection method has been described previously (felten et al., 2017) . in 25/34 cats fip diagnosis was achieved by post-mortem examination, including full body necropsy with histopathological examination. diagnosis of fip was confirmed when typical histologic lesions where detected (surfacebound multi-systemic pyogranulomatous and fibrinonecrotic disease with venulitis with or without high-protein exudate). in 17/25 cats with full body necropsy immunohistological staining for fcov-antibody was done on tissue sections and returned a positive result. immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in 20/34 cats, and all samples returned a positive result. a summary of the cases in the fip group can be found in the supplementary table 1 . cats were included in the control group (n = 37) if they were definitively diagnosed with a disease other than fip that explained the effusion. cats of the control group suffered from neoplasia (n = 20), decompensated cardiac diseases (n = 12), inflammatory diseases (n = 2), such as bacterial peritonitis and pleurisy, or other diseases (n = 3). one cat had chronic thoracic chylous effusion of unknown origin and secondary fibroplastic pleurisy. in another cat, an end stage kidney disease caused effusion, and one cat had thoracic effusion after subcutaneous urethral bypass placement, which resolved after treatment. the diseases of the cats of the control group (n = 37) were definitively confirmed ante-mortem (n = 18) or at necropsy with histopathological examination (n = 19). ante-mortem diagnosis was established by echocardiography for cardiac diseases (n = 8), and by cytology for neoplasia (n = 10). immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in 11/37 cats, with three positive and eight negative results. all effusions of the cats in the control group were tested for fcov by rt-pcr, and all results were negative. the rt-pcr detection method has been described previously (felten et al., 2017) . a summary of the cases in the control group can be found in supplementary table 2 . body cavity effusion samples of all cats were obtained ante mortem with ultrasound guidance for diagnostic purposes. the use of samples for this study was approved by the institutional animal care and use committee ('ethikkommission des zentrums für klinische tiermedizin'), permission number 32-25-06-2014. samples were stored at -80°c in a 1.5 ml eppendorf safe-lock microcentrifuge tube until assayed. all samples were centrifuged for 20 s at 15,000 × g. the supernatant of centrifuged thoracic and abdominal fluids was used for rna extraction. when using fresh fluid samples, omission of the centrifugation step should be considered to include intact cells with a high viral burden (pedersen et al., 2015) . in thawed samples, cell integrity is lost and cell debris can be removed. viral rna was isolated using the commercial zr viral rna kit™ (zymo research corp.) following the manufacturer's instructions. briefly, 100 μl aliquots of samples were mixed with a buffer that facilitates viral particle lysis and allows for rna adsorption onto the matrix of the zymo-spin™ column. then the rna was washed and eluted with 15 μl of rnase free water. extracted rna aliquots were stored at −80°c in an eppendorf 1.5 ml safe-lock microcentrifuge tube until further processing. the rt-lamp primer design was assisted by the software primerexplorer (https://primerexplorer.jp/e/). based on sequence analysis, the gene for the membrane protein (m) was selected as a target because it is highly conserved among fcov strains. the dna sequence from position 26,500 to 27,000 of the fcov strain black (genbank accession number: eu186072.1) was used to design the rt-lamp primers used in this study. a set of six primers, including two outer primers (forward primer f3 and backward primer b3), two inner primers (forward inner primer fip and backward inner primer bip), and two loop primers (forward loop primer loopf and backward loop primer loopb) were selected as the target sequence ( fig. 1 and table 1 ). detection of fcov was performed using rt-lamp. two different commercial reaction mixtures (isothermal mastermix by optigene ltd., uk, pcrun™ molecular detection mix by biogal, israel,) were compared using the same set of primers. for the amplification following the isothermal mastermix protocol, the total volume of 25 μl per reaction tube included 15 μl isothermal master mix, 5 μl template, 5 μl primer mix and 0.1 μl superscript® iii reverse transcriptase (thermo scientific). the primer mix consisted of 5 pmol each of f3 and b3 primers, 20 pmol each of fip and bip primers and 10 pmol each of loopf and loopb primers. for negative control, 5 μl water were added instead of 5 μl template. the reaction mix was incubated at 62°c for 75 min in a 7500 real-time pcr system (applied biosystems). during rt-lamp, fluorescence of dna products was measured once every minute (fam detection channel, λ max 518 nm), and the time to threshold crossing was analyzed. a positive sample (positive in rt-pcr and sequenced for mutations) was included in every run. all samples run in the 7500 realtime pcr system were subjected to a melt curve analysis after the run. a single sharp peak in the melt curve analysis demonstrates amplification of a single pcr product. the positive samples all showed a single peak and the same melting temperature as the positive control sample. following the pcrun™ molecular detection protocol the reaction mix contained 7.5 μl of luminescent reagent, 5 μl of template, 5 μl of primer mix and additional 0.1 μl superscript iii reverse transcriptase (thermo scientific). the primer mix consisted of 7.5 pmol each of f3 and b3 primers, 30 pmol each of fip and bip primers and 15 pmol each of loopf and loopb primers. for negative control, 5 μl water were added instead of 5 μl template. the reaction mix was incubated at 61°c for 90 min in a pcrun™ reader (biogal). amplification was detected by measuring bioluminescence twice every minute (fig. 2) . pcr products of both methods were verified on an agarose gel showing a typical pattern of multiple bands of different molecular weights (fig. 3) . the results of the 71 samples in the fcov rt-lamp assays using two different commercial reaction mixtures are shown in table 2 . two samples tested false positive using the isothermal mastermix and one sample tested false positive with the pcrun™ molecular detection mix. sensitivity and specificity for the reported sample groups of both rt-lamp assays are shown in table 3 . the pcrun™ molecular detection mix performed better both in sensitivity and specifity than the isothermal mastermix. the amplification times for the isothermal mastermix positives ranged between 4 and 39 min and for the pcrun™ molecular detection mix positives between 18-77 min. in the present study, rt-lamp assays were evaluated as a diagnostic tool for detection of fcov, in order to distinguish cats with and without fip that are presented with body cavity effusions. time from sample to result was kept to a minimum by isolating rna in about 10 to 15 min using a simple rna extraction kit. both commercially available reaction mixtures allowed an easy and fast preparation of the amplification reaction. with lamp assays, direct detection methods built into the amplification device are preferred, since opening lamp reaction tubes after amplification is not advisable to decrease the risk of carry-over contamination (parida et al., 2008; zanoli and spoto, 2013) . in the present study, a portable device results was used for the pcrun™ molecular detection mix. positive and negative amplification reactions were indicated by the device with '+' and '-', making it compatible as a point-of-care instrument. the isothermal mastermix is intended for use with a portable device, which was not part of this study. the machine used instead replicates the reaction conditions with a constant block temperature and uses a comparable fluorescence detection system. both assays tested have similar demands concerning handling skills and preparation time. detection of positive samples took about half the time with isothermal mastermix compared to the pcrun™ molecular detection mix, yet both tests require less than 90 min. the specificity of both methods for the reported sample group was comparable, with 94.6% and 97.3%, respectively. however, false positive results in a test that diagnoses a fatal disease are very critical and should not occur. a cross-reaction of the lamp-primers or carry-over contamination might be the cause of these false positive results. however, the negative controls without sample material did not show any indication for carryover contamination and stayed negative in the lamp assays. another possibility for false positive results is the detection of fcov in cats without fip, since systemic spread of fcov does occur, but does not inadvertently result in fip (porter et al., 2014) . this explanation is not very likely for the three samples of cats without fip that were positive by rt-lamp, since all three were negative by rt-pcr. the sensitivity of the pcrun™ molecular detection mix was superior for the reported sample group with 58.8% compared to 35.3% of the isothermal mastermix, using the same primers and pcr conditions. since the samples for the rt-pcr and for the rt-lamp had the same preanalytical treatment (frozen, thawed, and centrifuged), the results can be directly compared and showed that the rt-pcr for fcov performed much better than the rt-lamp in our sample group. this is in agreement with a study on other coronaviruses, where rt-lamp also exhibited a lower analytical sensitivity compared to rt-pcr (bhadra et al., 2015) . false negative results can occur in samples with a lower viral burden. the fcov viral load determined by rt-pcr in effusions has been found to be quite low in some samples of fip-suspected cats (lorusso et al., 2017) . in our study, the results for rt-pcr were only returned as 'positive' or 'negative' without quantification, leaving open the question whether only effusions with a high viral load resulted in a positive rt-lamp detection. another possible reason for the lack of sensitivity might be that sequences of current fcov strains show sequence variations compared to the sequences deposited in the genbank database, which were used to design the rt-lamp primers. although the primers were chosen to bind in highly conserved regions, variations can occur, which might impair binding and eventually lead to low or no amplification, resulting in poor sensitivity. reliable primers for rt-pcr target the 3′ utr of the fcov sequence, but the rt-lamp primers that were suggested by the primerexplorer software in this region included more sequence differences than the m region that we selected. modifications of the primers might enhance binding and improve sensitivity. two studies on lamp-based identification of fcov have been published to date. a study from thailand tested 63 samples of body cavity effusions from cats that were suspected to have fip both by rt-pcr and by rt-lamp. more samples tested positive with the rt-lamp than with the rt-pcr (44% vs. 38%) (techangamsuwan et al., 2013) . however, the inclusion criteria for cats to be suspected of having fip were not described in that study. their control samples consisted of plasma and fecal samples from healthy cats without any contact to other cats. the samples from this healthy control group also had more positive results by rt-lamp than by rt-pcr (50% vs 30%). the authors mention high rates of false positives in the negative controls (no template controls) when using rt-lamp, so it remains unclear whether their rt-pcr was less sensitive or their rt-lamp was prone to unspecific amplification. in the second study, different sample types of cats with a clinical suspicion of fip and fecal samples for screening for fcov-shedding cats were tested (stranieri et al., 2017) . in most sample types, including effusions, their rt-pcr had about twice as many positive results as their rt-lamp, and none of the rt-pcr-negative samples was positive in the rt-lamp method. in agreement with our findings, the sensitivity of the rt-lamp appears to be inferior to the rt-pcr. for detection of amplification, both studies used gel electrophoresis and one study (stranieri et al., 2017) additionally used detection of color change from violet to blue with hydroxynaphtol blue. while gel electrophoresis is quite time-consuming, the color change can be difficult to detect in samples with low amplification. amplification detection with fluorescence or luminescence as used in the present study is preferable, since the results can be read immediately and could be easily converted to a quantitative format with a standard curve. as a perspective for the future, rt-lamp reactions for virus detection could be run on new devices that integrate nucleic acid extraction, amplification and detection in a miniature format to achieve a true point-ofcare diagnosis (stumpf et al., 2016) . in conclusion, the rt-lamp in the present study was relatively specific but not very sensitive. the sample type was restricted to effusions of cats unequivocally diagnosed with fip and of cats without fip but with clinical signs indicative of fip. this is a realistic setting in which a veterinarian would use a test for detection of fcov in the effusion sample. the rt-lamp assay with the pcrun™ molecular detection mix can be used in a clinical setting to a certain extent. however, before it can replace conventional rt-pcr methods, sensitivity and specificity have to be enhanced by optimizing primers and amplification conditions. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the pcrun™ molecular detection mix and the pcrun™ reader were provided by biogal free of charge. biogal was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. the fcov rt-pcr and subsequent mutation detection was done by idexx free of charge. idexx was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. a study of naturally occurring feline coronavirus infections in kittens real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) spike protein fusion peptide and feline coronavirus virulence virus taxonomy: ninth report of the international committee on taxonomy of viruses comparison of realtime reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis comparison of different tests to diagnose feline infectious peritonitis clinical symptoms and diagnosis of feline infectious peritonitis development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus feline infectious peritonitis: still an enigma? discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis accelerated reaction by loop-mediated isothermal amplification using loop primers rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification loop-mediated isothermal amplification of dna loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases a review of feline infectious peritonitis virus infection: 1963-2008 levels of feline infectious peritonitis virus in blood, effusions, and various tissues and the role of lymphopenia in disease outcome following experimental infection amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis reverse transcriptase loopmediated isothermal amplification for the detection of feline coronavirus labdisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza a h3n2 virus simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (lamp) development and application of reverse transcription loop-mediated isothermal amplification (rt-lamp) for feline coronavirus detection feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses isothermal amplification methods for the detection of nucleic acids in microfluidic devices we thank biogal for providing the pcrun™ molecular detection mix and the pcrun™ reader for this study. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jviromet.2018.03.003. key: cord-258374-qht98q0l authors: takano, tomomi; azuma, natsuko; satoh, miyuki; toda, ayako; hashida, yoshikiyo; satoh, ryoichi; hohdatsu, tsutomu title: neutrophil survival factors (tnf-alpha, gm-csf, and g-csf) produced by macrophages in cats infected with feline infectious peritonitis virus contribute to the pathogenesis of granulomatous lesions date: 2009-04-03 journal: arch virol doi: 10.1007/s00705-009-0371-3 sha: doc_id: 258374 cord_uid: qht98q0l feline infectious peritonitis (fip) is a feline coronavirus (fcov)-induced fatal disease of domestic and wild cats. the infiltration of neutrophils into granulomatous lesions is unusual for a viral disease, but it is a typical finding of fip. this study aimed to investigate the reason for the lesions containing neutrophils in cats with fip. neutrophils of cats with fip were cultured, and changes in the cell survival rate were assessed. in addition, the presence or absence of neutrophil survival factors was investigated in specimens collected from cats with fip. furthermore, it was investigated whether macrophages, one of the target cells of fipv infection, produce neutrophil survival factors (tnf-alpha, gm-csf, and g-csf). we showed that virus-infected macrophages overproduce neutrophil survival factors, and these factors act on neutrophils and up-regulate their survival. these observations suggest that sustained production of neutrophil survival factors by macrophages during fcov infection is sufficient for neutrophil survival and contributes to development of granulomatous lesions. feline coronavirus (fcov) belongs to group i of the family coronaviridae. fcov consists of three major proteins: nucleocapsid (n) protein, membrane (m) protein and peplomer spike (s) protein [23] . fcov is classified into serotypes i and ii according to the amino acid sequence of its s protein [19, 20] . both serotypes consist of two biotypes: feline infectious peritonitis (fip) virus (fipv) and feline enteric coronavirus (fecv). thus, there are types i and ii fecv and fipv in fcov. fecv is asymptomatic in cats, but fipv causes fip. it has been proposed that fipv arises from fecv by mutation [8, 29, 37] , but the exact mutation and inducing factors have not yet been clarified. fip is a fatal disease, characterized by vasculitis associated with granulomatous inflammation containing b cells, neutrophils and macrophages. the infiltration of neutrophils into granulomatous lesions is unusual for a viral disease, but it is a typical finding of fip [28] . macrophages/monocytes play an important role in the pathogenesis of fip. it has been reported that the difference in the proliferation of macrophages/monocytes is related to the difference in pathogenicity between fecv and fipv [4, 31] . the possibility of feline vascular endothelial cell injury caused by metalloproteinase-9 and tnf-alpha produced by fipv-infected monocytes has also been reported [12] . we reported that virus replication in macrophages induced tnf-alpha production, and the tnf-alpha produced was involved in aggravation of the fip pathology: tnf-alpha produced by fipv-infected macrophages was involved in lymphopenia and an increase in the level of the cellular receptor of type ii fipv, aminopeptidase n [34] . tnf-alpha reportedly inhibits neutrophil apoptosis [22] , suggesting its involvement in the infiltration of neutrophils into granulomatous lesions in cats with fip, but this has not yet been clarified. it is also unclear whether fipv-infected macrophages/monocytes produce factors other than tnfalpha that are related to the survival of neutrophils. in this study, we investigated the reason for the granulomatous lesions containing neutrophils in cats with fip. neutrophils of cats with fip were cultured, and changes in the survival rate were examined. the presence or absence of neutrophil survival factors in specimens from cats with fip was also investigated. furthermore, whether macrophages, one of the target cells of fipv, produce neutrophil survival factors was assessed. type ii fipv strain 79-1146 (10 4 tcid 50 /ml) was administered orally to 6-to 8-month-old spf cats. nine cats that developed fip symptoms (fip cats), such as fever, weight loss, peritoneal or pleural effusion, dyspnea, ocular lesions, and neural symptoms, and nine 6-to 8-month-old spf cats administered a medium as mock infection controls were used in this study. fip diagnoses were confirmed upon postmortem examination, revealing peritoneal and pleural effusions and granulomatous lesions in major organs. all experiments were performed in accordance with the guidelines for animal experiments of kitasato university. felis catus whole fetus-4 (fcwf-4) cells were grown in eagle's minimum essential medium containing 50% l-15 medium, 5% fetal calf serum (fcs), 100 u/ml penicillin, and 100 lg/ml streptomycin. feline neutrophils and alveolar macrophages were maintained in rpmi 1640 growth medium supplemented with 10% fcs, 100 u/ml penicillin, 100 lg/ml streptomycin, and 50 lm 2-mercaptoethanol. type ii fipv strain 79-1146 was grown in fcwf-4 cells at 37°c. fipv strain 79-1146 was supplied by dr. m. c. horzinek of state university utrecht, the netherlands. mab 6-4-2 (igg2a) used in the present study recognizes the s protein of type ii fipv, as demonstrated by immunoblotting. it has been reported that mab 6-4-2 has virusneutralizing activity in assays carried out in fcwf-4 and crfk cells, but an enhancing activity in feline macrophage cultures, depending on the reaction conditions [10] . blood collected from spf and fip cats using a heparinized syringe was centrifuged at 3,000 rpm for 10 min, and the supernatant was used as a plasma sample. ascites fluid was collected from fip cats using a heparinized syringe and centrifuged at 3,000 rpm for 10 min, and the supernatant was collected. heparinized blood (10 ml) from spf and fip cats was diluted in twofold steps with phosphate-buffered saline (pbs) and subjected to ficoll-hypaque density gradient centrifugation at 1,700 rpm for 20 min. after the removal of peripheral blood mononuclear cells and supernatant by aspiration from the top layer, the pellets were mixed with an equal volume of saline containing 6% dextran for granulocyte separation and allowed to stand for 45 min at 37°c. the top clear layer was centrifuged at 400g for 10 min, and the pellet was mixed with 4 ml of 0.2% nacl for 2 min to eliminate contaminating erythrocytes and then mixed with 4 ml of 1.6% nacl. the cells were washed three times with pbs and resuspended with growth medium. cell purity was assessed to be more than 98% neutrophils by the examination of a smear stained with wright/giemsa solutions. to examine the effect of specimens from cats on the survival rate of neutrophils, feline neutrophils (1 9 10 5 cells/100 ll) were seeded into 96-well plates and cultured in the presence of fip-cat-derived ascites fluid (final concentration of 1:20), plasma (final concentration of 1:20), and spf-cat-derived plasma (final concentration of 1:20) for 24 h. prior to and after incubation, 10 ll of wst-8 solution (wst-8 cell proliferation assay kit; kishida chemical co., ltd, japan) was added. wst-8 is a tetrazolium salt that reacts with mitochondrial dehydrogenases, forming the formazan dye. expansion of viable cell numbers results in an increase in the activity of the mitochondrial dehydrogenases in the cells, corresponding to an increase in formazan dye metabolism. after the cells were returned to the incubator for 4 h, the absorbance of the formazan produced was measured at 450 nm with a 96-well spectrophotometric plate reader, as described by the manufacturer. the percent viability was calculated using the following formula: cell viability (%) = (after incubation od/prior to incubation od) 9 100. feline alveolar macrophages were obtained from spf and fip cats by broncho-alveolar lavage with hank's balanced salt solution (hbss) as described previously by hohdatsu et al. [9] . rna isolation and cdna preparation rna isolation and cdna preparation were performed employing the method of takano et al. [34] . determination of levels of feline gapdh mrna, tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n gene expression cdna was amplified by pcr using primers specific for feline gapdh mrna, tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes. the primer sequences are shown in table 1 . pcr was performed using the method of takano et al. [35] . the band density was quantified under appropriate uv exposure by video densitometry using scion image software (scion corporation, usa). tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes were quantitatively analyzed in terms of the relative density value compared to the mrna for the housekeeping gene gapdh. confluent fcwf-4 cell monolayers in 24-well multi-plates were inoculated with 100 ll of the sample dilutions. after virus adsorption at 37°c, the cells were washed with hbss, and 1 ml of growth medium containing 1.5% carboxymethyl cellulose was added to each well. the cultures were incubated at 37°c for 2 days, fixed in 10% buffered formalin, and stained with 1% crystal violet. inoculation of feline alveolar macrophages with fipv viral suspension (fipv strain 79-1146, 2 9 10 3 tcid 50 / 0.1 ml) and mab 6-4-2 solution were mixed in an equal volume ratio and allowed to react at 4°c for 1 h, and 0.1 ml of this reaction solution was used to inoculate feline alveolar macrophages (2 9 10 6 cells) cultured in each well of 24-well multi-plates. as controls, medium alone and virus suspension alone were added to feline alveolar macrophages. after virus adsorption at 37°c for 1 h, the cells were washed with hbss and 1 ml of growth medium. the cells and culture supernatant were collected every 24 h thereafter. the cells were used for measurement of the tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes. tnfalpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes were quantitatively analyzed in terms of the relative density value compared to the mrna for the housekeeping gene gapdh. the culture supernatant was employed for determination of the virus titer. data were analyzed by student's t test. the data in fig. 1a , b were also analyzed using the mann-whitney test. p values \ 0.05 were considered to indicate a significant difference between compared groups. the neutrophil counts in peripheral blood of fip cats were examined and compared with those of uninfected spf cats. the count of neutrophils at the time of blood sampling is shown in fig. 1 . the blood neutrophil counts in spf and fip cats were 5,252 ± 1,493 ll -1 (mean ± sd) and 9,425 ± 3,996 ll -1 , and median values were 4,921 and 8,093 ll -1 , respectively (p \ 0.05). to investigate the cause of increases in the neutrophil counts in fip cats, neutrophils were isolated from spf and fip cats, and the survival rates after 24-h culture were compared. the survival rate of neutrophils from fip cats was increased, but not significantly (p = 0.122), compared to that of spf cats (fig. 2) . the survival rate of neutrophils in the presence of specimens from fip cats was significantly higher than those of neutrophils cultured with spf cat-derived plasma and medium (fig. 3) . tnf-alpha, gm-csf, and g-csf mrna and fcov n gene expression levels in macrophages of spf and fip cats tnf-alpha, gm-csf, and g-csf mrna and fcov n gene expression levels were increased in alveolar macrophages derived from fip cats (fig. 4) . the virus titer was significantly higher in the culture supernatant of macrophages infected with a mixture of fipv and mab 6-4-2 than in that of macrophages cultured with medium and fipv alone. the neutrophil survival rates were significantly increased in the presence of the culture supernatant of macrophages infected with the mixture of fipv and mab 6-4-2 compared to those in the presence of other supernatants (fig. 5) . when spf-cat-derived alveolar macrophages were infected with a mixture of fipv and mab 6-4-2, the intracellular tnf-alpha, gm-csf, and g-csf mrna levels increased (fig. 6 ). neutrophils are important for host defense against pathogens. viral infection generally reduces the number of neutrophils. the transfer of peripheral blood neutrophils to marginal tissues, destruction of neutrophils due to excess antibody production, and direct cell death caused by viral infection are considered to be the causes of neutropenia [3, 16, 18, 19] . in human immunodeficiency virus and canine parvovirus infections, a reduced neutrophil count has been suggested to allow severe bacterial infection [14, 15] . feline immunodeficiency virus and feline panleucopenia virus infections also affect stromal cells in the bone marrow, thus leading to a decreased production of neutrophils [17, 26] . in contrast, severe acute respiratory syndrome (sars) coronavirus, which belongs to the same family as fipv, can also cause neutrophilia [39] . neutrophilia has been also reported in cats with fip [25] . paltrinieri [24] reported that cytokines accelerate the delivery of neutrophils to the inflamed lesions, thereby prolonging the lifespan of circulating neutrophils. he also reported that the apoptosis of neutrophils is delayed by cytokines, thus increasing the life of neutrophils in lesions. it is likely that neutrophilia in cats with fip is associated with the infiltration of neutrophils into granulomatous lesions. however, there seems to be no established theory to explain the infiltration of neutrophils into granulomatous lesions in cats with fip. in humans, the lifespan of neutrophils is short. when neutrophils are isolated from peripheral blood and cultured in vitro, apoptosis is induced, and about half of the cells die within 24 h [27] . when neutrophils isolated from peripheral blood of spf cats were cultured for 24 h, more than 60% died, suggesting that feline neutrophils also die due to apoptosis, similar to human neutrophils. furthermore, coculture with specimens from fip cats increased the survival rate of neutrophils, suggesting that the production of factors involved in the survival of neutrophils is enhanced in fip cats, and these factors act on neutrophils and prolong their lifespan. the tnf-alpha, gm-csf, and g-csf mrna levels were increased in macrophages of fip cats. these cytokine mrna levels were also elevated in macrophages infected with fipv and mab 6-4-2, clarifying the presence of neutrophil survival factors in the macrophage culture supernatant. it was suggested that: (1) fipv-infected macrophages release tnf-alpha, gm-csf, and g-csf in response to virus replication, and (2) these cytokines act on neutrophils and prolong their survival. we previously reported that fipv-infected macrophages produced tnfalpha and b-cell differentiation/survival factors, and these factors may have been involved in lymphopenia and hypergammaglobulinemia [33] [34] [35] . kipar et al. [12] suggested that fipv-infected monocytes are involved in feline vascular endothelial cell injury. cytokines and chemical mediators produced by fipv-infected macrophages/monocytes may play an important role in the pathogenesis of fip in cats. fig. 5 the culture supernatant of fipv-infected macrophages promotes neutrophil survival. spf-cat-derived alveolar macrophages (2 9 10 6 cells) were cultured with medium alone, fipv, or fipv and mab 6-4-2. the culture supernatant was collected after 72 h. the virus titer in the culture supernatant was measured by the plaque assay method (left). the neutrophils of spf cats (1 9 10 6 ml -1 ) were cultured at 37°c for 24 h in the presence of each culture supernatant (final dilution of 1:5), and cell viability was assessed by the wst-8 assay (right). n = 6. nd not detected fig. 6 relationship between tnf-alpha, gm-csf, and g-csf mrna expression and fipv replication in macrophages. spf-catderived alveolar macrophages (2 9 10 6 cells) were cultured with medium alone, fipv, or fipv and mab 6-4-2. the cells were collected at 0 h (as a control) and 72 h. the intracellular tnf-alpha, gm-csf, and g-csf mrna expression levels were measured by rt-pcr. tnf-alpha, gm-csf, and g-csf mrna were quantitatively analyzed in terms of the relative density value to mrna for the housekeeping gene gapdh. n = 6 neutrophil survival factors produced by macrophages in cats 779 tnf-alpha, gm-csf, and g-csf are cytokines inhibiting the apoptosis of neutrophils [5, 13, 21, 22] . these cytokines are secreted by macrophages and t cells. when these cytokines act on neutrophils, the intracellular apoptosis-inhibitory protein level increases, whereas the apoptosis-promoting protein level decreases, prolonging the survival of neutrophils [5, 38] . in inflammatory diseases, such as cystic fibrosis and kawasaki disease, the pathological condition is aggravated as the survival time of neutrophils is prolonged [5, 36] . moreover, the pathological aggravation of inflammatory diseases is associated with protease and reactive oxygen species produced by neutrophils [6, 32] . these mediators may also be produced in excess and aggravate the granulomatous lesions in fip cats. we are now progressing with studies on the expression of apoptosis-inhibitory and -promoting proteins in neutrophils of fip cats. the enhancement of cathepsin b production in macrophages by elastase produced by neutrophils has been reported [7] , while fipv reportedly requires cathepsin b to invade cells [30] , suggesting that neutrophils are also involved in a viral replication enhancement mechanism, different from antibody-dependent enhancement (ade), in fipv infection. addie et al. [1] reported that fcov re-infection of anti-fcov antibody-positive domestic cats might not result in the development of ade. the involvement of neutrophils in the enhancement of fipv production in macrophages and the influence of macrophages on neutrophil survival should be investigated further. virus production and tnf-alpha, gm-csf, and g-csf mrna expression were markedly enhanced in macrophages infected with fipv and mab 6-4-2 compared to macrophages infected with the virus alone. we used immunofluorescence to detect fipv antigen: 10-20% of cells were positive when spf cat-derived alveolar macrophages were infected with fipv and mab 6-4-2, whereas 1-2% of cells were positive when alveolar macrophages were infected with fipv [9, 11] . it seems that the increased number of virus-infected macrophages leads to the overexpression of neutrophil survival-factor-associated mrna in macrophages. in cats with fip, virus can be detected in macrophages of granulomatous lesions. to elucidate the mechanism of the infiltration of neutrophils into granulomatous lesions, we used alveolar macrophages, which can be readily collected in appropriate numbers. in addition, alveolar macrophages can be cultured without activation treatment, unlike monocytes. in this study, we showed that virus-infected macrophages overproduce neutrophil survival factors, and these factors act on neutrophils and up-regulate their survival. these observations suggest that sustained production of neutrophil survival factors by macrophages during fcov infection is sufficient for neutrophil survival and contributes to the development of granulomatous lesions. these findings may be important for elucidating the pathogenesis of fip. risk of feline infectious peritonitis in cats naturally infected with feline coronavirus gamma interferon/interleukin 10 balance in tissue lymphocytes correlates with down modulation of mucosal feline immunodeficiency virus infection immune and idiopathic neutropenia replication of 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spike protein during entry of serotype ii feline coronaviruses acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein the role of eosinophils and neutrophils in inflammation a ''possible'' involvement of tnf-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis tnf-alpha, produced by feline infectious peritonitis virus (fipv)-induced macrophages, upregulates expression of type ii fipv receptor feline aminopeptidase n in feline macrophages b-cell activation in cats with feline infectious peritonitis (fip) by fip-virus-induced b-cell differentiation/survival factors delayed apoptosis of circulating neutrophils in kawasaki disease feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses bcl-xl-and baxalpha-mediated regulation of apoptosis of human neutrophils via caspase-3 haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis neutrophil survival factors produced by macrophages in cats 781 acknowledgments this work was supported by grant for encouragement of young scientists (no. e0801) from the school of veterinary medicine, kitasato university. key: cord-306829-88nihy7q authors: sharif, saeed; arshad, siti suri; hair-bejo, mohd; omar, abdul rahman; zeenathul, nazariah allaudin; alazawy, amer title: diagnostic methods for feline coronavirus: a review date: 2010-07-28 journal: vet med int doi: 10.4061/2010/809480 sha: doc_id: 306829 cord_uid: 88nihy7q feline coronaviruses (fcovs) are found throughout the world. infection with fcov can result in a diverse range of signs from clinically inapparent infections to a highly fatal disease called feline infectious peritonitis (fip). fip is one of the most serious viral diseases of cats. while there is neither an effective vaccine, nor a curative treatment for fip, a diagnostic protocol for fcov would greatly assist in the management and control of the virus. clinical findings in fip are non-specific and not helpful in making a differential diagnosis. haematological and biochemical abnormalities in fip cases are also non-specific. the currently available serological tests have low specificity and sensitivity for detection of active infection and cross-react with fcov strains of low pathogenicity, the feline enteric coronaviruses (fecv). reverse transcriptase polymerase chain reaction (rt-pcr) has been used to detect fcov and is rapid and sensitive, but results must be interpreted in the context of clinical findings. at present, a definitive diagnosis of fip can be established only by histopathological examination of biopsies. this paper describes and compares diagnostic methods for fcovs and includes a brief account of the virus biology, epidemiology, and pathogenesis. feline coronaviruses (fcovs) are enveloped viruses with a large, capped, polyadenylated rna genome of about 29, 190 nucleotides. the fcovs are group 1 coronaviruses, recently designated as members of subgroup 1a in the family coronaviridae. other members of this subgroup include transmissible gastroenteritis virus (tgev), canine coronavirus (ccv), raccoon dog coronavirus (rdcov), and chinese ferret badger coronavirus (cfbcov) [1] . the order of the genes encoding the viral polymerase (pol) and the four structural proteins (the spike, envelope, membrane, and nucleocapsid proteins) is 5 -pol-s-e-m-n-3 . these genes are present in all coronaviruses. the feline coronavirus genome also includes additional genes (3a, 3b, 3c, 7a, and 7b) that encode nonstructural proteins. the functions of these gene products are not fully understood [2] . two biological types of fcovs are known: feline infectious peritonitis virus (fipv) and feline enteric coronavirus (fecv). in the widely accepted "in vivo mutation" theory, fipv arises by mutation from parental fecv in the gastrointestinal tract of an infected cat, spreads systemically and causes fip [3] [4] [5] . the mutation sites are not fully understood, but some accessory genes (3c and 7b) are candidates for the site of the critical mutations responsible for fip [6, 7] . an alternative hypothesis is the "circulating virulent/avirulent" theory, which suggests that both virulent and avirulent strains circulate in cat populations, and susceptible individuals exposed to the virulent strains develop the disease. this hypothesis was proposed after sequence analysis of four genes (pol, s, m and 7b) from fcov-infected healthy cats and cats with fip. phylogenetic analyses revealed that sequences of the m and 7b genes in viruses obtained from healthy cats were distinct from those obtained from sick cats and suggest the coexistence of both biotypes in cats [8] . however, as these viruses undergo mutation readily [7] and genetic differences in the 7b gene were not correlated with pathogenicity in another study [9] , the epidemiology of fipv is yet to be clarified. regardless of the source of fipv and uncertainty about the significance of genetic differences, the relationship between virulence and macrophage/monocyte tropism has been firmly established [7] . while both fipv and fecv may cause viraemia [10] [11] [12] , only fipv replicates in macrophages 2 veterinary medicine international and causes the disease [5, 13] . complex immune reactions between the virus, antiviral antibodies, and complement cause disseminated vasculitis, which is the hallmark of fip [14, 15] . based on their antigenic relationship with canine coronavirus, sequence analyses of the s gene, and their growth characteristics in vitro, fcov strains can be classified into serotypes i and ii. fcov serotype i strains are wholly feline. they are difficult to grow in cell culture and cause a slowly developing cytopathic effect. fcov serotype ii strains seem to have arisen by recombination between fcov serotype i and ccv. they grow more rapidly than serotype i viruses and induce a lytic cytopathic effect. fipv and fecv strains can be serotype i or ii [6, [16] [17] [18] . fcov infection is extremely common in cat populations. antibodies against fcov are found in 20%-60% of pet cats and up to 100% of cats in catteries or multi-cat households [14, [19] [20] [21] [22] [23] [24] . fcovs are highly infectious and spread predominantly by the faecal-oral route. about 75%-100% of cats in multi-cat environments shed the virus [14, 25, 26 ]. fecv infections are usually associated with mild disease at most. many cases remain asymptomatic, and in young kittens mild transient diarrhoea of several days duration is generally the only sign. vomiting occurs in a smaller proportion of cases and is not usually a prominent feature. infection with fecv rarely causes disease of sufficient severity to require specific diagnosis of the underlying aetiological agent. the virus can be demonstrated in the faeces of infected kittens by electron-microscopic examination or by reverse transcriptase polymerase chain reaction (rt-pcr) assay. however many healthy cats and kittens will also shed fcov in their faeces. thus, other than for detection of carriers or demonstrating the presence of fcov infection in a colony of cats, such investigations have limited value [15, 27] . fipv variants of fcov cause fatal peritonitis. cats with a poor cell-mediated immune response develop the effusive or "wet" form of disease, which is an immune complex vasculitis that causes leakage of protein-rich fluid from the blood vessels into the abdominal cavity, leading to a distended abdomen. in cats with partial cell-mediated immunity, the non-effusive or "dry" form develops, with pyogranulomatous or granulomatous lesions in multiple tissues. dry fip may become effusive in the terminal stages of the disease, when the immune system collapses [28, 29] . ante-mortem diagnosis of fip is difficult and frustrating. difficulties in definitively diagnosing fip arise from the lack of specific clinical signs, the lack of pathognomonic biochemical abnormalities, and the low sensitivity and specificity of tests routinely used in practice. fip diagnosis is based on assimilation of the history, haematology, and other supportive diagnostic tests, including serology and findings from imaging, tissue biopsies, and pcr [8, 15] . a typical history of fip cases includes acquisition of the cat from a cattery and a fever that waxes and wanes and does not improve with antimicrobial therapy. most commonly, kittens are infected between the ages of 6 and 8 weeks, at a time when maternally derived antibodies wane, mostly through contact with faeces from their mothers or other fcov-excreting cats [27] . fip is a disease with extremely diverse clinical manifestations. in the "wet" form, the most characteristic sign is a considerable amount of intracavitary effusion ( figure 1 ). the typical lesions of effusive fip are pyogranuloma and fibrinous plaques on the serosal surfaces of abdominal organs (figures 2 and 3) . dyspnea, mild pyrexia, and muffled heart sounds are common. ocular involvement in fip can include uveitis, keratic precipitations, and changes in the coloration of the iris. in the non-effusive form, lesions commonly occur in eyes and cns, but granulomas may also be found in the peritoneal cavity, leading to more diverse, and often more vague, clinical signs [15, 29] (figure 4) . there are a number of laboratory findings that are common in cats with fip, but none of them is pathognomonic. although it is often stated that lymphopaenia and neutrophilia are typical of fip, this change can be interpreted as a typical "stress leukogram" that occurs in many severe systemic diseases in cats [32, 33] . the most consistent laboratory finding in fip is an increase in total serum protein concentration. this is found in approximately 50% of cats with effusive disease and 70% of cats without effusive disease. the increase in total protein is caused by an increased concentration of globulins, mainly γ-globulins. a γ-globulin concentration of more than 32% is characteristic of fip [33] [34] [35] [36] . changes in the serum protein profile lead to a decreased albumin-to-globulin (a : g) ratio. an a : g ratio of less than 0.5 is strongly correlated with fip [37] . acute phase proteins (apps) are a large and varied group of glycoproteins in the serum, concentrations of which increase (or decrease) during certain inflammatory disorders. the concentrations of apps such as feline α1acid glycoprotein (fagp) and serum amyloid a (saa) can be measured and facilitate the diagnosis. saa and fagp concentrations increase in fip, but they are not specific for this disease. while moderately elevated levels of acute-phase proteins are found in several inflammatory conditions, high levels of fagp (>1.5 g/l) and saa in plasma or effusions can indicate fip and may be useful supportive tests [38] [39] [40] [41] . sampling from effusions is an important diagnostic step for fip because tests on effusions have much higher diagnostic value than tests performed on blood samples. effusions are typically clear yellow and viscous and may form fibrin strands. however, the presence of this type of fluid in body cavities alone is not diagnostic. involvement of peritoneal and plural cavities has been reported in 58% and 11% of fip cases, respectively [7, 15] . the effusion seen in fip is classified as a modified transudate to exudate with a very high-protein content (>3.5 g/dl) and moderate cellular content. fip effusions can be examined using a simple and cheap method called the rivalta test. adding a small drop of effusion to a test tube containing distilled water and a drop of 8% acetic acid can cause precipitation because of the high-protein content. this test seems to be useful for differentiation between effusions caused by fip and effusions caused by other diseases. however, false-negative results can be obtained in cats with bacterial peritonitis and false-positive results in cats with lymphoma [37, 38] . cytological evaluation of the effusion in cats with fip reveals macrophages and neutrophils in a dense proteinaceous background. neutrophils are non-degenerate or may show mild nuclear degenerative changes. lymphocytes and plasma cells may also be found in the fluid. in a study by hartmann and colleagues [37] , immunofluorescent staining of intracellular fcov antigen in macrophages in effusions had a positive predictive value of 100%, but the negative predictive value was not high (57%). this could be due to the small numbers of macrophages in the smear or masking of the antigen by competitive binding of anti-fcov antibodies in the effusion [15] . other laboratory parameters (e.g., liver enzymes, bilirubin, urea, and creatinine) can be variably increased, depending on the degree and localization of organ damage, but they are not helpful in making an aetiological diagnosis [15, 27, 38] . measurements of antibody in serum are useful diagnostic tools for detection of fcov infection. however, since a large percentage of healthy cats have antibodies against fcov, antibody testing is more helpful in the management of fcov infection (e.g., creating an fcov-free cattery) [15] . the sensitivity and specificity of a commercially available in-practice test kit for detection of fcov antibodies (immunocomb fcov antibody test kit, biogal, israel) were 95% and 83%, respectively [42] . antibody testing in cats suspected to have fip has limited value in confirmation of the diagnosis and results should be interpreted carefully. some cats with the wet form of fip have low titres or even no antibodies against fcov. this is because the large amounts of virus in the cat's body bind to antibodies and render them unavailable to antigen in the test or because the antibodies are lost in effusions [15] . the use of anti-7b proteins for serology does not appear to offer any significant advantage [43] . since fip is an immune-mediated disease, antibodyantigen complexes may circulate in the serum and effusions. the circulating complexes can be detected using a competitive enzyme-linked immunosorbent assay (elisa). however, the utility of this assay is limited because the positive predictive value of the test is not high (67%) and there are many false-positive results [37, 44] . there are several reports of detection of fcov by rt-pcr. some used primers targeted at conserved regions of the viral genome, such as the pol [45] [46] [47] , the 7b gene [43, 48, 49] , and the 3 untranslated region (3 utr) [50] [51] [52] . rt-pcr assays using these primers are able to detect most, if not all, fcov strains and could be a valuable tool for screening for the virus in cat populations. the sensitivity and specificity of the rt-pcr assay could be increased using real-time rt-pcr techniques [53] . as the sequence of the s gene differs between serotypes i and ii, some rt-pcr assays have targeted the s gene to differentiate the fcov serotypes [18, 54] . since specific genetic determinants of fcov biotypes are unknown and the genome contains various single nucleotide polymorphisms (snps) [7, 55, 56] , it is not possible to design pcr primers to distinguish between fipv and fecv [11] and thus discriminate between fip cases and fcovpositive healthy cats. in 2005, simons and colleagues [57] introduced a new pcr-based approach for detection of fip. the approach was based on the key pathogenic event in fip, viral replication in macrophages and monocytes. the primers targeted the conserved region of the m gene and the leader sequence to detect replicating virus in the blood. the assay had high sensitivity and specificity [57] and is currently used in some diagnostic laboratories [28, 58] . however, in a study on 26 cats, can-ş ahna et al. (2007) found the specificity of the assay using same primers to be poor [59] . the reason for the high-false-positive rate in healthy cats (53%) in this second study is not clear, but the different rna extraction kits used in these studies may have affected the quality of the template rna and the rt-pcr outcome [47] . veterinary medicine international 5 figure 6 : kidney of a cat with fip. severe degenerative and advanced necrotic changes within the lining endothelium of the convoluted tubules (mostly cytoplasmolysis). frank patchy interstitial nephritis, as indicated by the heavy infiltration of lymphocytes, plasma cells and some dead neutrophils, together with dilatation and congestion of the interstitial blood vessels. the photograph was kindly provided by dr. diane addie. moreover, since both studies used conventional rt-pcr techniques, they were not able to quantify the replicating mrna in blood of infected cats. therefore, a quantitative real-time rt-pcr assay that could determine the amount of viral mrna in blood may be able to better differentiate fcov-positive healthy cats from fip cases. rt-pcr assays have been used to detect fcov in faecal samples and are sensitive and useful for documenting that a cat is shedding fcov in faeces. faecal samples must be carefully handled, kept frozen, and protected from the rnadegrading enzymes that are present in most environments. rt-pcr should be performed as soon as possible after sampling, and even freezing samples may result in false negative results. the strength of the rt-pcr signal in faeces correlates with the amount of virus present in the intestine [15] . comparisons between rna samples extracted from faecal suspensions and fcov-infected cell culture supernatants showed that the presence of faecal factors significantly inhibited the reverse transcription reaction [47] . however, pedersen et al. [26] , found no evidence for faecal inhibitors in their rt-pcr assay. the virus can be detected in various tissues and ascitic fluid [50, 56, 60, 61] . liver (48%) and spleen (42.3%) samples appear more likely to contain detectable fcov than the kidneys (21.1%). in addition, the amounts of rna extracted from fresh tissues were significantly higher than from tissue fixed in formalin, ethanol or bouin's solution [61] . primers designed to detect fip in ill cats were also found to be able to amplify fcov in healthy cats [12, 37, 50] . thus, rt-pcr results should be interpreted in conjunction with the clinical status of the cat and cannot be used as the sole test to diagnose fip. there are several plausible explanations for false-negative rt-pcr results, including degradation of rna, failure of the reverse transcription reaction, and variation in the nucleotide sequences of fcovs. infection with ccv or tgev could also contribute to false-positive results because they share similar conserved regions [8, 54]. histopathological confirmation of fip has been used to define cases and has been regarded as the "gold standard" for diagnostic test comparisons. haematoxylin and eosin (h & e) stained sections typically have localized inflammation with macrophages, neutrophils, lymphocytes, and plasma cells ( figure 5 ). vascular lesions may be found surrounded by proliferation of inflammatory cells and this is characteristic for wet fip. pyogranulomas are mainly associated with fibrinous necrosis and may be large and consolidated or numerous and small. focal accumulations of inflammatory cells and necrotic-proliferative lesions are typical of the granulomatous lesions of dry fip [18, 27, 58, 62] (figure 6 ). immunohistochemical tests, such as immunoperoxidase staining, may enable the detection of fcov antigen in tissue. immunostaining cannot differentiate between fecv and fipv, but as fipv replicates more actively, higher concentrations of the viral antigen are found in fip cases [15] . viral antigen concentrations are lower in lesions in cats with dry fip than in those of cats with wet fip [14] . there are no pathognomonic clinical signs or specific laboratory tests for fip in cats. the presence of antibodies does not indicate fip and the absence of antibodies does not exclude it. many authors agree that serological data alone have limited diagnostic value. pcr assays are able to directly detect the fcov genome but, although they appear to be more sensitive for detection of coronaviral infection in cats, the results must be interpreted in conjunction with other clinical findings and cannot be used as the sole criterion for diagnosis of fip. a definitive diagnosis of fip should be confirmed by histopathology or detection of intracellular fcov antigen by immunofluorescent or immunohistochemical staining. evolutionary insights into the ecology of coronaviruses coronaviridae an enteric coronavirus infection of cats and its relationship to feline infectious 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peritonitis virus isolates of malaysia a mrna pcr for the diagnosis of feline infectious peritonitis feline infectious peritonitis: typical findings and a new pcr test the detection of feline coronaviruses in blood samples from cats by mrna rt-pcr development of a nested pcr assay for detection of feline infectious peritonitis virus in clinical specimens detection of feline coronaviruses in cell cultures and in fresh and fixed feline tissues using polymerase chain reaction feline infectious peritonitis the authors wish to thank dr. diane addie (www.catvirus.com) for providing the photograph and proofreading the paper. key: cord-336730-hqgwj8vs authors: fehr, daniela; holznagel, edgar; bolla, stefania; hauser, beat; herrewegh, arnold a.p.m.; horzinek, marian c.; lutz, hans title: placebo-controlled evaluation of a modified life virus vaccine against feline infectious peritonitis: safety and efficacy under field conditions date: 1997-07-31 journal: vaccine doi: 10.1016/s0264-410x(97)00006-6 sha: doc_id: 336730 cord_uid: hqgwj8vs abstract a modified live virus vaccine against feline infectious peritonitis (fip) was evaluated in a double blind, placebo-controlled field trial in two high-risk populations. the vaccine was found to be safe and efficacious in one population of cats that had low antibody titre against feline coronavirus (fcov) at the time of vaccination. although clinically healthy at the time of vaccination, retrospectively some vaccinees that later came down with fip were found to be rt-pcr positive for fcov in plasma and showed changes in blood parameters consistent with early stage of fip. it is concluded that vaccination can protect cats with no or low fcov antibody titres and that in some cats vaccine failure was probably due to pre-existing infection. feline infectious peritonitis (fip) is a normally fatal disease of cats caused by infections with feline coronaviruses (fcov) which are antigenically related to a respiratory coronavirus strain of man (hcv 229e), transmissible gastro-enteritis virus (tgev) of swine and canine coronaviruses13'. in switzerland, infections with fcov in domestic cats are widespread. about 80% of the cattery cats and 50% of all cats with access to outdoors were found to be seropositive3. five to 12% of these develop lethal fwt. certain cat populations seem to be more susceptible to fip. young cats are especially prone: 54% of all fip cases affected cats younger than 12 months of age and 70% cats younger than 4 year?. a genetic disposition in certain breeds and in cheetahs was described5-', and cats living in multiple-cathouseholds such as catteries or cat shelters and cats with access to outdoors are more likely to get exposed to fcov and develop fip than animals from single-cathouseholds'. clinical signs include the effusive or the non-effusive, granulomatous form of fip; both can also appear together. characteristic laboratory findings in fip 1996; accepted 12 december 1996) are anaemia, neutrophilia, lymphopenia, increase of total serum protein, hyperglobulinemia and decreased albumin5. in 1981, a low virulent fcov type, called feline enteric coronavirus (fecv), which caused only mild gastrointestinal and respiratory diseases mainly in kittens, was described'. antibodies to these fecv and the virulent fip-causing viruses (feline infectious peritonitis virus: fipv) do crossreact. these authors formulated the hypothesis that most of these seropositive cats are actually infected with fecv and that fipv is just a mutant of fecv which has the ability to infect macrophages. at this time, no molecular or immunological differences are known between fecv and fipv which can explain the difference of virulence between these coronaviruses". therefore, it appears to be justified to generally designate them as fcov and to consider every fcov infection in cats as a potential risk4,". several observations point out the important role of the cell mediated immunity (cmi) in fip patho-genesis12-16, but the detailed immune mechanisms for controlling fcov infection remain unknown. under experimental conditions humoral immunity does not lead to protection. on the contrary, after experimental fip infection seropositive cats develop fip after a much shorter incubation period than seronegative control cats1'-19. this antibody dependent enhancement (ade) is thought to occur when virus-antibody complexes are formed and bound to the fc receptors of macrophages. macrophages are then more efficiently infected by the fc receptor-mediated endocytosis than by the virus alone2+22. when we initiated this study, this modified live virus vaccine (primucell fip@) was already commercially available in the usa, but many questions concerning safety and efficacy under field conditions were still unanswered. the safety of the vaccine was confirmed under experimental and field conditions23, but vaccinated cats showed ade when challenged with a high dose of heterologous virus strain24. the efficacy of the vaccine was assessed only under experimental conditions. with this trial the safety and efficacy of the vaccine was evaluate under field conditions in two high risk populations. a preliminary report has been presented at the fecv/fipv-workshop in davis, ca in 1994 and published in the proceedings". the study was performed as a placebo-controlled double blind assay. neither the investigators, nor the cat owners, knew which of two colour coded vials contained the vaccine. the code was not opened to the investigators, veterinarians and cat owners, until all cats terminated the 12 month observation period. two populations with a high risk for fcov infection and fip were included in this trial. the first population consisted of 138 cats from 15 catteries with fip problems. in all of these catteries, fip cases had occurred in the last 18 months prior the beginning of this trial either in the cattery itself or in kittens which had been re-homed to new owners. we expected that some of these cats had been already exposed to fcov. the second population consisted of 609 cats < 12 months of age, which were vaccinated by veterinarians in switzerland. as already mentioned, this 2ge group is more susceptible to fip than older cat?,-. the cats of each population were further subdivided into two groups, vaccine and placebo, respectively, which were comparable regarding age, sex, breed and living conditions. only clinically healthy cats older than 16 weeks of age were vaccinated and pregnant queens were excluded from the study. in week 0 and 3-4 weeks later the cats were vaccinated intranasally with either the coded vaccine or the placebo. after the vaccination the two coded groups were kept separately for 48 h to prevent spread of the vaccine virus to cats of the placebo group. in both populations a blood sample was collected before vaccination (week 0) and tested for felv and fcov-antibodies. in cattery cats only, haematology and clinical chemistry were done in week 0, 8 and 30 and in 20 cats each of the vaccinated and of the placebo group, cd4+/cd8+-t-cells were measured in week 0,8 and 30. fiv-tests were carried out in week 0 in the cattery cats. of sick cats, a blood sample was collected and haematology, clinical chemistry, felv and fcov-antibodies were determined. the modified live virus vaccine was developed by gerber et a1.'7 briefly, fipv-df2 was attenuated in 99 cell culture passages on the norden laboratories feline kidney (nlfk) cell line. passages 61-99 were propagated at 31°c. the 99th passage was exposed to ultraviolet irradiation. the vaccine has been shown to induce iga antibodies in the mucosa and to stimulate the cell mediated immune response2'. the serial of the vaccine used in this study was a commercial batch (serial number 54851020) with a titre of 106.' tcid,,. the placebo consisted of supernatant of non-infected nlfk cell culture. the vaccine and placebo were provided by the manufacturer in identical vials coded with coloured labels. the code was not broken to the veterinarians and the cat owners until all cats had finished the 12 months observation period. the characteristics of the cattery cats and young pet cats are summarized in table 1 . animals of the placebo and the vaccine groups in both, the cattery cats and the young pet cats, did not differ significantly with respect to age, sex, breed and living conditions. antibody titres to fcov were measured by indirect immunofluorescence using pd-5 cells of swine origin infected with tgev as antigen. plasma dilutions of 1:25, 1: 100, 1:400 and 1: 1600 were tested. plasma samples of all cats were examined for circulating feline leukemiavirus (felv) p27 antigen29 and plasma samples of the cattery cats were also examined for antibodies to feline immunodeficiency virus (fiv) by indirect immunofluorescence using fiv-infected fl-4 cells as antigen . 3o samples with positive fluorescence results were subjected to western blotting for confirmation3'. in 20 cattery cats cd4+/cd8+-t-cells were measured by flow cytometry as described3*. of all cats dying of fip, 100 ~1 of plasma samples taken at the time of first vaccination were retrospectively examined for presence of fcov-rna by polymerase chain reaction (pcr)33. the mean of the haematological and clinical chemistry parameters between the vaccine and placebo group were analysed for significant differences by the mann-whitney u test, changes of laboratory values obtained from different cats over time were examined by the wilcoxon test. frequencies of fcov antibody titres in the placebo and vaccine group were compared using the x2 test. to determine differences in the frequencies of fip in the vaccine and placebo group, the exact test of fisher was performed34. the results are presented separately for the cattery cats and the population of the young pet cats. the side-effects reported after the vaccination in the cattery cats are summarized in table 2 . during the 12-21 months of observation, 13 cats of the vaccine group and 11 of the placebo group died due to various causes. five cats of the vaccine group and six cats of the placebo group died due to non fip-related causes. all cases, except one cat of the vaccine group which died 14 months after the vaccination with liver problems and two cats of the placebo group which died 12 and 22 months after vaccination due to an accident and joint problems in a 14-year-old cat, respectively, were submitted to necropsy and fip was excluded. fip cases occurred in six catteries. the characteristics of all cattery cats which died of fip are summarized in table 3 . some of these cats, though clinically healthy, showed changes in blood parameters at the time of vaccination. to our knowledge, the safety of the vaccine in breeding cats has not been investigated so far neither under experimental nor under field conditions. therefore, all data collected from queens which had kittens after the vaccination are summarized in table 4 . no differences were found between the parameters evaluated. with respect to the laboratory parameters no differences were found between those in the vaccine group and the placebo group at the different time points (haematology, clinical chemistry, cd4+/cd8+lymphocytes). however, in both the vaccine and placebo groups, changes in some of the laboratory parameters were observed at the different time points. both groups showed a decrease in albumin in weeks 8 and 30 compared to week 0 and an increase in plasmaprotein in week 30 compared to weeks 0 and 8 (pco.05) (data not shown). at the beginning of this trial, all cattery cats had tested negative for felv and fiv-antibodies, but 98.6% and 95.6% of the cats in the vaccine and placebo group showed fcov antibody titres of 25 or higher. the frequency of the fcov titres in cats of the vaccine and placebo group at different time points after vaccination (weeks 0, 8 and 30) is shown in figure 1 . there was no statistically significant difference in the distribution of the fcov antibody titre in the vaccine and. placebo group at the different time point, but the vaccine group as well as the placebo group showed a transient increase of titres in week 8 compared to week 0 (pco.05) followed by a decrease in week 30 compared to week 8 (pco.05). retrospectively, plasma samples collected from cats at the time of first vaccination, which were stored frozen, were submitted for rt-pcr for fcov (table 3) . of 13 plasma samples tested, three were positive. the side-effects reported in the population of the young pet cats are summarized in table 2 . the observation period in this population was between 12 and 19 months. the health condition of the cats at the end of the observation period is summarized in table 5 . thirteen cats of the vaccine group and 18 cats of the placebo group died from fip. all, except one in each group, were confirmed by necropsy (table 6) . two cats in the vaccine group were already ill at the time of (table 6 ). of 30 samples tested, 10 were found positive. in one cat shelter with high fip incidence, 25 cats were vaccinated (placebo 13 cats, vaccine 12 cats), of which 15 cats developed fip (placebo 9, vaccine 6). the frequency and distribution of antibody titres to fcov at the time of first vaccination is presented in figure 2 . more than 50% of these clinically healthy young cats had already been exposed to fcov in the first year of life. the distribution was identical in the vaccine and placebo group. domestic shorthair cats showed statistically significantly lower fcov antibody titres than pure-bred cats of the same age (p<o.ool) (figure 3) . cats of the placebo group, which showed a titre of 100 or higher, had a significantly higher risk for developing fip in the next 12 months (10.7%) than cats with a titre of 25 or lower (3.3%) (pzo.016) (figure 4) . though seven cats of the vaccine group and six of the placebo group were felv positive at the time of first vaccination, three more cats of each group tested positive during the observation period. one of these felvpositive cats of the vaccine group and three of the placebo group died due to fip which was confirmed by necropsy. the aim of this study was to evaluate the efficacy and safety of a modified live virus vaccine in a double-blind study under field conditions in two cat populations with higher risk for fip. not all cats infected with fcov develop lethal fip and the incidence of fip in a cat population can hardly be predicted'. in this study, the placebo and vaccine group were indistinguishable regarding distribution of age, sex and living conditions and no statistically significant difference in laboratory parameters (haematology, clinical chemistry, serology, cd4+/cd8+-lymphocyte subsets) were observed at the time of first vaccination. therefore, cats in the vaccine and placebo group had about the same probability to become infected with fcov and to develop fip, and differences in the frequency of fip cases or changes in laboratory parameters must be attributed to the vaccine. the vaccinated cats did not show an accelerated form of fip (tables 3 and 6, figure 6 ). one cattery cat aged 4.3 months was euthanized 12 days after vaccination, but this cat, though clinically inconspicuous, showed growth retardation compared to litter mates, anaemia (pcv: 27%), increase of total serum protein and viremia with fcov at the time of first vaccination. two young pet cats were euthanized 5 and 7 days after vaccination. cat 1, which died 5 days after vaccination had shown chronic nasal discharge and anorexia and had been treated with antibiotics and corticosteroids the last 2 weeks before vaccination. the fcov antibody titre was high (1600) at the time of vaccination and in retrospect this cats was positive for fcov in the rt-pcr. cat 2, which died 7 days after vaccination had also been anorectic for some time and had been treated by the veterinarian. this cat too had a high titre at the time of vaccination (1600) and was pcr positive. these two cats were vaccinated by mistake and should not have entered the study in the first place. they remained in the study for the sake of completeness and because our study represent the real situation in the field where cats with obvious clinical signs may be vaccinated by mistake. it can be argued that in these three cats vaccine induced acceleration of fip may have happened. however, if the incidence of fip cases during the first 150 days after vaccination were compared in vaccinated vs placebo cats, there is absolutely no difference in that 12 cats in the vaccine group and 11 cats in the placebo group died during this time. in the population of the cattery cats where prevalence of fcov antibodies was especially high (95%) the average time of death after vaccination was 3.5 weeks in the vaccine group and 20 weeks in the placebo group. from this, the lack of side-effects and effect on fertility it was concluded that the vaccine is safe. as expected, most of the cattery cats showed antibodies to fcov, indicating prior infection. not only the vaccine group, but also the placebo group showed an increase of titres in week 8 compared to week 0 and a decrease in week 30 compared to week 8 (figure 1 ). increase of antibodies could be explained by vaccination in the vaccine group, but not in the placebo group. as animals of the vaccine and placebo groups were separated for 48 h after the vaccination, spreading of the vaccine virus to cats of the placebo group seems unlikely. during the 8 weeks between vaccination and the collection of a second blood sample, antibody titres increased in cats of catteries, where fip cases occurred. a similar rise of antibody titres was observed in cattery cats where no fip cases were seen during this time period. it is well known, that stress can influence the immune system. therefore, we speculate that the stress caused by the handling during vaccination and the collection of blood samples led to a weakening of the cm1 and to a reactivation of a persistent fcov infection and an increase of the antibody titres. surprisingly 50% of the clinically healthy pet cats younger than 12 months showed antibodies to fcov (figure 2) . pure-bred cats showed higher titres and were more frequently seropositive for fcov than domestic cats (figure 3) , which is probably due to different living conditions. pure-bred cats are normally raised in multiple-cat-households with close contact of the cats to each other, whereas domestic cats are often kept in single-cat-households or in small groups. when vaccination was performed according to the recommendations of the manufacturer, the vaccine showed no overall efficacy in these cattery cats. in view of the fact that in all catteries fip cases had occurred during the last 18 months prior this study, it has to be concluded that some of the cattery cats, which later died of fip during the observation period, had already been vaccine 1997 volume 15 number 10 1105 field trial of an fip vaccine: d. fehr et al. infected with virulent fcov and were vaccinated during the incubation period. this conclusion is also supported by the facts that all cats showed antibodies to fcov, that some cats showed changes in blood parameters consistent with fip (anaemia, low serum albumin, high serum protein, high bilirubin), and that retrospectively some cats were rt-pcr positive for fcov at the time of first vaccination (table 3) . vaccination itself did not inauence laboratory parameters such as haematology, clinical chemistry or cd4+/cd8+-lymphocyte subsets (data not shown). in the population of the young pet cats no reduction of fip cases in the vaccinated cats was observed in the first 150 days after infection ( figure 6 ). however, after this time point until the end of the observation period, the vaccinated cats showed significantly less fip cases in one cat shelter the incidence of fip was 50% in the vaccine group (six out of 12 cats) and 69% in the placebo group (nine out of 13 cats). it can be concluded that the exposure to fcov was extremely high for these cats. as these animals were vaccinated after they had already been housed in the shelter for some time, it can be concluded that most of the vaccine failures in this population were due to previous infection with fcov. if cats with high fcov antibody titres at the time of first vaccination (400 or higher) were excluded and only cats with titres of 100 or lower at the time of first vaccination were examined in our study, vaccination significantly decreased the incidence of fip (p=o.o30) ( figure 5 ). this observation suggests that low antibody titres are related with some degree of immunity, probably through a strong cell-mediated immune response via the thl pathway35,36 and presumably with a lower virus load compared to cats with titres of 400 or higher. this interpretation would also be in agreement with the finding that high titres (in the placebo group) are associated with a higher risk for the development of fip (figure 4) . it can be concluded that vaccination of cats, which were already infected with fcov, cannot prevent or alter the course of the disease. however, vaccination of cats with no or low antibody titres seems to be efficient. these findings confirm the results of a placebocontrolled trial in which seronegative animals were vaccinated, before they were entered into a cat shelter with fip problems37. to increase the efficacy of the vaccine changes in husbandry and management conditions may be initiated that lead to a decrease in coronaviral load in the kittens4,". under experimental conditions, vaccination of fcov naive cats was also found to reduce the incidence and the severity of infections with the low virulent viruses called fecv38. since every fcov infection has to be considered a potential risk for fip4,", reducing these infections by vaccination may also help to reduce the fip incidence. cats of the placebo group with a titre of 25 showed a significantly higher risk for developing fip in the following 12 months (10.7%), than cats with lower titres (3.3%) (pzo.016, figure 4 ). this finding and the observation that many of these cats were pcr positive at the time of first vaccination suggest that they were already infected with fcov and were vaccinated during the incubation period. this would explain why vaccination showed a low efficacy in the young pet cats. however, it is difficult to apply the classic term of an incubation period to fip. according to pedersen and co-workers more virulent mutants of fcov capable of infecting macrophages can emerge spontaneously any time during an fcov infection39. as mutations in viruses occur randomly, it can be concluded that a high virus load increases the probability that such mutants arise. a functioning immune system can control the virus load at a low level. therefore, all factors which suppress the immune response, may increase the virus load and the genesis of virulent mutants. thus, a cat can develop fip many months or even years after an fcov infection. in conclusion, the use of a live modified vaccine against fip was found to be safe in high risk populations under field conditions. efficacy was clearly shown in cats with low fcov antibody titre, but overall was rather low in high risk populations. to optimize its efficacy, changes in management and husbandry should help to prevent fcov exposure prior to vaccination. our laboratory for their help as well as to the cat breeders, veterinarians and cat owners that participated in this trial. cornell vet. 1992, 82, 117 scott, f.w., corapi, w.v. and olsen, c.w. evaluation of the safety and efficacy of primucell-fip vaccine. feline hlth top. 1992, 7, 6 fehr, d., holznagel, e. and bolla, s. et a/. evaluation of the safety and efficacy of a modified live fipv vaccine under field conditions. feline pratt. 1995, 23, 83 pedersen, n.c. feline infectious peritonitis and feline enteric coronavirus infections. part i, feline enteric coronavirus. feline pratt. 1983, 13, 5 the biology and pathogenesis of coronaviruses antigenic homology among coronaviruses related to transmissible gastroenteritis virus das feline immunschwlchevirus in der schweiz: klinik und epidemiologie im vergleich mit dem leukbmie-und dem coronavirus a study of naturally occurring feline coronavirus infections in kittens die diagnostik dir felinen infektioesen peritonitis (fip): retrospektive und prospektive untersuchungen feline coronavirus infections of cheetahs field safety studies of an intranasal fipv vaccine serologic studies of naturally occurring feline infectious peritonitis an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis a comparison of the genomes of fecvs and flpvs and what they tell us about the relationships between feline coronaviruses and their evolution risk of feline infectious peritonitis in cats naturally infected with feline coronavirus role of thymus-dependent lymphocytes and antibodies in feline infectious peritonitis after oral infection role of circulation antibodies and thymus-dependent lymphocytes in production of effusive type of feline infectious peritonitis after oral infection virologic and immunologic aspects of feline infectious peritonitis virus infection delayed-type hypersensitivity skin response associated with feline infectious peritonitis in two cats evaluation of immunity to feline infectious peritonitis in cats with cutaneous viral-induced delayed hypersensitivity immunologic phenomena in the effusive form of feline infectious peritonitis early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoclonal antibodies characterization of an attenuated temperature sensitive feline infectious peritonitis vaccine virus protection against feline infectious peritonitis by intranasal inoculation of a temperature-sensitive fipv vaccine monoclonal antibodies to three epitopic regions of feline leukemia virus p27 and their use in enzyme-linked immunosorbent assay of ~27 felines t-lymphotropes lentivirus (ftlv): experimentelle lnfektion und vorkommen in einigen laendern europas. kleintierpraxis specificity assessment of feline t-lymphotropic lentivirus serology detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr two types of murine helper t cell clones. i. definition according to profiles of lymphokine activities and secreted proteins thl and th2 cells: different patterns of lymphokine secretion lead to different functional properties vaccination against naturally occurring fip in a single large cat shelter the potential use of a modified live fipv vaccine to prevent experimental fecv infection experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd2, fipv-ucd3, and fipv-ucd4 the study was supported by a grant from pfizer animal health inc. the authors thank drs alasdair wiseman and serge martinod from pfizer animal health inc. for their assistance. we are indebted to the technicians of key: cord-253498-w6qfzpi4 authors: paltrinieri, saverio; cazzaniga, stefania; da cunha, nazarè pinto; giordano, alessia title: electrophoretic fractionation of creatine kinase isoenzymes and macroenzymes in clinically healthy dogs and cats and preliminary evaluation in central neurologic disease date: 2010-08-02 journal: vet clin pathol doi: 10.1111/j.1939-165x.2010.00242.x sha: doc_id: 253498 cord_uid: w6qfzpi4 background: information about the electrophoretic distribution of ck‐mm, ck‐mb, and ck‐bb, serum creatine kinase (ck) isoenzymes that are indicators of skeletal muscle, cardiac muscle, and brain lesions, respectively, and ck macroenzymes (macro‐ck1 and macro‐ck2) in dogs and cats with and without central neurologic disease is scant and equivocal. objectives: the objectives of this study were to describe the electrophoretic distribution of ck isoenzymes and macroenzymes in healthy dogs and cats and to provide a preliminary assessment of the utility of ck enzymatic electrophoresis in dogs and cats with central neurologic disease. methods: electrophoretic separation of serum ck isoenzymes and macroenzymes was performed on freeze‐thawed serum samples from 20 healthy dogs and 3 dogs with central neurologic disease and from 14 healthy cats and 6 cats with neurologic feline infectious peritonitis (fip). electrophoretic separation was also performed on supernatants of homogenized brain, skeletal muscle, and cardiac muscle from both species, to assess the tissue distribution of isoenyzmes in dogs and cats. results: ck‐mm was the predominant isoenzyme in the serum of healthy dogs and cats, followed by macro‐ck2 and ck‐bb in dogs and by both macroenzymes in cats. in dogs, ck‐mb was essentially absent from both serum and homogenized hearts. ck‐bb increased in dogs with neurologic disease. in cats, ck‐bb was essentially absent from serum, but was present in brain homogenates. two of 6 cats with fip had increased macro‐ck1 and increased ck‐bb activity. conclusions: this study identified the electophoretic distribution of ck isoenzymes and macroenzymes of dogs and cats and provided encouraging data about the possible use of ck‐bb as a biomarker for canine neurologic disorders, but not for fip. creatine kinase (ck; ec 2.7.3.2) catalyzes the reversible reaction between adp and phosphocreatine to form creatine and atp. phosphocreatine acts as a phosphate donor for the formation of atp, which is needed for muscle contraction. at rest ck contributes to restoration of phosphocreatine concentration using atp in excess of that needed by muscle. in people, tissues with the highest ck activity include skeletal muscle and cardiac muscle, followed by neural tissue, although its activity per gram of neural tissue is low owing to the large amount of myelin in the central nervous system (cns). low ck activity can be detected in other tissues, such as gastrointestinal tract, urinary bladder, kidney, and thyroid. 1 ck is primarily cytoplasmic and leakage from injured or inflamed tissues leads to increased ck activity in blood, permitting assay of plasma or serum ck activity as a biomarker of tissue damage. ck is a dimer composed of b monomers, m monomers, or both, with a moderate degree of structural homology between the 2 subunits and between different species. 2 combinations of these monomers generate isoenzymes ck-bb, ck-mb, and ck-mm, which can be found predominantly in brain, cardiac muscle, and skeletal muscle, respectively. 3 ck-bb, ck-mb, and ck-mm are also called ck1, ck2, and ck3, respectively, according to their electrophoretic migration. in addition to the 3 dimeric ck isoenzymes, there is a structurally different mitochondrial isoform, ck-mt or ckm, detectable in tissues, and 2 macroenzymes, macro-ck1 and macro-ck2, detectable in blood. macro-ck1 is dimeric ck-bb bound to immunoglobulins macro-ck2 consists of oligomers of ck-mt. 3 total ck activity can be measured in plasma or serum using an enzymatic method that utilizes ck-nacetylcysteine (ck-nac). 4 although this method does not identify specific isoenzymes or isoforms, it provides an estimate of ck-mm activity, which is responsible for the majority of plasma ck activity in many species. ck-mb can be measured by immunoenzymatic methods designed for diagnostic purposes in people with myocardial diseases; these methods have also been used in dogs, although definitive validation studies have not been done. [5] [6] [7] by contrast, ck-bb and macroenzymes can be measured only by more expensive and labor-intensive methods, such as chromatography, immunoprecipitation, immunoinhibition, differential enzyme activation, and electrophoresis. 3 the limited information about ck isoenzymes in dogs is focused on ck-mm and ck-mb. [5] [6] [7] [8] [9] [10] [11] [12] [13] only one study reported serum ck-bb activity in dogs with neurologic disease. 12 to our knowledge there are no studies of canine and feline macro-ck1 and macro-ck2, which have been associated with immune-mediated and neoplastic conditions, respectively, in people, 14 or of electrophoretic fractions of ck in feline serum. the aim of this study was to describe the electrophoretic distribution of ck isoenzymes and macroenzymes in healthy dogs and cats and to determine if changes in ck distribution in a low number of animals with central neurologic disorders justifies future studies on ck-bb in animals with cns diseases. healthy dogs and cats were identified based on normal physical examination and lack of abnormalities detected on laboratory tests, including cbc, serum biochemical profile, serum protein electrophoresis (spe), and urinalysis. inclusion criteria for dogs and cats with central neurologic disease were the presence of neurologic signs and relevant laboratory test results as detailed below. blood samples were collected into tubes containing edta and tubes without anticoagulant (venoject, terumo italia srl, rome, italy) by the referring veterinarians from client-owned animals after obtaining informed consent from the owners as requested by the institutional animal care committee (comitato etico tutela degli animali). samples were submitted to the department of veterinary pathology, hygiene and public health, unit of general pathology and parasitology, university of milan for routine screening or diagnostic tests. samples that were grossly hemolyzed or lipemic were excluded from the study. serum was obtained by centrifugation (1100g â 8 minute) of blood collected in plain tubes. after collection and separation of serum the basic panel of diagnostic tests described below was immediately performed, and then samples were stored at à 201c. electrophoretic separation of ck isoenzymes was performed within 1 month on batched samples to reduce possible interassay variability. a cbc and a panel of serum biochemical tests, including, urea, creatinine, glucose, total protein, albumin, calcium, and phosphorus concentrations and alkaline phosphatase (alp), alanine aminotransferase (alt), lactate dehydrogenase, g-glutamyl transferase (ggt), and ck (ck-nac method) activities, was performed on all samples using an automated spectrophotometer (cobas mira, roche diagnostic, basel, switzerland) with reagents from real time diagnostic system (viterbo, italy). spe on agarose gel strips using an automated apparatus (hydrasis, sebia italia srl, florence, italy) was performed for 1 dog with suspected leishmaniasis and for all cats suspected of having the dry form of feline infectious peritonitis (fip). for these cats, serum concentration of a 1 -acid glycoprotein (agp) was also measured using a commercially available radial immunodiffusion kit (tridelta development ltd., maynooth, ireland). the diagnosis of leishmaniasis was based on detection of increased a 2 and g-globulins by spe, increased protein concentration and mixed pleocytosis in csf, high-antibody titers (direct immunofluorescence assay), and positive polymerase chain reaction test. the diagnosis of fip was based on detection of increased a 2 and g-globulins by spe and increased concentration of serum agp, 15 in most cases associated with renal failure (based on increased concentrations of creatinine, urea, and phosphate) or hepatobiliary damage (based on increased alt, alp, and ggt activities), on detection of typical gross and microscopic lesions, and on positive immunohistochemical staining for feline coronavirus within the lesions. in order to assess tissue distribution of isoenzymes and to confirm that isoenzymes have the same electrophoretic migration of their human counterparts, electrophoretic separation was performed on tissues homogenized using a modification of the original procedure. 1 specifically, slices of brain (temporal cortex and parietal regions), skeletal muscle (semitendinosus and semimebranosus), and cardiac muscle (left ventricular wall), approximately 1 cm 3 each, were collected during necropsy from a cat with chronic renal failure and a dog with a ruptured splenic hemangiosarcoma. slices were immersed in liquid nitrogen immediately after sampling and stored frozen at à 201c until use 1 day later. after thawing, tissues were placed in 1 ml of saline solution and manually homogenized using a tissue grinder (glass/teflon potter elvehjem wheaton, millville, nj, usa). following homogenization, fluid was transferred to an eppendorf tube and centrifuged at 2500g for 15 minutes. supernatant was then transferred to another tube and frozen at à 201c until use 1 week later. according to the manufacturer's instructions, if total ck activity is 4 750 u/l the sample should be diluted before electrophoretic fractionation of isoenzymes to prevent overlap of large bands and permit identification of single bands. therefore, the total ck activity of each supernatant was determined using the ck-nac assay, and each sample was diluted to obtain ck activity of approximately 500 u/l. electrophoresis of the supernatants of homogenized tissue was then performed using the same procedure for serum samples described below. electrophoresis was performed according to the manufacturer's instructions using a commercially available kit (hydragel iso-ck, sebia italia srl) and an automated apparatus (hydrasys, sebia italia srl) equipped with specific accessories (standard mask accessories for iso-ck/ld). the principle of the assay is that isoenzymes and macroenzymes have characteristic motility owing to the electric charge of the subunits. after migration, ck fractions are visualized based on a reaction catalyzed by all the ck fractions and based on the conversion of creatine phosphate to a chromogenic compound (formazan) through a series of intermediate reactions involving substrates and cofactors included in the kit. briefly, 200 ml of serum or supernatant from homogenized tissues were mixed with 2 ml of activating solution containing b-mercaptoethanol and incubated for 10 minutes at approximately 201c. for feline samples, 10 ml of activated serum or supernatant from homogenized tissues were placed in the wells of the applicator provided with the kit. for canine samples, 20 ml were applied as preliminary testing revealed that weak bands were generated when 10 ml were applied. agarose gel (8%, ph 8.40 ae 0.05, included in the kit) and the applicator were placed in the migration chamber, and the automated migration program was then selected. after migration (10-20 w, 27 v h, 201c) ck substrate containing chromogenic solution was applied and the reaction was stopped using blocking solution. gels were then washed, dried by heating, and placed on the scanner provided with the instrument. scanned images were analyzed using phoresis software (sebia italia srl) and visually inspected for possible errors in separation, which, if present, were manually corrected using the appropriate software utilities. intra-and interassay precision of the method was assessed in test runs on pooled canine and feline serum samples, randomly received by our diagnostic laboratory, irrespective of the presence or absence of disease or laboratory abnormalities. specifically, intraassay imprecision was tested by running each pooled serum on 3 different lanes of the same gel. interassay imprecision was tested by running aliquots of each pooled serum in 3 different runs, with 2 intervals of approximately 15 days. in the first run fresh pooled serum was used; aliquots frozen at à 201c were thawed and used in the other 2 runs. mean activities and standard deviations (sds) were calculated for both intra-and interassay precision tests, and coefficients of variation (cv) were calculated using the formula: cv% = (sd/mean) â 100. statistical analyses were done using an excel spreadsheet (microsoft corp., redmond, wa, usa) and analyse-it software (analyse-it software ltd., leeds, uk). activities were expressed as median values, with additional reporting of minimum and maximum activities. although this approach does not provide precise information about reference intervals, it has been suggested to estimate reference intervals in small groups of animals. 16 after determining that the data were not normally distributed, the results from dogs and cats with central neurologic disease were compared with those of the healthy control animals using a mann-whitney u-test with p .05 considered significant. year-old male) had neurologic leishmaniasis. fourteen healthy cats (7 female and 7 male, 1-10 years with a median age of 3.5 years) were included in the study. six cats (3 female and 3 male, 1-7 years with a median age of 1.5 years) were diagnosed with neurologic disease secondary to fip. necropsies were performed on the 6 cats, and in all cases lesions consistent with fip were present along with immunohistochemical positivity for the virus in at least 1 abdominal organ. however, examination of the brain for typical pyogranulomatous lesions was investigated in only 3 cats. analysis of homogenized tissue supernatants revealed that ck-bb and ck-mm were present in brain and skeletal muscle, respectively, in both dogs and cats ( figure 1 ). supernatants from feline brain also contained 2 weak bands with migration distances similar to those of ck-mb and ck-mm. supernatants from homogenized cardiac tissue from both cats and dogs mainly expressed ck-mm. a minor ck-mb band was detectable in cats, but not in dogs; however, ck-mb was detected in both dogs and cats in undiluted samples (ck activity 4 500 u/l; data not shown). macro-ck2 was detectable in supernatants from homogenized cardiac and skeletal muscle in both species. intra-and interassay imprecision for total ck activity in canine and feline serum was comparable (table 1 ). in dogs, both intra-and interassay cvs for fractionated enzymes were o 3% except for the fractions with the smallest quantities (ck-mb and macro-ck1). in cats, both intra-and interassay assay cvs were 6% except for the fractions with the smallest quantities (ck-bb and ck-mb). activities of fractionated ck of healthy animals were compared with those of dogs and cats with central neurologic disease ( table 2 , supporting information figures s1 and s2 ). ck-mm was the predominant electrophoretic fraction in serum samples from healthy dogs followed by macro-ck2 and ck-bb, whereas ck-mb and macro-ck1 were essentially absent. ck-mm was the predominant electrophoretic fraction in serum samples from healthy cats. macro-ck1 and macro-ck2 were detected in varying proportions, whereas ck-bb and ck-mb were essentially absent in healthy cats. individual dogs and cats with central neurologic disease had variable changes in the ck fraction activities in serum (table 3) . serum samples from all 3 dogs had absolute ck-bb activities higher than those of samples from healthy dogs (table 3, figure 2 ) and the median ck-bb activity (37 u/l) was significantly higher than the median ck activity of healthy dogs (7 u/l, p =.006). total ck activity was significantly greater in cats with fip (p =.006). fractionated ck activities for fip cats were not statistically different from those of healthy cats, but individual increases were observed (table 3 , figure 2 ). this study demonstrated that ck isoenzymes and macroenzymes can be separated by electrophoresis in canine and feline serum that has been frozen and thawed. the low activity of ck in canine serum required a modification of the manufacturer's protocol and a doubling of the amount of serum applied to achieve visible bands. the creation of a specific ''canine iso-ck'' program with a longer application time, increasing the amount of serum transferred to the gel, is advisable in the future. this low ck activity may have been a storage artifact as a previous study 17 and preliminary experiments in our laboratory (data not shown) demonstrated that freezing and thawing decreases ck activity. this decrease, however, accounts for o 10% of native ck activity. low total ck activity in our canine group was similar to that reported in previous studies, 8, 11 and discrepancies between the serum ck activities in healthy animals, especially cats, in this study and other studies 18, 19 may result from differences in analytical methods rather than storage artifacts. similarly, analysis of interassay imprecision for isoenzyme fractions in pooled serum samples did not reveal a common trend (neither an increase nor a decrease of percentages) associated with prior storage at à 201c. it should be emphasized that a complete validation study, which would have provided more detailed information about possible storage artifacts on isoenzyme electrophoresis, was not performed. specifically, the number of replicates in our preliminary testing was less than is recommended to assess both intra-and interassay imprecision, and stability should be assessed using a different approach. 20 despite these limitations, cvs indicated acceptable repeatability and reproducibility of isoenzyme determinations, comparable with those reported by the kit manufacturer for ck-bb, ck-mb, and ck-mm in human serum, 21 independent of storage conditions. a more detailed evaluation of storage-related artifacts, however, is recommended before suggesting clinical application of automated ck gel electrophoresis. apart from these technical aspects, this study provides useful information on both the distribution of ck isoenzymes and macroenzymes in freeze-thawed serum from healthy animals and on potential applicability of the technique to animals with central neurologic disease. in healthy dogs, ck-mm was the most abundant electrophoretic fraction, but significant ck-bb activity was also detectable. other studies have reported similar activities of ck-mm or ck-bb, 9,10 but 1 study demonstrated greater activities of ck-bb than ck-mm. 8 the most likely explanation for this discrepancy is that the technique used in the latter study was based on electrophoretic methods that did not differentiate macroenzymes, which are known to interfere with identification of isoenzymes. 22, 23 alternatively, ck-mm and ck-bb are, respectively, the most and the least stable isoenzymes in serum, 9 and storage, temperature, or ph variations could have caused reductions in ck-bb. however, this is unlikely because in our study all serum samples had identical storage conditions, and ck-bb was clearly detectable in some samples. however, ck-bb could have deteriorated during storage conditions and may have been subsequently detectable in those samples in which ck-bb activity was markedly increased before freezing. another possible explanation is individual variability, which was detected both in this and in previous studies. [8] [9] [10] individual characteristics of the sampled populations, possibly influenced by physiologic conditions, such as exercise and diet, could have caused discrepant results. ck-mb was essentially absent in the freezethawed canine serum in this study. this contrasts with studies that reported detection of ck-mb in canine serum. [9] [10] [11] in these studies, however, the presence of macroenzymes may have falsely increased ck-mb activity. 22, 23 in support of this hypothesis, another study in which ck-bb activity was possibly overestimated by interference from macroenzymes found the activity of ck-mb was low. 8 in addition, ck-mb was also present in small amounts in supernatants from homogenized heart, in agreement with another study. 9 the evaluation of ck-mb in pathologic conditions is outside the scope of this study, but our results suggest that the clinical utility of ck-mb measurement is low compared with that of other biomarkers. 6 ck-bb activity increased in all 3 dogs with central neurologic disease, as reported previously using a different method. 12 despite the low number of cases, which theoretically limits the statistical power of the comparison, the difference in ck-bb activity between healthy and diseased dogs was statistically significant. unfortunately, information about histologic brain lesions in the dogs, types of tumors, or degree of invasiveness of the tumors is lacking, precluding conclusions about the utility of ck-bb as a diagnostic or prognostic biomarker for specific cns diseases in dogs. however, these preliminary results suggest that studies to evaluate which types of cns disorders are characterized by increases in ck-bb activity would be valuable. in freeze-thawed serum samples from cats, ck-mm was the largest fraction, followed by macro-ck1 and macro-ck2. previous reports demonstrated that some areas of feline brain express ck-bb. 24 analysis of supernatants from homogenized feline brain in our study also confirmed that ck-bb is present in brain tissue and has the same migration pattern as in dogs. interestingly, ck-bb seems to be almost absent, or below detection limits, in serum from healthy animals and from most cats with fip. total ck activity was greater in cats with fip than in healthy cats, but this finding has poor diagnostic specificity as increased ck activity occurs in several pathophysiologic conditions, including anorexia, 17 myopathy, 18 metabolic syndromes, 25 and cns disorders. 26 the lack of consistent alterations in ck-bb activity in cats with fip may have several explanations. neurologic fip is a pyogranulomatous meningitis, and cns tissue is only secondarily affected. 16 although the blood-brain barrier is usually damaged, it is possible that leakage of ck-bb from neurons is minimal. another possible explanation is that ck-bb is released by cns cells but binds to immunoglobulins that are abundant in the serum of cats with fip 16 and also can be found in cerebrospinal fluid. 27 this would explain the increase of macro-ck1, which is a dimer of ck-bb and igg. 14 further studies on central neurologic diseases in cats are required to investigate the potential utility of the ck-bb assay, but based on these results, ck-bb does not appear to have clinical utility in supporting a diagnosis of fip with cns involvement. macroenzymes were identified based on their migration pattern, which likely corresponds to the migration pattern recorded for human macroenzymes as a high degree of interspecies homology has been reported. 2 it is interesting to note that macroenzymes, which in people are essentially absent from plasma except in cases of severe disease, 14 can be found in serum from healthy dogs and cats. the possible diagnostic utility of these macroenzymes should be assessed by future studies on animals with a broad spectrum of pathologic conditions. in conclusion, our study demonstrated that electrophoretic separation of ck isoenzymes and macroenzymes in dogs and cats is feasible, although modification of the original method is required for canine samples, and that ck-mm is the main electrophoretic fraction, followed by ck-bb and macro-ck2 in dogs and by macro-ck1 and macro-ck2 in cats. promising results about the possible use of ck-bb as a biomarker of central neurologic disorders in dogs were obtained, whereas in cats with fip the quantification of ck-bb does not seem to have clinical utility. future studies to investigate the type of cns disorders responsible for increases in ck-bb activity in dogs and to explore feline central neurologic disorders other than fip in cats are indicated to define the diagnostic and prognostic potential of ck-bb in a larger number of cases. additionally, studies on the diagnostic utility of electrophoresis of ck isoenzymes and macroenzymes in cerebrospinal fluid collected from dogs and cats with central neurologic disease may be warranted. creatine kinase in human tissues complete nucleotide sequence of dog heart creatine kinase mrna: conservation of amino acid sequence within and among species creatine kinase isoenzymes ck-nac''-complete activation biochemical serum profiles in dogs experimentally infected with angiostrongylus vasorum (baillet, 1866) cardiac troponins as indicators of acute myocardial damage in dogs characterisation of lipid profiles in dogs with parvoviral enteritis creatine kinase and its isoenzymes in dog sera creatine kinase and its isoenzymes in dogs serum creatine kinase activities in dogs with dirofilariasis creatine kinase in the dog: a review an elevation of brain-type creatine kinase isoenzyme in dogs with brain disease brain-type creatine kinase isoenzyme in young dogs relevance of macro creatine kinase type 1 and type 2 isoenzymes to laboratory and clinical data a review of feline infectious peritonitis virus infection: 1963-2008 reference values effects of storage time and freezing temperature on clinical chemical parameters from canine serum and heparinized plasma correlation between serum creatine kinase activities and anorexia in cats hepatozoon species infection in domestic cats: a retrospective study method validation diagnostic products corporation. hydragel, 7, 15 & 30 iso-ck s operation manual analytical patterns and biochemical properties of macro creatine kinase type 2 iga-ck-bb complex with ck-mb electrophoretic mobility can lead to erroneous diagnosis of acute myocardial infarction the regional variability of enzymes in the brain: relevance to csf enzyme determinations creatine kinase activity in dogs and cats with metabolic diseases clinical, cerebrospinal fluid, and histological data from thirtyfour cats with primary noninflammatory disease of the central nervous system use of albumin quotient and igg index to differentiate bloodvs brain-derived proteins in the cerebrospinal fluid of cats with feline infectious peritonitis this study was partially funded by a 2008 f.i.r.s.t. grant from the university of milan. the authors thank professor mauro di giancamillo and dr. davide zani from the radiology unit of the department of veterinary clinical science, university of milan.disclosure: the authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article. additional supporting information may be found in the online version of this article: figure s1 . dot plots showing the distribution of total ck and ck fraction activities (u/l) for dogs that were healthy and dogs with central neurologic disease. figure s2 . dot plots showing the distribution of total ck and ck fraction activities (u/l) for cats that were healthy and cats with central neurologic disease associated with feline infections peritonitis.please note: wiley-blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. key: cord-304616-k92fa15l authors: izes, aaron m.; kimble, benjamin; norris, jacqueline m.; govendir, merran title: assay validation and determination of in vitro binding of mefloquine to plasma proteins from clinically normal and fip-affected cats date: 2020-08-05 journal: plos one doi: 10.1371/journal.pone.0236754 sha: doc_id: 304616 cord_uid: k92fa15l the antimalarial agent mefloquine is currently being investigated for its potential to inhibit feline coronavirus and feline calicivirus infections. a simple, high pressure liquid chromatography assay was developed to detect mefloquine plasma concentrations in feline plasma. the assay’s lower limit of quantification was 250 ng/ml. the mean ± standard deviation intraand inter-day precision expressed as coefficients of variation were 6.83 ± 1.75 and 5.33 ± 1.37%, respectively, whereas intraand inter-day accuracy expressed as a percentage of the bias were 11.40 ± 3.73 and 10.59 ± 3.88%, respectively. accordingly, this validated assay should prove valuable for future in vivo clinical trials of mefloquine as an antiviral agent against feline coronavirus and feline calicivirus. however, the proportion of mefloquine binding to feline plasma proteins has not been reported. the proportion of drug bound to plasma protein binding is an important concept when developing drug dosing regimens. as cats with feline infectious peritonitis (fip) demonstrate altered concentrations of plasma proteins, the proportion of mefloquine binding to plasma proteins in both clinically normal cats and fip-affected cats was also investigated. an in vitro method using rapid equilibrium dialysis demonstrated that mefloquine was highly plasma protein bound in both populations (on average > 99%). successful treatment of feline infectious peritonitis (fip), a fatal, virulent coronavirus infection affecting predominantly younger cats, remains difficult [1] . reviews describing the virus responsible for fip as well as issues with diagnostics and therapeutics are available [1, 2] , providing an explanation as to why re-purposing drugs used in human medicine has been largely unsuccessful in treating fip infected cats [1] . recently, the use of antiviral therapies to alter the replication of virulent forms of feline coronavirus known as feline infectious peritonitis virus (fipv) has shown enormous hope for clinicians [3, 4] . however, veterinary access to these antiviral treatments is currently limited, leading to the global emergence of expensive, unregistered versions of these drugs without the necessary quality control assurances [4, 5] . accordingly, the need for inexpensive, safe, antiviral medications to treat fipv-infected cats remains. plos one | https://doi.org/10.1371/journal.pone.0236754 august 5, 2020 1 / 10 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 mefloquine is currently used for both prevention (as a monotherapy) and treatment (either alone, or in combination with artesunate) of chloroquine-resistant plasmodium falciparum malaria in humans [6] [7] [8] . it also has been shown to reduce the viral load of fipv and feline calicivirus (fcv) at low concentrations in infected crandell rees feline kidney cells without cytotoxic effects [9, 10] . using an in vitro assay, our team demonstrated that while mefloquine undergoes some phase i metabolism in cats, it does not undertake phase ii glucuronidative metabolism in this species [11] . in anticipation of conducting in vivo clinical trials of mefloquine to treat fipv-infected cats, mefloquine's plasma concentrations must be monitored to optimise the dosage regimen. knowledge of the amount of drug bound to plasma proteins is also important when optimising dosage regimens as only the unbound (or free) fraction is therapeutically active [12, 13] . although the plasma protein binding of mefloquine has been determined to be > 98% in humans [14] , its binding percentage in cats has not been reported. this information is particularly germane in any investigation of a potential fip antiviral as fip-affected cats have altered concentrations of the plasma proteins albumin (decreased) and globulins (elevated) compared to clinically normal cats [1, 15] . consequently, the aim of this study was two-fold: first, to develop and validate a high pressure liquid chromatography (hplc) method to detect mefloquine in feline plasma, and second, to determine the in vitro plasma protein binding of mefloquine in both clinically normal and fip-affected cats. mefloquine hydrochloride and verapamil hydrochloride (as the internal standard [is]) were purchased from sigma-aldrich (castle hill, nsw, australia). hplc grade methanol, hplc grade acetonitrile, phosphate buffered saline ph 7.4 (pbs) and triethylamine were purchased from thermo fisher scientific (macquarie park, nsw, australia). mefloquine and verapamil were quantified in feline plasma based on modified hplc method [16] . the hplc system consisted of a shimadzu lc-20at delivery unit, dgu-20a3 ht degassing solvent delivery unit, sil-20a auto injector, spd-20a uv detector and cto-20a column oven. shimadzu lc solution software version 1.24 (kyoto, japan) was used for chromatographic control, data collection and data processing. chromatographic separation was performed with a microsorb-mv c18 column (250 mm × 4.6 mm i.d., 5.0 μm; varian, mulgrave, vic, australia) with a 1.0 mm optic-guard c18 pre-column (choice analytical, thornleigh, nsw, australia) under a pressure of 1,900 psi at 25˚c. the isocratic mobile phase consisted of a mixture of 25 mm sodium phosphate buffer with 1.0% triethylamine adjusted to ph 2.5 and acetonitrile and methanol (50:25:25, v/v/v) at a flow rate of 1.0 ml/min. the injection volume was 10 μl per sample. the diode array detector was set at a wavelength of 220 nm. the retention times of verapamil and mefloquine were approximately 8.5 and 14.5 minutes, respectively. stock solutions of mefloquine and verapamil were prepared in 100% methanol and 100% acetonitrile, respectively, both at a concentration of 1.0 mg/ml. further working solutions of mefloquine were prepared in 100% methanol to achieve final concentrations of 500, 1,000, 2,500, 5,000, 10,000, 25,000 and 50,000 ng/ml. similarly, working solutions of verapamil were prepared in 100% acetonitrile to yield final concentrations of 12.5 μg/ml for the validation study and 5.0 μg/ml for the plasma protein binding study. stock solutions and working solutions were stored at 4.0˚c prior to use. blank, clinically normal, feline plasma was pooled (n > 5) and stored at -20˚c prior to use for preparation of standards and quality control (qc) samples. the origin of the plasma samples is described below. mefloquine standard samples (50, 100, 250, 500, 1,000, 2,500 and 5,000 ng/ml) and three quality control samples (qc) (250, 1,000 and 5,000 ng/ml) were prepared by spiking 10 μl of prepared working solutions of mefloquine into 90 μl of blank feline plasma. the proteins within the plasma samples were extracted through simple protein precipitation. specifically, 100 μl of acetonitrile, containing either 12.5 μg/ml of the is for the validation study or 5.0 μg/ml of the is for the plasma protein binding study, was added to the 100 μl feline plasma samples. the samples were then vortexed and centrifuged at 14,000 × g for ten minutes to remove any particulates. the supernatant was injected into the hplc system. assay selectivity was established by analysing pooled (n > 5) blank, clinically normal, feline plasma to ensure that there were no endogenous interference peaks around the retention times of mefloquine and verapamil. mefloquine concentrations were measured via standard curves performed on three replicates of each concentration of mefloquine (100, 250, 500, 1,000, 2,500 and 5,000 ng/ml) on three consecutive days. a weighting factor (1/x) was used to assign the relative importance of the observations in the regression; in this case, to ensure that larger observations were not over-fitted. the theoretical lower limit of detection (llod, the lowest analyte concentration that can be distinguished from the assay background noise) and the lower limit of quantification (lloq, the lowest concentration at which an analyte can be reliably detected at a specified level of accuracy and precision) were estimated as follows [17] : llod = 3.3 x σ /s, where σ is the standard deviation of the y-intercepts from the regression lines and s is the mean slope of the calibration curves. thereafter, the lloq was calculated using the formula lloq = 3 x llod. acceptance criteria for the lloq was defined as precision with a cv � 15% and accuracy within ± 20% of nominal concentration with repeated analyses [18] . intra-and inter-day precision, expressed as cv (%), were analysed from triplicates of qc samples (250, 1,000 and 5,000 ng/ml), both within a single day and on three consecutive days, respectively. intra-and inter-day accuracy, expressed as bias, were determined by a percentage difference between the estimated value and the nominal value of mefloquine as follows: bias (%) = 100 -(estimated value / nominal value × 100). absolute recovery of mefloquine and is were determined by comparing the peak area of pre-spiked plasma samples (n = 9) at concentrations of 250, 1,000 and 5,000 ng/ml with corresponding concentrations of mefloquine and is in mobile phase. each assay was conducted in triplicate. the linearity of the data was assessed through linear regression analysis with graphpad prism software version 8.1.1 (graphpad software, inc., ca, usa). with owner verbal or written consent, blood was collected from both clinically normal and fip-affected cats. the use of the residual plasma from this patient for this study was approved by the university of sydney animal ethics committee (protocol: 2016/1027). the animals were considered clinically normal based on inclusion criteria modified from that of norris et al. (2007) [19] . specifically, the cats designated for inclusion as clinically normal subjects were systemically well and had their blood collected for purposes other than investigating a current illness. examples of clinically normal cats included those presenting for annual examinations, routine vaccinations or for routine screening tests prior to sedation or general anaesthesia for de-sexing, grooming, routine dental scaling or radiography post-acute trauma [19] . in contrast, a cat's fip status was confirmed by direct immunofluorescence of effusion samples and immunohistochemistry on tissue samples (n = 5), or simply immunohistochemistry (n = 5) using methods outlined by worthing et al. (2012) [20] . additionally, other haematological tests were performed including quantification of albumin: globulin which was consistent with the hyperglobulinaemia observed with cats infected with fipv [15] . all plasma samples were stored at -20˚c prior to analysis. plasma samples from both clinically normal and fip-affected cats were provided by the paddington cat hospital (sydney, nsw) and the veterinary pathology and diagnostic service, sydney school of veterinary science. the in vitro plasma protein binding (ppb) of mefloquine was determined by the rapid equilibrium dialysis (red) method. the red assay was performed according to manufacturer's (thermo fisher scientific, macquarie park) instructions. pooled plasma samples (n > 5) were assigned to one of two experimental groups, depending on health status, i.e., either clinically normal cat plasma or fip-affected plasma. prior to experimentation, the ph of the pooled plasma (approximately ph 8) was adjusted to ph 7.3 [21] , to mimic the physiologic ph of cats. likewise, the total protein (tp), albumin and globulin concentrations of the pooled plasma samples were quantified prior to the assay by the vpds using a thermo fisher scientific konelab prime 30i analyzer (scoresby, vic, australia) employing conventional protocols. for each experimental group, two final concentrations of mefloquine (either 5,000 or 10,000 ng/ml) were tested using three replicates for each concentration. these concentrations were selected as they are comparable to the 10 μm concentration (10 μm = 3.78 μg/ml or 3,780 ng/ml) tested by mcdonagh et al. (2014) [10] in fipv-infected crandell rees feline kidney cells. accordingly, 300 μl of plasma pre-spiked with mefloquine was added to the redringed chamber of the red device whilst 550 μl of pbs was added to the white-ringed chamber. the unit was covered with sealing tape (thermo fisher scientific, scoresby, vic, australia, product no. 15036) and incubated on an orbital shaker at 250 rpm for four hours. the temperature was set at 38˚c to mimic the normal body temperature of cats. following incubation, 100 μl of post-dialysis samples were removed from the red-ringed chambers. these samples were then placed in separate microcentrifuge tubes and precipitated using 100 μl of chilled acetonitrile pre-spiked with the is (5.0 μg/ml verapamil). after vortexing, the mixtures were centrifuged at 14,000 × g for ten minutes. the supernatant was injected into the hplc system. similarly, 200 μl volumes of post-dialysis samples were removed from the whiteringed chambers. these samples also underwent hplc analysis. all samples were prepared and analysed in triplicate. the ppb of mefloquine was determined by the following equation: %free ¼ ðconcentration buffer chamber=concentration plasma chamberþ � 100% %bound ¼ 100 à %free statistical data analysis was performed using graphpad prism software version 8.1.1 (graph pad software, inc., ca, usa). prior to parametric testing, a shapiro-wilk normality test performed on the mefloquine ppb data indicated a normal distribution. likewise, an inspection of a normal quantile-quantile (qq) plot of this data further corroborated its normality. a twoway anova evaluating the effects of health status and drug concentration on mefloquine ppb was performed. results were considered statistically significant at p < 0.05. based on the uv spectra, the greatest area under the mefloquine peak was at a wavelength of 220 nm. the retention times of the is and mefloquine were approximately 8.5 and 14.5 minutes, respectively. no endogenous interference was observed at the retention times of mefloquine and the is. chromatograms of feline plasma pre-spiked with mefloquine and the is as well as blank feline plasma are shown in fig 1. selectivity. pooled blank feline plasma and feline plasma pre-spiked with mefloquine (250, 1,000 and 5,000 ng/ml) and the is (12.5 μg/ml) were used to check the selectivity of this method. due to the limited volume of fip-affected feline plasma available, only blank plasma of clinically normal cats was used. as demonstrated in fig 1, no endogenous plasma components interfered with elution of analytes. linearity, llod, lloq, accuracy and precision. the plasma peak ratio (area of mefloquine divided by the is area) versus the concentration was plotted and determined to be linear for the concentration range used (100 to 5,000 ng/ml). the mean regression standard curves (n = 3) for mefloquine were described as y = 9.374 e-005 x + 0.001417, with a weighting factor of 1/x. the correlation coefficient value (r 2 ) for each curve � 0.97. estimated from standard curves, the llod for mefloquine was 20.5 ng/ml and the lloq was 61.5 ng/ml. however, as mefloquine concentrations < 250 ng/ml were not accurate (>20% accuracy), the lloq was set as 250 ng/ml. intra-and inter-day precision expressed as cvs ranged from 5.30 to 8.74% and 3.77 to 6.32%, respectively. intra-and inter-day accuracy expressed as a percentage of the bias ranged from -12.67 to 13.96% and -15.01 to 9.04%, respectively. these values satisfied the guidelines regarding assay reliability [18] . intra-and inter-day precision and accuracy are summarised in table 1 . drug recovery from plasma. the absolute recovery rates of mefloquine expressed as percentages ± standard deviation (s.d.) from the 250, 1,000, and 5,000 ng/ml qc samples (n = 3) were 107.82 ± 5.93; 99.91 ± 5.58; and 105.21 ± 6.32, respectively. the absolute recovery rate of the is expressed as a percentage ± s.d. was 123.74 ± 3.77 (n = 9). in vitro plasma protein binding of mefloquine in healthy and fip-affected cats. the measured concentrations of total protein, albumin and globulin in the pooled plasma for the respective experimental groups are provided in table 2 . although reference intervals (ri) vary depending on the laboratory and the methodology used for quantification, reference intervals from cornell university's animal health diagnostic center [22] were used as none were provided by the testing laboratory. the results of in vitro plasma protein binding of mefloquine in clinically normal and fipaffected cats are provided in table 3 . a significant difference was found between the plasma protein binding of mefloquine in clinically normal and fip-affected cats (p = 0.0004), even though mefloquine was determined to be highly plasma protein bound (on average > 99%) in both groups. moreover, a significant interaction effect between health status and mefloquine concentration was identified (p = 0.0007). in contrast, no significant difference was discovered between the plasma protein binding of the two concentrations of mefloquine (p = 0.11). there has been recent interest in the suitability of mefloquine as an antiviral treatment for fip [9, 10, 23] . the majority of publications that detect and quantify mefloquine use liquid chromatography. liquid chromatography demonstrates good sensitivity (i.e., it can detect low concentrations of mefloquine), is highly specific and the machinery is affordable for veterinary diagnostic laboratories. although an hplc assay to detect mefloquine in an in vitro matrix has been reported [23] , in contrast, this study describes an assay to quantify mefloquine table 1 . intra-and inter-day precision and accuracy for the qc samples (250, 1000 and 5000 ng/ml of mefloquine) tested in triplicate for each concentration. to the authors' knowledge, this is the first report of mefloquine ppb in a non-human species. this study provides the novel finding that mefloquine is highly plasma protein bound in cats. in humans, mefloquine is also highly plasma protein bound (> 98%) [14] , which may account, in part, for its long half-life of approximately three weeks in healthy human subjects [24] . high plasma protein binding can result in the drug-protein complex acting as a reservoir for the physiologically active free drug concentration and consequently prolonging its duration of action [12, 25] . likewise, it should not be assumed that a drugs' ppb is constant across species [25] . in a comparative species study investigating the plasma protein binding of 574 compounds, drugs tend to be slightly more bound in human plasma proteins in comparison to the plasma proteins of rats, mice and dogs [26] . some of the compounds that showed significant interspecies differences in plasma protein binding included diazepam (98% ppb in humans versus 84.8% in rats), prazosin (94.7% ppb in humans versus 65% in rats versus 63% in dogs) and sildenafil (93.6% ppb in humans versus 74% in dogs) [26] . diseases, such as fip, can alter the concentrations of plasma proteins and in turn impact upon a drug's plasma protein binding and ultimately its therapeutic efficacy [27] . for example, in humans, the proteins albumin and α 1 -acid glycoprotein (aag) provide the largest contribution to the protein binding of drugs [27, 28] . yet, with acute falciparum malaria, serum concentrations of aag in non-immune human patients increase two-fold within 24 hours whereas plasma levels of albumin decrease by 30% [29] . likewise, in fip-affected cats, common serum protein abnormalities may include elevated aag [30] and globulin levels [1, 15] as well as decreased albumin levels and a low a: g ratio [15] . here, as described in the literature, the confirmed fip plasma samples demonstrated elevated globulin and decreased albumin levels as well as a low a: g ratio. mefloquine has a high affinity for aag binding, preferentially binding to aag over albumin [27] . thus, if more aag is present in fip-affected cats, it may be preferentially bound by mefloquine. yet, small changes in the unbound drug fraction of highly protein bound drugs can have a significant therapeutic impact [28] . for example, the reduction of ppb from 99.9% to 99.8% can lead to a doubling of therapeutically active unbound drug concentration in plasma [28] . here, although a significant difference was found between the plasma protein binding of mefloquine in clinically normal and fip-affected cats, due to the unknown biological variability of the assay, it is likely that this difference is equivocal. fundamentally, it is difficult to generalise from two pooled plasma samples to two populations of individuals since the biological variability in each population is unknown. the variability (standard deviation) used to evaluate differences in ppb were derived from the assay method and do not represent biological differences. yet, it is also unlikely that further in vitro mefloquine feline plasma protein binding studies will resolve this issue. on the contrary, observing the clinical response of mefloquine in fip-affected animals ultimately may decide mefloquine's therapeutic efficacy in this patient population. in vitro studies to quantify plasma protein binding have some limitations. whilst the major advantage of the red method include its speed, simplicity, reliability and cost-effectiveness [31] , its drawbacks, which also serve as limitations to this study, involve nonspecific membrane binding of the drug [31] as well as changes in oncotic pressure leading to an overestimation of unbound drug concentration [32] . moreover, in vitro ppb may not mirror in vivo ppb in the live animal as the structure of the plasma proteins and their affinity to bind substrates can vary with age, disease and /or presence of competing endogenous and exogenous compounds such as dietary constituents or other therapeutic drugs [33, 34] . furthermore, the drug-protein dissociation rate, which can also greatly affect highly ppb drugs [35] , was not determined in this study. likewise, an additional limitation to this project was the scarcity of confirmed fipaffected plasma as this impacted upon its availability for use in both the hplc validation and ppb protocols. finally, given mefloquine's purported mechanism of action as a schizonticide, another potential limitation concerns the effects of the intraerythrocytic accumulation of mefloquine on plasma protein binding. however, previous studies have demonstrated that red blood cells do not serve as a significant depot for mefloquine [36, 37] . recently, the broad spectrum coronavirus protease inhibitor, gc376 [38] , and the adenosine nucleoside analogue, gs-441524 [3, 4] have been shown to be safe and efficacious antiviral agents for use against fipv. yet, as neither of these agents has obtained registration for veterinary use, investigations into more readily available drugs with anti-fipv activity, such as mefloquine, are urgently required for this invariably fatal disease. this study has validated an accurate and reliable assay to detect mefloquine in feline plasma and demonstrated that mefloquine is highly plasma protein bound in both clinically normal and fip-affected cats. further studies describing mefloquine's pharmacokinetic profile in the cat should be progressed. an update on feline infectious peritonitis: diagnostics and therapeutics a review of feline infectious peritonitis virus infection: 1963-2008 the nucleoside analog gs-441524 strongly inhibits feline infectious peritonitis (fip) virus in tissue culture and experimental cat infection studies efficacy and safety of the nucleoside analog gs-441524 for treatment of cats with naturally occurring feline infectious peritonitis fifty years' fascination with fip culminates in a promising new antiviral mefloquine for preventing malaria in pregnant women the position of mefloquine as a 21 st century malaria chemoprophylaxis patient age does not affect mefloquine concentrations in erythrocytes and plasma during the acute phase of falciparum malaria antiviral effect of mefloquine on feline calicivirus in vitro identification and characterisation of small molecule inhibitors of feline coronavirus replication in vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush-tailed possum (trichosurus vulpecula) predicting plasma protein binding of drugs: a new approach drug-protein binding: a critical review of analytical tools clinical pharmacokinetics of mefloquine positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population a high performance liquid chromatographic assay of mefloquine in saliva after a single oral dose in healthy adult africans defining limit of detection and limit of quantitation as applied to drug of abuse testing: striving for a consensus international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use prevalence of feline immunodeficiency virus infection in domesticated and feral cats in eastern australia risk factors for feline infectious peritonitis in australian cats ph adjustment of human blood plasma prior to bioanalytical sample preparation chemistry (cobas) reference intervals in vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush-tailed possum clinical application of mefloquine pharmacokinetics in the treatment of p. falciparum malaria in vitro binding of cefovecin to plasma proteins in australian marsupials and plasma concentrations of cefovecin following single subcutaneous administration to koalas (phascolarctos cinereus) species differences in drug plasma protein binding selective plasma protein binding of antimalarial drugs to α1-acid glycoprotein plasma protein binding and blood-free concentrations: which studies are needed to develop a drug? serum protein concentrations in plasmodium falciparum malaria critical assessment of the diagnostic value of feline α1-acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach protein binding of antimicrobials: methods for quantification and for investigation of its impact on bacterial killing errors in estimating the unbound fraction of drugs due to the volume shift in equilibrium dialysis clinical pharmacology: plasma protein binding of drugs what is the true clinical significance of plasma protein binding displacement interactions? drug safety characterization of plasma protein binding dissociation with online spe-hplc characterization of in vivo metabolites of wr319691, a novel compound with activity against plasmodium falciparum studies of the disposition and metabolism of mefloquine hcl (wr 142,490), a quinolinemethanol antimalarial, in the rat reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor professor michael court (washington state university, college of veterinary medicine) provided invaluable insight into the interpretation of the mefloquine plasma protein binding results. we also thank the veterinary pathology and diagnostics services, sydney school of veterinary science and dr. randolph baral (the paddington cat hospital, sydney, nsw), for providing the feline plasma samples. key: cord-270414-gh9agf4x authors: fischer, y.; ritz, s.; weber, k.; sauter‐louis, c.; hartmann, k. title: randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis date: 2011-10-12 journal: j vet intern med doi: 10.1111/j.1939-1676.2011.00806.x sha: doc_id: 270414 cord_uid: gh9agf4x background: currently there is no drug proven to effectively treat cats with feline infectious peritonitis (fip). hypothesis: propentofylline (ppf) can decrease vasculitis, and therefore prolong survival time in cats with fip, and increase their quality of life. animals: twenty‐three privately owned cats with fip. methods: placebo‐controlled double‐blind trial. fip was confirmed by histology or immunostaining of feline coronavirus (fcov) antigen in effusion or tissue macrophages or both. the cats were randomly selected for treatment with either ppf or placebo. all cats received additional treatment with glucocorticoids, antibiotics, and low molecular weight heparin according to methods. results: there was no statistically significant difference in the survival time of cats treated with ppf (8 days, 95% ci 5.4–10.6) versus placebo (7.5 days, 95% ci 4.4–9.6). the median survival time of all cats was 8 days (4–36 days). there was neither a difference in quality of life (day 7, p = .892), in the amount of effusion (day 7, p = .710), the tumor necrosis factor‐alpha (tnf‐α) concentration (day 7, p = .355), nor in any other variable investigated in this study, including a complete blood count, and a small animal biochemistry profile. conclusions and clinical importance: this study did not detect an effect of ppf on the survival time, the quality of life, or any clinical or laboratory parameter in cats with fip. therefore, ppf does not appear to be an effective treatment option in cats with a late stage of the disease fip. f eline infectious peritonitis (fip) is one of the most frequent causes of death in young cats. [1] [2] there is no proven record of cats with a confirmed diagnosis having recovered from fip. 3 thus, fip is usually lethal; no controlled study has verified the success of any treatment used to date. 1,4-6 therefore, providing objective evidence of the effectiveness of any treatment against this disease is important. several case reports can be found in the online veterinary information network (http://www.vin.com) that describe a positive effect of the methylxanthine derivative pentoxifylline (ptx) (trental a ) on the survival time in cats with fip. several veterinarians and well-known specialists in feline medicine have suggested that the use of ptx can be effective in treating cats with fip. 4, [6] [7] [8] according to those reports, ptx does not cure but is suggested to prolong the life of these cats. [4] [5] 8 in these reports it has been suggested that ptx is likely to decrease vasculitis, which is responsible for the majority of clinicopathological findings of fip. 1 the mode of action of the methlyxanthine derivatives is not fully understood, and the mechanism remains unknown. [9] [10] the ptx inhibits several cytokines, such as interleukines and tumor necrosis factor-alpha (tnf-a). 9 there are studies in rats and humans (in vivo and in vitro) describing the inhibition of cytokines by ptx, [11] [12] [13] [14] [15] and ptx and other methylxanthine derivatives seem to suppress tnf-a synthesis. 15 these proinflammatory cytokines play a major role in the pathogenesis of vasculitis. 16 therefore, it has been suggested that vasculitis may be effectively controlled with ptx because of its effect in neutralizing or suppressing these cytokines. [11] [12] [13] [14] [15] propentofylline (ppf) and ptx have mainly been trialed for use in people with peripheral vascular diseases, [17] [18] [19] [20] cerebrovascular diseases (such as alzheimer's disease, brain ischemia, or cerebrovascular insufficiency), 9, [20] [21] [22] endotoxemia, 14, 23 and ischemic heart disease. 20, 24 tnf-a also induces fibrinogen synthesis, [25] [26] [27] and is responsible for an increased production of free radicals alt alanine aminotransferase ap alkaline phosphatase ci confidence interval fcov feline coronavirus felv feline leukemia virus fip feline infectious peritonitis fipv feline infectious peritonitis virus fiv feline immundeficiency virus ifat immunofluorescent antibody technique ppf propentofylline ptx pentoxifylline rbc red blood cells spss statistical package for the social sciences tnf-a tumor necrosis factor-alpha tp total protein wbc white blood cells which cause endothelial cell damage. 28 by inhibiting the synthesis of tnf-a by activated monocytes, ptx can probably decrease fibrinogen levels, a common component of the effusion in cats with fip. 10, 19 it was previously postulated that high fibrinogen levels could be an index of tnf-a levels. this finding is supported by a close correlation between decreased fibrinogen levels and clinical improvement. 19 a study into geriatric cachexia in humans additionally showed that ptx may decrease cachexia by down-regulating proinflammatory cytokines, such as tnf-a, interleukin 1 and 6, serotonin, and interferon-c. because cats with fip are often anorectic, this was considered to be another positive effect of the methylxanthine derivatives on the well-being of cats with fip. 24, 29 ppf, another methylxanthine derivative, is licensed in several european countries (including germany) for veterinary use in dogs. it is very similar to ptx (which is not licensed in germany for veterinary use) in its chemical structure as well as in its pharmacological effects. 30-31 both ptx and ppf inhibit several cytokines, such as interleukins and tnf-a. 9 furthermore, ppf has already been applied securely and effectively to cats with feline asthma. 32 therefore, ppf instead of ptx was used in this study. the aim of this study was to evaluate the efficacy of ppf on the survival time and quality of life in cats with a confirmed diagnosis of fip in a placebocontrolled double-blind trial. the study included 23 client-owned cats. inclusion criterion to enter the study was the definitive diagnosis of fip. all cats were presented to the clinic of small animal internal medicine, lmu university of munich, germany. an informed consent of participation signed by the owners was obtained for all cats. this study fulfilled the general german guidelines for prospective studies with owners' consents and was approved by the ethics committee and the animal protection officials of the regierung von oberbayern, germany (permission no. 55.2-1-54-2531-127-09). consecutive cases of cats with confirmed fip that had owners willing to participate in the study, presented to the clinic between april 2009 and december 2010, were entered into the study. diagnosis of fip was either confirmed by detection of feline coronavirus (fcov) antigen in macrophages in the effusion using direct immunofluorescence 33 (n = 9), by histopathological examination of tissue, positive immunohistochemical staining of fcov antigen in macrophages 34 (n = 22), or by both. cats with feline immundeficiency virus (fiv) or progressive feline leukemia virus (felv) infection were not included in the study (snap felv/ fiv test b ). cats with severe clinical signs (karnofsky's score 35 <30%) or a survival time less than 72 hours after treatment initiation were retrospectively excluded (2 cats). one cat had to be excluded in retrospect because of a lack of owner compliance. seventeen of the 23 cats (74%) were european shorthair cats, 2 (9%) were british shorthair cats, and there was one (4%) of each of the following breeds: birman, persian, norwegian forest cat, and persian crossbred. the youngest cat was 13 weeks old and the oldest cat 2.8 years (mean, 0.9 years; median, 0.7 years; interquartile range, 0.42-1.25 years). fifteen (65%) cats were younger than 12 months; 20 (87%) cats were younger than 2 years. seventeen (74%) cats were male (5 neutered), and 6 (26%) female (2 neutered). the study was designed as a placebo-controlled, double-blind randomized trial. cats were randomly assigned to the ppf (n = 7) or placebo group (n = 16). the dosage of ppf was based on the dosage used of ptx to treat cats with fip in the literature and anecdotal case reports of different authors. in those reports, 10-15 mg/kg or 100 mg/cat every 12 hours was given po. 6 according to studies in humans, ppf and ptx are used at the same dosage. 36 cats in this study therefore received a median dosage of 18-25 mg/kg ppf c,d during the whole study period. alternatively, cats received the similar amount of tablets of placebo e (containing lactose, magnesium stearate, and cellulose) every 12 hours po. the ppf and the placebo pills were coded. therefore, veterinarians and owners giving the pills were blinded to identity of the treatment. the code was broken after 23 cats had been treated. all results (including survival time, karnofsky's score, blood and effusions variables, and volume of collected effusion) were obtained blinded. all cats were also treated with glucocorticoids. in case of effusion at day of presentation (n = 21), dexamethasone f (1 mg/kg) was given intraperitoneally or intrathoracically (depending on the location of effusion) every 24 hours for 6 days after thoraco-or abdominocenthesis. cats without effusion (n = 2) received dexamethasone f (1 mg/kg) sc for 6 days. after this period, all cats were treated with oral prednisolone g,h (2 mg/kg) every 24 hours until death. in addition, cats received amoxicillin/clavulanic acid i (12.5 mg/kg iv every 12 hours) for 7 days; dalteparin sodium j (75 iu/kg sc every 12 hours) for 5 days, which was gradually tapered within the next 2 days (day 6: 36 iu/kg, day 7: 18 iu/ kg); as well as fluid and nutritional treatment if necessary during the hospitalization. if the cats were not properly vaccinated, they were treated sc with one dose (4 ml) of immunglobulins k (a product containing antibodies against feline panleukopeniavirus, feline herpesvirus, and feline calicivirus). this product was given to decrease the risk of acquiring an infectious disease because of immune suppression by glucocorticoid treatment and hospitalization. glucocorticoids were given, because it is currently the only treatment thought to have a beneficial effect on cats with fip although there are no controlled studies. 3, 37 antibiotics were administered to minimize the risk of bacterial infection because paracentesis was performed daily (if effusion was present), and because of the high dosage of glucocorticoid treatment. cats also received low molecular weight heparin (dalteparid sodium) to minimize the risk of a disseminated intravascular coagulation (dic), which is often observed in cats with fip. 1,38-39 all cats were either hospitalized during the 1st 7 days after treatment initiation or had to be presented to the clinic daily. physical and ultrasound examinations were performed daily. the general condition was characterized by the karnofsky's score. the index enables judgment of quality of life and well-being in cats by means of a score of 0% (dead) to 100% (absolutely healthy and happy). 35 on day 0 (day of inclusion in the study) as well as on the control days (day 7, 14, and 28), a complete physical examination was performed, and blood was collected. a cbc was performed with an automatic analyzer (cell-dyn l ), the small animal biochemistry profile (see table 1 ) was examined using an automatic analyzer (hitachi m ). aliquots of the serum samples were preserved at -80°c for detection of tnf-a. if present, effusion was aspirated, and the amount was recorded. depending on their health status, cats were returned to their owners after day 7. the owners were asked to fill in a provided diary recording temperature, respiratory rate, weight, general condition (duration of sleeping time, eating, playing, and grooming behavior) every day, as well as any problem noticed by the owners. follow-up examinations in the clinic were scheduled on days 7, 14, and 28, including physical examination, examination of a cbc, a small animal biochemistry profile, and ultrasound to detect the presence of effusion. tnf-a was measured in the serum (on day 0, 7, 14, and 28) using an elisa. n because the elisa is only validated for cell culture supernatants, a spiking experiment using serum samples was performed. serum components can impact the accuracy of elisa results and may interfere with antibody binding or show cross-reactivity. to assess recovery of serum samples and to assess accuracy of measured values, 200 pg tnf-a were spiked into a serum sample of a healthy cat. the sample was diluted in sample diluent (pbs o + 10% fetal calf serum p ) 2-fold to yield samples containing 100, 50, and 25 pg. as a control, sample diluent was spiked and diluted accordingly. the spiked undiluted control yielded results in the expected range (89%). the spiked undiluted serum sample showed recovery of 65%, indicating inhibitors of detection in the serum. the serum at a 1 : 2 dilution showed recovery of 73% compared to 88% of the diluted con-trol. the recovery loss of 15% was considered acceptable, and interference of inhibiting components appeared to be not severe; all serum samples were therefore diluted 1 : 2 for detection of tnf-a. the elisa was performed according to the manufacturer's instructions. a 96-well microplate n was coated with capture antibody by overnight incubation. the next day, the wells were washed and samples (diluted 1 : 2) and standards n were incubated for 2 hours at room temperature. after washing, the detection antibody n was added and incubated for another 2 hours at room temperature. for detection, streptavidin-hrp n was used with tetramethylbenzidin n as a substrate solution. the reaction was stopped after 10 minutes with 0.5 m sulfuric acid. n the elisa was measured with a bio tek reader, q and the data analysis was performed using gen5 data analysis software. r all cats were randomly assigned to 2 groups, the ppf group and the placebo group. a power analysis had been performed before starting the study (using pass, 2008; http://www.ncss. com/pass). for this analysis, a clinical relevant difference in median survival time was set at 21 days, assuming that animals treated with ppf would survive at least 21 days longer than animals receiving placebo. these differences could have been detected with 18 animals per group, using a power of 80% and a significance level of 5%. however, an interim analysis on the survival time was performed after 23 cats had been treated, because most cats in the study at that time point have survived for less than 29 days, and the median survival time was not significantly different between the groups (median survival time ppf: 8.0 days; placebo: 7.5 days). therefore, it was decided to terminate the study prematurely for reasons of animal welfare, as the expected clinical relevant differences and the difference of the survival time were clearly not achievable. statistical analysis was performed using statistical software spss version 17.0 (http://www.spss.com). variables compared between both groups (ppf or placebo group) included survival time, karnofsky's score, red blood cells (rbc), hemoglobin, hematocrit, platelets, white blood cells (wbc), monocytes, lymphocytes, banded neutrophils, mature neutrophils, alanine aminotransferase (alt), alkaline phosphatase (ap), bilirubin, total protein (tp), albumin, albumin to globulin ratio, urea, creatinine, glucose, and the volume of effusion. a difference in the survival time between both groups was evaluated using a log-rank test. differences between the parameters of the 2 groups at day 0, day 7, and day 14 were investigated using a mann whitney u test. p-values <.05 were considered significant. a bonferroni correction was performed to rule out multiple test interference. a 5% significance level was assumed for all variables, and thus the p-value of .05 was divided through the number of tests performed (n = 20). therefore, a final value of p .0025 for each variable was considered significant. there was no statistically significant difference in any blood parameter or in the amount of effusion at any time point between cats treated with ppf, and those that received placebo ( table 1 ). the karnofsky's score of both groups also showed no statistically significant difference at the start of the study. cats survived between 4 and 36 days (median, 8 days). the median survival time of cats in the ppf group was 8 days (range 4-36; 95% confidence interval [ci] 5.4-10.6), and of cats in the placebo group the median survival time was 7.5 days (range 4-22; 95% ci 4.4-9.6). the difference in survival time between the 2 groups was not significantly different (p = .665) (fig 1) . twenty-two of 23 (96%) cats survived less than 29 days. these 29 days were preset as expected minimum survival time in cats receiving ppf. in a previous study, a median survival time of 8 days was detected in cats with fip. 3 in the present study, it was assumed that cats treated with ppf would live at least 21 days longer than those receiving placebo (with a median survival of 8 days), as this makes a relevant difference for the owners. 3 no statistically significant differences of any blood parameter or of effusion were apparent after the 7 and 14 day period of treatment between the ppf group and the placebo group. the karnofsky's score of both groups on the evaluated control days (day 7 and day 14) also showed no significant difference. on day 7, only 14 cats remained in the study. two of them improved 10% in the karnofsky's score, 5 cats showed no difference and the karnofsky's score of 7 cats deteriorated for at least 70%. on day 14, only 4 cats remained in the study and the karnofsky's score of all these cats had deteriorated for at least 80% compared to day 0. no statistical evaluation was performed after day 14 because only 1 cat was alive at day 28 (next control day). only in 6 cats (4 of the ppf group, 2 of the placebo group) serum samples of more than one time point were available for the comparison of the tnf-a concentration during treatment with ppf. a significant decrease was not found in any of these cats; conversely, most cats even showed increased tnf-a serum levels during the study period. in this study, there was no statistically significant difference in the survival time of cats treated with ppf versus placebo. there was also no statistically significant difference in any other variable evaluated between both groups, including the cbc and a small animal biochemistry profile (as shown in table 1 ). the median survival time (8 days) of cats with fip after definitive diagnosis in this study was nearly identical to the median survival of the study of ritz et al. 3 there are no other reports on median survival times of cats after fip was confirmed. however, in the study of ritz et al, 3 several cats survived longer than 4 weeks, which was not the case in the present study. unfortunately, the desired effect of ppf was not observed. it has been proposed that ppf may decrease the volume of effusion, by inhibiting cytokines (particularly tnf-a) and thereby reducing resulting vasculitis. there was neither a significant difference in the amount of effusion between the ppf and the placebo group, nor a decrease in tnf-a in any of the cats in which serial measurement was performed. a cure was never the ultimate goal in the use of ppf in cats with fip, because it is not an antiviral drug. however, because of the pharmacological features it was assumed to have positive effects on the well-being of the cats and the survival time. ppf was meant to inhibit the tnf-a production, 9 which is involved in the development of vasculitis. [11] [12] [13] [14] [15] the tnf-a concentration, however, did not decrease in the cats treated with ppf, but increased instead. most likely, ppf was not even able to exhibit its function within this short period of time, and the tnf-a increase mainly reflected severe progression of the disease. reasons for the lack of efficiency can be multifaceted. the most probable reason is that treatment may have been initiated too late. if signs of vasculitis were apparent, the immune-mediated process in cats with fip might have been progressed too far to be delayed by ppf. in the present study, 21 of the 23 cats already showed effusion at the day of presentation. as shown in an experimental trial, signs of fip become apparent 1-2 weeks after inoculation of the mutated feline infectious peritonitis virus (fipv). 40 as the effect of ptx is described by the manufacturer information a to be seen after 2-4 weeks after treatment initiation, and it is an assumption that the same time frame would apply to ppf, most of the cats were already dead before an effect could be reached. a further reason for lack of ppf efficiency in this study could be that the treatment intervals could have been too long. an application of ppf every 12 hours was used in this trial following the anecdotal reports describing an effect of ptx in cats. 4, 6, 32, 41 the manufacturer instruction recommends administration of ptx 3 times daily in human medicine, 20 because of a relatively short plasma half-life of 0.4-0.8 hours of the drug. a pharmacokinetic study in dogs indicated that ptx be administered every 8 hours. 42 the reasons behind the reported beneficial effects of ptx described in case reports (http://www.vin.com) are currently unknown. treatment might have been initiated earlier in these cats. alternatively, fip was not confirmed by histopathology or immunofluorescent antibody technique (ifat) in most of these cases; so, these cats could have suffered from other diseases. some of the cats might have had a "non-effusive" form of fip, which is considered to have longer survival times than in cats with effusion. 43 drug interactions between ppf and the other medications (especially the glucocorticoids) in this study are a possibility, but are not reported. in addition, in a recent study in cats with asthma, glucocorticoids and ppf were safely used in combination with no adverse interactions. the glucocorticoid dose can be reduced by addition of ppf to treatment. 32 therefore, no adverse effects were expected with the combination ppf and glucocorticoids in the present study. glucocorticoids are routinely used in cats with fip, 1,3,44-47 as it is not the virus itself that causes major damage but the cat's own immune reaction that leads to the fatal consequences. there are no evidence based studies that glucocorticoids have a positive effect in cats with fip. 37 the cats of the present study received an immunosuppressive dose of glucocorticoids (2 mg/kg). together with the stress caused by hospitalization and daily paracenthesis, glucocorticoids might be a more confounding factor. [48] [49] potentially, a lower dose of glucocorticoids, or no glucocorticoids at all, might be better for "long-term" treatment. the beneficial effects of glucocorticoids in the treatment of fip must be questioned given the median survival time is 8-9 days in the present and in the previous study, 3 both in treatment and placebo groups. 3 therefore, future treatment study protocols should include a 3rd group of cats that receive no glucocorticoids. alternatively a doubleblinded study just evaluating glucocorticoids as treatment option for fip could be performed. there was no statistically significant difference in the karnofsky's score during the treatment period between the two groups. few cats showed an increased wellbeing shortly after participating in the study. this was most likely induced by the corticosteroids and was not the effect of ppf, because this phenomenon could be observed in both groups. however, the improvement in the general condition was not long-lasting, and cats deteriorated rapidly between 4 and 21 days after treatment initiation. this study had several limitations. the 1st limitation is the unequal distribution of cats to the ppf and the placebo group. as this was a blinded, randomized trial, the distribution could not be influenced. another limitation might be the small number of cats (only 2) without initial effusion. definitive diagnosis in cats without effusion, however, is much more difficult to obtain. 33 the ppf might be more useful in cats without effusion, as it may have a chance to prevent vasculitis and therefore effusions feline infectious peritonitis feline infectious peritonitis and feline enteric coronavirus infections. part 1 effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis feline infectious peritonitis: what's new? available at: www.ancats.com.au/pdf files/feline infectious peritonitis summary diagnosis and treatment of fip in the real world 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macrophages and lymphocytes summary of product characteristics, karsivan (propentofylline) use of propentofylline in feline bronchial disease: prospective, randomized, positive-controlled study comparison of different tests to diagnose feline infectious peritonitis immunohistological demonstration of feline infectious peritonitis virus antigen in paraffin-embedded tissues using feline ascites or murine monoclonal antibodies karnofsky's score modified for cats effect of propentofylline and pentoxifylline on cerebral blood flow using 123i-imp spect in patients with cerebral arteriosclerosis feline infectious peritonitis. abcd guidelines on prevention and management disseminated intravascular coagulation in experimentally induced feline infectious peritonitis jacobse-geels he, daha mr, horzinek mc. antibody, immune complexes, and complement activity fluctuations in kittens with experimentally induced feline infectious peritonitis a retrospective study of canine and feline cutaneous vasculitis pharmacokinetics of pentoxifylline in dogs after oral and intravenous administration clinicopathological findings and disease staging of feline infectious peritonitis: 51 cases from 2003 to 2009 in taiwan infectious peritonitis in a cat that subsequently developed a myeloproliferative disorder beitrag zur therapie der fip effect of thromboxane synthetase inhibitor on feline infectious peritonitis in cats use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus die diagnositik der felinen infektio¨sen peritonitis (fip): retrospektive und prospektive untersuchungen we thank the intervet deutschland gmbh, unterschleißheim, germany, for partial support of this study. key: cord-327352-cbnjsrmt authors: kipar, a; bellmann, s; kremendahl, j; köhler, k; reinacher, m title: cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis date: 1998-10-23 journal: vet immunol immunopathol doi: 10.1016/s0165-2427(98)00158-5 sha: doc_id: 327352 cord_uid: cbnjsrmt twenty-three cats with spontaneous feline infectious peritonitis (fip) were examined by light microscopy including immunohistology and histochemistry in order to determine the cellular composition and the expression of viral antigen in lesions in fip. furthermore, the presence of plasma-cells producing coronavirus-specific antibodies was evaluated in situ. macrophages and neutrophils were demonstrated by an antibody against calprotectin (leukocyte protein l1, myeloid/histiocyte antigen), neutrophils were recognized due to their chloroacetate esterase activity, and band t-lymphocytes were identified by antibodies against the cd3 antigen and the cd45r antigen, respectively. expression of viral antigen was immunohistologically demonstrated by a monoclonal antibody (mab) against coronavirus while coronavirus-specific antibodies in situ were identified by the application of feline coronavirus prior to the coronavirus antibody. lesions were classified as diffuse alterations at serosal surfaces, granulomas with areas of necrosis, granulomas without extended necrosis, focal and perivascular lymphoplasmocytic infiltrates, and granulomatous-necrotizing vasculitis. diffuse alterations on serosal surfaces were represented either by activated mesothelial cells with single coronavirus antigen-bearing macrophages or by layers of precipitated exudate containing single to numerous granulomas with areas of necrosis. in liver and spleen, the exudate was often underlaid by a small band of subcapsular b-cells with an occasional plasma-cell producing coronavirus-specific antibodies. in other locations, a variably broad band of b-cells and plasma-cells, often infiltrating between underlying muscle fibers, separated the exudate from the unaltered tissue. some of these plasma-cells were positive for coronavirus-specific antibodies. in granulomas with areas of necrosis, the central necrosis was surrounded by macrophages usually expressing considerable amounts of viral antigen. few b-cells and plasma-cells were found in the periphery. in granulomas without extended necrosis, the number of macrophages were lower. only few macrophages expressing low amounts of viral antigen were present. b-cells and plasma-cells formed a broad rim. few plasma-cells stained positive for coronavirus-specific antibodies. in both types of granulomas, few neutrophils were found between macrophages. few t-cells were seen scattered throughout the lesions. focal and perivascular lymphoplasmocytic infiltrates were mainly seen in omentum and leptomeninx. b-cells were the predominant cells; some plasma-cells were positive for coronavirus-specific antibodies. viral antigen was not readily detected in these alterations. granulomatous-necrotizing vasculitis was occasionally found in kidneys and leptomeninx. it was dominated by macrophages which often stained strongly positive for coronavirus antigen. different types of alteration were often seen in the same animal and even the same tissue. there was no obvious correlation between the cat's age, gross pathological changes, and the histological types of alteration. single plasma-cells positive for coronavirus-specific antibodies were found around blood vessels distant from inflammatory alterations, within the lung parenchyma, as infiltrating cells in the mucosa of the small intestine, and in spleen and mesenteric lymph node. results show that alterations in fip are heterogeneous concerning cellular composition and expression of viral antigen. the dominance of b-cells in part of the lesions together with the presence of plasma-cells positive for coronavirus-specific antibodies indicate that these cells may play a role in the maintenance of inflammatory processes in fip. the histopathological findings in spontaneous and experimental feline infectious peritonitis (fip) have been described in several previous reports on effusive and noneffusive fip (wolfe and griesemer, 1966; montali and strandberg, 1972; ward et al., 1974; hayashi et al., 1977 hayashi et al., , 1980 . although usually characteristic, both macroscopic and histological alterations were not always indicative of fip. therefore, differential diagnoses can sometimes not be readily excluded by routine post-mortem histopathology, and the diagnosis of fip has to be confirmed by immunohistological demonstration of coronavirus antigen within the lesions (tammer et al., 1995) . we performed a systematic examination on the various lesions in naturally infected animals in order to further investigate cellular composition and content of immunohistologically detectable coronavirus antigen, and to detect the production of coronavirus-specific antibodies in situ. the study was performed on 23 cats with fip. animals had been routinely necropsied at the department of veterinary pathology, leipzig university, leipzig, germany. tissue samples were fixed in 10% unbuffered, neutralized formalin for 16±18 h and were embedded in paraffin. five mm-thick sections were either stained with hematoxylin±eosin or used for immunohistological and histochemical examinations. additionally, feline leukemia virus (felv) infection was diagnosed by immunohistology as described by kovacevic et al. (1997) . immunohistology was performed on fip lesions, lymphatic tissues (spleen and mesenteric lymph nodes), and bone marrow. peroxidase-anti-peroxidase (pap) and avidin±biotin peroxidase complex (abc) methods were performed as described (sternberger et al., 1971; hsu et al., 1981) . briefly, sections were deparaffinized in xylene and rehydrated through graded alcohols. endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide in methanol at room temperature for 30 min. sections were washed with trisbuffered saline (tbs, 0.1 m tris±hcl with 0.9% nacl, ph 7.6). to demonstrate coronavirus antigen, sections were treated with target unmasking fluid (tuf; dianova gmbh, hamburg, germany) for 10 min at 968c. for the demonstration of the cd45r antigen of b-cells (monteith et al., 1996) , sections were incubated in citrate buffer (10 mm, ph 6.0) at 968c for 30 min. to demonstrate the myeloid/histiocyte antigen of monocytes, macrophages, and neutrophils (leukocyte protein l1, calprotectin; dale et al., 1983; flavell et al., 1987) and the cd3 antigen of t-cells (beebe et al., 1994) , sections were pretreated with 0.5% protease (type xxiv: bacterial, sigma chemie deisenhofen, germany) diluted in phosphate-buffered saline (ph 7.2) at 378c for 5 min. after pretreatment, 50% swine serum in tbs were applied for 10 min at room temperature prior to rabbit anti-human cd3, while 10% rat serum in tbs were used prior to the monoclonal mouse antibodies and undiluted normal horse serum prior to rat antimouse cd45r, respectively. slides were then incubated for 12±16 h at 48c with the primary antisera: mouse anticoronavirus (fcv3-70; 1:100 in tbs; custom monoclonals international, west sacramento, ca), mouse anti-human myeloid/histiocyte antigen (leukocyte protein l1, calprotectin; 1:1600 in tbs; dako diagnostika gmbh, hamburg, germany), rat antimouse cd45r (b220 (ly5), clone ra3-6b2; 1:1000 in tbs; cedar lane laboratories, hornby, canada), and rabbit anti-human cd3 (1:500 in 20% swine serum in tbs; dako diagnostika gmbh). for rabbit anti-cd3, swine anti-rabbit igg (1:100 in 20% swine serum in tbs; dako diagnostika gmbh) was used as a link antibody, followed by rabbit pap complex (1:100 in 20% swine serum in tbs; dako diagnostika gmbh). for the mouse mabs, rat antimouse igg (1:100 in tbs; dianova gmbh) and mouse pap complex (1:500 in tbs; dianova gmbh) were applied, while biotinylated rabbit anti-rat igg (1:100 in tbs; vector laboratories, burlingame, ca), followed by the abc (a and b each 1:100 in tbs; vector laboratories) were used for the demonstration of the cd45r antigen. incubations were performed at room temperature for 30 min each. between each incubation step slides were washed with tbs. sections were incubated for 10 min with 0.05% 3,3 0 -diaminobenzidine tetrahydrochloride (dab; serva, heidelberg, germany) in 0.1 m imidazole/hcl buffer (ph 7.1) and counterstained with papanicolaou's hematoxylin (1:20 in aqua dest.; e. merck, darmstadt, germany). for the demonstration of coronavirus-specific antibodies, slides were incubated with feline coronavirus (df-2 fipv; purified pelleted culture supernatant, 10-fold concentrated and stored in pbs; 1:25 in tbs) for 12±16 h at 48c and washed with tbs prior to the primary mouse anti-coronavirus antibody which was then detected by the pap method as described above. negative controls for pabs and mabs were incubated with normal rabbit or rat serum or a non-reacting mouse mab directed against chicken lymphocytes (domingo et al., 1986) , respectively. negative controls for the detection of coronavirus-specific antibodies implied replacement of feline coronavirus by infectious bursitis disease virus (serotype i, purified, pelleted culture supernatant, 2-fold concentrated and stored in pbs; 1:25 in tbs), and either the application of the non-reacting mabs directed against chicken lymphocytes mentioned above or a mouse anti-canine distemper virus mab (dv2-12; 1:100 in tbs; custom monoclonals international) instead of the mouse anti-coronavirus mab. one case each of plasma cell-dominated gingivitis, conjunctivitis and otitis externa were examined for the presence of coronavirus antigen and plasma-cells producing coronavirus-specific antibodies. formalin-fixed and paraffin-embedded feline tissues (spleen, lymph nodes and bone marrow) served as positive controls for the leukocyte markers. in order to allow a clearcut differentiation of the cells in areas with caryorhexis, neutrophils were identified according to their chloroacetate esterase activity. the demonstration of the chloroacetate esterase was performed as previously described (osbaldiston et al., 1978; schaefer, 1983) . animals ranged from 5 months to 6 years of age. fourteen cats were male and nine were female. reported clinical findings had been variable. in six cases, however, fip was suspected. information about the duration of the disease were available in three cases: animals had shown clinical symptoms for 2, 3, and 10 days, respectively. gross pathology was variable. thirteen cats showed peritoneal effusion which was in one case accompanied by both pleural and pericardial effusion. another three cats only exhibited pleural effusion and, in two of these cases, pericardial effusion. in most of the cases (n18), fibrinous and/or granulomatous peritonitis was observed, involving abdominal wall, omentum, and the serosa of various tissues in a variable frequency. eight of these cats showed parenchymal granulomas which were most often located in the kidney (n7); another three cats exhibited necrotizing lymphadenitis of the mesenteric lymph nodes, and in further two cats fibrinogranulomatous pleuritis were additionally observed. two animals showed pleuritis and pneumonia besides granulomatous hepatitis and lymphadenitis of the mesenteric lymph nodes or granulomatous hepatitis, nephritis, and leptomeningitis, respectively. in one cat each, lesions were restricted to fibrinousgranulomatous pleuritis and granulomas in both kidneys. correlations between the cat's age and gross pathological findings could not be stated. in 14 cats, felv co-infection was diagnosed by immunohistology. in lymphoid tissues, cd3 antigen-expressing t-lymphocytes, displaying a dark brown reaction at the cell periphery, were located in the paracortical region of lymph nodes and the periarteriolar sheath regions in the spleen. cd45r antigen-positive b-lymphocytes exhibited a similar staining pattern and were found in lymphoid follicles of lymph nodes and spleen. monocytes, neutrophils, and macrophages showed a strong cytoplasmic staining for the myeloid/histiocyte antigen (leukocyte protein l1, calprotectin) in all tissues. chloroacetate esterase activity was represented by a faint red cytoplasmic staining of neutrophils in tissues and myelomonocytic precursors in bone marrow, and a strong granular cytoplasmic staining of mast cells. staining for coronavirus antigen was represented by a granular precipitate in the cytoplasm of macrophages or, to a lesser extent, free within areas of necrosis (figs. 1(a), 1(b) and figs. 2, 3). after application of fipv prior to the demonstration of coronavirus antigen, staining for both coronavirus antigen and coronavirus-specific antibodies were seen in the same slide (figs. 1 and 4(a)). however, as the presence of coronavirusspecific antibodies were represented by an almost homogeneous cytoplasmic staining in plasma-cells which could be identified according to their morphology, both types of reaction were readily distinguishable (figs. 1 and 4(a)). lesions were variable and were classified according to their distribution and cellular composition as diffuse alterations at serosal surfaces, granulomas with areas of necrosis, granulomas without extended necrosis, focal and perivascular lymphoplasmocytic infiltrates, and granulomatous-necrotizing vasculitis. diffuse alterations of serosal surfaces were variable. mainly on the spleen, enlarged activated mesothelial cells, recognizable due to their cuboidal shape, were seen in combination with few macrophages in the edematous serosa (fig. 2) . coronavirus antigen expression was restricted to the infiltrating macrophages (fig. 2) . on liver and spleen, layers of precipitated exudate containing few foci composed of macrophages which expressed variable amounts of viral antigen, were also seen (fig. 3) . mainly in the liver, the protein exudate was underlaid by a small band of subcapsular b-cells and plasma-cells (fig. 3) . occasionally, a single plasma-cell stained positive for coronavirusspecific antibodies. in many cases, serosal surfaces were covered with layers of precipitated exudate containing numerous granulomas with areas of necrosis. these granulomas were dominated by macrophages frequently only expressing small amounts of viral antigen ( fig. 4(a) , (c)). neutrophils were rarely seen. the precipitated exudate was separated from the unaltered parenchyma by few to many layers of b-cells and plasma-cells which occasionally infiltrated between underlying muscle fibers ( fig. 4(a) , (b)); some of these plasma-cells were positive for coronavirus-specific antibodies ( fig. 4(a) ). few t-cells were scattered throughout the alteration (fig. 4(d) ). in some cases, diffuse almost pure b-cell and plasma-cell infiltration of pleura or peritoneum was seen. on the pleura, this was occasionally accompanied by proliferation of fibroblasts. these lesions contained single to numerous plasma-cells positive for coronavirus-specific antibodies. viral antigen expression was in these cases restricted to single granulomas with areas of necrosis which were only detected after extended examination. granulomas with areas of necrosis were seen within diffuse alterations of the serosa ( fig. 4; see above) , within the omentum, and within parenchymas (fig. 1) . a variably large area of central necrosis was surrounded by inflammatory cells which were almost completely identified as macrophages (figs. 1(b) and 4(b) ). viral antigen expression were mostly restricted to a variable number of macrophages ( figs. 1 and 4(a) ). single to few neutrophils were scattered between macrophages. in the periphery of and close to the lesions, b-cells and plasma-cells were found; some of these plasma-cells stained positive for coronavirus-specific antibodies ( fig. 1(a) ). few t-cells were scattered throughout the granulomas. granulomas without extended areas of necrosis were mainly observed in the kidneys (fig. 5 ). the small center comprised of macrophages which only rarely exhibited faint staining for viral antigen (fig. 5(a) ). they were intermingled with single neutrophils. the lesions were dominated by a broad rim of b-cells and plasma-cells ( fig. 5(b) ); the latter were in part positive for coronavirus-specific antibodies. t-cells were again rare and scattered throughout the lesion. focal and perivascular lymphoplasmocytic infiltrates were mainly found in omentum and leptomeninx (fig. 6) ; perivascular infiltrations were also seen occasionally in the brain of cats with granulomatous leptomeningitis (fig. 7(a) ). some of the plasma-cells stained positive for coronavirus-specific antibodies (figs. 6(b) and 7(a)). macrophages and t-cells comprised a minority; neutrophils were rarely observed. granulomatous-necrotizing vasculitis was occasionally seen, mainly in the leptomeninx and the kidneys. macrophages comprised the dominant cell population. they often showed a strong positive reaction for coronavirus antigen (fig. 7(b) ). single to few neutrophils were seen in proximity to the vessel wall. t-cells were few and scattered throughout the infiltrates. different types of the lesions described above were frequently seen in the same animal and even in the same organ. single plasma-cells producing coronavirus-specific antibodies were also found around blood vessels in tissues without further alterations: in the liver portal trias, the interstitium of the renal medulla, the lung, and the leptomeninx. they were also seen scattered in the wall of alveoli in the lungs and around bronchi. furthermore, numerous plasma-cells producing coronavirus-specific antibodies were identified among infiltrating cells of the mucosa of the small intestine. epithelial cells did not express coronavirus antigen. mesenteric lymph nodes without granulomatous-necrotizing lesions generally showed follicular hyperplasia with an increased number of cd45r antigen-positive b-cells and a large number of sinus histiocytes. they contained single to numerous plasma-cells positive for coronavirus-specific antibodies which were located in medullary cords and in or outside the lymph node capsule. in the paracortical t-cell zone the number of cd3 antigen-positive t-cells were slightly to moderately reduced. in the spleen, plasma-cells with coronavirus-specific antibodies were rarely seen in the periphery of granulomatousnecrotizing lesions, within the subcapsular band of lymphocytes, and were diffusely disseminated. in spleens without granulomatous-necrotizing lesions, follicular hyperplasia and a slight reduction of t-cells in the periarteriolar sheath region was observed; viral antigen were not expressed. bone marrow was generally hyperplastic containing all cell lines. part of the cells were positive for the cd3 or the cd45r antigen, respectively. in four cases, focal accumulations of b-cells were additionally seen. about half of the bone marrow cells expressed the myeloid/histiocyte antigen. in most cases, the number of neutrophils were mildly to massively increased. neither coronavirus antigen nor plasma-cells positive for coronavirus-specific antibodies were found. in three cases, information on the time period of clinical disease were available. the two cats showing clinical symptoms for 2 or 3 days prior to death exhibited fibrinous exudate on the peritoneum containing granulomas with areas of necrosis and moderate expression of viral antigen. plasma-cells positive for coronavirus-specific antibodies were seen in the periphery of those lesions. the animal who had been ill for 10 days showed bcell-and plasma-cell-dominated omentitis and serositis of the small intestine with few granulomas with areas of necrosis. viral antigen were moderately expressed. in many cases, mild to moderate b-cell-dominated focal interstitial infiltration of the renal cortex was found. there, neither viral antigen nor plasma-cells positive for coronavirus-specific antibodies were demonstrated. negative controls for leukocyte antigens and coronavirus antigen as well as for coronavirus-specific antibodies did not show any positive staining. in neither case of plasma cell-dominated inflammation (gingivitis, conjunctivitis and otitis externa) coronavirus antigen or plasma-cells positive for coronavirus-specific antibodies were found. based on immunohistological and histochemical characterization of inflammatory cells as well as the presence of coronavirus antigen and plasma-cells producing coronavirus-specific antibodies in the lesions of 23 cats with spontaneous fip, this study describes the composition of alterations observed in fip after natural infection. as previously described, coronavirus antigen can be demonstrated in fip lesions by immunofluorescence or other immunohistological methods (pedersen and boyle, 1980; tammer et al., 1995) . we applied a coronavirus-specific mouse mab usable on formalinfixed and paraffin-embedded samples for immunohistology. in this study, lesions which had first been generally described by wolfe and griesemer (1966) , were classified according to their distribution and cellular composition. diffuse alteration at serosal surfaces included activation of mesothelial cells and layers of precipitated exudate containing granulomas with areas of necrosis and macrophages expressing viral antigen. b-cells and plasma-cells comprised underlying cell layers. there, some plasma-cells were positive for coronavirus-specific antibodies. according to the amount of necrosis, granulomas were divided into those with areas of necrosis and those without extended necrosis. while the first were dominated by macrophages with only few surrounding b-cells and plasma-cells, the latter exhibited a small center with macrophages and a broad rim of b-cells and plasma-cells. expression of viral antigen was nearly exclusively restricted to intact macrophages and were stronger in granulomas with areas of necrosis. in granulomas without extended necrosis, viral antigen was often not abundantly present, thereby rendering an immediate immunohistological diagnosis of fip more difficult. single plasma-cells positive for coronavirus-specific antibodies were found in the periphery of and close to both types of granuloma. findings indicate that b-cells form a band between granulomas and unaltered tissue on serosal surfaces and progressively infiltrate granulomas, hence replacing macrophages. the presence of plasma-cells bearing coronavirus-specific antibodies between b-cells in close proximity to granulomas with necrosis and serosal exudate layers further indicates, that the immune response against the coronavirus starts before these fip-specific alterations develop. focal accumulations and perivascular infiltrations of b-cells and plasma-cells were often observed in the omentum and sometimes in the leptomeninx. some of these plasmacells were positive for coronavirus-specific antibodies. this indicates that they represent a fipv-specific immune response as it is also supported by the demonstration of plasmacells bearing coronavirus-specific antibodies around blood vessels distant from granulomas. the presence of plasma-cells producing coronavirus-specific antibodies in cats with fip in both lung tissue and intestinal mucosa without fip lesions is presumably due to local immune response to oronasal coronavirus infection. the immunohistological staining of macrophages in granulomas and acute vasculitis with a mab against coronavirus indicates viral replication in macrophages. this has been demonstrated in cultivated macrophages (jacobse-geels and horzinek, 1983) and by experimental studies which showed that aerosol application of fipv results in infection of macrophages and persistent cell-associated viremia (weiss and scott, 1981a, b) . electron microscopical examinations demonstrated virus replication in peritoneal macrophages, mesothelial cells, and degenerating macrophages in inflammatory lesions (zook et al., 1968; hayashi et al., 1978; weiss and scott, 1981b) . however, in our study, even activated mesothelial cells did not stain positive for coronavirus antigen. viral antigen was frequently not readily detectable in granulomas without extended necrosis where the number of macrophages were also comparatively low. the question then arises whether the remaining macrophages do not contain nearly any virus or whether the lower number of macrophages decreases only the statistical chance to detect viral antigen in immunohistology. it also seems possible that b-cells and plasma-cells totally replace macrophages, thereby leading to the development of mononuclear infiltrates as they are often seen in the renal cortex in cats with fip regardless of their age. however, although these infiltrates were mainly comprised of b-cells, plasma-cells positive for coronavirus-specific antibodies were never seen in those infiltrates in this study. t-cells comprised a minority in all types of lesions. the reason for this might be that t-cells do not play a major role in the pathogenesis of fip lesions or that animals show a t-cell depletion. the latter was demonstrated in a previous study which also showed that t-cell depletion in fip develops due to apoptosis (haagmans et al., 1996) . neutrophils were found among infiltrating cells in necrotizing lesions but were generally rarely seen. they did not contain viral antigen. this indicates that in fip lesions neutrophils do not have a pathogenic role other than lysis of necrotic material. information on the time of alteration development in the experimentally induced fip is not unequivocal and does not refer to the occurrence of lymphocytes and plasma-cells (hayashi et al., 1980; weiss and scott, 1981b; stoddart et al., 1988) . in naturally infected animals, correlation between the duration of clinical disease and the various types of fip lesions have not yet been evaluated (wolfe and griesemer, 1966) . in our study, the two cats showing 2 or 3 days of clinical disease prior to death exhibited fibrinous exudate containing granulomas with areas of necrosis on the peritoneum. viral antigen was moderately expressed in granulomas, in the periphery of which few plasma-cells producing coronavirus-specific antibodies were seen. the animal which had been ill for 10 days showed b-cell-and plasma-cell-dominated omentitis and serositis at the small intestine with few granulomas with areas of necrosis. the increased presence of b-and plasma-cells could be interpreted as signs of a prolonged course of the disease. in general, obvious correlation between the cat's age, the presence of abdominal or pleural effusions, and the histological type of alterations could not be stated. on the contrary, the various types of lesions were frequently seen in the same animal and even in the same organ. it seems possible that natural fip infection represents a slow disease progress with constant development of acute alterations, represented by granulomas with areas of necrosis. on the other hand, alterations might reflect different capacities of cellular immune response in affected cats. the small band of subcapsular b-cells below layers of precipitated exudate was seen mainly in the liver. this might be the result of the cellular immune response which is focused on b-cells and macrophages. all cats in our study showed follicular hyperplasia and sinus histiocytosis in lymphatic tissues as well as bone marrow hyperplasia as has been described previously (wolfe and griesemer, 1966; montali and strandberg, 1972; hayashi et al., 1980) . in 14 cases, felv co-infection was diagnosed by immunohistology. felv infection is often accompanied by fip (cotter et al., 1975; reinacher, 1989) and is, like other concurrent infections, considered as a potential predisposing factor for fcov infection (evermann et al., 1991) . felv has been shown to permit the reactivation of fip in healthy fcov carriers (pedersen, 1987) . in our study, differences could not be stated between felv-positive and felv-negative cats with fip concerning the expression of viral antigens, the number of plasma-cells producing coronavirus-specific antibodies, and the distribution and cellular composition of the lesions in fip. summarizing the results of our study, one can state that fip lesions are heterogenous as to their cellular composition and the intensity of viral antigen expression. plasma-cells producing coronavirus-specific antibodies were present in the periphery and vicinity of granulomas. it does not seem unlikely, that granulomas develop from foci of centrally degenerating macrophages expressing coronavirus antigen. neutrophils are rarely intermingled. few b-cells were seen in the periphery, but seem to progressively replace (degenerated) macrophages. the number of macrophages and the intensity of staining for coronavirus antigen decreases with the reduction of the areas of necrosis. finally, b-cells obviously dominate focal lesions which are often devoid of coronavirus antigen. fip lesions are thought to be induced by immune complexes which are deposited at the wall of blood vessels, activate complement and damage vascular tissues. furthermore, complement-mediated tissue damage may occur due to the binding of antibodies to viral antigen. enhanced uptake into and thereby virus replication in macrophages may follow opsonization of fipv by antibodies (pedersen and boyle, 1980; weiss and scott, 1981a, c; weiss, 1994) . this is corroborated by our finding that b-cells differentiating into plasma-cells which in part produce coronavirus-specific antibodies seem to play an important role in the maintenance of inflammatory processes in fip. the factors inducing b-cell accumulation and maturation of at least single b-cells into plasma-cells producing coronavirus-specific antibodies are not yet clearly defined. however, cultured peritoneal exudate cells (activated mesothelial cells, macrophages) from cats with fip have been shown to release high amounts of il-6 into the supernatant (goitsuka et al., 1990) . fipv-infected alveolar macrophages and peritoneal exudate cells are known to produce il-1 in situ (goitsuka et al., 1987 (goitsuka et al., , 1988 hasegawa and hasegawa, 1991) . il-1 and il-6 function as regulators of b-cell growth and differentiation and might therefore be responsible for maturation and immunoglobulin production of activated bcells and plasma-cells in fip lesions (kishimoto, 1989; goitsuka et al., 1987 goitsuka et al., , 1988 goitsuka et al., , 1990 hasegawa and hasegawa, 1991) . primary stage of feline immunodeficiency virus infection: viral dissemination and cellular targets association of feline leukemia virus with lymphosarcoma and other disorders in the cat purification and partial characterization of highly immunogenic human leukocyte protein, the l1 antigen monoclonal antibodies directed towards the two major cell populations in the bursa of fabricius of the chicken perspectives on the epizootiology of feline enteric coronavirus and the pathogenesis of feline infectious peritonitis identification of tissue histiocytes on paraffin sections by a new monoclonal antibody release of interleukin-1 from peritoneal exudate cells of cats with feline infectious peritonitis feline interleukin-1 production induced by feline infectious peritonitis virus il-6 activity in feline infectious peritonitis apoptosis and t-cell depletion during feline infectious peritonitis interleukin-1 alpha mrna-expressing cells on the local inflammatory response in feline infectious peritonitis systemic vascular lesions in feline infectious peritonitis detection of coronavirus-like particles in a spontaneous case of feline infectious peritonitis pathology of non-effusive type feline infectious peritonitis and experimental transmission use of avidin±biotin-peroxidase complex (abc) in immunoperoxidase techniques expression of feline infectious peritonitis coronavirus antigens on the surface of macrophage-like cells the biology of interleukin-6 immunohistochemical diagnosis of feline leukemia virus infection in formalin-fixed tissue extraperitoneal lesions in feline infectious peritonitis identification of monoclonal antibodies for immunohistochemical staining of feline b-lymphocytes in frozen and formalin-fixed paraffin-embedded tissues cytochemical demonstration of esterases in peripheral blood leukocytes immunologic phenomena in the effusive form of feline infectious peritonitis virologic and immunologic aspects of feline infectious peritonitis virus infection diseases associated with spontaneous feline leukemia virus (felv) infection in cats the unlabeled antibody-enzyme method for immunohistochemistry. preparation and properties of soluble antigen±antibody-complex (horseradish peroxidase±antihorseradish peroxidase) and its use in identification of spirochetes the sites of early viral replication in feline infectious peritonitis immunohistological demonstration of feline infectious peritonitis virus antigen in paraffin-embedded tissues using feline ascites or murine monoclonal antibodies feline infectious peritonitis: experimental evidence for its multiphasic nature pathogenesis of feline infectious peritonitis: nature and development of viremia pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever feline infectious peritonitis and other coronaviruses feline infectious peritonitis ultrastructural evidence for the viral etiology of feline infectious peritonitis the authors wish to thank mrs. r. bo èrner and mrs. p. herzog for technical assistance and mr. r. dobroschke for photographical assistance. we are grateful to dr. hermann nieper, institut fu èr virologie, universita èt leipzig, leipzig, germany, for kindly providing the feline coronavirus and infectious bursitis disease virus culture supernatants. key: cord-308537-i6um5iu2 authors: hoskins, johnny d. title: coronavirus infection in cats date: 1993-01-31 journal: veterinary clinics of north america: small animal practice doi: 10.1016/s0195-5616(93)50001-3 sha: doc_id: 308537 cord_uid: i6um5iu2 cats are susceptible to natural infection with several strains of feline coronavirus that result in either effusive and noneffusive feline infectious peritonitis or enteritis. excretion of coronavirus by infected cats into the environment occurs by way of feces, oronasal secretions, and possibly urine. clinical diagnosis of coronavirus infection is made by evaluating the case history, physical findings, laboratory results, and coronavirus antibody titers as well as ruling out analogous diseases. an intranasal temperature-sensitive feline infectious peritonitis coronavirus vaccine is available for use in healthy cats 16 weeks of age or older. in many species of animals, coronaviruses have a relatively restricted organ tropism. they infect the respiratory or gastrointestinal system, or both. 41 fip coronavirus, feline enteric coronavirus, canine coronavirus (ccv), transmissible gastroenteritis virus (tgev) of swine, and human respiratory tract coronavirus comprise an antigenic cluster of closely related viruses within the family coronaviridae. 34 the major structural proteins of these coronaviruses are antigenically similar to the extent that most investigators consider these coronaviruses as host-range variants rather than individual corona virus species. 5 strains of feline coronavirus that infect cats have been divided into those that cause fip and those that induce subclinical to severe enteritis (table 1) . 30 strains of fip coronavirus differ from those of feline enteric coronavirus in that fip coronavirus is able to escape from the gastrointestinal tract and spread to replication sites in other organs. strains of fip coronavirus and feline enteric coronavirus probably represent pathogenic variants of a single coronavirus type.' alternatively, strains of fip coronavirus may arise as mutants of feline enteric coronavirus strains. 33 current knowledge of the cell culture-adapted strains of feline coronavirus is based on their morphologic, structural, and antigenic relationship to tgev and ccv; the nature of the disease caused in the host; growth characteristics in cell culture systems; and the degree of relatedness to ccv in virus neutralization tests. 30 the most virulent strains of fip coronavirus, such as fipv-79-1146 and fipv-df2, cause fatal fip disease in most cats inoculated oronasally, whereas strains of intermediate virulence, such as fipv-ucdl, cause fip disease in cats undergoing extended exposure. strains of low virulence, such as fipv-ucd2 and fipv-ucd3, can become established as asymptomatic infections; fatal fip disease occurs only if the immunologic responsiveness of the host later becomes compromised. 33 most cases of fip disease result from infection with strains similar to fipv-tn406 and not with those strains of fip coronavirus that are closely related to ccv. 33 most asymptomatic cats with positive coronavirus-antibody titers have been previously infected by strains of feline enteric coronavirus or fip coronavirus, which usually do not cause fatal disease by natural routes of infection. asymptomatic cats, however, may later succumb if exposed to a more virulent strain of fip coronavirus. coronavirus antibody-positive cats that resist later challenge probably seroconvert as a result of a systemic infection with a virulent strain of fip corona virus. 25 various strains of fip coronavirus evidently survive in the environment much longer than originally was believed. in recent studies/ 6 fip coronavirus was recovered from contaminated dry surfaces for 3 to 7 weeks. the amount of infectious virus recovered decreases with time. because relatively large quantities of fip coronavirus are required to establish natural infection in a susceptible cat, strains of coronavirus probably are not contagious to cats for longer than 2 or 3 weeks after an environment, bedding, food bowl, or water bowl is contaminated. 36 infectivity of feline coronavirus strains is destroyed by most household disinfectants and detergents. 11 feline enteric coronaviruses, such as strains of fecv-79-1683 and fecv-ucd, are highly infectious by the oral route to all cats of any age and have an affinity for the apical columnar epithelia of the duodenum, jejunum, and ileum. 27 adult cats often endure subclinical infections; low-grade fever, vomiting, and diarrhea may be observed in newly weaned kittens. natural infection with strains of feline enteric coronavirus results in production of antibodies that presently are serologically indistinguishable from the antibodies produced by infection with fip coronavirus, ccv, or tgev. most cats infected with a strain of feline enteric coronavirus remain persistently infected and periodically shed coronavirus in their feces. 25 coronavirus antibodies do not, however, protect most cats from later exposure with virulent strains of fip coronavirus. these antibodies may actually sensitize cats to later exposures, thereby hastening the disease process caused by a virulent strain of fip coronavirusy investigators remain unsure of the routes of natural transmission by which feline coronaviruses are passed between cats. excretion of coronavirus by infected cats into the environment occurs by way of feces, oronasal secretions, and, possibly, urine. 25 initial infection of susceptible cats most likely results from ingestion and/or inhalation of the corona virus. 11 close contact between cats is probably required for the most effective transmission of coronavirus, although the possibility of virus transmission by fomites (contaminated clothing, bedding, and dishware) also exists. transmission of fip coronavirus in utero, as suggested by several reports/ 3 • 24 has not been convincingly shown to occur. domestic cat and other felidae, including lions, mountain lions, leopards, jaguars, cheetahs, lynxes, sand cats, and pallas cats, are susceptible to infection with fip corona virus. 14 • 28 in domestic cats, fip disease occurs predominantly in young animals, although all ages are susceptible. a higher incidence occurs between 6 and 12 months of age, whereas a lower incidence is noted from 5 to 13 years of age, followed by an increased incidence in cats 14 older. 26 male and female cats are. affected equally. fip occurs more frequently in purebred cats than in other breeds. after natural infection, localization and replication of fip coronavirus occur in large mononuclear cells of regional lymphoid tissue at or near the site of initial virus penetration (fig. 1 ). 43 • 44 a primary viremia results with free virus and virus-infected mononuclear cells being transported to other body organs, especially to the liver, spleen, and lymph nodes. these organs are subsequently involved because they contain large populations of macrophages, which are the principal target cells for fip corona virus infection. 43 a secondary macrophage-associated viremia then occurs, resulting in further spread of fip coronavirus throughout the body! the susceptibility of cats to fip disease may involve several predisposing factors, including age at time of exposure, genetic susceptibility, physical condition, stress, presence of concurrent disease (especially feline leukemia virus and feline immunodeficiency virus infections), challenge dose and strain of feline coronavirus, route of infection, previous sensitization with nonprotective corona virus antibodies, and cell-mediated immunocompetence. 2 • 4 if an effective cell-mediated immune response is exhibited during the primary phase of coronavirus infection, viremia will probably be terminated, thereby protecting the cat against fip disease. 28 most cats subsequently do not show clinical evidence of fip coronavirus infection. some cats, however, may develop a transient fever that lasts for 1 to 5 days and mild mesenteric iymphadenopathy. 30 if the infected cat is unable to mount an effective cellmediated immune response and produces coronavirus antibodies that do not neutralize the virus, infection progresses rapidly into a fulminating form of fip disease. a similar situation may occur if the cat has already acquired coronavirus antibodies through previous infection with a strain of feline enteric coronavirus or fip coronavirus of low virulence. 6 in the absence of an effective cell-mediated immune response, a strong nonprotective humoral response may result in effusive form of fip disease. cats that exhibit a partial cell-mediated immune response may develop noneffusive fip disease. u an effective cell-mediated immune response does not always eliminate fip coronavirus from the body. in some recovered cats, the infection may persist in various places in the body, such as the gastrointestinal tract or associated lymph nodes. 30 factors that depress cell-mediated immune responsiveness, such as feline leukemia virus and feline immunodeficiency virus infections, concurrent disease, and advanced age, may allow recrudescence of macrophageassociated infection with periodic shedding of fip corona virus, thereby resulting in effusive or noneffusive disease. 2 • 4 an exaggerated and nonprotective humoral response results in excessive levels of coronavirus antibodies and the formation of large immune complexes that are rapidly phagocytized by reticuloendothelial cells. 44 immune complexes deposited in small blood vessels fix and activate complement, thereby resulting in the release of the third component of complement. phagocytosis of aggregates containing coronavirus, immunoglobulin, and complement is aided by the presence of receptors for immunoglobulin and complement on the macrophage surface. 43 macrophages that are in perivascular locations ingest aggregates of intact fip coronavirus, immunoglobulin, and complement and thus encourage replication of the virus and the release of new virus and complement components. 20 uptake and processing of fip coronavirus in these macrophages may be enhanced by an impaired cell-mediated immune response. 5 recurrent complement-mediated damage results in the release of chemotactic complement components and the attraction of neutrophils. release of proteolytic enzymes from degenerated neutrophils exacerbates tissue damage. 5 in cases of fip disease, degenerative and proliferative changes occur in blood vessels, particularly in the endothelial and medial layers of small veins and arteries in the peritoneal serosa and in the interstitial connective tissue of parenchymatous organs. 18 the vascular lesion results in a pyogranulomatous lesion. 5 in effusive disease, complement-mediated damage to the vascular endothelium results in increased vascular permeability and leakage of a nonseptic exudate rich in fibrin and immunoglobulin. 5 severe damage to the vascular endothelium also contributes to disseminated intravascular coagulation. the development of multiple clotting abnormalities, including thrombocytopenia, increased quantity of fibrin-fibrinogen degradation products, and decreased activity of clotting factors vii, viii, ix, xi, and xii, is evident in fatal cases of fip disease. 42 although most cats undergoing the primary phase of fip coronavirus infection recover, many cats remain persistently infected, that is, they are persistent carriers of fip corona virus. a small number of these cats subsequently develop the fatal fip disease weeks to months after the primary phase of infection. 36 in the early phase of fip disease, cats with the effusive form may be presented with nonspecific signs, such as nonresponsive fever, anorexia, lethargy, weight loss, and pale mucous membranes. icterus may be seen in patients with severe liver involvement. 3 • 4 recurring episodes of diarrhea and constipation may be observed. progressive abdominal distention occurs from an accumulation of ascitic fluid in the peritoneal cavity. the volume of fluid present in cases of effusive disease varies and is generally a reflection of disease chronicity. 2 • 4 in chronic cases, a liter or more of fluid may accumulate within the peritoneal cavity. abdominal palpation usually elicits no response of pain. in some cats, the omentum may be palpated as a firm, contracted mass in the anteroventral abdomen. 2 • 4 pleural effusion with clinical signs of respiratory distress infrequently occurs in cats with effusive fip disease. these cats are presented with decreased exercise tolerance, dyspnea, and muffled heart and lung sounds. pericardia! effusion may be present. 38 ocular and central nervous system signs are seldom seen in cats with effusive disease. 2 • 4 cats that recover from effusive disease usually undergo a period of time with noneffusive disease before they show clinical evidence of complete recovery. 25 clinical diagnosis of the noneffusive fip disease usually is impeded by a lack of specific signs. onset of noneffusive disease is more insidious than is onset of the effusive form and is frequently associated with organ-specific signs resulting from disseminated pyogranulomatous lesions in various organs. nonspecific signs of weight loss, nonresponsive fever, and malaise may occur for several weeks before any organ-specific manifestations are present. 2 • 4 signs of renal or hepatic insufficiency and pancreatic, central nervous system, or ocular disease may be seen in cats with severe organ impairment! abdominal palpation may reveal mesenteric lymphadenopathy and nodular irregularities that are caused by surface-oriented pyogranulomata on various viscera, especially the kidneys. 2 • 4 pneumonia with clinical signs of respiratory distress infrequently occurs. when present, pulmonary lesions consist of mixed inflammatory cell infiltrates in peribronchiolar areas that may radiographically appear as ill-defined, patchy, interstitial, and peribronchiolar densities. 38 ocular involvement usually is associated with other clinical signs of noneffusive disease, but may be present as a single manifestation. ocular lesions result from a necrotizing and pyogranulomatous uveitis that localizes around vascular structures. 2 • 4 changes in the anterior chamber include corneal edema, aqueous flare, hypotonia, iritis, hyphema, hypopyon, and keratic precipitates. on ophthalmoscopic examination, flame-shaped or boat-shaped hemorrhages may be present. engorgement of retinal vessels and perivascular cuffing is often noted. choroidal inflammation may cause subretinal fluid accumulation and secondary bullous or linear retinal detachments. 21 neurologic signs in cats with noneffusive fip disease are variable and may include incoordination, posterior paresis, nystagmus, convulsions, intention tremors, cranial and peripheral nerve deficits, hyperesthesia, generalized ataxia, head tilt, behavioral changes, and urinary incontinence.n signs reflecting central nervous system and ocular involvement frequently occur together in noneffusive disease. lesions of central nervous system are usually multifocal or diffuse pyogranulomas and are located around the smaller blood vessels. the lesions are surface-oriented and primarily affect the choroid plexus, meninges, and ependyma. 2 • 4 in cases of noneffusive disease, many patients that do not show neurologic involvement have histopathologic evidence of central nervous system involvement.> 2 hemograms of cats with fip disease often show a mild to moderate normocytic, normochromic anemia and leukocytosis that may be associated with absolute neutrophilia. absolute leukopenia may develop in some cats, especially in more fulminating or fatal cases. lymphopenia is commonly observed and is profound in cats with concurrent feline leukemia virus infection . 39 on serum chemistry profiles, elevations in blood urea nitrogen and serum creatinine may be present in cats with either effusive or noneffusive fip and may reflect dehydration and inflammatory lesions in the kidneys. elevations in serum alanine transaminase and serum alkaline phosphatase as well as hyperbilirubinemia may occur if the inflammatory process involves the liver. 2 • 4 total serum proteins exceed 7.8 g/dl in 55% of cats with effusive disease and in 75% of cats with noneffusive disease because of varied increases in alpha-2, beta-2, and gamma globulins. this polyclonal gammopathy is not pathognomonic for fip disease but reflects its inflammatory nature. 25 hyperglobulinemia also may occur in other inflammatory conditions that are associated with persistent antigenic stimulation of antibody-producing cells. aspirated peritoneal fluid typically is clear to slightly opaque or pale yellow to golden as well as being viscous in consistency. a stable foam often develops after shaking, presumably reflecting a protein content in excess of 5 g/dl. 2 • 4 fluid specimens may contain fibrin strands and flakes that settle with time and may clot when exposed to room air. cellular count of fluid usually is between 1600 to 25,000 cells/ill. stained smears from a concentrated fluid sample commonly show a mixture of intact neutrophils, macrophages, plasma cells, lymphocytes, mesothelial cells, and a few erythrocytes. 9 cultures of aspirated fluid for fungi, bacteria, and mycoplasmas usually reveal no growth. clinical diagnosis of pip disease is made by evaluating the presenting history, physical findings, laboratory results, coronavirus antibody titers, and exclusion of analogous diseases. 2 • • these diagnostic maneuvers, however, do not provide conclusive evidence that a cat has pip disease, especially in cases of noneffusive disease in which accumulative abdominal fluid is not available for examination. 9 tissue biopsy is the only diagnostic procedure that definitively confirms the presence of pip disease in apparently healthy cats or sick cats with either effusive or noneffusive disease. 2 • 9 any diagnosis of pip disease made without tissue biopsy or eventual necropsy examination must be considered, at best, to be a presumptive diagnosis. several serologic test procedures are available for detecting coronavirus antibodies in cats, including biologic assays, such as virus neutralization, and nonbiologic assays, such as indirect immunofluorescent antibody test, enzymelinked immunosorbent assay, kinetics-based enzyme-linked immunosorbent assay, agar gel immunodiffusion, and passive hemagglutination techniques. 9 the target antigen used in these assays may be either a pip coronavirus (in liver sections of experimentally infected cats or in cell culture) or another coronavirus in the pip coronavirus antigenic cluster, usually tgev or ccvy coronavirus antibodies may be found in serum of apparently healthy cats, in cats with disorders other than effusive or noneffusive disease, and in cats with pip disease. excluding cats in catteries and in multiple-cat households, 10% to 40% of cats in the general feline population have coronavirus antibodies in their serum. if cats are housed together, the rate of seropositivity is completely absent or present in 80% to 90% of the cats, depending on whether feline corona virus is enzootic in that cat population. 5 there are several situations in which assay results for the detection of coronavirus antibody are potentially helpful to the clinician and the cat owner. serotesting for feline coronavirus may be used as a screening procedure to determine if coronavirus is enzootic in a cat population and as an adjunct in the diagnosis of pip disease. 5 most cats with histopathologically confirmed pip disease have high coronavirus-antibody titers. 5 titers greater than 1:3200 usually are associated with noneffusive disease, whereas titers of 1:100 to 1:3200 may be found in cats with effusive and noneffusive disease and in cats with feline enteric coronavirus infection. 25 in some cats with pip disease, negative titers or a decrease in titers may be seen terminally; this is a grave prognostic sign. in addition to correlating the positive coronavirus-antibody titer with other diagnostic information on the patient, the clinician should be aware that differences in assay results can be found between diagnostic laboratories and in the clinician's own interpretation of assay results. 2 a few cats with histopathologically confirmed pip disease may have negative coronavirus-antibody titers. 5 reasons for negative titers include disappearance of coronavirus antibodies in the terminal stages of pip disease, formation of immune complexes that leave little if any free coronavirus antibodies to react in the assay procedure, use of assay systems that are not sensitive enough to detect low levels of coronavirus antibodies, and presence of small amounts of coronavirus antibodies in fulminating cases of effusive diseasey in addition, bovine serum components found in some commercial feline vaccines may induce antibody production in cats vaccinated with those prod-ucts. these antibodies may react with antigenically similar bovine serum components in cell cultures that are used to propagate target viruses of the fip coronavirus antigenic cluster for immunochemical assays. resultant reactivity may be mistaken for coronavirus antibodies in serum of a recently vaccinated cat. because of this potential interference, it is recommended that elective serotesting of healthy cats for evidence of exposure to feline coronavirus should be delayed until at least 3 to 4 months after the last parenteral vaccine was given. 5 at the time of this writing, available serologic assays for detecting coronavirus antibodies do not identify the strain of coronavirus that is responsible for the eat's seroconversion; also, the presence of coronavirus-antibody titers merely indicates that the cat has been infected with a coronavirus in the coronavirus antigenic cluster. 5 • 36 most healthy cats with positive coronavirusantibody titers have probably been infected with strains of feline enteric coronavirus, with non-fip disease-producing strains of fip coronavirus, or with fip disease-producing strains. most healthy coronavirus antibody-positive cats are probably not immune carriers of highly virulent fip coronavirus. 33 it is impossible to predict accurately the long-term prognosis of healthy coronavirus antibody-positive cat. positive antibody titers do not indicate that a cat is protected against future development of fip disease, will later develop fip disease, or is definitely a hazard to other cats. 2 because effective treatment is unavailable for complet~ elimination of coronavirus infection, long-term prognosis for cats definitively diagnosed with fip disease is extremely poor. antiviral drugs are not available for effective treatment of affected cats, although research is ongoing to evaluate various antiviral compounds. most treatment regimens provide, at best, only shortterm remission. palliative therapy is effective for cats with fip disease that are in good physical condition, have a good appetite, and are free of severe anemia or neurologic deficits; 9 however, few cats with fip disease conform to these standards or are presented early enough in the course of disease to be given meaningful palliative therapy. effective drug therapy for fip disease relies on the use of systemic corticosteroids (such as oral prednisolone, 2 to 4 mg/kg daily) to decrease the disseminated corona virus antibody-mediated vasculitis. 9 although corticosteroids may provide short-term remission, long-term use is not curative in cats with disease resulting from defective cell-mediated immune response. the general well-being of cats that receive systemic corticosteroids should be monitored weekly to monthly. if the cat shows a favorable response to therapy during the first few weeks, treatment should be continued for at least 3 months. 9 if the cat is in complete remission at the end of 3 months, the corticosteroids may be slowly withdrawn. treatment should be reinstated, however, if signs of the disease recur. progressive physical deterioration of the cat during treatment is a poor prognostic sign. 9 cats that develop pyogranulomatous lesions that only affect the eyes, especially unilateral or bilateral uveitis, respond relatively well to topical and/ or systemic corticosteroid therapy 37 along with subconjunctival injection of methylprednisolone or triamcinolone acetonide (figure 2 ). if ocular inflammation is severe and sight has been lost, enucleation may be indicated. initial incidence of new cases of fip disease in catteries and multicat households may be reduced by promptly isolating all cats with signs associated with fip disease, removing all cats infected with feline leukemia virus or feline immunodeficiency virus infection, and reducing overcrowding as well as by improving hygiene and nutrition, selecting queens that have good mothering instincts and are able to raise healthy litters, and only admitting cats that have negative corona virus-antibody titers. 25 using coronavirus-antibody serotesting as part of a test-and-removal program to control fip disease (similar to successful programs used to reduce incidence of feline leukemia virus and feline immunodeficiency virus infections) cannot be recommended. serologic assays used in detecting coronavirus antibodies in cats have poor specificity compared with the high accuracy of enzymelinked immunosorbent assay and indirect fluorescent antibody testing procedures used to detect feline leukemia virus p27 antigen or levels of feline immunodeficiency virus antibodies. removal of healthy coronavirus antibodypositive cats from catteries or multicat household is justified only if strong evidence that the cat is a source of fip coronavirus infection for other cats exists. 3 a first-generation temperature-sensitive fip coronavirus (ts-fipv) vaccine that protects cat against fip coronavirus challenge is available (primucell-fip, smithkline beecham animal health, exton, p a) . the ts-fipv vaccine contains attenuated live virus and is derived by the following laboratory procedure. the original wild-type strain of fip coronavirus, known as fipv-df2 isolate, is attenuated by 99 passages in norden laboratories feline kidney (nlfk) cell line, of which passages 61 to 99 are propagated at 31°c. the 99th passage of fipv-df2 is made temperature-sensitive by exposure to ultraviolet irradiation. the ts-fipv is again propagated on nlfk cells for eight more passages and then lyophilized for subsequent vaccine·use. 16 the attenuated ts-fipv strain replicates primarily in the upper respiratory tract and associated regional lymph nodes (mandibular, medial retropharyngeal, and cervical) of cats (figs. 3 and 4) . the ts-fipv proteins stimulate local immunoglobulin a and a cell-mediated immune response, as well as a systemic cell-mediated immune response that may deter systemic dissemination of the fip corona virus. 12 • 16 • 17 the manufacturer (smithkiine beecham animal health) recommends administering the primucell-fip vaccine intranasally to healthy cats 16 weeks of age or older. initial vaccination is given with two doses 3 to 4 weeks apart. annual revaccination with a single dose is recommended. vaccinated pregnant cats, dexamethasone-suppressed cats, feline leukemia virus-infected cats, and feline enteric coronavirus-infected cats have not shown a febrile response or blood dyscrasias. 15 various feline enteric coronavirus strains produce clinical signs that resemble those produced by tgev in swine and by ccv in puppies. 25 feline enteric coronavirus infection is most severe in recently weaned kittens; most infected adult cats, however, remain apparently healthy.>' clinical signs in kittens include low-grade fever, intermittent vomiting, depression, and mild to moderately severe diarrhea that lasts for 2 to 5 days. kittens with severe disease may be anorectic for 1 to 3 days. 32 in kittens with more severe infection, a transient neutropenia may accompany the onset of diarrhea. 25 the most severe lesions, such as villous atrophy, fusion of adjacent villi, and sloughing of the mature columnar epithelium from the upper portion of the villi, occur in the mature columnar epithelia of the duodenum, jejunum, and ileum. 27 • 32 mortality is negligible, and nearly all affected kittens recover. 25 diagnosis a definitive diagnosis of feline enteric coronavirus infection is difficult to obtain. clinical resemblance of feline enteric coronavirus infection to other enteritides can confound diagnosis." electron microscopy of stool specimens to search for coronavirus particles is expensive, time-consuming, and fraught with false-negative and false-positive results. furthermore, true-positive results provide only presumptive evidence of the cause of diarrhea. 8 coronavirus antibody assays do not provide the information for a definitive diagnosis of feline enteric coronavirus infection. 25 • 27 histopathologic lesions also are not sufficiently specific for the diagnosis, and viral isolation is impractical for routine use. 8 supportive care is the only treatment option for feline enteric coronavirus infection in kittens. 25 -27 food and water should be withheld during the more severe phases of infection, and a balanced electrolyte solution be given parenterally if there is moderate to severe dehydration. antimicrobial therapy usually is not required . in a study at louisiana state university, primucell-fip vaccine was administered to 9-week-old, specific-pathogen-free kittens according to the manufacturer's instructions. a similar group of nonvaccinated animals served as controls. all of the kittens were oronasally challenged with virulent feline enteric coronavirus 2 weeks after final vaccination. the extent of clinical signs caused by feline enteric coronavirus infection in the vaccinated and nonvaccinated kittens is shown in figure 5 . the vaccinated kittens experienced milder enteric disease after challenge exposure than did age-matched nonvaccinated kittens. the febrile response on days 1 and 3 postchallenge were significantly lower in the vaccinated kittens (p < 0.05). there was also a significant reduction (p < 0.05) in the amount of feline enteric coronavirus isolated from the jejunum, duodenum, and mesenteric lymph nodes of vaccinated kittens. morphometric analysis of the duodenum and the jejunum indicated that villus height was less affected in the vaccinated kittens than in nonvaccinated kittens ( figure 6 ). there was a significant difference (p < 0.05) in villus height in the jejunum but not in the . morphometric results of the small intestines from ten nonvaccinated kittens and ten kittens vaccinated twice intranasally with ts·fipv vaccine following feline enteric coronavirus challenge. five kittens served as age-matched (nonvaccinated-nonchallenged) controls. duodenum of the vaccinated group. in addition, the nonvaccinated kittens lost significantly more weight than the vaccinated kittens. the results of this study indicated that the ts-fipv vaccine partially protected the kittens against disease caused by feline enteric coronavirus infection. various feline enteric coronavirus strains are very common in the general cat population. the virus is found in virtually every cattery and multicat household and in approximately 25% of all outdoor cats. 25 thus, it is nearly impossible to prevent kittens and adult cats from being exposed to feline enteric coronavirus. nature limits the severity of feline enteric coronavirus infection by providing young kittens with maternal antibody protection until 6 to 12 weeks of age. 25 cats are susceptible to natural infection with several strains of feline coronavirus that may result in either effusive and noneffusive fip disease or in subclinical to severe enteritis. investigators are still unsure of the routes by which strains of coronavirus are transmitted between cats. excretion of coronavirus by infected cats into the environment occurs by way of feces, oronasal secretions, and, possibly, urine. fip coronavirus remains stable outside the host for as long as 3 to 7 weeks and is rapidly inactivated by most household disinfectants. clinical diagnosis of coronavirus infection is made by evaluating the presenting history, physical findings, laboratory results, coronavirus antibody titers, and by excluding analogous diseases. the presence of coronavirus antibodies can be used to screen cats for the presence of coronavirus infection and as an adjunct in diagnosing clinical coronavirus infection. a intranasal ts-fipv vaccine that protects against natural coronavirus challenge is available for healthy cats 16 weeks of age or older. a study of naturally occurring feline coronavirus infections in kittens feline viral diseases preventive health care and infectious disease control feline infectious peritonitis: an immune-mediated coronaviral vasculitis barlough je: cats, coronaviruses and coronavirus antibody tests do feline coronavirus antibody tests provide a conclusive diagnosis serodiagnosis aids and management practices for feline retrovirus and coronavirus infections infectious diseases of the dog and cat feline infectious peritonitis experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus feline infectious peritonitis characterization of a temperature sensitive feline infectious peritonitis coronavirus cdna cloning and sequence analysis of the gene encoding the peplomer protein of feline infectious peritonitis virus diagnostic features of an epizootic of feline infectious peritonitis in captive cheetahs new approaches to feline infectious peritonitis prevention in feline infectious peritonitis: current status. lawrenceville, nj, veterinary learning systems protection against feline infectious peritonitis by intranasal inoculation of a temperature sensitive-fipv vaccine characterization of an attenuated temperature sensitive feline infectious peritonitis vaccine virus systemic vascular lesions in feline infectious peritonitis the virology and pathogenesis of feline infectious peritonitis. brief review antibody immune complexes and complement activity fluctuations in kittens with experimentally induced feline infectious peritonitis intraocular inflammation in cats as a manifestation of systemic diseases feline infectious peritonitis. the central nervous system form epigenetic transmission of feline infectious peritonitis feline infectious peritonitis coronavirus diseases (coronavirus enteritis, feline infectious peritonitis) virologic and immunologic aspects of feline infectious peritonitis virus infection feline infectious peritonitis and feline enteric coronavirus infections. part i. feline enteric coronaviruses feline infectious peritonitis and feline enteric coronavirus infections. part ii. feline infectious peritonitis attempted immunization of cats against feline infectious peritonitis pathogenic differences between various coronavirus isolates immunologic phenomena in the effusive form of feline infectious peritonitis an enteric coronavirus of cats and its relationship to feline infectious peritonitis experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd2, fipv-ucd3, and fipv-ucd4. compend contin educ pract vet antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species coronaviruses: structure and genome expression update on fip the real and unreal feline coronaviruses feline infectious peritonitis the veterinary annual, issue 26 early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization the biology and pathogenesis of coronaviruses disseminated intravascular coagulation in experimentally induced feline infectious peritonitis pathogenesis of feline infectious peritonitis . nature and development of viremia pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence cross protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis virus key: cord-315094-pzixgqcy authors: benetka, viviane; kübber-heiss, anna; kolodziejek, jolanta; nowotny, norbert; hofmann-parisot, margarete; möstl, karin title: prevalence of feline coronavirus types i and ii in cats with histopathologically verified feline infectious peritonitis date: 2004-03-26 journal: vet microbiol doi: 10.1016/j.vetmic.2003.07.010 sha: doc_id: 315094 cord_uid: pzixgqcy feline coronaviruses (fcov) vary widely in virulence causing a spectrum of clinical manifestations reaching from subclinical course to fatal feline infectious peritonitis (fip). independent of virulence variations they are separated into two different types, type i, the original fcov, and type ii, which is closely related to canine coronavirus (ccv). the prevalence of fcov types in austrian cat populations without fip has been surveyed recently indicating that type i infections predominate. the distribution of fcov types in cats, which had succumbed to fip, however, was fairly unknown. pcr assays have been developed amplifying parts of the spike protein gene. type-specific primer pairs were designed, generating pcr products of different sizes. a total of 94 organ pools of cats with histopathologically verified fip was tested. a clear differentiation was achieved in 74 cats, 86% of them were type i positive, 7% type ii positive, and 7% were positive for both types. these findings demonstrate that in fip cases fcov type i predominates, too, nonetheless, in 14% of the cases fcov type ii was detected, suggesting its causative involvement in cases of fip. feline infectious peritonitis (fip) is a fatal, immune-mediated disease of domestic and wild fe-et al., 1982; wege et al., 1982) . barlough et al. (1985) showed that an infection with ccv caused seroconversion but no clinical signs in the cats examined, neither was the course of the subsequently experimentally induced fip disease more severe. in contrast to these findings, mcardle et al. (1992) demonstrated that after infection with ccv the course of fip disease was more severe, and that ccv induced in some cats similar symptoms as in the dog. furthermore, one ccv strain caused in a cat clinical symptoms which were not discernible from fip. after all, the importance of ccv for the cat remains uncertain (sparkes et al., 1992) . two biological types of fcovs are known, feline infectious peritonitis virus (fipv) and feline enteric coronavirus (fecv) (pedersen, 1976b (pedersen, , 1983 (pedersen, , 1987 pedersen et al., 1981) . the genome of some fecv strains proved to be 0.3 kb shorter, suggesting a deletion of 300 bp at the 3 -end (vennema et al., 1992) . molecular studies showed that fipv arises by mutation from fecv (pedersen et al., 1981; evermann et al., 1991; hök, 1993; poland et al., 1996; herrewegh et al., 1997; vennema et al., 1994 vennema et al., , 1998 . both fipv and fecv may, depending on their virulence, cause viremia (herrewegh et al., 1995; fehr et al., 1996; gunn-moore et al., 1998; horzinek, 2000) . fcovs are separated into two different types based upon their growth ability in vitro, their antigenic relationship to ccv, their neutralisation reactivity with sprotein-specific mabs (fiscus and teramoto, 1987a,b; hohdatsu et al., 1991 hohdatsu et al., , 1992 and upon sequence analysis of the s-protein gene (motokawa et al., 1995) . while type i shows no or little replication in cell culture (fipv ucd1, ucd2, ucd3, ucd4, tn-406, nw1, yayoi, ku-2, dahlberg, fecv ucd), type ii induces a lytic cytopathic effect (fipv 79-1146, nor15 (df2) , cornell-1, fecv 79-1683) . the ability of an fcov strain to propagate in cell culture does not correlate with its virulence in vivo (mochizuki et al., 1997) . among the fcov types i and ii, both fipv and fecv strains are represented. the s-protein gene of type ii is closely related to those of tgev and ccv, showing a similarity of the nucleotide sequence of 91 and 81%, respectively, but of only 46% with the s-protein gene of type i (motokawa et al., 1995) . herrewegh et al. (1998) demonstrated that fcov type ii resulted from recombination of fcov type i with ccv. recent studies indicate that type ii uses the feline aminopeptidase n (fapn), a cell-surface metalloprotease on the intestinal, lung and kidney epithelial cells, as receptor, and that fapn may also bind ccv, tgev and hcv. it is not clear whether or not this receptor specificity of type ii plays a role in the pathogenesis or pathological alterations of fip (williams et al., 1991; de groot and horzinek, 1995; hohdatsu et al., 1998; tresnan and holmes, 1998) . the prevalence of types i and ii has been surveyed in two studies from austria and japan, respectively, both suggesting that the majority of fcov infections is due to type i (hohdatsu et al., 1992; posch et al., 1999 posch et al., , 2001 . fcovs are ubiquitous in the cat population, highly infectious by the oronasal route and therefore endemic in multi-cat households, catteries and shelters. investigations showed that a high percentage of cats without fip symptoms from exposed environments were positive for fcov infection: 39-85% were seropositive, 37-95% viremic and 73-81% excreted virus in their faeces (addie and jarrett, 1992b; sparkes et al., 1992; herrewegh et al., 1995; foley et al., 1997a,b; gunn-moore et al., 1998) . posch et al. (1999 posch et al. ( , 2001 found 71% seropositive cats in austrian cat populations without signs of fip, 26% of these cats tested positive for fcov nucleic acid in blood. there is strong evidence for the existence of persisting and chronic infections, with virus persisting in the intestine and other organs of healthy cats. asymptomatic carriers may excrete virus over a period of months or even years (foley et al., 1997a; herrewegh et al., 1995 herrewegh et al., , 1997 . asymptomatic carriers and shedders represent coronavirus reservoirs and therefore the main problem in the prevention of fip in multi-cat environments (addie and jarrett, 1992a,b; addie et al., 1995 addie et al., , 1996 foley et al., 1997a,b; herrewegh et al., 1997) . approximately 5-10% of seropositive cats develop fip, with the highest incidence in cats between 6 months and 5 years of age, and the majority of cases occurring in cats ≤1 year of age (scott, 1991; addie and jarrett, 1992a) . the higher incidence of fip among purebred cats (scott, 1991) , cheetahs (evermann et al., 1988) and cats from fip-susceptible bloodlines may be an indication for a genetic predisposition. in addition, the sex of the host may influence the outbreak of the disease. while pedersen (1976a) found no generic disposition, potkay et al. (1974) and binder and hartmann (2000) observed a higher incidence of fip among males than among females. although serological testing by immunofluorescence assay (ifa) (moestl, 1983) or elisa (mochizuki and furukawa, 1989 ) is a helpful tool for fip diagnosis, results can only be interpreted in correlation with clinical symptoms (sparkes et al., 1991) . at present, the only conclusive fip diagnosis can be established by histopathological examination of a biopsy or post mortem material. the recently developed reverse transcriptase polymerase chain reaction (rt-pcr) assays, using primers targeted to highly conserved regions of the viral genome (3 -utr (untranslated region) (herrewegh et al., 1995; fehr et al., 1996) , or s-protein gene (li and scott, 1994; gamble et al., 1997) ), which are common to all fcov strains, became a valuable tool for the detection of fcov nucleic acid in blood, body cavity effusions, faeces and tissue samples of infected cats. in particular the n-terminal domain of the s-protein gene allows a differentiation between the two types i and ii. posch et al. (1999 posch et al. ( , 2001 developed an rt-pcr using primers targeted to the s-protein gene to study the prevalence of the two fcov types in cats without fip symptoms, and showed that 55% of the pcr-positive cats proved positive for type i, 28% for type ii and 17% for both types. with the retrospective study presented here we investigated the prevalence of the two types of fcovs in cats with histopathologically verified fip using nested and seminested rt-pcr assays, with primers targeted as well to the s-protein gene. the aim of this study was to investigate the distribution of the two fcov types in fip diseased cats. furthermore-since fcov types i and ii may use different receptors-we wanted to investigate whether the two types are associated with differences in the clinical course of the disease and/or distinct histopathological changes. finally we intended to get more information about the importance of ccv for the cat. ccv itself may infect the cat, or it may be involved indirectly, regarding the possibility that recombinations between fcov type i and ccv may happen in the field at any time (horzinek, 2000) . between 1997 and 2000 a total of 1754 cats were examined at the institute of pathology and forensic veterinary medicine of the university of veterinary medicine, vienna, and 154 of these cats were diagnosed with fip. the analysis of breed, gender and age of the 154 cats with fip compared to the 1600 cats without fip symptoms is shown in table 1 . the statistical evaluation of the parameters breed, gender and age in the two groups "cats with fip" and "cats without fip" was carried out by χ 2 -test using the program spss for ms windows, version 8.0. from 94 of the 154 cats with histopathologically confirmed fip organ samples (lung, liver, spleen, kidney, gut) were available, either formalin-fixed paraffinembedded tissues (pet) (n = 65, 1997-1998) or fresh organ samples (n = 29, 1999-2000) . pet samples had been fixed in buffered formaldehyde for 48 h and were then embedded in paraffin. fresh organ samples were taken during section and either processed immediately or stored at −80 • c until used. the preparation of pet samples was carried out essentially as described by sorg and metzler (1995): four to six 5 m thick sections of paraffin-embedded organs from each cat were pooled and deparaffinised by incubating for 30 min in xylene and washing twice for 5 min in ethanol at room temperature. after centrifugation and air drying for 10 min at 37 • c, 25-50 l proteinase k and 200-400 l (depending on the sample size) buffer atl (qiagen, valencia, ca, usa) were added and the samples were then incubated at 37 • c for 5 days. when necessary, another equivalent of proteinase k and buffer atl was added at the second or third day of incubation. after inactivation of proteinase k at 95 • c for 8 min and centrifugation, rna was extracted from the upper aqueous phase using a commercially available kit (qiaamp viral rna mini kit, qiagen, valencia, ca, usa). the extracts were then stored at −80 • c. one to three grams of each organ sample were pooled and homogenised with sterile sand, and resuspended in 2-3 ml diethyl pyrocarbonate (depc)treated water. after centrifugation the rna was the general screening for fcov was carried out by rt-and nested (n) pcr as described by herrewegh et al. (1995) using the primers p205 and p211 for rt-pcr and p204 and p276 for npcr, respectively. samples positive in these assays were submitted to further analysis employing oligonucleotide primers, which had been designed in regions of the s-protein gene allowing a differentiation between fcov types i and ii. to improve the sensitivity of the pcr assays, a second round of amplifications (npcr with primers b, seminested with primers a) was carried out following rt-pcr. the primers were selected with the help of the primer designer program (scientific and educational software, version 3.0) and are shown in table 2 . rt-pcr was carried out as a single-tube assay with a reaction volume of 25 l (22.5 l pcr mixture and 2.5 l template) using a commercially available kit (access rt-pcr system, promega, madison, wi, usa). the mgso 4 concentration was optimised at 1 and 2 mm using the primers fecv1 and fecv2, respectively. negative samples were re-tested by employing the one step rt-pcr kit from qiagen (valencia, ca, usa). cycler schemes were carried out following the instructions of the manufacturers. an amount of 2.5 l of the rt-pcr product was added to 22.5 l of the master mix for npcr and seminested pcr, respectively, containing 10 mm tris-hcl (ph 9 at 25 • c), 50 mm kcl, 0.1% triton x-100, 200 m each 2 -deoxynucleoside 5 -triphosphate, 1.5 mm mgcl 2 , 1.25 u taq polymerase and 40 pm of each primer. forty-five cycles of denaturation at 94 • c, primer annealing at 60 • c and primer extension at 72 • c, 30 s each, were employed. as the possibility of false positive results due to carryover of amplification products in particular during npcr cannot be ruled out, a number of precautions were taken to minimise the risk of contamination. these included the physical separation of all pcr procedures, the use of at least four negative controls of rnase free water for each assay and of three or more primer pairs for each sample. the rt-pcr amplification product was added to the master mix for the npcr in a laboratory specifically installed for this purpose. finally sequence analysis of 15 amplification products served as an additional control. twenty microlitres of each pcr product were analysed by electrophoresis in a 1% agarose gel for 1 h 10 min at 90 v, and visualised by ethidium bromide staining. the 100 bp ladder (amersham pharmacia biotech inc., piscataway, nj, usa) served as molecular weight marker. bands were visualised with uv illumination and photographed using the eagle eye tm ii uv gel imaging system (stratagene, la jolla, ca, usa). sequence analysis was performed after gel extraction of the amplified product (qia quick gel extraction kit, qiagen, valencia, ca, usa) and sequencing pcr (abi prism big dye tm terminator cycle sequencing ready reaction kit, perkin elmer, alameda, ca, usa) using the sequence analyser abi prism 310 genetic analyser (pe applied biosystems). partial nucleotide sequences (a stretch of 108 bp within the s-protein gene region) of selected 11 type i and 2 type ii positive samples, as well as one of the five samples which had tested positive for both types, were determined; their alignment is shown in fig. 1 . extracts of cell culture supernatants from five different fcov-strains (type i: fipv ku2, fipv nw1; type ii: fipv 79-1146, fecv 79-1683, fipv df2) were submitted to rt-pcr, nested and seminested pcr employing the primers fecv1a, fecv1b, fecv2a and fecv2b. gel extracts of the strains fipv ku2 and fipv 79-1146, containing 8.85 and 6.69 pmol/l dna, respectively, obtained after rt-pcr with the primers fecv1b and fecv2b, were diluted in rnase free water with a concentration of 1% trna, and served as template for npcr. as far as antibody titres had been recorded in the case histories, they were compared to the pcr results. the results of the pathological examination were analysed according to the following criteria: during section the presence and amount of fip typical effusion (low, medium, high amount) and of fip suspicious granulomas and pyogranulomas (granulomas yes, no and localisation) were recorded. the subsequent histopathological examination confirmed the diagnosis fip only in the presence of the typical vasculitis with central necrosis and perivascular infiltration with plasma cells, macrophages, lymphocytes and neutrophils. general data of 154 fip-diseased cats were analysed and compared to those of 1600 cats without fip symptoms examined during the same period of time (1997) (1998) (1999) (2000) at the institute of pathology and forensic veterinary medicine of the university of veterinary medicine in vienna (table 1 ). the statistical examination showed that the incidence of fip was significantly higher among males versus females (p = 0.035), among purebred versus domestic short hair cats (p = 0.000) and among young animals up to 1 year (p = 0.000). a significantly greater number of males among fip-diseased cats was found in the age category 0-1 year (p = 0.04), but in cats older than 1 year this trend could not be observed (p > 0.05). while 6 of the 65 pet samples tested negative, nucleic acid could be detected in all 29 fresh organ samples with the primers described by herrewegh et al. (1995) . the differentiation of the two types was accomplished in 47 of the 59 pet samples which tested positive for fcov (the pcr result of one additional sample was questionable) and in 27 out of 29 fresh organ samples (one additional questionable result). in total, a differentiation was possible in 74 samples. among these, 64 (86%) tested positive for type i, 5 (7%) for type ii and 5 (7%) for both types i and ii (table 3) . of the 69 samples positive for type i, 68 tested positive with the fecv1b primers, 25 with both the fecv1a and fecv1b primer pairs, and 1 with the fecv1a primers only. of the 2 samples with a questionable pcr result, 1 tested questionably positive with the fecv1a primer pair and negative with the fecv1b primers, the second one vice versa. of the 10 samples positive for type ii, 1 tested positive employing the fecv2a primers and 9 using the fecv2b primers. of these results 60 (58%) were already achieved after rt-pcr. extracts of cell culture supernatants of five different fcov strains, the type i strains fipv ku2 and fipv nw1, and the type ii strains fipv 79-1146, fecv 79-1683 and fipv df2, were subjected to rt-pcr and nested or seminested pcr with the primer pairs fecv1a, fecv1b, fecv2a and fecv2b. employing the primers fecv1a and fecv1b, the type i strains ku2 and nw1 showed amplification products of the estimated size, whereas the type ii strains 79-1683, 79-1146 and df2 tested negative (fig. 2, primers fecv1b ). with the primers fecv2a and fecv2b the strains 79-1683, 79-1146 and df2 tested positive whereas the type i strains remained negative (fig. 3 , primers fecv2b). gel extracts of the strains fipv ku2 and fipv 79-1146, obtained after rt-pcr with the primer pairs fecv1b and fecv2b, tested positive in the nested pcr assays up to a dilution of 10 −6 and 10 −9 , respectively. antibody titres to fcov were known from the case history for 25 cats, 20 of them had titres of ≥1:400, 3 of 1:100, 1 of 1:10 and 1 was indicated as serologically negative. in the group of cats with fcov antibody titres of ≥1:400 twelve tested positive for fcov nucleic acid of type i and were negative for type ii, and one was positive for type ii but negative for type i; for seven cats a differentiation between the two types could not be achieved. all three cats with titres of 1:100 tested positive for type i and negative for type ii. the single cat with a titre of 1:10 and the sero-negative cat tested both negative for fcov nucleic acid. partial nucleotide sequences of the s-protein gene of 11 type i and 2 type ii positive samples as well as of one sample positive for both types were deter-mined. the sequences were compiled (resulting in a readable stretch of 108 bp) and aligned using the sequence of the type i strain fipv ku2 as a reference. in the alignment, also the corresponding sequences of the fcov type ii reference strain 79-1146 and of the ccv reference strain insavc-1 were included. the analysis of the samples revealed a nucleotide identity of 86-91% for the type i specimens, and of 73-75% for the type ii samples, respectively, in reference to the type i strain fipv ku2 (fig. 1) and of 77 and 78% for the type ii specimens in reference to the ccv strain insavc-1. histopathologic examination exhibited no differences related to the type of fcov detected. both types were found in effusive and non-effusive fip as well as in cases with signs of both forms; a statistical evaluation was not possible due to the small number of type ii positive samples. the comparison of the two groups, cats with fip (n = 154) and cats without fip symptoms (n = 1600) examined in the years 1997-2000 at the institute of pathology and forensic veterinary medicine of the veterinary university in vienna showed significant differences. the statistical evaluation using the χ 2 -test of the parameters breed, gender and age in the two groups showed that the incidence of fip was significantly higher among purebred cats, males, and among cats 1 year of age or younger. the percentage of purebred cats in fip-diseased cats was more than twice as high as in the comparative group (33.6% versus 13.5%, p ≤ 0.001). these findings are sustained by earlier studies (pedersen, 1983) . foley and pedersen (1996) observed a higher susceptibility for fip in purebred cats when a first degree relative succumbed to fip. due to inbreeding a genetic predisposition may have evolved in certain breeds of cats, which may allow fcov to propagate more efficiently in these cats than in cats with a wider genetic history. in the group of the cats with fip, the majority was male (62.4%), only 37.6% were female. although in the comparative group the percentage of males was slightly higher as well (53.4% males versus 46.6% females), the difference between the groups was significant (p ≤ 0.05). these findings are in contrast to pedersen (1976a) , who did not find a sexual predisposition, but in accordance with potkay et al. (1974) and binder and hartmann (2000) , who also reported a higher incidence of fip among males. the majority of the cats with fip was 1-year-old or younger (52.1%), in the comparative group only 33.1% were in this age class. addie and jarrett (1992a) found as well as scott (1991) a higher incidence of fip in cats of up to 1 year. when comparing the incidence of males and females in the age groups 0-1 year and older than 1 year between the cats with and without fip, we found that among younger cats the incidence of males was significantly higher in the cats with fip than in the comparative group. the role of sex-specific differences in the immune system, in particular the cell mediated immunity and the importance of these factors in neutered animals (hormonal influence) are still not clear. in a total of 93.6% of the samples, fcov nucleic acid could be detected. only six pet samples tested negative, whereas all fresh organ samples tested positive. in respect to the expected lower rna concentration in the pets due to the formalin fixation procedure on one hand and due to the long storage time (2-3 years) on the other hand, we chose primers which amplified, compared to those employed by posch et al. (1999 posch et al. ( , 2001 , a smaller segment of the viral genome. with these primers we achieved a differentiation of the two types in 47 of 65 pet specimens and in 27 of 29 fresh organ samples, in addition two samples exhibited a questionable pcr result. as expected, the percentage of positive pcr results was lower in the pet samples than in the fresh organ samples. specificity was tested on five different fcov strains with four different primer pairs (figs. 2 and 3, primers fecvb). we found no false positive results and all amplification products showed bands of the expected size. despite the use of different primer pairs, in some samples the pcrs remained negative, probably due to the variability in the s-protein gene of fcov. the oligonucleotide primers employed in the pcr assays exhibited high sensitivity, the fecv1b primers proved to amplify specific nucleic acid up to a dilution of 10 −6 , and the fecv2b primers showed amplification even up to a dilution of 10 −9 . since the original rna concentration was similar in both samples, these findings indicate a higher sensitivity for the detection of type ii viruses. thus, in those samples, in which the differentiating pcr was unsuccessful, fcov type i may predominate as well. on the other hand, due to the lower sensitivity of the type i pcr, we cannot exclude a causative involvement of type i in the type ii positive cats. of the 74 samples in which a differentiation was achieved, 64 (86%) tested positive for type i, 5 (7%) for type ii and 5 (7%) for both types i and ii, thus identifying type i as the causative agent in the majority of the fip cases we examined. whereas posch et al. (1999 posch et al. ( , 2001 identified type ii in 45% (including those samples with both types) of fcov-positive cats without fip symptoms, we found type ii only in 14% of cats with fip involved, among them 7% showing a double infection with both types i and ii. these findings are in contrast to the results of hohdatsu et al. (1992) in japanese cats, in which none of the healthy cats tested positive for type ii, whereas among the chronically diseased cats without fip symptoms over 10% and among the fip-diseased cats even more than 30% were infected with fcov type ii. due to their close antigenic relationship in the s-protein gene and the need to choose primers from this region for a possible differentiation between the two fcov types, an infection with ccv would also have been detected (data not shown). therefore, a causative involvement of ccv in the type ii positive fip cases cannot be ruled out in this study. the importance of ccv for the cat still remains unclear (barlough et al., 1985; mcardle et al., 1992) , but the possible recombination between fcov type i and ccv horzinek, 2000) requires further investigation, in particular the role of ccv in double infections with both fcov types observed especially in multi-cat households. the temperature-sensitive fipv strain used as fip vaccine is a type ii strain (fipv df2). regarding the fact that the majority of the fip cases we examined was due to type i, the question arises whether this fact contributes to some of the observed vaccine failures, and whether the inclusion of also a type i strain in the vaccine should be considered. the sequencing results show even in the very short region, which had been sequenced, a clear discrimination between fcov type i, fcov type ii and ccv strains (fig. 1) . the 11 partial sequences of the austrian fcov type i samples show significant differences, compared to the reference strain ku2, which resulted in identity rates of (only) 86-91% to the reference strain; also within the austrian fcov type i samples several nucleotide changes can be noticed, indicative of a quite high mutation rate. the fcov type ii samples form an own group with an identity to the fcov type i reference strain of only 73-75%, respectively. ccv exhibits an identity to fcov-1 ku-2 of 72%; its much closer relationship to fcov type ii than to fcov type i can nicely be observed by the similarity of several nucleotide changes of fcov type ii and ccv. these findings support the observation that fcov type ii may arise from recombinations with ccv . nonetheless, ccv also exhibits several unique nucleotide changes. the sequencing data of one of the five samples showing double infections (fig. 1) demonstrate clearly the plausability of such co-infections. among the cats with known antibody titres, all cats positive for fcov nucleic acid showed antibody titres of 1:100 or higher. neither during section nor in the histopathologic examination any differences related to the fcov type detected could be identified. in the group of the type i positive cats 58% showed signs of both forms (effusive and non-effusive) of fip, followed by 28% with non-effusive and 14% with effusive fip. in the type ii positive samples all forms of fip were represented as well. these findings do not point towards a pathogenetic importance of the receptor-specificity of type ii (hohdatsu et al., 1998) , but emphasises the role of the immune response and the genetic predisposition of the individual in the outbreak of the disease. our findings suggest an involvement of each of both fcov types in fip which is in accordance with earlier reports that both types of fcov are able to cause fip; they also correlate with the results obtained in healthy fcov-infected cats, supporting the predominance of fcov type i infections in both fcov-infected healthy and fip-diseased cats. fcov type ii, the probable recombination between type i and canine coronavirus, was involved in 14% of the fip cases investigated. it has to be assumed that these recombinations occur in the field and therefore contact with dogs excreting canine coronavirus may play a role in the emergence of new type ii fcov. however, the samples positive for fcov type ii need further investigations with respect to their relationship and even differentiation to ccv. special interest should also be paid to cats with double infections concerning the role of field infections with ccv and their role in the development of fip. finally, no differences in the histopathological changes were found related to the fcov type detected. a study on naturally occurring feline coronavirus infections in kittens feline coronavirus antibodies in cats risk of feline infectious peritonitis in cats naturally infected with feline coronavirus feline coronavirus in the intestinal contents of cats with feline infectious peritonitis experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus klinik der felinen infektiösen peritonitis. 9 feline infectious peritonitis biological and pathological consequences of feline infectious peritonitis virus infection in the cheetah perspectives on the epizootiology of feline enteric coronavirus and the pathogenesis of feline infectious peritonitis nachweis feliner coronaviren mittels 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shift and genetic drift during feline coronavirus evolution the biology and pathogenesis of coronaviruses receptor for mhv is a member of carcinoembryotic antigen family of glycoproteins we cordially thank helga lussy, claudia pallan and dr. barbara bauder for their excellent technical assistance. key: cord-319685-dw0qsl4s authors: porter, emily; tasker, séverine; day, michael j; harley, ross; kipar, anja; siddell, stuart g; helps, christopher r title: amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis date: 2014-04-25 journal: vet res doi: 10.1186/1297-9716-45-49 sha: doc_id: 319685 cord_uid: dw0qsl4s recent evidence suggests that a mutation in the spike protein gene of feline coronavirus (fcov), which results in an amino acid change from methionine to leucine at position 1058, may be associated with feline infectious peritonitis (fip). tissue and faecal samples collected post mortem from cats diagnosed with or without fip were subjected to rna extraction and quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) to detect fcov rna. in cats with fip, 95% of tissue, and 81% of faecal samples were pcr-positive, as opposed to 22% of tissue, and 60% of faecal samples in cats without fip. relative fcov copy numbers were significantly higher in the cats with fip, both in tissues (p < 0.001) and faeces (p = 0.02). pcr-positive samples underwent pyrosequencing encompassing position 1058 of the fcov spike protein. this identified a methionine codon at position 1058, consistent with the shedding of an enteric form of fcov, in 77% of the faecal samples from cats with fip, and in 100% of the samples from cats without fip. in contrast, 91% of the tissue samples from cats with fip and 89% from cats without fip had a leucine codon at position 1058, consistent with a systemic form of fcov. these results suggest that the methionine to leucine substitution at position 1058 in the fcov spike protein is indicative of systemic spread of fcov from the intestine, rather than a virus with the potential to cause fip. feline coronavirus (fcov) infection is ubiquitous in domestic cats, particularly in multi-cat households where up to 90% of animals may be infected [1] [2] [3] . the majority of fcov infections are asymptomatic or are associated with mild enteric disease [4] . however, approximately 5-12% of infected cats develop the invariably fatal disease, feline infectious peritonitis (fip) [5] [6] [7] . one of the most important questions in fcov research is why some fcov-infected cats develop fip, whereas others remain healthy. one current model of fip pathogenesis proposes that cats are infected with fcov by the faecal-oral route. subsequently, the virus mutates into the virulent form. this form has an enhanced tropism for monocytes/macrophages, and in vitro studies suggest that this is reflected as sustainable replication in, and subsequent activation of, monocytes [8, 9] . these activated monocytes carry the virus in the blood and, as a result of complex interactions with endothelial cells, induce the granulomatous phlebitis that is the pathogenic hallmark of fip [10, 11] . the age, breed, gender, reproductive status and immune response of individual cats also influence the development of fip [12] . currently, there is intense interest in determining which mutations alter the virulence of fcovs. a recent paper published by chang et al. [13] derived full genome sequence data from a collection of fcovs obtained from the faeces of healthy cats and from the tissues of cats diagnosed with fip. they provided evidence of an association between fcov virulence and an amino acid substitution (methionine to leucine at position 1058, m1058l) within the putative fusion peptide of the fcov spike (s) protein. specifically, the authors concluded that the m1058l substitution distinguished fip from non-fip associated fcovs in 92% of cases. a second substitution, two amino acids downstream of m1058l (serine to alanine at position 1060, s1060a) distinguished a further 4% of fip from non-fip associated fcovs. the s protein fusion peptide is a critical element in the fusion of viral and cellular membranes during virus entry [14] and it is reasonable to think that amino acid substitutions within this peptide may alter the tropism of the virus. in addition, a study by licitra et al. has shown that it is possible to distinguish between fcovs from animals with and without fip on the basis of one or more substitutions in the amino acid sequence that comprises the furin cleavage motif within the s protein [15] . this furin cleavage site (consensus motif r-x-k/r-r, where r is the basic arginine residue, x is any residue and k is the basic lysine residue) delineates the border of the receptor-binding (s1) and fusion (s2) domains of the s protein and is distinct to the m1058l substitution site described above. mutation at this site is proposed to alter proteolytic cleavage of the s protein and modify s protein fusogenic properties, which again may relate to the tropism of the virus [15] . finally, pedersen et al. [16] concluded that truncating and non-truncating mutations in the 3c gene occur in a significant proportion of fcovs associated with fip. chang et al. [17] suggest that functional 3c protein expression is crucial for fcov replication in the gut but is dispensable for systemic replication. however, they also do not exclude the possibility that the loss or alteration of the 3c protein may enhance the fitness of the virus in the monocyte/macrophage environment. over the past 8 years, the university of bristol has collected a large number of post-mortem tissue and faecal samples from a cohort of thoroughly examined cats. these include cats with a definite diagnosis of fip, confirmed by the presence of the typical histological fip lesions, in which immunohistochemistry (ihc) demonstrated fcov antigen within macrophages [18] , and cats with diseases other than fip that completely lacked any histological changes consistent with fip. importantly, this long-term study has enabled both faecal and tissue samples to be collected from fcov-infected cats with and without fip, allowing comparable samples from naturally infected cats to be examined. samples were screened for fcov rna by quantitative reverse transcriptase-polymerase chain reaction (qrt-pcr) [19] and, if positive, were assessed for the m1058l substitution by pyrosequencing. post-mortem tissue samples, and faeces whenever possible, were collected from cats that were euthanized with suspected fip, or due to other diseases. fip was then definitively diagnosed or excluded by histopathology and, in the case of fip, the demonstration of fcov antigen in fip lesions by ihc [18] . tissues of cats without fip were also tested by ihc for the presence of viral antigen. tissue samples were collected into rnalater (life technologies) within 2 h of death for subsequent molecular analysis. the tissue samples were left in rnalater for 24-48 h at room temperature or 4°c before the rnalater was discarded, and the tissue samples stored at −80°c. the faecal samples were stored at −80°c until use. further samples were collected into 10% neutral-buffered formalin for histology and ihc. the tissues collected comprised primarily mesenteric lymph node, liver, kidney, spleen and omentum, while other tissues (e.g. intestine, brain, lung, pericardium, pancreas or other lymph nodes) were included based on gross pathological findings or reported clinical signs. the formalin-fixed tissue samples were subjected to standard processing for histopathology. they were embedded in paraffin wax and sections prepared and stained by haematoxylin-eosin. sections were examined by a boardcertified veterinary pathologist (mjd) at the university of bristol for histopathological changes. selected wax blocks were then sent to veterinary laboratory services, university of liverpool for ihc analysis as previously described [18] . for a cat to be assigned to the "fip group", it needed to have histopathological changes consistent with fip in which fcov antigen was demonstrated within macrophages in lesions [18] . for a cat to be assigned to the "non-fip group", histopathological changes consistent with fip needed to be completely absent, and fcov antigen within macrophages needed to be absent for all tissues. only individual tissue samples with a positive qrt-pcr result, and lesions consistent with fip on histopathology were used for pyrosequencing. faecal samples were classified on the basis of the diagnosis attributed to the cat from which the sample originated. total rna was extracted from 20 mg of tissue or 10 mg of faeces with a nucleospin rna ii kit (macherey-nagel) using methods based on previous work by dye and siddell [19, 20] . reverse transcription was done using a mj mini gradient thermal cycler and improm ii reverse transcriptase (promega). nine microlitres of total rna solution were combined with 4 μl improm ii 5× reaction buffer, 2.4 μl 25 mm mgcl 2 , 1 μl dntps (10 mm each), 1 μl random hexamers (0.5 μg/μl) and 1 μl improm ii reverse transcriptase. the reaction was made up to a total volume of 20 μl with rnase-free water. the following thermal profile was used; 20°c for 5 min, 42°c for 30 min, 70°c for 15 min and 4°c hold. the resulting 20 μl of cdna was added to 30 μl of rnase-free water and stored at −20°c. randomly selected samples were checked for inhibition of the rt reaction using an rna internal amplification control. no inhibition was detected (results not shown). the qpcr was done on an agilent mx3005p qpcr system (agilent technologies). a qpcr master mix was made for each reaction with 12.5 μl 2× gotaq master mix (promega), 0.5 μl of 10 μm forward and reverse primer (p009/p010) ( table 1) , 0.125 μl of 5 μm taqman probe (table 1 ), 1.25 μl 50 mm mgcl 2 and made up to 20 μl with rnase-free water. the primers and probe were produced by metabion (metabion international) and were described previously by dye et al. [19] . one-tenth of the randomly primed cdna (5 μl) was added to the pcr master mix. the reaction plate was heat sealed and the following thermal profile was used: 95°c for 2 min, 40 cycles of 95°c for 15 s, 55°c for 15 s and 72°c for 15 s. fluorescence was detected at 520 nm during the extension phase. feline cov cdna was used as a positive control and rnase-free water as a negative control. reactions that failed to reach the threshold cycle (ct) value by cycle 40 were deemed to be negative. a ct value of 40 was assigned a relative copy number of 1 [21, 22] , and the following equation, which takes into account the 96% efficiency of the qrt-pcr assay [19] , was used to calculate the relative copy number of each qrt-pcr positive sample: 1.96 (40-ct value) . all samples that were positive by fcov qrt-pcr underwent conventional pcr to amplify a 153 base-pair dna fragment encompassing position 1058 in the s protein gene. pcr was done using a mj mini gradient thermal cycler. briefly, for each reaction, a pcr mix was made that included 12.5 μl 2× gotaq master mix, 0.5 μl of 10 μm forward and reverse primer (f614/r766) (table 1) , 2 μl of randomly primed cdna reaction products and water to a volume of 25 μl. the following thermal profile was used; 95°c for 2 min, 40 cycles of 95°c for 15 s, 52°c for 20 s and 72°c for 20 s, before being held at 4°c. the pcr products were used for the pyrosequencing reaction or stored at -20°c until required. samples that failed to produce definitive sequence data were pyrosequenced, following repeat amplification using the same pcr protocol with 50 cycles of amplification. single strand sequencing templates were produced by binding the biotinylated pcr product to streptavidincoated sepharose beads (fisher), followed by chemical denaturation and neutralisation. for each sample, the following mix was prepared; 2 μl streptavidin beads, 40 μl pyromark binding buffer (qiagen) (ph 7.6 containing 10 mm tris-hcl, 2 m nacl, 1 mm edta, 0.1% tween 20) made up to 55 μl with water. the bead mixture was added to the pcr product and shaken at 1400 rpm for 10 min. the sequencing primer mix contained 0.75 μl 10 μm sequencing primer (table 1 ) and 24.25 μl pyromark annealing buffer (qiagen) (20 mm tris-oac, 5 mm mg-oac ph 7.6) for each sample. the control oligonucleotide (a self-sequencing oligonucleotide) mix contained 1 μl 10 μm oligonucleotide c4 and 24 μl annealing buffer. twenty five microlitres of the sequencing primer mix was added to the appropriate wells of a pyrosequencing plate, 25 μl of control oligonucleotide was added to one well, and the plate was placed on the pyrosequencing workstation. the pyrosequencing workstation was prepared with trays containing wash buffer (10 mm tris-oac ph 7.6), denaturing buffer (0.2 m naoh), 70% ethanol and distilled water. the streptavidin bead bound pcr product was taken up using a vacuum pump and the pyrosequencing bead collector. the bead collector was then placed in the 70% ethanol for 5 s, in the denaturing buffer for 5 s and in the wash buffer for 10 s. after turning off the vacuum, the bead collector was placed in the pyrosequencing plate and agitated for 30 s to dislodge the beads. the pyrosequencing plate was heated on a plate holder at 80°c for 2 min, before being placed into the pyromark q24 (qiagen) and left to cool for 5 min. while the plate was cooling, the pyrosequencing cartridge was prepared. pyromark gold q24 enzyme, substrate and dntps (qiagen) were added into the appropriate wells of the cartridge. volumes were as outlined by the pyromark q24 software. during the experimental set up, the dispensation order of the nucleotides was defined as; cgctcatg. the cartridge was placed into the pyromark q24 instrument and the protocol run. all primers used in the pyrosequencing assay were designed using a combination of pyromark assay design software (qiagen), primer 3' software [23] and mfold [24] , and were made by eurofins (mwg operon) ( table 1 ). the primer positions were based on those used by chang et al. [13] , and numbered according to the fcov c1je genome [genbank:dq848678]. degeneracies were added to the primers, and the location of the primers optimised, based upon a sequence alignment comprised of all available type i fcov genomes (data not shown). conventional pcr to amplify a 153 base-pair dna fragment encompassing position 1060 in the s protein gene was carried out as described above, (see pyrosequencing methods) on samples that did not show a m1058l substitution in the pyrosequencing assay. the pcr primers (f614/r766) were then used as sequencing primers in a standard sanger sequencing protocol (eurofins, mwg operon). the fcov relative copy numbers were entered into a database (excel 2010, microsoft) and exported into ibm spss statistics software (version 19.0). the data sets were evaluated for normal distribution using the kolmogorov-smirnov (k-s) test. non-normally distributed data were described as median and range (minimum and maximum values). data evaluating fcov relative copy numbers in tissue and faecal samples from cats with and without fip were analysed using a multilevel modelling approach (mlwin v2.27) [25] , to account for the repeated measures within cats, and a non-parametric mann-whitney u test. the conclusions drawn from both analyses were in full agreement, so the simpler mann-whitney u test analysis is presented here. relative copy numbers were compared between the fip and non-fip samples for tissue and faecal samples combined, for faecal samples only, and for tissue samples only. significance was assigned at a level of p < 0.05. historical samples were collected with full informed consent from owners that samples could be used for research purposes. the project has been approved under ethical review by the university of bristol animal welfare and ethical review board (vin/14/013). a total of 112 samples were analysed and full details of the samples and results are shown in table 2 . in cats with fip, the diagnosis was confirmed by histopathology and the demonstration of fcov antigen within macrophages in fip lesions by ihc. in cats without fip, the diagnosis was made by histology; neoplasia (e.g. lymphoma, astrocytoma, chemodectoma, or biliary cystadenoma), and inflammatory processes (e.g. chronic lymphoplasmacytic infiltrates of unknown aetiology in liver and kidney, and bronchopneumonia) predominated. a total of 26 faecal samples were analysed by fcov qrt-pcr, and 19 (73%) were positive. these comprised 13 of 16 (81%) faecal samples from cats with fip, and 6 of 10 (60%) faecal samples from cats without fip (table 2) . a total of 86 tissue samples were analysed by fcov qrt-pcr, and 52 (60%) were positive. these comprised 43 of 45 (95%) tissue samples from cats with fip, and 9 of 41 (22%) tissue samples from cats without fip (table 2) . relative fcov rna copy numbers were not normally distributed (p < 0.001). the relative copy numbers in pooled faecal and tissue samples in the fip group (median; range: 44 347; 0-16 547 217) were significantly higher (u = 241.0, p < 0.001) than in the non-fip group (0, 0-10 090). when only tissue samples were considered, the relative copy numbers in the fip group (75 976; 0-16 574 217) were also significantly higher than those in the non-fip group (0; 0-2 146) (u = 72, p < 0.001). finally, analysis of faecal samples alone showed that the relative copy numbers in the fip group (9 062; 0-11 819 441) were significantly higher than those in the non-fip group (34; 0-10 090) (u = 36.5, p = 0.02). the 19 faecal and 52 tissue samples with positive qrt-pcr results were subjected to the pyrosequencing assay. of the 19 faecal samples successfully sequenced, 10 were obtained using the 40 cycle pyrosequencing assay, whereas 9 required the 50 cycle assay. of the 52 tissue samples successfully sequenced, 34 were obtained using the 40 cycle pyrosequencing assay, whereas 18 required the 50 cycle pcr. in 15 of the 19 (79%) faecal samples positive by qrt-pcr, a methionine (aug) codon alone was found at position 1058 (table 2 and figure 1a ). in 3 of the 19 (16%) samples, a leucine codon (uug) alone was found at position 1058 (table 2 and figure 1b) . additionally, 1 (5%) sample (cat 60, faeces) showed a mixed population of rna coding for either methionine (aug) or leucine (cug) at this position (table 2 and figure 1c ). importantly, methionine and leucine codons were identified in faecal rna samples from both cats with and without fip (table 2) . specifically, a methionine codon was identified in 10 samples from 10 fip cats, and a leucine codon was identified in 3 samples from 3 fip cats. overall, a methionine codon was found in the majority (10/13; 77%) of faecal samples from cats with fip, but a significant number had a leucine codon (3/13; 23%). similarly, a methionine codon was identified in 6 samples from 6 cats without fip (including one mixed infection), and a leucine codon was identified in 1 sample from 1 cat without fip (which had the mixed infection). overall, a methionine codon was found in all 6 faecal samples from cats without fip, and 1 sample in 1 cat had a leucine codon (1/6; 17%, which also had a methionine as a mixed infection). in 47 of the 52 (90%) tissue samples positive by qrt-pcr, a leucine codon (44 uug, 3 cug) alone was found at position 1058 (table 2 and figures 2a and 2b ). in the remaining 5 of the 52 (10%) tissue samples, a methionine codon (aug) was found at this position ( table 2 and tissue types are defined by superscripts: a omentum, b liver, c kidney, d lung, e stomach, f lymph node, g cerebrum, h abdominal lymph node, i heart, j spleen, k colon, l mesenteric lymph node, m brain, n mesentery, o pleura, p pericardium, q medulla, r pons, s small intestine, t intestine. the amino acid coded at position 1060 in the s protein of fcov rna from tissue samples without the m1058l substitution is defined by the superscripts: 1 ucu (ser) and 2 gcu (ala). figure 2c ). no mixed infections were found in tissue samples. importantly, leucine and methionine codons were identified in rna from tissue samples from both cats with and without fip (table 2) . specifically, a leucine codon was identified in 39 samples from 23 fip cats, and a methionine codon was identified in 4 samples from 2 fip cats. overall, a leucine codon was found in the majority (39/43; 91%) of fip tissue samples, but a significant number had a methionine codon (4/43; 9%). similarly, a leucine codon was identified in 8 samples from 6 cats without fip, and a methionine codon was identified in 1 sample from 1 cat without fip. overall, a leucine codon was found in the majority (8/9; 89%) of tissue samples from cats without fip, with a minority (1/9; 11%) having a methionine codon. in one specific case (cat 70), we noted that the sample from one tissue (spleen) contained a leucine codon, whereas samples from three other tissues (liver, lung and mesenteric lymph node) all contained a methionine codon. conventional rt-pcr amplification products of rna from 5 tissue samples that did not contain the m1058l substitution (from cats 37, 56 and 70) were analysed by sanger sequencing for the s1060a substitution. one of the 5 samples (cat 37, pleura) showed an alanine codon at position 1060. the remaining four samples (1 from cat 56 and three from cat 70) all showed a serine codon at this position (table 2 ). the most important finding in this study is that the m1058l substitution in the fcov s protein does not correlate with fip disease phenotype, as was proposed by chang et al. [13] . we reach this conclusion because of two observations. first, although a leucine codon was found in the majority (91%) of tissue samples with fip lesions, a leucine codon was also found in the majority (89%) of non-fip tissue samples. second, a significant number (9%) of fip tissue samples had a methionine codon at this position. tissue samples from naturally fcov infected cats without fip have not been previously evaluated and provide an important insight into fcov infection in the absence of fip. we believe that the m1058l substitution is more likely to be a marker of systemic fcov infection, as opposed to a marker of fip or the development of disease. as our tissue samples were collected post-mortem, we cannot exclude the possibility that the 6 cats without fip (8 samples, from cats 33, 49, 51, 54, 56 and 57) and the leucine codon at position 1058, would have gone on to develop fip if they had not been euthanized due to other reasons. however, histopathological changes consistent with fip were absent in all 6 cats, and ihc did not identify fcov antigen. the finding that the majority of tissue samples from both cats with and without fip have a leucine codon at position 1058 does not challenge the idea that systemic spread of fcov is an important step in the development of fip. indeed, the latter is supported by the findings of our study, since fcov rna was found in a far greater proportion of tissue samples with fip lesions (95%) than tissue samples from cats without fip (22%), and, in those samples that were qrt-pcr positive, significantly higher fcov relative copy numbers were found in the fip samples, as has been found in a previous study in naturally infected cats [26] . however, as sampling in our study took place post-mortem, it could also be argued that the elevated fcov levels in fip tissues were a consequence of the massive immunological dysregulation that results from the disease, rather than a contributing factor towards the development of disease. another viewpoint is that the low levels of fcov rna from the tissues of cats without fip are a result of fcov infecting only fully differentiated macrophages and monocytes, among which are tissue-specific macrophages. this view is supported by our results as ihc, a detection method of low sensitivity, did not detect fcov antigen anywhere in the pcr-positive tissues from cats without fip, providing further evidence of low level viral infection either of tissue macrophages (in persistently infected animals) or in monocytes that were in vessels in the respective organ at the time of sampling [27] . these findings are also in accordance with recent in vivo and in vitro studies that showed only the virulent form of fcov can effectively and sustainably replicate in monocytes [9, 28, 29] . the m1058l substitution was not the only s protein substitution that was proposed by chang et al. [13] to correlate with the fip disease phenotype. they also showed that a second substitution, s1060a, distinguished a further 4% of fip from non-fip associated fcovs. we confirmed this result in so far as rna obtained from one of five tissue samples that did not show the m1058l substitution showed the s1060a substitution. we believe that, as was proposed by chang et al. [13] , changes such as the m1058l and s1060a substitutions, and potentially others, could be representative of a class of mutations that influence the fusogenic activity of the fcov s protein and, as such, deserve particular attention with regard to the pathogenesis of fip [13] . it is noteworthy that the substitutions identified by licitra et al. [15] that are proposed to distinguish between fcovs from animals with and without fip are also suggested to have an effect upon the fusogenic activity of the s protein. with regard to faecal samples, our study found that an unexpectedly high percentage (81%) of faecal samples from cats with fip were fcov qrt-pcr positive and their relative copy numbers were significantly higher than those of faecal samples from cats without fip. moreover, the majority (77%) of fcov rna sequences in faecal samples from cats with fip had a methionine codon at position 1058 in the fcov s protein gene, suggesting that these animals were shedding an enteric form of the virus. it seems reasonable to suggest that these cats were infected with an enteric, and a systemic, virulent form of fcov. whether one form was derived from the other following a single infection or whether these cats were infected twice with different fcovs cannot be determined. it has been proposed that the severe immune dysregulation in cats with end-stage fip might create an opportunity for super-infection by enteric fcov circulating in surrounding carriers [17] . more interestingly, a smaller but significant proportion of faecal samples from cats with fip (23%) provided fcov rna samples that encoded leucine at position 1058. the current model of fip pathogenesis proposes that once the enteric form of the virus has mutated to a virulent form, it is generally no longer horizontally transmitted via the faeces [12, 16] . this view has been challenged [30] , and the current results also suggest that a systemic form of the virus can be found in the faeces of fip cats. however, we accept that this does not mean that excreted virus is necessarily able to infect further cats by the enteric route, as has recently been shown in some experimental studies [16] . further research is necessary to resolve these issues. in contrast to the pattern shown by the analysis of faecal samples from cats with fip, the analysis of the faecal samples from cats without fip seemed more straight-forward. all "non-fip" faecal samples that were fcov qrt-pcr positive encoded methionine at position 1058, indicative of infection with the enteric form of the virus. also, as found in our study, a shedding proportion of 60% by cats without fip is not unexpected [12, 31] . an interesting faecal sample from a cat without fip (cat 60) showed a mixed population of rnas encoding for either methionine or leucine at position 1058. there was no evidence of fcov rna in the single tissue sample taken from cat 60, and, therefore, one interpretation could be that the m1058l substitution in the faecal sample was a relatively recent event and the virus had not yet spread systemically. however, this interpretation has to be considered as tentative because we have observed examples of negative qrt-pcr results in tissue samples from cats that were clearly fcov infected due to their fip grouping; cat 58, liver; cat 61, liver. as histopathology results and ihc for these two liver samples showed changes consistent with fip, these negative qrt-pcr results are likely to have arisen due to an absence of fcov in the particular samples taken for molecular analysis, as a variable distribution of fcov in affected tissues has been reported [18] . our study importantly also demonstrated that a pcrbased pyrosequencing [32] approach is a rapid and accurate method to identify single nucleotide differences at a specific position within a dna fragment, or in our case a viral genome. however, it has some limitations. some samples required 50, rather than 40, cycles of pcr amplification to generate adequate amounts of dna for sequencing. this was especially true for pcr products generated from samples that contained low amounts of viral rna, e.g. tissue samples from cats without fip. there were also several samples which, despite containing quantities of viral rna measurable by qrt-pcr, did not produce sufficient pcr products for pyrosequencing, even after 50 amplification cycles. these samples were excluded from the results as they did not contribute any additional sequence data to the study. however, one explanation may be that, despite the degeneracy of the primers used (f614/r766), differences in the viral primer binding sites may have precluded efficient amplification in these samples. in summary, we have used a pyrosequencing assay to determine the distribution of a specific m1058l substitution in the s protein of fcov rna obtained from a large number of post-mortem tissue and faecal samples from cats with and without fip. additionally, sanger sequencing was used to determine whether the s1060a substitution was present in tissue samples that did not contain the m1058l substitution. this represents the first study that compares similar samples from cats with and without fip with regard to the viral phenotype. our results contribute to a better understanding of fcov genomic mutations and how they may, or may not, be used as markers of the virus phenotype. the results also make clear that the relationship between the viral genotype and the development of fip is complex. we are currently using an approach that involves the deep sequencing of complete fcov genomes in clinical samples, in order to throw further light on this relationship. feline coronavirus antibodies in uk cats serologic studies of naturally occurring feline infectious peritonitis isolation and characterization of viruses related to the sars coronavirus from animals in southern china feline infectious peritonitis. abcd guidelines on prevention and management clustering of feline coronaviruses in multicat households common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus a study of naturally occurring feline coronavirus infections in kittens activation of p38 mapk by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells replication of feline coronaviruses in peripheral blood monocytes morphologic features and development of granulomatous vasculitis in feline infectious peritonitis altered expression of adhesion molecules on peripheral blood leukocytes in feline infectious peritonitis a review of feline infectious peritonitis virus infection: 1963-2008 spike protein fusion peptide and feline coronavirus virulence the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex mutation in spike protein cleavage site and pathogenesis of feline coronavirus feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis evaluation of real-time rt-pcr for the quantification of fcov shedding in the faeces of domestic cats genomic rna sequence of feline coronavirus strain fcov c1je development and use of real-time pcr to detect and quantify mycoplasma haemocanis and "candidatus mycoplasma haematoparvum" in dogs use of real-time quantitative pcr to detect chlamydophila felis infection primer3 on the www for general users and for biologist programmers mfold web server for nucleic acid folding and hybridization prediction mlwin version 2.1. in centre for multilevel modelling natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats sites of feline coronavirus persistence in healthy cats acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein a mrna pcr for the diagnosis of feline infectious peritonitis an outbreak of feline infectious peritonitis in a taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type ii feline coronavirus use of a reverse-transcriptase polymerase chain reaction for monitoring the shedding of feline coronavirus by healthy cats pyrosequencing: an accurate detection platform for single nucleotide polymorphisms amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis the authors thank the veterinary practices, cat breeders and rescue centres that helped in the acquisition of samples used in this study. we also thank our colleagues, dr emi barker, dr chris palgrave, louise dawson and debra fews at the feline centre and the veterinary pathology unit, langford veterinary services, university of bristol, who have assisted in obtaining post mortem samples. we would also like to thank members of the histology laboratory, veterinary laboratory services, school of veterinary science, and university of liverpool for technical assistance. professor toby knowles of the school of veterinary sciences, university of bristol, is also thanked for his help with statistical analyses. this research was supported by a project grant from the petplan charitable trust. the authors declare that they have no competing interests. key: cord-348746-yaf61cmx authors: foley, janet e.; leutenegger, christian title: a review of coronavirus infection in the central nervous system of cats and mice date: 2008-06-28 journal: j vet intern med doi: 10.1111/j.1939-1676.2001.tb01572.x sha: doc_id: 348746 cord_uid: yaf61cmx feline infectious peritonitis (fip) is a common cause of death in cats. management of this disease has been hampered by difficulties identifying the infection and determining the immunological status of affected cats and by high variability in the clinical, pathological, and immunological characteristics of affected cats. neurological fip, which is much more homogeneous than systemic effusive or noneffusive fip, appears to be a good model for establishing the basic features of fip immunopathogenesis. very little information is available about the immunopathogenesis of neurologic fip, and it is reasonable to use research from the well‐characterized mouse hepatitis virus (mhv) immune‐mediated encephalitis system, as a template for fip investigation, and to contrast findings from the mhv model with those of fip. it is expected that the immunopathogenic mechanisms will have important similarities. such comparative research may lead to better understanding of fip immunopathogenesis and rational prospects for management of this frustrating disease. f eline infectious peritonitis (fip) is a fatal, immune-mediated disease produced as a result of infection of macrophages by mutant feline coronavirus strains (fipvs). the severity of fip is determined by virus strain and by host-specific, partially heritable immune responses. 1 the causative agent, fip virus (fipv), is a macrophage-tropic mutant of the ubiquitous feline enteric coronavirus (fecv). [2] [3] [4] these feline coronaviruses are closely related to transmissible gastroenteritis virus (tgev) of pigs and more distantly related to mhv (a coronavirus of rodents), human respiratory coronavirus, and others. as with most rna viruses, a high rate of point or other small-scale mutations and larger scale recombination events occur. type ii fecvs are viruses that arise as a result of recombinations between type i fecv and canine coronavirus (ccv). type ii fecvs acquired a canine s gene and express a canine s gene product. 5 mutations are common in the 7b open reading frame (orf) of both type i and type ii fecvs and may be associated with reduced virulence. 4, 6 the 7b orf arose in the fecv/ccv lineage and encodes a nonstructural secretory glycoprotein of undetermined function, which is not necessary for viral replication. 7 deletions, point mutations, and frame-shift mutations leading to early truncation of the 3c orf also are common. 4, 6 because fip-defining mutations may occur in the s, 3c, and 7b genes, polymerase chain reaction (pcr) with these genes as a target cannot discriminate between benign fecv and fatal fipv. [2] [3] [4] murine hepatitis virus shares most genes with the fecvs, including m (membrane glycoprotein), e (small membrane), n (nucleocapsid), and s (spike glycoprotein), which is post-translationally modified to s1 and s2. 8 the mhv genome, however, also codes for an he protein and does not contain a 7b orf. mutations in the e and s proteins lead to attenuation of mhv. 9 a third coronavirus, hcv-229e, has been implicated in neurological disease in people. 10 fip occurs most frequently in cats younger than 3 years of age from multiple-cat homes (shelters and breeding catteries). 11, 12 one-quarter to one-third of cats with noneffusive fip have either primary neurological fip or neurological abnormalities as a part of their overall disease presentation (foley and pedersen, unpublished data). 13 the immunological and pathological characteristics of neurological fip, however, are much more stereotypic than those of systemic fip. thus, neurological fip may be useful for studying basic mechanisms of fip pathogenesis. the extent to which genetic differences among viral strains confer relative neurotropism is unknown, but some cats with severe neurological disease have mild or undetectable systemic disease. both neurological and generalized fip may present first as a nonspecific illness, with clinical signs including weight loss, weakness, fever, and lethargy. abdominal abnormalities are detected commonly on physical examination of cats with neurological fip, including mesenteric lymphadenopathy and irregular splenic and renal surfaces. 14, 15 common historical findings in cats with neurological fip include dementia, pica, seizures, inappropriate elimination, incontinence (fecal and urinary), and compulsive licking. 15, 16 neurological examination may identify ataxia, hyperesthesia, reduced consciousness, hyperreflexia, crossed-extensor reflexes, reduced conscious proprioception, caudal paresis, cerebellar-vestibular signs, or cranial nerve deficits. 14, 15, [17] [18] [19] ophthalmic lesions also are common in neurological fip, including anterior uveitis, keratic precipitates, flocculent debris in the anterior chamber, retinitis, and anisocoria. 15 murine hepatitis virus, like fipv, can produce either systemic infection or disease primarily affecting the liver. well-defined neurotropic genetic variants of mhv also occur, including a well-studied variant designated mhv-jhm, which is responsible for progressive encephalomyelitis and death in infected mice and rats. intranasal or intracranial inoculation of mhv-jhm in balb/c or c57bl/6 mice leads to rapid, fatal encephalitis. 20 the syndrome in survivors (either because the virus is attenuated or the host is resistant, vaccinated, or a pup from a vaccinated dams) consists of chronic demyelination and hindlimb paralysis 21 and has been proposed as a model for multiple sclerosis. 22 determinants of neurovirulence are found in the mhv s gene. 23, 24 a second variant of mhv, mhv-oblv60, leads to neuronal infection specifically in the anterior olfactory bulb after intranasal challenge in mice. 25 neurological disease the definitive lesion of fip is a pyogranuloma that results from immune-mediated phenomena secondary to coronaviral infection of macrophages. the most common sites are serosal, pleural, meningeal, ependymal, or uveal membranes. in the earliest stages of abdominal fip, diffuse alterations with activated mesothelial cells and a few coronavirus-infected macrophages or an exudative precipitate may be detected on serosal surfaces. 26 larger pyogranulomas become grossly visible, ranging from small lesions, often on the renal capsular surface, to severe, generalized miliary granulomatous lesions that distort renal surfaces, disseminate throughout the omentum and gastrointestinal serosa, and invade splenic and hepatic parenchyma. gross lesions of fip in the cns may be subtle, with ependymitis, thickening and opacification of meninges, and ventricular dilatation, usually in the fourth ventricle and least often in the lateral ventricles. 15 lesions occur most commonly on the ventral surface of the brain, often accompanied by secondary obstructive hydrocephalus. histopathologically, lesions in the brains of cats with fip consist of meningitis, ependymitis (ranging from mild ependymal infiltration to complete effacement of the ependymal lining by a heavy infiltrate of histiocytes and lymphocytes), periventriculitis, and choroiditis of varying severity, often superficial and oriented around the ventricles, with dense infiltrates of lymphocytes, plasma cells, neutrophils, and macrophages. 14, 15, [27] [28] [29] [30] [31] meningitis may be more severe on the ventrocaudal surfaces of the brain, especially at the base of the cerebellum and the brainstem, including the medulla oblongata. in the meninges, the inflammatory cells may have a predominantly perivascular distribution, forming cuffs around arteries (periarteritis) and infiltrating the wall of veins and venules (phlebitis), with exudation of a cell-and protein-rich edema fluid, and periventricular reactive astrocytosis. the inflammation may extend into the superficial neuropil, as well as into cranial nerve roots. 15 if lesions are deep, they usually are perivascular with scattered glial nodules. 27 hydrocephalus is seen in association with leptomeningitis, meningeal fibrosis, accumulation of cellular debris, and obstruction of the cerebrospinal fluid flow. pathologic lesions after mhv infection in the brains of rats and mice are variable, depending on viral dose and route of administration and rodent genotype, age, and immunological characteristics. 32 in severe acute jhm encephalitis, necrotizing lesions are found in the gray and white matter, with axonal changes, including disintegration of neurofilaments. 32 within the necrotic areas, perivascular neutrophilic infiltrates occur especially in the subependyma, choroid, and meninges. in acute mhv-a59 infection in cd8ϩ t-cell deficient mice, periventricular encephalitis occurs with lymphocytic infiltration into the choroid plexus, ependyma, and subependymal brain tissue. 33 lesions in mhv-oblv60 consist of local neuronal infection, some mitral cell destruction, and t-cell inflammation with astrocytosis. 25 some evidence suggests a pathological component of vasculitis, with mouse endothelial cell lines susceptible to mhv-jhm exhibiting cytopathology within several days of infection. 34 the subacute to chronic immune-modulated pathological changes that occur with manipulation of the mhv-jhm system are particularly relevant to the murine model of fip. depending on mouse strain and immunological status, mhv-jhm produces meningeal inflammation associated with t-cells and macrophages and demyelination but relatively little disease in axons. demyelination occurs within and adjacent to areas of inflammation and astrocytic proliferation. 32, 35 neutrophils and monocytes have been observed infiltrating through endothelial cell junctions and participating in the phagocytosis of myelin debris. 32 perivascular cuffing by macrophages is observed in chronic encephalitis, as in fip. the route of entry of fipv into the cns is unknown. the fipv virus probably travels hematogenously in macrophages, and 1 study reported a cat with positive immunohistochemical staining for fipv in monocytes in blood vessels of the choroid plexus. 36 once in the cns, there is little evidence that fipv enters any cells other than macrophages. foley et al 15 reported positive immunohistochemical staining (by means of a mouse monoclonal antibody against the fipv n protein) for fipv, primarily in macrophages in fip granulomas but also in some lymphocytes. virus-infected cells were numerous in some areas of intense ependymitis and choroiditis and free within the ventricular lumen, with very few positive cells in the meningeal infiltrates. no staining was observed in vascular basement membranes or in cells of the neuropil. macrophages in necrotic regions and in the center of lesions often are not infected with coronavirus. 37 neurotropic mhv strains have several routes of entry into the cns. some studies show that mhv-jhm travels up the olfactory nerve and enters the cns. 38 this is not surprising, because coronaviruses generally are epitheliotropic, and neurons share embryological origin with epithelial cells. cns infection with jhm also may occur after peripheral infection and viremia. 39, 40 receptors for neurotropic human coronavirus hcv-229e have been detected not only in human lung cells but also in human neuron, astrocyte, and oligodendrocyte cell cultures. 41 hcv-229e also infects macrophages and endothelial cells, however, suggesting hematogenous introduction into the cns, 40, 42 similar to fip. once inside the cns, the major targets for mhv are glial cells and neurons. 35 especially in neuroattenuated strains, mhv-jhm infects primarily oligodendrocytes 43 and has been reported in astrocytes. 44 mhv-4 infects mainly neurons. 20 subsequent events in chronic disease pathogenesis include recruitment of inflammatory cells and the interactions of immune cells with the virus-infected cells. macrophages, activated t-cells, and some b-cells may cross into the intact cns through the blood-brain barrier, with the potential for major cytokine upregulation. 45, 46 the immunopathogenesis of neurologic coronavirus infections after establishment of fipv in the cns, mechanisms of disease are primarily immune-mediated, involving humoral and cell-mediated immunity (cmi). coronavirus-infected macrophages can trigger massive complement activation and deposition of c3 on affected surfaces, disseminated intravascular coagulopathy, vessel necrosis, and effusion. 37, [47] [48] [49] however, immunopathogenic events in neurological fip have not been well described. both in systemic and neurological fip, antibodies (especially to the spike protein) contribute to the opsonization of viral antigen and have the capacity to mediate or enhance disease. [50] [51] [52] [53] [54] antibodies to fecv and fipv are identical. anti-fipv igg and igm-producing b-cells are present in fip lesions, at the interface of healthy tissue and granulomas, and in the serum and csf of cats with neurological fip. 26, 37 apparently, some antibody production occurs locally in the cns, in response to viral antigen in the brain. in one study, serum coronavirus titers of 16 cats with neurological fip were positive, with a median titer of 1 : 400, whereas csf titers were positive in 15 cats, with a median titer of 1 : 100. 15 two cats had high protein concentrations and increased cells, predominantly lymphocytes and neutrophils. the titer in csf was not statistically correlated with serum titer, and the ratio of serum : csf titer was not correlated with the serum : csf protein ratio. if passive leakage of protein from serum into the csf were invoked to explain the presence of antibodies, it would be expected that the total protein : anti-fipv igg ratios would be similar in both serum and csf. in contrast, csf igg titers tended to be proportionally much higher than serum titers. this finding suggests that anti-fipv igg may have been produced in the tissues of the brain in response to a locally replicating virus. although almost nothing is known about the mechanism, cell-mediated immunity has been hypothesized to be protective against fip. [50] [51] [52] [53] [54] cd4ϩ t-cells commonly are observed in fip granulomas. 37 during acute experimental multisystemic fip, apoptosis and t-cell depletion were observed in the spleen and mesenteric lymph nodes. 55 this effect was induced by heat-treated effusion fluid (presumably containing some cytokines) but not tissue culture fluid. likewise, little is known about the induction of cytokines during the course of fip. preliminary findings suggest that development of fip appears to be associated with a switch to predominantly th2 immunity, with increases in il-10 concentrations. 56 goitsuka et al 57 described increases in il-6 and il-1, but gunn-moore et al 58 reported reductions in th 2 and th 1 cytokines including il-2, il-4, il-10, and il-12, which they attributed to general immunosuppression. mildly increased amounts of il-1␣ mrna are detected inconsistently in association with lesions in cells of many organs in cats with fip. 59 roles of il-1 in this setting may include vascular endothelial activation, regulation of macrophages, il-8 and il-6, and chemotaxis. il-6 and il-1 can increase b-cell growth and differentiation and could exacerbate humoral contributions to the severity of fip. the inflammatory cytokines tnf-␣, il-1␣, and il-6 could circumvent the blood-brain barrier during systemic fip, or they could cross the blood-brain barrier after reorganization of the endothelial actin cytoskeleton. 60, 61 no information is available regarding cytokine production in neurological fip, and how cytokines in the cns compare to those in abdominal tissues of cats with generalized or effusive fip is unknown. mhv neurological disease can range from severe, acute, necrotizing, rapidly fatal encephalitis to chronic immunemediated demyelination with little encephalitis, depending on mouse strain, age, and immunocompetence. the appreciation of the principal role of the immune system in producing demyelination emerged from a series of experiments performed over several decades. profoundly immunosuppressed mice develop high concentrations of virus in the cns, but demyelination and clinical signs are minimal. immunocompetent mice have variable or even low concentrations of virus but develop marked disease. immune reconstitution in immunocompromised mice results in the development of severe demyelinating disease. if mice are pretreated with passive infusions of antibodies or t-cells or if they receive neuroattenuated mhv strains, they develop chronic, but not fatal, disease after mhv-jhm infection. 62, 63 immunocompetent c57bl/6 mice clear mhv-jhm virus from the brain but develop severe immune-mediated demyelination and paralysis. 22 in contrast, severe combined immunodeficient (scid) mice have persistent viral loads but no neurologic impairment or detectable lesions. gamma-irradiated immunocompromised mice similarly were resistant to chronic demyelination, but reconstitution of the immune system with adoptive transfer of splenocytes restored the immune-mediated lesions. 64 both cd4ϩ and cd8ϩ t-cells apparently are required to clear mhv from the cns. cd8ϩ t-cells are the predominant infiltrating leukocyte in lewis rats with mhv-jhm and paralytic disease, 65 whereas infiltration in clinically normal mhv-jhm-positive brown norway rats consists of cd4ϩ cells. ctls may kill some virally infected cells and protect mice from fatal disease, but they do not completely eliminate virus. 66 when cd8ϩ-depleted mice are reconstituted, virus load is reduced, and infection is not detected in most infected cell types, except for oligodendroglial cells. 66 mice with nucleocapsid or spike proteinspecific cd8ϩ t-cells develop chronic demyelinating disease. 21, 67 in the mhv-oblv60 model, depletion of cd8ϩ cells is associated with delayed clearance of oblv60 (but infected mice did recover). 25 ctls probably exacerbate lesions by contributing to tissue damage. mhv is relatively labile genetically (a common characteristic of rna viruses), and mutant strains with recognition sites that can evade ctl-mediated viral killing apparently increase and become the predominant viral strains in response to ctl-mediated natural selection. this feature results in disease progression in immunocompetent, but not immunosuppressed, hosts infected with these mutants. 68 several studies have suggested that apoptosis may be important in clearing virus from the cns. specific ctl recognition promotes apoptosis of mhv-infected cells. 69 in experimental allergic encephalomyelitis in rats, apoptosis appears to help control inflammation. 70 the role of cd4ϩ cells in mhv infection is also complex. nucleocapsid or spike protein-specific cd4ϩ t-cells have been shown to protect mice from coronaviral encephalomyelitis in the absence of cd8ϩ t-cells. 71 if mice with mhv-oblv60 had cd8ϩ but not cd4ϩ cells, they developed persistent infection. if they were cd8ϩ deficient, but cd4ϩ cells were normal, they had delayed clearance of the virus, suggesting a primary role for cd4ϩ cells in clearing this virus. 25 sussman et al 72 and williamson and stohlman 73 documented the requirement for cd4ϩ cells for clearance of jhm from mice. however, ␥-irradiated mice that were reconstituted with cd4ϩ cells responded to mhv-jhm challenge with earlier and more severe onset of neurological disease. 74 cd4ϩ knockout mice had less inflammation (with fewer macrophages and microglial cells) and less demyelination than did cd4ϩ competent mice. 75 the presence of mhv-jhm in astrocytes triggers a cytokine cascade that contributes to demyelination. 76 sun et al 77 documented production of tnf-␣, il-1␤, and il-6 by astrocytes in the spinal cords of mice that were chronically infected with mhv-jhm, localized to areas of virus infection and demyelination. tnf-␣ and il-6 are also produced in the brains of acutely infected mice, but the major cell producing these cytokines is the macrophage. these cytokines may help recruit t-cells and monocytes and may increase vascular permeability. 78 il-1␤ promotes leukocyte adhesion to endothelial cells, and tnf-␣ is toxic to oligodendrocytes. 63 the role of il-6 is unclear. effects attributed to il-6 include recruitment and activation of t-cells and macrophages, expansion of ctls, modulation of plasma cell differentiation, increased vascular permeability, downregulation of acute phase proteins, and contributions to immune-mediated destruction in the cns. [79] [80] [81] [82] in a study of cytokine profiles in lethally compared to sublethally affected mice with jhm, both th 1 -and th 2type cytokines, including ifn-␥, il-4, and il-10, were induced in all mice. 83 in the mice that died, tnf-␣ was induced more rapidly, and il-1␣ was increased. in mice with nonlethal infections, il-12 and il-1␤ were increased, and il-6 was expressed early. minor differences were observed in the patterns of inflammation in il-10-deficient compared to syngeneic mice, but the outcome of infection (eg, mortality and virus load) was not affected. 84 these results, combined with those of sun et al, 77 suggest that il-1␤ may allow mice to survive the early stages of the disease but may not allow for clearance of chronic infection. interferon-␥ also appears to be important for clearance of mhv infection. ifn-␥-deficient mice developed persistent jhm infection with increased clinical signs and mortality compared to mice competent to produce ifn-␥. 85 antiviral antibody and ctl responses were normal in ifn-␥-negative mice, despite the fact that ifn-␥ modulates antibody production. viral antigen occurred in oligodendrocytes and in association with cd8ϩ t-cells, suggesting that ifn-␥ is necessary for control of viral replication in oligodendrocytes. treatment of mice with anti-ifn-␥ antibody increased the mortality rate of mice, whereas immunotherapy with ifn-␥ reduced mortality and virus load. 86 mhv-oblv60 infection in immunocompetent mice induced transient mrna upregulation for cytokines il-1␣, il-1␤, il-6, tnf-␣, and ifn-␥. 25 nude mice differed in their cytokine profiles in that they lacked mrna for ifn-␥, whereas concentrations of the other cytokines remained persistently high. the authors suggested that the increased cytokines in the nude mice were produced by cns glial cells and possibly cd4ϩ and cd8ϩ subsets. ifn-␥ treatment of human neuronal cell cultures markedly increased the susceptibility of cells to infection with hcv-oc43 (although cells were susceptible to 229e without ifn-␥ treatment). 87 in this study, the hypothesized role of ifn-␥ was induction of mhc class i expression. ifn-␥ may increase superoxide dismutase and protect against oxygen radicals, kill virus directly, and increase the cytotoxicity of other effector cells. the chemokine crg-2 is expressed primarily by astrocytes during mhv infection and co-localizes with areas of viral rna and demyelination. 88 the function of crg-2 is not known, but this chemokine is also found in simian immunodeficiency viral (siv) encephalitis 89 and lymphocytic choriomeningitis. 90 with very little information available about the immunopathogenesis of neurologic fip, research performed in mice can be used as a model for fip and to contrast findings from the mhv model with those in fip. the immunopathogenic mechanisms are expected to have important similarities, but the demyelination that occurs in mhv is a more extensive pathological sequela of coronaviral infection (but not more fatal for the patient) than the focal granulomas that occur in fip. it can be hypothesized that in fip, upregulation of cytokines il-1␤, il-6, ifn-␥, and tnf-␣ may be important in mediation of destructive inflammation, but cytokines il-6 and ifn-␥ may be important in controlling viral infection, despite adverse inflammatory effects. cytokine and chemokine alterations in the brains of mice and cats with coronavirus infections may provide valuable clues to the immunopathogenesis of these diseases, as well as possible diagnostic markers and targets for immunological treatment of disease. it is tempting to consider cytokine modulation in the treatment of fip, but this application is premature without information regarding the cytokine profiles of affected cats. in addition to characterizing the concentrations of cytokines in the cns of cats with fip, it will be important to describe the timing of upregulated cytokine transcription. in mice with hmv-jhm, treatment with recombinant ifn-␥ resulted in reduced virus load in the liver but not in the brain. 86 if specific pro-inflammatory cytokines are found to be consistently high in cats with neurological fip, use of cytokine antagonists may eventually be of benefit in treatment. successful management of fip may consist of better methods of prevention as well as management of disease once it occurs. a commercial fip vaccine is available that consists of a mucosally delivered, temperature-sensitive mutant form of fipv. the vaccine virus is supposed to undergo replication only in outer oronasal cavities at low temperatures, thus triggering protective antibodies but not fip. a few controversial studies have documented reduction in fip as a result of vaccination. 91, 92 many vaccinated cats, however, fail to seroconvert with either igg or iga (foley and pedersen, unpublished data) , and independent studies failed to identify vaccine efficacy. 93, 94 other vaccines, including dna vaccines, have not progressed beyond experimental stages either because of lack of seroconversion, lack of protection, or both. in contrast with fip vaccines, vaccines against mhv have been shown to protect mice from challenge with virulent virus. a vaccine with purified spike protein 95 and synthetic s2 led to neutralizing antibodies in vivo. 96, 97 recombinant subunits against s in tobacco mosaic virus were given subcutaneously or intranasally and were protective against mhv-jhm. 98 the current standard of care for immunomodulation of fip is immunosuppressive doses of prednisone, to sustain an acceptable quality of life for as long as possible. the effects of steroids include lymphocytolysis, inhibition of arachidonic acid metabolism, reduction in cytokine rna transcription, and nitric oxide synthesis inhibition. 99 other potentially useful antiinflammatory drugs include antileukocyte antibodies and antioxidants. these treatments are only palliative. a better understanding of the fundamental pathogenesis of fip will be necessary to offer better treatment and, ultimately, cure of this disease. the inheritance of susceptibility to feline infectious peritonitis in purebred catteries two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus a comparison of the genomes of fecvs and fipvs and what they tell us about the relationships between feline coronaviruses and their evolution feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses feline coronavirus type ii strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type i and canine coronavirus the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf7a/7b transcription unit of different biotypes a novel glycoprotein of feline infectious peritonitis coronavirus contains a kdel-like endoplasmic reticulum retention signal proteolytic 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interferon gamma potentiates human coronavirus oc43 infection of neuronal cells by modulation of hla class i expression dynamic regulation of alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease chemokine expression in simian immunodeficiency virus-induced aids encephalitis chemokine gene expression in the brains of mice with lymphocytic choriomeningitis vaccination against naturally occurring fip in a single large cat shelter overview of the development of a modified live temperature-sensitive fip virus vaccine independent evaluation of a modified live feline infectious peritonitis virus vaccine under experimental conditions (university of liverpool experience) independent evaluation of a modified live fipv vaccine under experimental conditions (cornell experience) protection from lethal coronavirus infection by affinity-purified spike glycoprotein of murine hepatitis virus, strain a59 vaccination against lethal coronavirus-induced encephalitis with a synthetic decapeptide homologous to a domain in the predicted peplomer stalk immunogenic peptide comprising a mouse hepatitis virus a59 b-cell epitope and an influenza virus t-cell epitope protects against lethal infection protective immunity against murine hepatitis virus (mhv) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an mhv epitope adjunctive therapy for bacterial meningitis: rationale for use, current status, and prospects for the future key: cord-323805-9n63ms3c authors: pedersen, niels c.; liu, hongwei; gandolfi, barbara; lyons, leslie a. title: the influence of age and genetics on natural resistance to experimentally induced feline infectious peritonitis date: 2014-11-15 journal: vet immunol immunopathol doi: 10.1016/j.vetimm.2014.09.001 sha: doc_id: 323805 cord_uid: 9n63ms3c naturally occurring feline infectious peritonitis (fip) is usually fatal, giving the impression that immunity to the fip virus (fipv) is extremely poor. this impression may be incorrect, because not all cats experimentally exposed to fipv develop fip. there is also a belief that the incidence of fip may be affected by a number of host, virus, and environmental cofactors. however, the contribution of these cofactors to immunity and disease incidence has not been determined. the present study followed 111 random-bred specific pathogen free (spf) cats that were obtained from a single research breeding colony and experimentally infected with fipv. the cats were from several studies conducted over the past 5 years, and as a result, some of them had prior exposure to feline enteric coronavirus (fecv) or avirulent fipvs. the cats were housed under optimized conditions of nutrition, husbandry, and quarantine to eliminate most of the cofactors implicated in fipv infection outcome and were uniformly challenge exposed to the same field strain of serotype 1 fipv. forty of the 111 (36%) cats survived their initial challenge exposure to a type i cat-passaged field strains of fipv. six of these 40 survivors succumbed to fip to a second or third challenge exposure, suggesting that immunity was not always sustained. exposure to non-fip-inducing feline coronaviruses prior to challenge with virulent fipv did not significantly affect fip incidence but did accelerate the disease course in some cats. there were no significant differences in fip incidence between males and females, but resistance increased significantly between 6 months and 1 or more years of age. genetic testing was done on 107 of the 111 infected cats. multidimensional scaling (mds) segregated the 107 cats into three distinct families based primarily on a common sire(s), and resistant and susceptible cats were equally distributed within each family. genome-wide association studies (gwas) on 73 cats that died of fip after one or more exposures (cases) and 34 cats that survived (controls) demonstrated four significant associations after 100k permutations. when these same cats were analyzed using a sib-pair transmission test, three of the four associations were confirmed although not with genome-wide significance. gwas was then done on three different age groups of cases to take into account age-related resistance, and different associations were observed. the only common and strong association identified between the various gwas case configurations was for the 34.7–45.8 mb region of chromosome a3. no obvious candidate genes were present in this region. naturally occurring feline infectious peritonitis (fip) is usually fatal, giving the impression that immunity to the fip virus (fipv) is extremely poor. this impression may be incorrect, because not all cats experimentally exposed to fipv develop fip. there is also a belief that the incidence of fip may be affected by a number of host, virus, and environmental cofactors. however, the contribution of these cofactors to immunity and disease incidence has not been determined. the present study followed 111 random-bred specific pathogen free (spf) cats that were obtained from a single research breeding colony and experimentally infected with fipv. the cats were from several studies conducted over the past 5 years, and as a result, some of them had prior exposure to feline enteric coronavirus (fecv) or avirulent fipvs. the cats were housed under optimized conditions of nutrition, husbandry, and quarantine to eliminate most of the cofactors implicated in fipv infection outcome and were uniformly challenge exposed to the same field strain of serotype 1 fipv. forty of the 111 (36%) cats survived their initial challenge exposure to a type i cat-passaged field strains of fipv. six of these 40 survivors succumbed to fip to a second or third challenge exposure, suggesting that immunity was not always sustained. exposure to non-fip-inducing feline coronaviruses prior to challenge with virulent fipv did not significantly affect fip incidence but did accelerate the disease course in some cats. there were no significant differences in fip incidence between males and females, but resistance increased significantly between 6 months and 1 or more years of age. genetic testing was done on 107 of the 111 infected cats. multidimensional scaling (mds) segregated the 107 cats into three distinct families based primarily on a common sire(s), and resistant and susceptible cats were equally distributed within each family. genome-wide association studies (gwas) on 73 cats that died of fip after one or more exposures (cases) and 34 cats that survived (controls) demonstrated four significant associations after 100k permutations. when these same cats were analyzed using a sib-pair transmission test, three of the four associations were confirmed although not with genome-wide significance. gwas was then done on three different age groups of cases to take into account age-related resistance, and different associations were observed. the prevalence and severity of infectious diseases among multi-cat populations is a product of many diverse factors that affect the host/pathogen interaction (pedersen, 1991) . environmental factors include things such as population density, sanitation, and interchange of animals while agent factors include virulence, dose, and route of exposure. host factors include developmental and heritable anomalies in the immune system and age at the time of exposure and intercurrent illnesses. many of these diverse cofactors have been implicated in fip. foley et al. (1997) studied a number of environmental risk factors for fip in seven catteries and found that cat numbers (density) and husbandry procedures had no influence on fip incidence while age, high coronavirus antibody titers, and the proportion of cats shedding coronavirus were significantly associated with fip risk. all of these risk factors are interrelated, because fecal coronavirus shedders are much more likely to have antibody titers >1:100 and younger cats are more likely to shed fecv at higher levels and for longer periods (pedersen et al., , 2008 . the stresses of placing young cats into shelters have also been shown to greatly increase the levels of fecv shedding . field strains of fipv are known to vary intrinsically in virulence and this virulence may be further affected by the route of administration (pedersen et al., 1984; pedersen and floyd, 1986) . the dose of virus used also can alter disease outcome although a dose that causes lethal infection in one cat may be insufficient to infect another (pedersen and black, 1983) . virulence may be influenced by the exact fip-inducing mutations that are present. the known fipassociated mutations in fecv 3c and the s1/s2 cleavage site are highly variable and unique to each isolate while the two single nucleotide mutations in the fusion domain are common to all fipvs (pedersen, 2014) . mutations in 7b can also alter virulence in some tissue culture-adapted strains, but do not play a role in the fecv-to-fipv mutations in nature (pedersen, 2014) . additional mutations may await discovery and their singular or collective roles in fip remain to be determined. several host factors have been implicated in fip. the stress of surgery, especially when performed at a young age, may increase susceptibility of cats to fip development (kass and dent, 1995) . co-infections with felv will greatly increase the incidence of fip by interfering with fip immunity; more than one-third of all fip cases occurred in cats that were persistently infected with felv (cotter et al., 1973; pedersen et al., 1977) . feline immunodeficiency virus (fiv) can also compromise host immunity and increase fip prevalence under experimental conditions (poland et al., 1996) . the present study was designed to eliminate as many potential agents, environmental, and host risk factors for fipv infection as possible. the same field strain and infectious dose of virus were used for challenge exposure, a uniform standard of care was provided with no extraneous pathogen exposure, and the cats originated from the same breeding stock. the study was then concentrated on two potential risk factors that have been poorly studied, age at the time of exposure and genetic susceptibility. the effect of age on fipv infection has not been directly addressed, even though it has been previously discussed (pedersen, 2009 ) and well documented for pathogens such as feline leukemia virus (felv) (hoover et al., 1976) . kittens are born with immature immune systems, and the period between 4 and 16 weeks of age is when igg and iga systems are being compensated by passive local and systemic immunity (pedersen, 1987a) . immaturity of the immune system may also play a role in the ability to vaccinate kittens to fip; a commercially marketed attenuated live fipv vaccine only demonstrated sufficient efficacy for licensing when given to kittens 16 weeks or older (gerber et al., 1990) . field and laboratory studies indicate that some sort of maternal or innate resistance to fecv infection is present in neonatal kittens and that fecv fecal shedding usually does not occur until 9 weeks of age, even among kittens born to infected queens (pedersen et al., 2008) . most cases of fip occur in cats between 4 and 18 months of age (reviewed pedersen, 2009 ) suggesting that some infections may remain subclinical for an extended period of time. the possible role of genetics in fip resistance has been implied from a number of studies. fip did not exist before the 1950s (holzworth, 1963) , suggesting that cats may not have had time to genetically adapt, thus explaining why morbidity and mortality are so high in experimental fipv infections. pedigreed cats are more likely to develop fip than random-bred cats (robison et al., 1971; rohrbach et al., 2001; pesteanu-somogyi et al., 2006; worthing et al., 2012) , and certain breeds are also more likely to succumb to fip (bell et al., 2006; norris et al., 2005; pesteanu-somogyi et al., 2006; worthing et al., 2012) . one study of persian catteries and pedigrees indicated that susceptibility to fip was at least 50% heritable . resistance to fip in birman cats also appears to have a genetic component as determined by gwas (golovko et al., 2013) . natural resistance to fip has also been observed in up to one-third of random-bred cats used as controls in vaccine studies (baldwin and scott, 1997; gerber et al., 1990; glansbeek et al., 2002; hohdatsu et al., 2003; kiss et al., 2004; pedersen and black, 1983; wasmoen et al., 1995) . the cats, infection outcome data, and dna used in the present study originated from studies on type 1 fipv and fecv conducted over the last several years with other objectives. over the course of these studies, 111 cats of various age and gender were exposed one or more times to virulent strains of fipv and their disease course closely monitored and cause of death confirmed to be fip. forty of the 111 cats resisted a single challenge exposure and 34 remained resistant after repeated infections. the studies were unique in that all of the cats were housed in identical facilities, cared for in an identical manner, and maintained free from other feline pathogens. therefore, they were not affected by many of the agents, environmental, and host factors that might affect the incidence of fip in nature. this allowed for an uncomplicated assessment of risk factors such as age, gender, and genetic susceptibility on disease outcome. cats were obtained from the specific pathogen free (spf) breeding colony of the feline nutrition and pet care center, university of california, davis (uc davis) (uc davis iacuc #16988). the colony was established in 1976 with a small number of cats derived aseptically by cesarean section and records on all matings have been maintained to the present time. mating pairs were selected based on degree of relatedness and outcrossing to enhance genetic diversity done on two occasions, 1995 and 1999. the relationships of all cats were known from the colony records. cats used for this study were housed in the feline research laboratory of the center for companion animal health under conditions required by usda regulations. fifty-four of the 111 of cats were coronavirus naïve while 57 had previous fecv or non-virulent fipv exposure (pedersen et al., 2008 (pedersen et al., , 2012 . experimental infection studies were conducted under uc davis institutional animal care and use committee protocol #16637. the origins of type i fipv-i3c2 and fipv-m3c2 and the preparation of cell-free infectious inoculates have been previously described (pedersen et al., 2012) . a total of 1 ml of a 1:5-1:10 dilution of a 25% cell-free suspension of diseased omentum was given by either the intraperitoneal (ip) or oronasal (on) route. this proved infectious to 100% of cats by either route based on the occurrence of disease and/or seroconversion. cats were sedated with ketamine hydrochloride and inoculated either intraperitoneally (ip) or on (0.5 ml orally, 0.5 ml nasally) with the various virus stocks. rectal temperatures were recorded starting 1-2 days prior to inoculation and at 1-2 day intervals thereafter. cats were examined daily for signs of disease, such as fever, inappetance, depression, diarrhea, dehydration, ascites, hyperbilrubinemia, hyperbilirubinuria, and jaundice. affected cats were euthanized with an intravenous overdose of pentabarbital/ phenytoin as soon as their disease course was deemed terminal. antibodies to feline coronavirus were titrated by indirect immunofluorescence using crandell-rees feline kidney cells infected with fipv-79-1146 as an antigen substrate (pedersen, 1976) . whole edta-treated blood was available from 107 of 111 cats and genomic dna isolated using the qiagen (valencia, ca) gentra puregene blood core kit. gwas was performed using the illumina infinium iselect feline dna array (illumina inc., san diego, ca). the arrays were tested by geneseek inc. (lincoln, ne). snp genotyping rate and minor allele frequency (maf) was evaluated using plink (purcell et al., 2007) . snps with a maf < 5%, genotyping rate < 90%, and individuals genotyped for <90% of snps were excluded from downstream analyses. an mds with two dimensions was performed on 41,004 snps in plink to evaluate population substructure within cases and controls. inflation of p-values was evaluated by calculating the , and assessed with a q-q plot. a casecontrol whole genome association analysis was performed and corrected with 100,000 t-max permutations (-mperm 100,000) with significance at −log10 (pgenome) ≥ 1.3. the transmission disequilibrium test among sib-pairs (sib-tdt) (spielman and ewens, 1998) was performed on 18 phenotypically discordant sib-pairs using the function (-dfam). the sib-tdt analysis was conducted without including the founders in frequency calculation (-nonfounders). one hundred eleven cats were experimentally infected with virulent fipv either by the ip or on routes, and the disease outcome ultimately confirmed either by necropsy or seroconversion. there was no difference in challenge outcome between the two routes (data not shown). fifty-seven cats had one or two prior exposures to fecv, non-infectious fipv mutants, or sub-infectious doses of virulent fipv. thirty-three of these 57 (58%) cats developed fatal fip, compared to 38 of 54 (70%) of naïve cats after experimental infection with virulent fipv (fig. 1) , which was not significantly different (p = 0.24, fisher's exact test). the strength of immunity was tested by re-challenge. twenty two of 24 (92%) of the pre-sensitized survivors and 14 of 16 (88%) survivors without prior coronavirus exposure were still resistant after a second challenge-exposure (fig. 1) . thirteen survivors from both groups were then exposed to fipv a third time, and 11 of 13 (85%) remained resistant (fig. 1) . one cat survived a fourth infection and another survived five exposures (fig. 1) . the onset of disease after fipv infection always coincided with the appearance of fever (fig. 2) , which was rapidly followed by other signs such as inappetence, lethargy, cessation of grooming, hyperbilirubinemia, hyperbilirubinuria, jaundice, and ascites. in contrast, cats that resisted disease showed virtually no febrile response, remained outwardly normal, and seroconverted (fig. 2) . pre-sensitization to non-disease-causing feline coronaviruses did not significantly alter the mortality rate although cats with prior exposure were somewhat more likely to develop accelerated disease (fig. 3) . all of the cats with prior coronavirus exposure became terminally ill within 31 days while five cats without prior exposure survived from 33 to 105 days. four of these five slow progressors died of non-effusive of fip and one of effusive fip. survival rates were examined for cats of different gender and age. no difference was observed in fip incidence between male and female cats (data not shown). there was a progressive and significant (p = 0.0008) decrease in mortality from 6 months to greater than 1 year of age (fig. 4) . over 80% of cats younger than 6 months of age died compared to less than 45% of cats infected at greater than 1 year of age. cats from 6-12 months of age were intermediate in susceptibility. fip is known to persist in a subclinical form for some period of time following survival from challenge exposure to fipv (pedersen, 1987b) and this has confounded the interpretation of survival data in past fip vaccine studies (baldwin and scott, 1997; hoskins et al., 1994) . to rule out subclinical infections among resistant cats in the present study, six individuals that had survived two or more challenge exposures were necropsied after 4-6 months and examined for subclinical lesions. no gross evidence of subclinical disease was found. therefore, most cats that survived fipv challenge will eventually clear the infection if given enough time. genome-wide association studies were conducted on 107 of the 111 infected, including 73 cases and 34 controls. plink analysis showed snps with genome-wide significance on chromosomes a3, b1, b4, and c1 (fig. 5) . a fifth snp with genome-wide significance was present among non-annotated snps (uk) but was not further investigated. in order to determine any effect of relatedness on gwas of the total population, family-related substructure was determined by multidimensional scaling (mds). mds segregated all case and control cats into three separate families (a, b, c) (fig. 6) . cats from family a were sired by multiple related cats while cats in families b and c were each descended from a single sire. there was no significant difference in how fip resistant and susceptible cats segregated between and within families (fig. 6) . the family substructure identified by mds was amended using a sib-tdt analysis with 18 phenotypically discordant nuclear families. after permutation, none of the snps remained genome-wide significant although strong associations were again observed on chromosomes a3, b1, and c1, the association on b4 was lost, and two new associations occurred on chromosomes c2 and d3 (fig. 7) . it was apparent that age at the time of exposure was a significant independent risk factor for disease outcome. therefore, an attempt was made to compensate for age in the selection of cats used for gwas (fig. 8) . the control group of cats remained the same based on the assumption that if a cat survived fipv infection at <6 months of age, it would also survive exposure at 6 months and older. conversely, a cat that died when exposed at <6 months of age might have survived if infected at >6 months of age fig. 7 . sib-transmission/disequilibrium test of 54 cats that died of fip and 24 survivors using the -dfam and 100,000 permutation command in plink. five peaks of strong association were identified on defined chromosomes. four of the five associations were near potential candidate genes relevant to fip immunopathogenesis. independent on any genetic factors. although the population size of case and controls was similar for each age group tested by gwas, there were marked differences in the major genome-wide associations seen on manhattan plots depending on the age of the case cats at the time of fipv exposure (fig. 8) . the only strong association in common with these three age-adjusted gwas studies and the total case/control population was for a region on chromosome a3 that extended from 34.7-45.8 mb (table 1) . it was also noteworthy that a disproportionate number of the highest 25 ranking snps fell into this region, regardless of the configuration of case and controls based on age (fig. 8) , relatedness (fig. 7) , or on neither of these factors (fig. 6) . based on ensembl, this region contains 41 protein-coding genes and 9 novel protein-coding transcripts. none of the 41 genes appeared to be obvious candidates for immune or inflammatory processes involved in fip. the goal of this study was to identify cofactors that were most strongly involved with natural resistance to fipv infection. this was accomplished by negating as many potential cofactors as possible using a standardized virus challenge, cats from the breeding facility, optimal husbandry, providing a uniform environment and diet, minimizing extraneous stresses, and eliminating the effects of other common infections that might occur in multi-cat environments such as catteries or shelters. after minimizing the agent, environmental, and host cofactors, the opportunity existed to study host-related factors such as genetics, age, and gender on fip resistance. the present study also dealt with the strength of immunity, which does not appear to be absolute. about 10% of cats that survived one fipv infection succumbed to a second or third exposure. a similar occurrence was observed by wasmoen et al. (1995) ; one of five cats that had successfully resisted a challenge exposure that killed 4 of 5 non-vaccinates developed fip upon a second exposure. this type of immunity is different from that established by feline panleukopenia, a parvovirus disease. panleukopenia immunity is usually solid and is more dependent on humoral than cellular responses (scott, 1987) . therefore, fipv immunity more closely resembles immunity to its parent virus, feline enteric coronavirus (fecv). fecv-infected cats shed virus in their feces for weeks or months before sufficient immunity develops to stop shedding, but after shedding ceases, antibody levels fall and many of the cats become susceptible to reinfection (pedersen et al., 2008) . subclinical disease is also known to linger after initial natural and experimental infection in some cats as demonstrated by felv activation (pedersen, 1987b; pedersen et al., 1977) . this was the first study documenting the significance of age at time of exposure on fipv outcome, even though it has been frequently cited as a disease cofactor (pedersen, 2009 ). immunity to experimental fipv infection increased progressively from 4 months of age through adulthood. gerber et al. (1990) also reported an age-related response to an attenuated live fipv vaccine, with significant protection only observed when vaccination was started at 16 weeks of age. the effect of age on disease outcome is well known for infectious disease agents such as feline leukemia virus (hoover et al., 1976) . age resistance to felv increases dramatically during kittenhood as the immune system matures and has confounded felv vaccine duration of immunity studies (wilson et al., 2012) . gender, in particular intact males, has been reported as a risk factor for fip in other studies (norris et al., 2005; pesteanu-somogyi et al., 2006; rohrbach et al., 2001) . we did not see a gender bias in the present study, nor was it seen in an earlier study of purebred and random-bred cats (foley et al., 1997) . a large component of the present study involved attempts to associate fip resistance to specific genetic markers by gwas. previous experience with a large cohort of inbred birman cats (golovko et al., 2013) suggested that this approach could be applied to the present cohort of randomly bred cats. however, the same population substructure problems encountered in the birman study were faced in this study. gwas comparing all cases and controls demonstrated four significant genome-wide associations on several chromosomes and some possible candidate genes. however, there was considerable population substructure as revealed by mds and attributed to separate male founder effects. population substructure due to relatedness in a case-control study can be overcome by using different types of analysis, such as the transmission disequilibrium test (tdt) or the sib-tdt that was employed in this study. a previous gwas study localized the autosomal recessive locus associated with hypokalemia in cats by analyzing as few as 35 cases and 25 controls (gandolfi et al., 2012) . however, the present study was conducted on random-bred cats, which are known to have less linkage disequilibrium than within pedigreed cats (alhaddad et al., 2013) . the study was further confounded by the polygenic appearance of the inheritance. inheritance to fip resistance/susceptibility in a similarly sized cohort of birman cats also appeared to be polygenic and there were no common regions of association, which would have reinforced both studies (golovko et al., 2013) . to compensate for family-related substructure, a transmission disequilibrium test among sib-pairs using the statistics of spielman and ewens (1998) was then performed. eighteen discordant sib-pair nuclear families were identified within the cohort, which was more than the 13 phenotypically discordant sib-pairs that successfully detected the association with a cone-rod dystrophy in dogs (wiik et al., 2008) . based on sib-tdt on the fip cohort, five strong snp associations on different chromosomes were identified, but none reached genomewide significance. snps on chromosomes a3, b1, and c1 were shared by the two different analyses while two new associations on chromosomes c2 and d3 appeared. although the associations detected by sib-tdt did not reach genome-wide significance after permutations, similar regions were suggested by both analyses within the three overlapping chromosomes. it is possible that these regions could reach genome-wide significance if more discordant sib-pairs are added to the association analysis. an attempt was also made to compensate for age at the time of exposure as an independent and presumably non-genetic risk factor for fip resistance. unfortunately, the number of case and control cats challenge exposed after 1 year of age was too low, so cats exposed at >6 months and >1year were combined. the control population remained the same for all gwas configurations based on the premise that kittens surviving exposure at less than 6 months of age would still resist exposure as they aged. as was expected based on previous gwas configurations, relatively small changes in the case populations had a marked effect on observed associations. after comparing the results of gwas based on age, gwas of the total population, and gwas based on family structure, only one peak of strong, but not genome-wide significant, associations were present on chromosomes a3 in a region between 34.8 and 46 mb. thirty five annotated genes were present within this region, but none appeared to be strong candidates for fip resistance. it can be concluded from these various gwas studies that resistance to fip in this population of relatively random-bred spf cats was not influenced by a single or even small number of genes. as in an earlier study with a much more inbred birman population (golovko et al., 2013) , any genetic component of resistance is likely to be polygenic and divergent between various populations. although mutations in a single gene have been identified that confer resistance to infectious disease, such as the ccr5 mutation for hiv infection (dean et al., 1996) , susceptibility and resistances to infectious agents clearly involve complex host/virus/environment interactions that make genetic studies difficult. this has been shown in diseases such as human and ruminant tuberculosis (chimusa et al., 2014; le roex et al., 2013) , a disease that closely resembles the dry form of fip. the existence of additional risk factors, involving the environment, host, and agent, is perhaps one of the most daunting problems in the search for genetic influences on infectious diseases. this study removed a large number of those confounding factors but was still unable to identify specific genes that might be involved in fip resistance. unfortunately, even highly inbred breeds, such as birman, with significant linkage disequilibrium and closed colonies, such as the one in this study, suffer from high genomic inflations. even so, the strong associations demonstrated in this relatively small gwas employing a relatively low-density array indicate that fip resistance is influenced in some part to genetic factors. the heritability of these genetic factors remains a subject of ongoing breeding studies. we did not interrogate one region on chromosome a3 that was consistently found to differ in association between all of the various gwas configurations. hopefully, the present data can be reanalyzed as the cat genome annotation improves and more dense arrays become available. next-generation and whole exome sequencing are also becoming cost accessible and might be preferable ways to search for complex genetic associations and specific mutations. it might also be fruitful to mate immune cats to see if resistance is heritable and if so, to do gwas or next-generation sequencing on their offspring. the objective of this study was to define natural immunity to fip among randomly bred specific pathogen-free cats bred for laboratory purposes under conditions that would eliminate as many extrinsic disease cofactors as possible. cats were housed free of other feline pathogens and fed and cared for in a uniform manner. this emphasized the relative influence of age at the time of exposure, strength of immunity as gauged by repeated challenge exposure, and possible genetic resistance. one-third of random-bred laboratory cats used in various studies over the last decade appeared to be resistant to infection with type i field strains of fipv. however, immunity was not absolute and a small number of cats died after a second and even third challenge. age at the time of exposure seemed to be the most significant predictor of resistance; cats under 6 months of age were most apt to develop fip, cats 6-12 months were intermediate, and cats over 12 months of age demonstrated significant resistance. strong genetic associations were identified by gwas in regions of several chromosomes, especially when comparing all cats that died of fip with all survivors. however, all but one of these regional associations changed when gwas was adjusted for family substructure or age at the time of fipv exposure. this confirmed previous gwas studies on fip resistance in birman cats (golovko et al., 2013) ; both studies showed inheritance of fip resistance to be highly complex and confounded by considerable population stratification. future breeding studies will hopefully confirm the heritability of fip resistance. the authors declare no conflicts of interest. extent of linkage disequilibrium in the domestic cat, felis silvestris catus, and its breeds attempted immunization of cats with feline infectious peritonitis virus propagated at reduced temperatures the relationship between the feline coronavirus antibody titre and the age, breed, gender and health status of australian cats genome-wide association study of ancestry-specific tb risk in the south african coloured population multiple cases of feline leukemia and feline infectious peritonitis in a household genetic restriction of hiv-1 infection and progression to aids by a deletion allele of the ckrs structural 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infectious peritonitis in catteries disease outcome and cytokine responses in cats immunized with an avirulent feline infectious peritonitis virus (fipv)-ucd1 and challenge-exposed with virulent fipv-ucd8 bovine tb in livestock and wildlife: what's in the genes? clinicopathological findings associated with feline infectious peritonitis in sydney, australia: 42 cases an update on feline infectious peritonitis: virology and immunopathogenesis a review of feline infectious peritonitis virus infection: 1963-2008 feline husbandry. in: diseases and management in the multiple-cat environment basic and clinical immunology virologic and immunologic aspects of feline infectious peritonitis virus infection serologic studies of naturally occurring feline infectious peritonitis attempted immunization of cats against feline infectious peritonitis using either avirulent live virus or sublethal amounts of virulent virus experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd2, fipv-ucd3, and fipv-ucd4. compendium feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats significance of coronavirus mutants in feces and diseased tissues of cats suffering from feline infectious peritonitis pathogenesis of feline enteric coronavirus infection common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus pathogenic differences between various feline coronavirus isolates studies of naturally transmitted feline leukemia virus infection prevalence of feline infectious peritonitis in specific cat breeds two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus plink: a tool set for whole-genome association and population-based linkage analyses naturally occurring feline infectious peritonitis: signs and clinical diagnosis epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals viral diseases (panleukopenia) a sibship test for linkage in the presence of association: the sib transmission/disequilibrium test protection of cats from infectious peritonitis by vaccination with a recombinant raccoon poxvirus expressing the nucleocapsid gene of feline infectious peritonitis virus a deletion in nephronophthisis 4 (nphp4) is associated with recessive cone-rod dystrophy in standard wire-haired dachshund difficulties in demonstrating long term immunity in felv vaccinated cats due to increasing agerelated resistance to infection risk factors for feline infectious peritonitis in australian cats funds for this study were provided over a period of many years by organizations such as the center for companion animal health, school of veterinary medicine, uc davis, winn feline health, and the cat health network grant d12fe-516 (a consortium of the morris animal foundation, the winn feline foundation, the american association of feline practitioners and the american veterinary medical foundation). we are also grateful for the many private donations that have been made by individuals and private groups such as save our cats and kittens fip (sock fip). key: cord-268492-0rbmqarx authors: alberer, martin; von both, ulrich title: cats and kids: how a feline disease may help us unravel covid-19 associated paediatric hyperinflammatory syndrome date: 2020-09-02 journal: infection doi: 10.1007/s15010-020-01515-3 sha: doc_id: 268492 cord_uid: 0rbmqarx nan to the editor, with great interest we have been following latest news and reports on clusters of paediatric patients displaying clinical features of hyperinflammatory shock and a possible association with a sars-cov-2 infection [1] . although most children show only a mild and uncomplicated course of covid-19, in a small subset of paediatric patients severe symptoms including hyperinflammatory state, persistent fever, circulatory shock, and evidence of organ dysfunction have been reported. this novel syndrome is still a puzzle for clinicians and scientists since its underlying pathology is poorly understood making targeted treatment and preventive measures difficult. similarities to children presenting with kawasaki disease (kd) have been reported in some of these critically ill children while some of them predominantly display features of toxic shock, such as seen in severe staphylococcal or streptococcal infection. the rcpch and cdc have published a case definition and scientists refer to this novel but still very rare severe clinical condition in children as "paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2" (pims-ts). while reflecting on this syndrome and its characteristic features, some interesting similarities come to mind when comparing the clinical course of pims-ts cases and the specific features of a disease in cats called feline infectious peritonitis (fip) caused by the feline coronavirus (fcov), an alphacoronavirus [2] . both diseases show a predominance for the young. cats are predominantly affected between 4 and 16 months of age which would relate to children and young persons in humans. initially, a seemingly harmless viral gastrointestinal infection of the cat turns into a lifethreatening systemic infection. of particular note in this context, in two case series gastrointestinal symptoms have been the predominant feature of early pims-ts disease in almost all children [1, 3] . in cats, enterocytes are initially infected causing mostly mild gastrointestinal disease; but in a subset of infected cats (about 5%) a severe systemic infection arises after a variable period of time ranging from 2 weeks up to several months. in fip, fibrinous and granulomatous serositis, protein-rich serous effusion in body cavities and/or granulomatous lesions develop [2] . most of the children in the pims-ts case series of riphagen et al. also showed ascites and pleural effusions [1] . histopathologically, a granulomatous vasculitis is seen in fip which relates to features of the kawasaki syndrome although only small and medium sized vessels are primarily affected [2] . furthermore, fip shares additional features with severe covid-19. like in the human host, overexpression of inflammatory cytokines has been shown in fip, particularly for tnf-α, il-1β and il-6 [2, 4] . infected and subsequently activated monocytes/macrophages have been proposed to play a key role in this fulminant pathology [2] . in human covid-19, these cytokines have been associated with a severe course of disease and deemed to be the hallmark cytokines of the cytokine storm. infection of monocytes/macrophages with sars-cov-2 has been described, but it is still unclear whether these cells allow a permissive infection of the virus [5] . on the other hand, even non-permissive infection of macrophages and dendritic cells by sars-cov has been associated with significant overexpression of pro-inflammatory cytokines [6] . in addition, similar findings in white blood cell count, such as lymphopenia possibly caused by tnf-α-mediated apoptosis, are equally detected in both conditions and have been described in many human covid-19 cases as well as in fip [2] . the underlying pathophysiological mechanisms of fip are still unclear. an in vivo mutation of fcov affecting the viral spike protein (s-protein) and the occurrence of different pathogenic strains have been proposed in this context. on this note, it would be of great interest to see whether mutations in the viral genome, particularly in regions affecting the s-protein of sars-cov-2, could lead to a change in cell tropism enabling the virus to more effectively infect and replicate within human monocytes/macrophages subsequently leading to the clinical picture of pims-ts. unfortunately, results on the pathological and histopathological examinations on patients with pims-ts, i.e. post-mortem pathology, are still missing. closing this knowledge gap could help to clarify the underlying mechanisms of this challenging condition. finally, rna expression profiling of these cases in comparison to milder ones, like currently underway in the diamonds study (https ://www.diamo nds20 20.eu), will certainly add critical information on this aspect in the near future. despite the fact that sars-cov-2 belongs to the genus of betacoronavirus and fcov to the genus of alphacoronavirus the common link for both disease manifestations is the occurrence of multi-system vasculitis involving monocytes and macrophages as possible key players in the pathogenesis. with regards to other coronavirus infections, only for ferret systemic coronavirus (frsc) a similar disease entity has been reported which closely resembles the non-effusive (dry) form of fip. interestingly, also in sars-cov infection a vasculitis with infiltration of monocytes and involvement of small veins has been described, a characteristic feature of fip [7] . other human and animal coronaviruses usually cause gastrointestinal (e.g. canine coronavirus, transmissible gastroenteritis virus) and/or respiratory disease (e.g. canine respiratory coronavirus, human coronavirus oc43) varying in individual degree of severity [8] . infection of cats with sars-cov-2 is possible but up to now no infected animals showing symptoms and signs resembling fip or pims-ts have been reported. even though currently known receptors for both viruses, ace-2 and aminopeptidase n, are different, it has not been described that binding to these receptors triggers a receptorspecific effect besides mediating cell entry. furthermore, the receptor for the type i of fcov is not known yet. although exact pathophysiological pathways of both viral infections are unfortunately still unclear, both viruses seem to have the potential to manipulate and evade the innate immune response by means such as the production of accessory proteins (e.g. accessory proteins 7a, 3a, 3b and non-structural protein 5 (nsp5) in the case of fcov) or by interfering with the antiviral type i interferon response resulting in a hampered or delayed activation of this process [9, 10] . in the case of nsp5, this effect is mediated via negatively affecting the rig-i-like receptor pathway which has also been shown for sars-cov and is also likely to play a role in sars-cov-2 infection [10, 11] . in our opinion, both viruses essentially manipulate the infected monocyte/macrophage leading to enhanced cytokine production. this results in an optimized environment for viral replication due to induced t-cell apoptosis and upregulation of both ace-2 and aminopeptidase n, respectively. from a monocytic perspective, the cell is essentially locked in cytokine overdrive. interestingly, remdesivir, currently the only drug with proven benefit on the clinical course of covid-19, has also been successfully been used in the treatment of fip. we propose that fip could be a promising veterinary disease to learn more about sars-cov-2 activity in monocytes/macrophages and help to elucidate the mechanisms underlying the profound production of cytokines resulting in a severe cytokine storm and pims-ts. author contributions ma and ub designed structure and focus of the correspondence, undertook respective literature search and wrote the manuscript. funding open access funding provided by projekt deal. no external funding was received. conflict of interest both ma and ub declare that there are no conflicts of interest. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. hyperinflammatory shock in children during covid-19 pandemic infectious diseases of the dog and cat. amsterdam: elsevier kawasaki-like multisystem inflammatory syndrome in children during the covid-19 pandemic in paris, france: prospective observational study feline infectious peritonitis as a systemic inflammatory disease: contribution of liver and heart to the pathogenesis pathological inflammation in patients with covid-19: a key role for monocytes and macrophages dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice the clinical pathology of severe acute respiratory syndrome (sars): a report from china coronaviruses in cats and other companion animals: where does sars-cov-2/covid-19 fit? orf7-encoded accessory protein 7a of feline infectious peritonitis virus as a counteragent against ifn-alpha-induced antiviral response feline infectious peritonitis virus nsp5 inhibits type i interferon production by cleaving nemo at multiple sites covid-19: immunology and treatment options key: cord-271078-zyy8gx25 authors: sharif, saeed; arshad, siti s; hair-bejo, mohd; omar, abdul r; zeenathul, nazariah a; fong, lau s; rahman, nor-alimah; arshad, habibah; shamsudin, shahirudin; isa, mohd-kamarudin a title: descriptive distribution and phylogenetic analysis of feline infectious peritonitis virus isolates of malaysia date: 2010-01-06 journal: acta vet scand doi: 10.1186/1751-0147-52-1 sha: doc_id: 271078 cord_uid: zyy8gx25 the descriptive distribution and phylogeny of feline coronaviruses (fcovs) were studied in cats suspected of having feline infectious peritonitis (fip) in malaysia. ascitic fluids and/or biopsy samples were subjected to a reverse transcription polymerase chain reaction (rt-pcr) targeted for a conserved region of 3'untranslated region (3'utr) of the fcov genome. eighty nine percent of the sampled animals were positive for the presence of fcov. among the fcov positive cats, 80% of cats were males and 64% were below 2 years of age. the fcov positive cases included 56% domestic short hair (dsh), 40% persian, and 4% siamese cats. the nucleotide sequences of 10 selected amplified products from fip cases were determined. the sequence comparison revealed that the field isolates had 96% homology with a few point mutations. the extent of homology decreased to 93% when compared with reference strains. the overall branching pattern of phylogenetic tree showed two distinct clusters, where all malaysian isolates fall into one main genetic cluster. these findings provided the first genetic information of fcov in malaysia. feline infectious peritonitis (fip) is a highly fatal disease of cats caused by generalized infection with a feline coronavirus (fcov). fcovs belong to subgroup 1a of coronaviruses in the family coronaviridae, order nidovirales. other members of this subgroup include porcine transmissible gastroenteritis virus, canine coronavirus, raccoon/dog coronavirus and chinese ferret badger coronavirus [1, 2] . fcovs are enveloped, positive-strand rna viruses with a large, capped and polyadenylated rna genome of about 29 kb. the cap structure at the 5' end of genome is followed by an untranslated region (utr). at the 3' end of the genome is another utr of 275 nucleotides, followed by the poly (a) tail. the sequences of the both 3'-and 5'-utr are important for rna replication and transcription [3] . two biotypes of fcov are described in cats: feline infectious peritonitis virus (fipv) and feline enteric coronavirus (fecv). infection with fecv is usually unapparent or manifested by a transient gastroenteritis. in contrast, fipv infection causes a fatal immune-mediated disease with a wide spectrum of clinical signs. fip refers to the more common effusive (wet) form of the disease characterized by peritonitis and/or pleuritis. the effusive form is caused by complement-mediated vasculitis and results in inflammatory exudation into body cavities. in some fip cases, partial cell-mediated immunity cause non-effusive (dry) form which is characterized by granulomatous involvement of various organs particularly central nervous system and eyes. however, the fip forms can transform to each other [4] [5] [6] . it has been suggested that virulent fipv arises by mutation from parental fecv in the individual, persistently infected host [4, 7, 8] . it is not yet clear which alterations in the fcov genome are responsible for the generation of fipv from fecv [3, 6] . fip occurs worldwide and is ubiquitous in virtually all cat populations [6] . the disease was reported as a major factor of kitten mortality in uk [9] and it is currently one of the leading infectious diseases causing death among young cats from shelters and catteries [6] . the first case of fip in malaysia was reported in 1981 [10] and the feature of cats with fip were described in a retrospective study [11] . antibodies against fcovs were found in 100% of cats living in malaysian catteries [12] and the virus was detected in 84% of healthy cats using rt-pcr [13] . in present study, a conserved region of 3'untranslated region (3'utr) is used to detect fcov and determine the descriptive distribution and phylogeny of local isolates in fip-suspected cats. abdominal fluids and/or tissue samples of 28 cats suspected of having the effusive form of fip were obtained from the university veterinary hospital, universiti putra malaysia (uvh-upm) over the period of three years (2007) (2008) (2009) ). ascitic fluids were diluted 1:10 in phosphate buffer solution (pbs), aliquoted and stored at -70°c until used. organ samples were homogenized in 1:10 of pbs. insoluble components were removed by centrifugation for 10 min at 3000 g and the supernatant fraction was collected and kept at -70°c. two fcov reference strains (fecv 79-1683; atcc® no.vr-989™ and fipv79-1146; atcc® no. vr-216™) were used for rt-pcr optimization. virus stocks were propagated in confluent crandell feline kidney cells. the viruses were harvested when the infected cells showed 80% cytopathic effects. the virus suspension was freezed-thawed three times and stored at -70°c until used. rna was extracted from the infected cell culture supernatants and clinical samples using trizol® reagent (invitrogen, carlsbad, california, usa) according to the manufacturer's instructions. the partial 3'utr was amplified by rt-pcr using previously described primers [7] . one-step rt-pcr was performed using access rt-pcr system and rnasin® ribonuclease inhibitor (promega, madison, wisconsin, usa). the reaction was optimized on a thermal cycler (mj research, waltham, massachusetts, usa). pcr products of 223 bp were analyzed using electrophoresis on a 2% agarose gel, stained with ethidium bromide and observed under uv light. pcr products of 10 positive cases were selected randomly, purified using pcr sv protocol (geneall®, seoul, south korea) and sequenced in both direction with the primers (medigene, selangor, malaysia). data analysis was performed using statistical tables calculator, which is available online at http://faculty.vassar.edu/lowry/odds2x2.html. age, breed and gender differences were compared by calculating positivity rate, odds and 95% confidence intervals. the rt-pcr assay amplified the target band in 25 out of 28 cats' samples (89%). although, the pcr results must be interpreted in conjunction with clinical or pathological findings, detection of the virus in fip-suspected cats may be useful to confirm fip. since fcovs are ubiquitous in cats with high seroprevalence [5, 6, 12] , pcr provides the obvious advantage over serology by directly detecting fcov genome rather than documenting a previous immune system encounter with the coronavirus. the primers of this pcr assay were chosen from a highly conserved region of 3'utr of the fcov genome to detect most, if not all of the fcov strains. the usefulness of these primers for a general screening test has been reported previously [14] [15] [16] . fcov-positivity rate in cats younger than two years old (64%) was higher than older cats, but they are not significant. however, the result is consistent with other studies demonstrating higher incidence of fip in cats below 2 years of age [5, 11, 14] and agree with the fact that fip is a disease of young cats. typical clinical cases are first appear during the postweaning period, but most deaths from fip occur in cats 3-16 months of age [6] . most of the fcov-positive cats in our study were males (80%). higher incidence of fip among males was previously reported [14, 17, 18] . as the pathogenesis of the disease is still not fully understood, the relation of gender and incidence of fip is not clear. about 56% of fcov-positive cases were dsh, 40% persian, and 4% siamese cats. in the present study, the majority of cats (96%) diagnosed with fip were dsh or persian. this finding is in accordance with a previous report on fip in malaysia showing that 69.7% and 27.3% of cats diagnosed with fip were dsh and persian cats, respectively [11] . however, these studies did not conclude that these two breeds were more susceptible to fip because of limited variation in cat breeds presented at uvh-upm and lack of clinical cases of fip in different breeds in malaysia. furthermore, in a study on the prevalence of fip in specific cat breeds, dsh and persian cats were at low risk compared to others [18] . age, breed and gender distribution in fcov-positive cats are shown in figure 1 and statistical analysis is summarized in table 1 . out of 25 pcr positive cases, 10 isolates were selected for further sequence analyses. all 10 field isolates designated as upm1c/07 to upm10c/09 with accession no. fj897745 to fj897754, respectively were deposited in the genebank (table 2 ). these sequences were aligned with published sequences of fcov using clustalw multiple alignment (bioedit version 7.0.9). the sequences of four malaysian fcov isolates which have been isolated from healthy cats in a previous study [19] were also included in the alignment (table 2) . homology matrix and phylogenetic trees were constructed using neighbor-joining method (bioedit) and treetop-phylogenetic tree prediction (genebee-molecular biology server the sequences of ten local isolates showed 96% homology and when compared to published sequences of fcov, the homology decreased to 93%. the homology between partial sequences of fcov isolates from malaysia were higher than those from different geographical origin (32 strains from usa, 13 strains from netherlands, two strains from taiwan, and one strain from uk). these findings support previous observations showing a correlation between different fcov biotypes with similar geographic background [8] . multiple sequence alignment showed a few point mutations and single-nucleotide deletions in the sequences of local isolates ( figure 2 ). these findings indicate single nucleotide polymorphisms (snps) in fcovs as described previously [6, 20] . no particular pattern of mutation or deletion was found in this part of fcovs genome. phylogenetic tree constructed by cluster algorithm showed that the sequences were genetically separated in two distinct clusters; all local sequences fell into one main cluster and suggested they may derived from a common ancestor ( figure 3 ). however, a whole genome sequence is needed to determine genetic pattern of malaysian fcovs. phylogenetic tree constructed by neighbor-joining method showed the phylogenetic relations of the sequences in an unrooted-tree algorithm (figure 4 ). in conclusion, the present study indicated that males and young cats are more likely to be diagnosed with fip. the homology of partial sequences of 3'utr of fcov isolates in malaysia was shown to be higher than those from the other regions. coronaviridae. fields virology philadelphia: lippincott williams & wilkinsknipe dm, howley pm evolutionary insights into the ecology of coronaviruses genome organization and reverse genetic analysis of a type i feline coronavirus an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis feline infectious peritonitis. veterinary clinics of north america small animal practice a review of feline infectious peritonitis virus infection: 1963-2000 detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr feline infectious peritonitis virus arise by mutation from endemic feline enteric coronaviruses kitten mortality in the united kingdom: a retrospective analysis of 274 histopathological examinations feline infectious peritonitis -two case reports. kajian vet malaysia retrospective examination of feline infectious peritonitis cases presented to university veterinary hospital, universiti putra malaysia (uvh-upm) between serological survey of catteries for cats infected with feline coronavirus prevalence of feline coronavirus in two cat populations in malaysia prevalence of feline coronavirus types i and ii in cats with histopathologically verified feline infectious peritonitis field strain feline coronaviruses with small deletions in orf7b associated with both enteric infection and feline infectious peritonitis genetic diversity and phylogenetic analysis of feline coronavirus sequences from portugal feline infectious peritonitis in a closed breeding colony prevalence of feline infectious peritonitis in specific cat breeds phylogenetic analysis of feline coronavirus isolates from healthy cats in malaysia quasispecies composition and phylogenetic analysis of feline coronaviruses (fcovs) in naturally infected cats genomic rna sequence of feline coronavirus strain fipv wsu-79/1146 the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf7a/7b transcription unit of different biotypes genomic organization and expression of the 3' end of the canine and feline enteric coronaviruses descriptive distribution and phylogenetic analysis of feline infectious peritonitis virus isolates of malaysia the authors would like to thank the staffs of the university veterinary hospital and cat owners who participate in this project. the study was funded by mosti project no. 02-01-04-sf0485: development of a rapid test for diagnosis of feline coronavirus. authors' contributions ssa designed and coordinated the study and helped in draft correction. ssh carried out the molecular studies, performed the rt-pcr assay and sequence analysis and drafted the manuscript. mhb, aro and naz participated in the sequence analysis and proof reading. lsf, nar, ha and shsh participated in the collecting of clinical samples. mahi helped in lab works. all authors read and approved the final manuscript. the authors declare that they have no competing interests.received: 2 september 2009 accepted: 6 january 2010 published: 6 january 2010 key: cord-351955-9l4786lb authors: pedersen, niels c.; liu, hongwei; dodd, kimberly a.; pesavento, patricia a. title: significance of coronavirus mutants in feces and diseased tissues of cats suffering from feline infectious peritonitis date: 2009-08-26 journal: viruses doi: 10.3390/v1020166 sha: doc_id: 351955 cord_uid: 9l4786lb the internal fecv→fipv mutation theory and three of its correlates were tested in four sibs/half-sib kittens, a healthy contact cat, and in four unrelated cats that died of fip at geographically disparate regions. coronavirus from feces and extraintestinal fip lesions from the same cat were always >99% related in accessory and structural gene sequences. snps and deletions causing a truncation of the 3c gene product were found in almost all isolates from the diseased tissues of the eight cats suffering from fip, whereas most, but not all fecal isolates from these same cats had intact 3c genes. other accessory and structural genes appeared normal in both fecal and lesional viruses. deliterious mutations in the 3c gene were unique to each cat, indicating that they did not originate in one cat and were subsequently passed horizontally to the others. compartmentalization of the parental and mutant forms was not absolute; virus of lesional type was sometimes found in feces of affected cats and virus identical to fecal type was occasionally identified in diseased tissues. although 3c gene mutants in this study were not horizontally transmitted, the parental fecal virus was readily transmitted by contact from a cat that died of fip to its housemate. there was a high rate of mutability in all structural and accessory genes both within and between cats, leading to minor genetic variants. more than one variant could be identified in both diseased tissues and feces of the same cat. laboratory cats inoculated with a mixture of two closely related variants from the same fip cat developed disease from one or the other variant, but not both. significant genetic drift existed between isolates from geographically distinct regions of the western us. feline infectious peritonitis (fip) was first introduced as an "important disorder of cats" by holzworth [1] and a clinico-pathologic conference on the disease was published the following year [2] . the incidence of fip rose progressively over the next two decades. the occurrence of fip among all cats seen at veterinary medical teaching hospitals in the usa from 1986-1995 was 1:200 among new feline visits, 1:300 among total cat accessions, and 1% of accessions at diagnostic laboratories [3] . the incidence is several times higher among kittens and young cats originating from catteries or shelters. the disease was thought to be viral when first described but no specific etiologic agent was identified at the time [4] . zook et al. [5] observed virus particles in the tissues of experimentally infected cats, however, the close similarities of fip virus (fipv) in tissues to members of the family coronaviridae was noted by ward [6] . the ability of fipv to cause either a non-effusive (dry, parenchymatous) or effusive (wet, non-parenchymatous) form of the disease was first reported by montali and strandberg [7] . the close genetic relationship of fipv to coronaviruses of dogs and swine was first recognized by pedersen et al. [8] . the existence of two serotypes, feline-or canine-coronavirus like, was described in 1984 [9] . fip was originally believed to be an uncommon clinical manifestation of a ubiquitous and largely nonpathogenic agent named feline enteric coronavirus (fecv) [10] . subsequent studies demonstrated that the agent of fip was distinct from fecv in disease potential but that both viruses co-existed in the same population and were antigenically identical [reviewed in 11, 12] . it was subsequently hypothesized that fipv might be a simple mutant of fecv [13] , and the two viruses were later described as biotypes of each other [14] . animal studies, with both natural [15] and experimental [16] infection, also suggest that fipvs arise spontaneously during the course of fecv infection. vennema et al. [17] demonstrated that all major structural and accessory genes of wild type fecvs were virtually identical to fipvs from the same or closely related cats. however, 85% of fipvs studied had deleterious mutations in a small accessory gene called 3c. these mutations, which were either deletions or introduced stop codons, were also found to be unique to each cat. in spite of indirect and direct supporting evidence for internal fecv→fipv mutation, the role of fecv mutation in fip, and especially in the 3c gene, has not been given much attention in the literature of fip [reviewed 11] . in fact, there is a general feeling that fipv and fecv are either the same virus, with disease being dependent on the nature of the host's immune response [reviewed 11] , or that the causative mutation is in other genes [18] . although the precise origin of fipvs is debated, there appears to be agreement regarding the relative cell tropisms of fecvs and fipvs. fecvs are thought to have greater tropism for the mature apical intestinal epithelium, while fipvs are believed to have a greater tropism for macrophages [reviewed 11 ]. this has led to the a strongly held belief that coronaviruses found in the feces are fecv-like, while viruses found in extra-intestinal (usually lesional) tissues are fipv-like [19] . the purpose of this study was to repeat the original work of vennema et al. [17] with a new and geographically diverse group of cats and to test the major tenant of the fecv→fipv theory and three of its possible correlates. the major tenant of the theory assumes that functional mutations in the 3c gene are somehow related to the fip biotype. the first correlate of this theory supposes that each fip cat will have its own unique 3c mutant which is not transmitted cat-to-cat. the second correlate assumes compartmentalization of enteric and fip biotypes to gut and internal tissues, respectively. the third correlate, if correct, should show fipvs to be as geographically diverse as the fecvs from which they arise complete structural (s, e, m, n) and accessory (3a-c and 7 a, b) gene sequences were obtained from diseased omentum of the four related cats that died of fip and the isolates designated were fipv-ucd11, 12, 13 and 14 ( table 1 ). the numbers of nucleotides sequenced for isolates fipv-ucd11 to ucd14 are shown in table 1 , while the relationship of the fipvs isolated from the 4 related scottish fold cats is shown in figure 1 . the overall sequence identity for the nine structural and accessory genes was ≥ 99% with only a small number of mutations among the four highly related viruses (table 1) . mutations consisted of minor snp changes, and less commonly deletions that appeared to be randomly scattered among the genes that were sequenced; about one half of the mutations resulted in amino acid changes ( table 2 ). among the 9 structural and accessory genes of the four related cats, the highest genetic variability was in the 3c gene, followed by the s and m genes ( table 2 ). the least variability was detected in the 3b, 7a, and 3a accessory genes. among all of the genes sequenced, only the 3c genes of the fipv isolates had snps that resulted in premature stop codons or deletions that caused frame shifts; both resulting in a variable truncation of the 3c protein ( figure 2 ). the omentum viruses from red consisted of two distinct variants, as determined by sequences obtained from multiple overlapping pcr products (table 1) . these variants were designated fipv-ucd11a and -ucd11b. there were only five snps scattered across the nine structural and accessory genes between the two variants. two variants were also sequenced from the omentum of toby, one with a non-functional 3c gene (fipv-ucd12) and one with a functional 3c gene (fecv-ucd5). these two variants were identical in sequence except for a single-base deletion in the 3c of one of the variants (fipv-ucd12). four additional unrelated cats (392312, 384062 and 388210) from paradise, menlo park, and san jose, ca, respectively, and cat-t from mountlake terrace, wa were included in the study. the three cats seen at the vmth suffered from the non-effusive form of fip, while the washington state cat died of effusive fip. the e, m, n, 3a-c and 7a, b genes were amplified from the omentum or organ granulomas of all four animals. viruses were readily detected in the diseased tissues of cats 388210, 388406, cat-t, and 392312 and designated fipv-ucd15 to ucd18, respectively (table 1) . fipv-ucd15a possessed a two-nucleotide deletion near the end of the 3c gene and a second deletion of 48nucleotide involving the terminus of 3b and beginning of 3c ( figure 2 ). mutations of the 3c gene in fipv-ucd16 and -ucd17 involved premature stop codons. two variants with six scattered snps and an identical deletion in the 3c genes were identified in organ granulomas of cat 392312 and designated fipv-ucd18a and -ucd18b (table 1 ). all of the structural and accessory genes that were sequenced for the eight different fip cats appeared to be intact, except for the 3c genes. the 3c genes from all eight isolates contained deletions or snps that either produced truncating frame shifts or premature stop codons ( figure 2 ). the sequence relationship of the four unrelated fipv isolates to each other and to the fipv isolates from the four related scottish fold cats is shown in figure 1 . the overall genetic similarity for the e, m, n, and 3a-c, 7a, b genes ranged from 89-99% among the 8 fipv isolates. the four fipvs from unrelated cats showed sequence identity of 89-92% to each other and to the fipvs from the four related cats. feces or colonic scrapings from the four related cats and a fifth unrelated housemate contained feline coronaviruses ( table 3 ). the amount of viral rna in feces in cats with fip was much lower than in diseased omentum and obtaining complete sequences of all 9 genes was not always possible. therefore, the actual genes sequenced for each fecal coronavirus isolate are shown in table 3 . coronaviruses isolated from the feces of two cats, tux and toby, were ≥99% identical and contained identical 3c gene mutations to the omental viruses from the same cats. the coronavirus isolated from lucy's feces (designated fecv-ucd3) had an intact (i.e., wild type or non-deliterious) 3c and its sequence was otherwise 99% identical to the sequence of fipv-ucd14 found in her diseased omentum. the sequence obtained from the fecal virus of simba, a housemate of lucy, also had an intact 3c gene and was designated fecv-ucd4. fecv-ucd4, was most closely related to the fipv isolated from lucy and was 99.7% related to the consensus nucleotide sequences of coronaviruses obtained from the four related fip cats ( figure 1 , table 2 ). c there were 7 snps between fipvs found in colonic scraping and the diseased omentum. d the biotypes of the virus isolated from the feces of these cats were not determined due to an inability to amplify the 3c gene. a similar finding was found for the cats that were unrelated to those described above and that were from disparate geographic regions. three of four fecal samples (388210, 388406 and cat-t) contained amplifiable rna and complete 3c genes were sequenced in 2/3 of these cats (388210 and 388406). the 3c gene sequence of the fecal virus of cat 388406 was intact and ≥99% related to the fipv found in diseased tissue ( table 2 ). this fecal isolate was designated fecv-ucd6. the 3c gene of 388210 fecal virus contained a deliterious two-nucleotide deletion near the end of the 3c gene and was designated fipv-ucd15b. this same deletion was also detected in the lesional fipv-ucd15a. however, fipv-ucd15a did not contain the 48-nucleotide deletion involving 3b and 3c of fipv-ucd15b ( figure 2 ). only the 7a, b genes were sequenced from the feces of cat-t and the sequence was 100% identical to the 7a, b sequence from the omental fipv-ucd17. this study of lesional and/or fecal coronaviruses from nine cats both supported and modified the previous conclusions of vennema et al. [17] . viruses from diseased tissues from all eight cats in this study had truncating mutations, either in the form of deletions leading to frame shifts or coding changes causing premature stop codons in the 3c gene. such damaging mutations were not present in other accessory and structural genes in this or in a previous study [17] . as with the earlier study [17] , all or almost all of the fecal isolates from diseased cats and a healthy contact control animal had intact 3c genes. taken as a whole, the present study supported a role for deleterious 3c gene mutations in the genesis of fipvs from fecvs. however, not all fipv isolates have deleterious 3c gene mutations. although 8/8 (100%) of lesional isolates in the present study had functional mutations in their 3c genes, only 11/13 (85%) of the fipvs reported by vennema et al. [17] had deliterious 3c gene mutations. we have also recently observed what appeared to be intact 3c genes in 12/31 random breed cats that were adopted from a large shelter in northern california and died of fip. however, several of these isolates contained mutated 3c genes as minor variants, and without animal inoculation studies it is not possible to say whether or not these or the remaining isolates were capable of causing fip. the existence of helper/defective virus replication in the latter situation also needs to be considered. animal inoculation studies to determine the biotype of a given feline coronavirus are critical for determining the ultimate biotype of any isolate, regardless of its sequence regularities or irregularities. it was therefore important to demonstrate herein that an isolate from the four related cats reported herein was capable of causing fip. since some fipvs appear to have intact 3c genes, it may be premature to ascribe the fip biotype solely to deleterious mutations in the 3c gene. however, what are the alternatives? it can be argued that mutations in the conserved replicase/transcriptase genes may have a similar effect; that small mutations in other structural and accessory genes, collectively or singly, will have the same effect; that fipv and fecv are identical viruses; or that deleterious 3c gene mutations are an effect of the disease and not its cause. involvement of the replicase/transcriptase genes is unlikely, because the replicase/transcriptase region is highly conserved among feline coronaviruses and unlikely to be involved in cell tropism or evasion of the host's immune response. one study of a natural serotype i fipv isolate (c1je) showed a high degree of sequence conservation within the replicase/transcriptase genes compared to other feline coronaviruses, while a premature stop codon limited the 3c gene product to the first 16 amino acids [19] . it is also unlikely that mutations in other accessory or structural genes are involved, even though such mutations have been frequently found in feline coronaviruses. firstly, 3c gene mutations in fipvs occur significantly out of proportion to mutations in other structural or accessory genes. secondly, there is little scientific evidence, especially based on animal inoculation, that other accessory genes are involved in fip. in the original report that proposed the internal mutation theory, 11/13 of the fipvs had 3c mutations, while 2/13 isolates had only 7b mutations [17] . however, both of the latter cats were related and had been experimentally infected with an identical fecv (fecv-rm); a third sibling cat from this group had the same 7b mutation but with a unique functional 3c mutation. variants were not tested at the time and it is possible that 3c mutants would have been present if the two discordant isolates had been adequately sequenced. earlier studies have also demonstrated an absence of 7b mutations in almost all fecvs and other fipvs and indicate that such mutations are most likely tissue culture artifacts [17, 20] . yet other studies suggest that 7a and 7b mutations occur in nature in both fip and enteric infections and are therefore not directly linked to pathogenicity [20, 21, 23] . there is a general belief that host and environmental factors, and not virus mutation, are the basic determinants of whether a cat develops fip or just a mild enteritis following exposure to the common feline coronavirus [24] [25] [26] [27] [28] . for such a theory to be correct, fecvs and fipvs would have to be identical in both genetic structure and virulence. the evidence that fecvs and fipvs cause very different diseases is strong [10] [11] [12] [13] . even though environmental and host factors are admittedly important in fip [29, 30] , lesional viruses from the eight fip cats in this study, even though highly related to fecal isolates, were easily differentiated from each other based on deliterious 3c gene mutations alone. moreover, an infectious inoculum made from the diseased omentum of one of the fip cats induced fip in 3 of 12 cats that were experimentally infected (see section 2.2.). confirmation of biotype by animal inoculation, such as described herein, is rarely done in published reports concerned with feline coronavirus infection [reviewed in 11] . the possibility that deleterious 3c gene mutations are an effect of the disease and not a cause also has to be considered. however, there is little precedence for this and given the ability of a lesional isolate from the present study to produce fip, it is counterintuitive for a functional 3c gene mutant to be both a cause and effect of its own disease. this theory would also not explain why all non-tissue culture adapted fecv strains used for experimental inoculation studies have intact 3c genes, while all tissue culture adapted and non-adapted strains have mutated 3c genes [17] . the existence of feline coronavirus variants was not a novel observation [23, 31, 32] , but their frequency and fate has not been previously addressed. variant forms were found in both extraintestinal tissues and feces of the 9 cats in this study, but only one variant became predominant upon experimental passage from one cat to another (see section 2.2). the infecting variant may have been merely the first virus into a macrophage, or its selection may have involved more complex host/virus interactions. we also found that subtle, and sometimes significant, genetic mutations (usually snps and deletions) occurred upon primary replication in a new host. therefore, genetic variation among feline coronaviruses occurs both within and between host cats. selective infection with a single variant can also rapidly lead to genetically distinct clades of coronavirus, especially when combined with a high intrinsic and extrinsic mutation rate. twelve laboratory cats were inoculated intraperitoneally with a cell-free inoculum prepared from the diseased omentum of red, which contained two variant forms of the virus (fipv-ucd11a and -ucd11b). three of these cats developed effusive fip within 2-4 weeks. viral rna was isolated from the omentum of each experimentally infected cat at the time of necropsy. the s (one cat) and e, m, n and 3a-c, 7a, b genes (all three cats) were sequenced. one of the cats was found to be infected with fipv-ucd11a, while two of the cats were each infected with fipv-ucd11b. each of these cats had a nearly identical variant of ucd-11a or ucd-11b in its diseased omentum ( table 4 ). the premature stop codon of parental 3c gene was preserved in fipv isolates from all three cats. however, fipv-ucd11b.2 isolated from one of the three cats had acquired two additional large deletions affecting both the 3b and 3c genes that were not in the infecting virus (table 4 and figure 2 ). table 4 . name and biotype designation of coronavirus isolates from three cats dying of experimentally induced fip. the genes that were sequenced, their mutability, degree of relatedness to the consensus sequence of fipv-ucd11a, b, and nature of the functional mutation in the 3c gene are given for each cat. it is important to determine by animal inoculation studies the true biotype of a feline coronavirus that is being reported, rather than always referring to a generic feline coronavirus [reviewed in 11] . feline coronaviruses that possess the fip biotype, such as fipv-ucd11a,b, will readily induce fip in from 25-100% of infected individuals, depending on the strain being tested [reviewed in 11]. however, bonifed (cat-to-cat passaged, non-tissue culture adapted) fecv strains will rarely induce fip in healthy cats [9, 12, 15, 17 ]. the present study adds to our knowledge of genetic drift among feline coronaviruses that inhabit the same cat, multi-cat household, cattery, or geographically distant region. all of the fipvs and fecvs isolated from the five cats that had close contact with each other in sonoma, california were ≥99% related (tables 1 and 3; figure 1 ). based on gene sequences and historical facts, it can be reasonably concluded that cat simba was infected with an fecv following contact with cat lucy. this supported another correlate of the internal mutation theory; fecvs are easily spread cat-to-cat, while fipvs are not. addie et al. [33] also noted that the same strain of coronavirus tended to persist among any given group of cats. however, coronaviruses within a closely housed group of cats, and even within the same cat, undergo continuous genetic drift. we observed sequence differences of 1-2% or less in cats from the same group, while genetic drift between cats from distant areas of the western us was on the order of 6-16%. herewegh et al. [34] also found that feline coronaviruses from individuals within the same environment had unique genetic fingerprints and fell within the same clade, while geographically distant isolates belonged to genetically unique clades. the notable mutational drift observed among feline coronaviruses across geographic regions, in the face of genetic conservation within stable groups of cats, is paradoxical. however, the evidence indicates that coronavirus infection in any group of cats originates from a single founder virus, that virtually every cat in a group is infected rapidly and efficiently, and that cats appear to resist superinfection with closely related strains [34] . the single founder virus effect was confirmed in the present study (table 4) . thus, marked genetic drift occurs when a single coronavirus strain is serially passed from one susceptible population to the next. this scenario was supported by our animal transmission studies; when cats were simultaneously infected with two closely related variants of fipv, each variant segregated into different cats. therefore, minor mutants may become predominate when passed cat-to-cat. the s sequences of fipv-ucd11 to 14 were compared to that of previously reported fipvs and to a purported fecv (wsu-79-1683) (data not shown). the s protein shared 98% sequence identity among the four fipv isolates, and 87-91% sequence identity to other published serotype i fipvs. however, when compared to the s protein of serotype ii feline coronaviruses wsu-79-1146 and wsu-79-1683, there was only 43-44% sequence identity (data not shown). based on the comparison of s proteins, fipv-ucd11 to 14 were classified as serotype i feline coronaviruses. however, these studies demonstrate that serotype designation of feline coronaviruses can be more easily made from comparisons of the much smaller 3a rather than significantly larger s sequences (71 vs 1471 amino acids in the respective gene products) (figure 3 ). similar to the s protein comparison, the 3a sequence of all four fipvs from the related cats shared 98-100% sequence identity to each other, and 84-94% sequence identity to the four fipvs from unrelated cats and from published serotype i fipvs ( figure 3 ). however, when compared to the 3a protein of serotype ii feline coronaviruses wsu-79-1146 and wsu-79-1683 or to 3a of canine coronavirus, there was only 65-70% sequence identity. these results indicate all eight fipvs from this study clearly belonged to serotype i based on their 3a protein sequences, while known serotype ii viruses and the canine coronavirus formed a separate group (figure 3 ). fipv is unique from most other viruses, because it is infrequently spread from animal-to-animal in a horizontal manner, yet it is highly infectious when extracts of diseased tissues or fluids are inoculated into naïve cats by a number of routes [reviewed in 11] . the general belief is that enteric biotypes are compartmentalized to the gut, while fip biotypes are found only within internal organs [19] . however, viruses with 3c mutations identical to fipvs from lesional tissues were present in the feces of some cats in this study (table 3) , thus making horizontal transmission theoretically possible in certain circumstances. there is also evidence that fipv may have been shed in urine of fipv infected cats [35] , and that coronavirus may be present in the blood, especially among younger cats [36] . there are also several reports of fip outbreaks of sufficient magnitude and acuteness to suggest horizontal transmission [reviewed in 11] . while this study did not answer the question as to the relative importance of vertical and horizontal transmission, it indicated the need to carefully study fecal and lesional virus isolates that are involved in explosive, large scale, epizootics of fip and not just the common enzootic form. amount of sequence homology among this protein, the similarities in their hydropathy profiles, both to each other and to the corresponding m proteins, as well as to the sars-cob 3a protein, are quite remarkable. nothing is known about these proteins, but it is clear that it will be interesting to learn more about their biological features." a great deal of research has been reported, and is being conducted, on the sars coronavirus 3a gene and protein and it is evident that this gene and its product play an important role in viral assembly, spread and pathogenesis, as well as to protective immunity [38] [39] [40] . if the 3c protein of feline coronavirus truly has an analogous function to sars coronavirus 3a protein, sars coronavirus research might be applicable to feline coronaviruses and how they cause disease. the authors have used the original names of fecv to refer the enteric biotype of feline coronavirus, and fipv for the fip biotype. published non-tissue culture adapted coronavirus isolates from the feces of healthy cats always possess an intact or wildtype 3c, while strains from fip diseased tissues have mutated 3c genes [17] . therefore, the designation of fecv or fipv in this study was applied to isolates with 3c genes that yielded either intact or truncated proteins, respectively. the generic term "coronavirus" or "feline coronavirus" was used herein when not referring to a specific biotype. four scottish fold kittens were born into the same cattery in sonoma, california; red, toby and lucy were from the same litter of three, while tux was born a week later in a litter of three to a sister queen and the same tom. simba, an 11 year-old american curl, was born in an unrelated cattery and resided in another sonoma household as a pet. lucy was placed into this household with simba when she was 17 weeks old, while red went to live in another home with two other older cats when at 14 weeks of age. tux and toby remained in their home cattery with several other cats. lucy, tux, red and toby first showed signs of indicative fip at 23, 33, 35 and 40, weeks of age, and were euthanatized with confirmed disease at 27, 37, 39 and 41 weeks of age, respectively. all other contact cats have remained healthy to this time. four additional cats were recruited from the western us. two of them were 26-and 60-month old burmese (388406 and 392312) from paradise and menlo park, ca, respectively. the third was a 16month old birman (388210) from san jose, ca, and the fourth was a 2-year old sphinx (cat-t) from mountlake terrace, wa (courtesy dr. tracy tomlinson). full necropsies on all cats, except cat-t were performed at the school of veterinary medicine teaching hospital (vmth), university of california, davis, ca. cat-t was necropsied at a private veterinary diagnostic laboratory (phoenix central laboratory, everett, wa). a definitive diagnosis of fip was confirmed on all eight cats by gross and microscopic examination of tissues and immunohistochemistry. the four related scottish folds and cat-t suffered from the effusive form of fip, while the two burmese and one birman cats suffered from non-effusive fip. samples of diseased omentum (effusive fip) or kidney granulomas (non-effusive fip), along with feces (or colonic mucus/mucosal scrapings from one cat) were collected at the time of necropsy and stored at -20°c. feces from the healthy sentinel cat, simba, were also collected. a cell free inoculum was made from the diseased omentum of red, one of the four related cats. omentum was frozen in liquid nitrogen and ground to a powder. the frozen omental powder was reconstituted in 0.25g/ml hbss (hanks buffered saline solution) and centrifuged twice at 2,000 x g for 30 minutes. the supernatant was stored at -70°c as viral stock. the viral stock was diluted 1:3 with hbss when used as inoculum for the fipv transmission study. adult specific pathogen free cats were obtained from the breeding colony of the feline health and pet care center, school of veterinary medicine, university of california, davis, ca. a total of twelve cats were inoculated intraperitoneally with 1 ml of cell free viral inoculum. three cats developed fip within 2-4 weeks and complete necropsies established that all three cats had effusive fip. diseased tissues and feces were collected for isolation of feline coronavirus rna. viral rna was extracted from omentum, granulomas of kidney, and colonic mucus/mucosal scrapings using qiagen raeasy mini kit (qiagen, usa). about 30 mg ground lesional tissues were lysed with 600 µl lysis buffer containing b-mercaptoethanol. after thoroughly mixing, the lysate was homogenized with qiashredder (qiagen, usa) and an equal volume of 70% ethanol was added to the homogenized lysate. the lysate mixture was applied to rneasy spin column and the rna binding to the column was achieved by centrifugation. the rneasy spin column was then washed and the rna was eluted with 50 µl rnase-free water and stored at -70°c. feces from 8/9 cats were suspended with 5 volumes of phosphate buffer saline (pbs) by vortexing. the suspension was centrifuged at 8,667 x g for 10 min and the supernatant transferred to a new tube and centrifuged at 54,174 x g for 30 min. the pellet containing the virus was suspended with 5 ml pbs and centrifuged again at 54,174 x g for 30 min. the pellet was suspended in 140 µl pbs and the viral rna extracted using a qiaamp viral rna mini kit (qiagen, usa). briefly, 560 µl lysis buffer containing carrier rna was mixed with the 140 µl viral suspension and incubated at ambient temperature for 10 min; 560 µl 100% ethanol was added to the lysate. the lysate mixture was applied to qiaamp mini spin column and the rna binding to the column was achieved by centrifugation. the column was then washed and the rna was eluted with 50 ml rnase-free water and stored at -70°c. the published sequences of feline coronaviruses in genbank were used to design the primers for a reverse transcripase polymerase chain reaction (rt-pcr). three primer pairs were designed from highly conserved regions and used to amplify three overlapping fragments containing the nine structural and accessory genes of feline coronavirus (figure 4 and table 5 ). the rt-pcr was carried out with qiagen longrange 2step rt-pcr kit (qiagen, usa). the viral rna was first denatured by incubating at 65°c for 5 min and then chilled on ice. the reverse transcription was carried out in 20 l reaction mixture containing 10 units of longrange reverse transcriptase, 0.8 unit of rnase inhibitor, 1 mm dntp, 1 mm oligo dt , and 5 l of denatured viral rna in 1x reaction buffer. the mixture was incubated at 42°c for 2 hr followed by 85°c for 5 min. the reverse transcribed cdna was stored at -20°c or used immediately in pcr amplification. the viral cdna was amplified in 20 l reaction mixture containing 2 l cdna, 1 unit longrange pcr enzyme mix, 0.5 mm dntp, 0.25 mm forward primer, and 0.25 mm reverse primer in 1x pcr buffer. the mixture was then incubated at 93°c for 3 min and amplified for 30 cycles at 93°c for 30 s, 60°c for 30 s, and 68°c for 1 min per kb of pcr product, followed by a final extension for 10 min at 68°c. the reverse transcribed viral rna from feces was amplified for 40 cycles under the same condition. the pcr products were electrophoresed in tae buffer on a 0.8% agarose gel. the pcr product was purified using a qiagen gel purification kit (qiagen, usa). sixty primers were ultimately used for sequencing, with the s gene requiring the most primer development and modification (primer sequences not shown). regions containing mixed sequences due to the presence of a minor variant were also resolved with overlapping primers. the purified overlapping pcr products encoding the nine structural and accessory genes were sequenced with a bigdye terminator v3.1 cycle sequencing kit (applied biosystems, usa) in 15 µl reaction containing 1 µl big dye terminator mix, 2 µl reaction buffer (5x), 35 ng sequencing primer, and 3 µl (out of 50 µl) gel purified pcr product. the sequencing reaction was incubated at 93°c for 2 min and then amplified for 40 cycles at 93°c for 20 s, 50°c for 20 s, and 60°c for 4 min. unincorporated dye terminators and dntp were removed by gel filtration based performa dtr ultra 96-well plate kit (edgebio, usa) and the cycle amplified products were analyzed by capillary electrophoresis using an abi 3730 genetic analyser (applied biosystems, usa). vector nti advance 10 software (invitrogen, usa) was used for alignment of sequence data. the percent sequence identity for pairwise alignment and the phylogenetic relationship among different fipv isolates was analyzed using clustalw2ii (www.ebi.ac.uk/tools/clustalw2/). tables 2 and 3 list the various fipv and fecv isolates that were studied and the genes that were sequenced for each. these sequences have been deposited in the database of genbank. the ubiquitous form of feline coronavirus is readily passed cat-to-cat by the fecal-oral route and is the cause of a mild or unapparent enteritis. like coronaviruses in general, feline enteric coronavirus (fecv) is undergoing constant mutation within its accessory and structural genes. feline infectious peritonitis (fip), which is a highly fatal systemic disease, is a sequel of fecv infection in a small proportion of cats. the virus isolated from the diseased tissues of cats with fip is highly related to the fecv identified in the feces. although snp and deletion mutations were common between isolates from the same cat, only the 3c gene was rendered non functional by such mutations. deleterious mutations in 3c tend to be found in diseased internal tissues, while viruses with intact 3c are found mainly in the feces. while deleterious mutations of the 3c gene were seen in all 8 fip cats in this study, and in virtually all previously reported fipvs, they are by no means a universal finding. however, there is compelling evidence that when they do occur, they are the cause of fip and not an effect of the disease. deleterious 3c gene mutants will readily cause fip when inoculated into laboratory cats, whereas their largely non-pathogenic fecal counterparts with intact 3c genes are readily transmitted from one cat to another. these gene 3c mutants, when they occur, are unique to each cat with fip, indicating that they arise independently in each host and not by mutation in one cat with subsequent horizontal spread to others. several minor variants can co-exist in both tissues and feces and when two variants are inoculated together into the same cat, one or the other, but not both, will predominate. the high mutability of feline coronaviruses leads to minor genetic differences between cats in one closely contained geographic area, while significant genetic differences are seen between isolates from geographically disparate regions. more in depth studies on the function of the feline coronavirus 3c gene will be important for determining its precise role in fip. some important disorders of cats clinico-pathology conference epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals feline infectious peritonitis ultrastructural evidence for the viral etiology of feline infectious peritonitis morphogenesis of a virus in cats with experimental feline infectious peritonitis extraperitoneal lesions in feline infectious peritonitis antigenic relationship of the feline infections peritonitis virus to coronaviruses of other species pathogenic differences between various feline coronavirus isolates. coronaviruses; molecular biology and pathogenesis an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis a review of feline infectious peritonitis virus infection: 1963-2008 pathogenesis of feline enteric coronavirus infection virologic and immunologic aspects of feline infectious peritonitis virus infection perspectives on feline coronavirus evolution elimination of feline coronavirus infection from a large experimental specific pathogen-free cat breeding colony by serologic testing and isolation two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein genomic rna sequence of feline coronavirus strain fcov c1je persistence and evolution of feline coronavirus in a closed catbreeding colony field strain feline coronaviruses with small deletions in orf7b associated with both enteric infection and feline infectious peritonitis deletions in the 7a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis belák s. preliminary studies on feline coronavirus distribution in naturally and experimentally infected cats feline leucocyte antigen class ii polymorphism and susceptibility to feline infectious peritonitis natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats natural feline coronavirus infection: differences in cytokine patterns in association with the outcome of infection high viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (fcov) type i and in naturally fcov-infected cats association between faecal shedding of feline coronavirus and serum alpha1-acid glycoprotein sialylation inheritance of susceptibility of feline infectious peritonitis in purebred catteries risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus quasispecies composition and pylogenetic analysis of feline coronaviruses (fcovs) in naturally infected cats. fems 32 antigenic and plaque variations of serotype ii feline infectious peritonitis coronaviruses persistence and transmission of natural type i feline coronavirus infection the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf7a/7b transcription unit of different biotypes feline infectious peritonitis: experimental studies the detection of feline coronaviruses in blood samples from cats by mrna rt-pcr glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and m severe acute respiratory syndrome coroanvirus 3a protein is a viral structural protein amino terminus of the sars coronavirus protein 3a elicits strong, potentially protective humoral responses in infected patients the severe acute respiratory syndrome (sars)-coronavirus 3a protein may function as a modulator of the trafficking properties of the spike protein this research was funded by the center for companion animal health, school of veterinary medicine, university of california, davis, ca 95616. we are also grateful for anonymous and named donations from owners of cats dying from fip. the authors are grateful for the assistance of sue weitendorf for allowing access to a critical group of cats used in this study. positive proof that the 3c protein is responsible for the fip phenotype, in at least a proportion of cats dying of fip, will require knowledge of its exact function, of which we currently know very little. a genblank blast search shows a 30% genetic homology between feline coronavirus 3c and sars coronavirus 3a (data not shown). moreover, the 3c protein of feline coronavirus also has an identical hydrophillicity profile to its own m protein and to the m and 3a proteins of sars coronavirus [37] . these similarities prompted oostra and colleagues [37] to state -"(…) it appears that all group 1 [corona] viruses expresss group-specific proteins predicted to be triple-spanning membrane proteins. examples are the feline orf 3c protein and the hcov-nl63 orf 3a protein (…) despite the small key: cord-322317-wsagoy52 authors: stranieri, angelica; scavone, donatella; paltrinieri, saverio; giordano, alessia; bonsembiante, federico; ferro, silvia; gelain, maria elena; meazzi, sara; lauzi, stefania title: concordance between histology, immunohistochemistry, and rt-pcr in the diagnosis of feline infectious peritonitis date: 2020-10-18 journal: pathogens doi: 10.3390/pathogens9100852 sha: doc_id: 322317 cord_uid: wsagoy52 histology, immunohistochemistry (ihc), and reverse transcription polymerase chain reaction (rt-pcr) have been used to diagnose feline infectious peritonitis (fip), but no information regarding the comparison of their diagnostic performances on the same organ is available. the aims of this study were to determine the concordance among these tests and to evaluate which combination of tests and organs can be used in vivo. histology, ihc, and nested rt-pcr (rt-npcr) for feline coronavirus (fcov) were performed on spleen, liver, mesenteric lymph node, kidney, large and small intestine, and lung from 14 fip and 12 non-fip cats. sensitivity, specificity, predictive values, likelihood ratios, and concordance were calculated. ihc and rt-npcr had the highest concordance in lung and liver, histology and ihc in the other organs. the sensitivity of histology, ihc, and rt-npcr on the different organs ranged from 41.7 to 76.9%, 46.2 to 76.9%, and 64.3 to 85.7%, respectively, and their specificity ranged from 83.3 to 100.0%, 100% and 83.3 to 100.0%. therefore, ihc is recommended when histology is consistent with fip. if rt-npcr is performed as the first diagnostic approach, results should always be confirmed with ihc. lung or liver provide accurate information regardless of the method, while ihc is preferred to rt-npcr to confirm fip in the kidney or intestine. feline coronavirus (fcov) is a widespread, highly contagious virus belonging to the species alphacoronavirus 1 of the subgenus tegacovirus (genus alphacoronavirus, subfamily orthocoronavirinae, family coronaviridae, and order nidovirales) [1] . fcov may induce a transient or persistent enteric infection, characterized by mild or absent clinical signs, or an invariably lethal systemic disease called feline infectious peritonitis (fip) [2] [3] [4] . according to their pathogenicity, fcovs can be divided into two different biotypes: feline enteric coronavirus (fecv) and feline infectious peritonitis virus (fipv), respectively [4] . it has been supposed that mutations of the viral genome could be responsible for the pathotypic switch, but they are more likely associated with a change in tropism from enterocytes to regarding the fip group, five cats showed non-effusive forms of the disease, with primarily neurological (n • 2, 3, 5) and ocular signs (n • 1, 4), along with fever, lethargy, and anorexia. the remaining nine cats were affected by effusive fip, with abdominal (n • 6, 7, 8, 10, 12, 13, 14) and/or thoracic (n • 9, 11, 12) effusion. when present, the effusion displayed features typical of fip, such as yellowish color, viscous and sticky consistency, often associated with the presence of fibrin clots [18] . the exceptions were represented by cats n • 12 and 14, whose effusions, collected in vivo, were initially consistent with fip, while at necropsy had a purulent appearance, confirmed by cytologic evaluation, which allowed the categorization of the effusion as bacterial purulent effusion. regarding the non fip group, in four cases, fip was considered as a possible differential diagnosis because of the presence of hyphemia (n • 18), body cavity effusion and icterus (n • 20), anisocoria and tremors (n • 23), neurological signs and thoracic effusion (n • 26). in eight cats, fip was not clinically suspected, and death occurred for severe injuries (n • 15, 19, and 22) or rodenticide poisoning (n • 17) confirmed by necropsy, which revealed fractures and/or bleeding. the remaining cats were affected by inflammatory (n • 16), infectious (n • 21), and cardiac (n • 24, 25) diseases. see supplementary table s1 for additional information regarding histologic lesions. histological examination was performed on 93 tissue samples obtained from fip cats and 83 obtained from non fip cats. supplementary tables s1 and s2 summarize histological findings recorded in each organ and results regarding macroscopic alterations; histology, ihc, and pcr recorded in all the tissues examined in this study, respectively. as reported in table s2 , histological lesions were also present in sections taken from organs not exhibiting evident macroscopic alterations. overall, 55/93 tissues (59.1%) from fip cats exhibited pathogens 2020, 9, 852 4 of 15 histological features consistent with the disease. different types of lesions consistent with fip were often simultaneously detected in the same animal and even within the same organ. most of the cats (11/14, 78 .6%) in the fip group, in fact, showed lesions consistent with fip in more than one organ, with different distribution based on the organ. fibrinous serositis was consistently found, particularly on the spleen, liver, and lungs, while mesenteric lymph nodes were prevalently affected by granulomatous lesions, often with vasculitis. on the other hand, kidneys were often affected by lymphoplasmacytic infiltrates, in association either with granulomatous/pyogranulomatous lesions or vasculitis. small and large intestine showed the various distribution of the above-mentioned lesions. in cat n • 2, which presented neurological signs, histological lesions consistent with fip were found only in the central nervous system (cns) with the diagnosis of severe, chronic, multifocal to coalescent lymphoplasmacytic meningoencephalitis, and ependymitis. not infrequently (38/93 tissues, 40.9%), the examined histological sections did not show either relevant lesions or lesions consistent with fip. in the fip group, the tissues that most often showed typical fip histological lesions (table 2) were the lung, kidney, and mesenteric lymph node, followed by the liver and spleen, while the small and large intestine were the organs less frequently affected by lesions imputable to fip. as regards the non fip group, the histological examination did not show lesions consistent with fip in 77/83 samples (92.7%), and, therefore, lesions potentially consistent with fip were found only in 6/83 tissues (7.2%), collected from 3/12 cats (25.0%). in this group, the organs that most frequently showed lesions possibly consistent with fip were the kidney, followed by the lymph node, spleen, and liver. lesions consistent with fip were never detected in the small and large intestine ( table 2) . in a few cases, histological results were compatible but not highly suggestive for fip and were categorized as negative or positive depending on findings in other tissues. in particular, 5/93 tissues from fip cats showed lesions suspected for fip, and three were categorized as negative (small intestine and lung of cat n • 6 and large intestine of cat n • 9) and two as positive (lymph node and kidney of cat n • 10 and 11, respectively). in only 1/83 samples from non fip cats, histology was non-conclusive, and the sample was categorized as positive considering findings in the other tissues (lung of cat n • 25). all the 12 cats (100.0%) assigned to the non fip group tested negative at ihc for fcov antigen in all the examined organs, with the exception of columnar enterocytes of cat n • 21. this result was anyway considered negative because viral antigen was not found in the context of a histological lesion. on the other hand, 13/14 cats (92.8%) assigned to the fip group tested positive in at least one of the examined organs, for a total of 53/93 immunohistochemically positive tissues (56.9%). the only cat in the fip group with negative ihc in all the organs systematically sampled for this study was the cat n • 2, in which lesions morphologically consistent with fip and immunohistochemically positive were instead restricted on the cns only. fcov immunodetection was more frequent in lesions located in the pathogens 2020, 9, 852 5 of 15 lung and lymph node, followed by the spleen, liver, and kidney, while the small and large intestine were instead less often found to be positive (table 2 ). as regards the fip group, all the cats showed a positive result in at least one of the examined tissues, and 70/92 samples (76.1%) gave a positive result, while the fcov genome was not found in 22/92 tissues (23.9%). the organs which more frequently tested positive were the kidney and mesenteric lymph node, followed by spleen, lung, and large intestine, while the small intestine and liver were the least frequently positive organs. considering the non fip group, 5/12 cats (41.7%) exhibited a positive result in at least one tissue, and overall, 6/83 samples (7.2%) were positive, while fcov rt-npcr was negative in 77/83 samples (92.8%). the organs displaying the most frequent detection of viral rna were the mesenteric lymph node and spleen. the kidney and small intestine were positive in only one case, while the large intestine, liver, and lung never tested positive ( table 2 ). the percentage of cases for which both compared methods classified the same sample as negative or positive, together with the corresponding cohen's kappa coefficient are reported in table 3 . the highest rate of concordance was found between histology and ihc either on the whole set of tissues or each specific tissue. exceptions were the lung, where the highest rate of concordance was found between ihc and rt-npcr, and the liver, on which ihc and rt-npcr had the same concordance of histology and ihc. the tissue with the highest rate of concordance (close to or higher than 90.0%, k > 0.8) was the small intestine, followed by the liver, large intestine, and lymph node, while the kidney, spleen, and lung had the lowest concordance (80-90%, k coefficients between 0.6 and 0.8, i.e., strong). the highest rate of concordance was found in tissues with a high prevalence of double negative results. with regard to discordant results, the analysis of individual data (table s2 ) revealed that the occurrence of negative ihc in tissues with lesions consistent with fip was more frequent than positive ihc in tissues without lesions consistent with fip. more specifically, ihc was negative in all the six samples from the non fip group and 2/56 samples from the fip group that were histologically consistent with fip. conversely, in the fip group, ihc was positive in only 1/37 samples on which no lesions histologically consistent with fip were found. in particular, the spleen of cat n • 9 showed a moderate increase in the plasmacytic component and perivascular macrophages, not organized in a typical fip lesion but immunohistochemically positive. as stated above, with the exception of the liver, that had an almost absolute concordance and a cohen's k coefficient that could be classified as "almost perfect", in all the other organs, the percentage of concordance varied between 70 and 90% and the level of concordance measured by the k coefficient was strong (0.60-0.80) for the kidney and large and intestine, but moderate (0.40-0.60) for the lymph node, small intestine, and lung. with regard to discordant results, the analysis of individual data (table s2 ) revealed that positive rt-npcr in tissues without lesions potentially consistent with fip occurred more frequently than negative rt-npcr in tissues with lesions potentially consistent with fip. in particular, rt-npcr was negative in all the six samples from the non fip group and in 1/56 samples from the fip group that had lesions histologically consistent with fip. curiously, this latter sample was anyway positive in ihc. conversely, rt-npcr was positive in 15 tissues from cats with fip and 6 tissues from the non fip group in which no lesions consistent with fip were found at histology. more specifically, 4/6 rt-npcr-positive tissues from the non fip group did not have histological lesions at all (spleen of cat n • 20 and n • 23, lymph node of cat n • 21, kidney of cat n • 19), while in 2/6 rt-npcr-positive cases lymphoid hyperplasia was found (lymph node of cat n • 20, small intestine of cat n • 24). in the fip group, no lesions at all were detected in six rt-npcr-positive cases (spleen of cat n • 3, kidney of cat n • 12, small intestine of cats n • 9 and 12, large intestine of cats n • 2 and 12), while eight rt-npcr-positive cases had lesions not consistent with fip, such as lymphocytic hyperplasia (spleen of cat n • 1, liver of cat n • 14, lymph nodes of cats n • 1, 2 and 14, large intestine of cat n • 9), interstitial nephritis (cat n • 13) and pulmonary emphysema (cat n • 14). the percentage of concordance was higher than 90%, and the level of concordance measured by the k coefficient was almost perfect only for the liver and lung. in all the other organs, the percentage of concordance varied between 80 and 90%, and the k coefficient was classified as strong (0.6-0.8). overall, double negative results (negative results in both rt-npcr and ihc) were more common than double positive results (positive result in both rt-npcr and ihc). as regards discordant results, the analysis of individual data (table s2) revealed that, with one single exception (lung of cat n • 11) of positive ihc and negative rt-npcr, in all the other discordant cases, rt-npcr was positive in tissues with negative ihc. in particular, this occurred in the same 6 cases from the non fip group and in 15/21 fip tissues in which histology was classified as negative and rt-npcr was positive (spleen of cats n • 1 and 3, liver of cat n • 14, lymph nodes of cats n • 1, 2, and 14, kidney of cats n • 5, 12, and 13, small intestine of cats n • 9 and 12, large intestine of cats n • 2, 9, and 12 and lung of cat n • 14), whose histological findings have been described above. additionally, ihc was negative, but rt-npcr was positive in the kidney of cat n • 4 and the large intestine of cat n • 14, which, however, had histological lesions consistent with fip. the diagnostic performance of histology, ihc, and rt-npcr for the diagnosis of fip on the different tissues analyzed in this study are reported in table 4 . histology showed absolute specificity and positive predictive value (ppv) only in the small and large intestine, while in all other organs except the kidney (83.3%), specificity was between 90.0 and 92.0%. the organs that less frequently showed lesions imputable to fip, leading to very low sensitivity, were the small and large intestine with a sensitivity of 41.7 and 53.8%, respectively. the other organs also showed low sensitivity, except lung (76.9%), which also showed the likelihood ratios (lr)-result closest to 0.00 (i.e., the lr− that indicates a good probability to exclude the disease), followed by the kidney and mesenteric lymph node. immunohistochemistry showed absolute specificity in all tissues and, consequently, a high ppv, while its sensitivity, as well as the negative predictive value (npv), were low, and the lr− was not close to 0.00. conversely, specificity and ppv of rt-npcr were absolute only for the liver, large intestine, and lung, although, for the kidney and small intestine, both the specificity and the ppv and especially the lr+ were high. however, sensitivity and the npv of rt-npcr, although not absolute and probably not relevant for diagnostic purposes, were higher than for ihc. in addition, in this case, however, the lr− was always distant from the optimal value of 0.00. this study was designed to achieve information about the concordance of different tests (histology, ihc, rt-npcr) performed on the same organs of cats with fip, aiming to find which one could be sampled in vivo to achieve an early diagnosis. cats with both the non-effusive and effusive form were included for a fair comparison between methods applied to the same tissues. in fact, despite the clinical distinction, mixed or transition forms are common [9] , and effusion and granulomatous lesions can be present to a greater or lesser degree and coexist [19] . of course, in the effusive form, analysis of the effusion is very useful for diagnosis, even though the sensitivity of immunostaining is not absolute, and rt-npcr lacks both sensitivity and specificity [20] . therefore, tests on tissues might be necessary for diagnosis confirmation, even when effusion is present. the organ panel was selected with the major aim of including organs that can be sampled in vivo when trying to reach an early diagnosis in clinical practice. organs reportedly affected by fip lesions and, at the same time, accessible from a surgical point of view, were selected, aiming to guide the clinician to perform targeted surgical biopsies in cats suspected to have fip. the analysis of every single test demonstrated, as expected that none of the tests have both absolute specificity and sensitivity. histological lesions most frequently detected in the fip group comply with the ones described in the literature as strongly consistent with fip [9, 21, 22] . in some organs from fip cats, histological lesions consistent with fip were not detected. however, cats with fip had histological lesions in more than one organ, except for two cats in which lesions were restricted to the kidney, which has been reported as one of the most frequently affected organs [9, 18] , and cns. these findings suggest that the collection of biopsies from more than one organ might increase the probability of diagnosing fip. however, histological lesions consistent with fip, such as granulomatous inflammation and lymphoplasmacytic infiltrates [23] , can also be detected in tissues from cats affected by diseases other than fip. these lesions, however, in the absence of granulomatous lesions, cannot be considered absolutely diagnostic for fip [21, 24] . differently from histology, ihc was positive only in cats with fip. in non fip cats, ihc positive signal was rarely found on villous superficial columnar epithelial cells of the large intestine. this may occur in non-symptomatic carriers since colonic enterocytes are thought to be the main site of viral persistence in the gut [7, 25] . the few immunohistochemical positive results in the small and large intestine are quite surprising, given the tendency of fip-related lesions to follow the course of the cranial mesenteric artery and their frequent involvement of the serosa of these organs [18] . however, ihc may also be negative in thoracic or abdominal organs when lesions are localized only in the cns, as occurred in one cat of our caseload. it is noteworthy that revising the spleen of cat n • 9 with a negative histological examination and positive ihc, only a mild increase in the perivascular number of macrophages was noticed multifocally, but was not considered as a typical lesion (figure 1a ). in light of the positivity to ihc (figure 1b) , these lesions could be considered as early lesions. this confirms that the presence of lesions potentially consistent but not absolutely diagnostic for fip should induce the pathologist to perform ihc since the combination of tests may be more informative than histology alone. pathogens 2020, 9, x for peer review 9 of 17 one organ, except for two cats in which lesions were restricted to the kidney, which has been reported as one of the most frequently affected organs [9, 18] , and cns. these findings suggest that the collection of biopsies from more than one organ might increase the probability of diagnosing fip. however, histological lesions consistent with fip, such as granulomatous inflammation and lymphoplasmacytic infiltrates [23] , can also be detected in tissues from cats affected by diseases other than fip. these lesions, however, in the absence of granulomatous lesions, cannot be considered absolutely diagnostic for fip [21, 24] . differently from histology, ihc was positive only in cats with fip. in non fip cats, ihc positive signal was rarely found on villous superficial columnar epithelial cells of the large intestine. this may occur in non-symptomatic carriers since colonic enterocytes are thought to be the main site of viral persistence in the gut [7, 25] . the few immunohistochemical positive results in the small and large intestine are quite surprising, given the tendency of fip-related lesions to follow the course of the cranial mesenteric artery and their frequent involvement of the serosa of these organs [18] . however, ihc may also be negative in thoracic or abdominal organs when lesions are localized only in the cns, as occurred in one cat of our caseload. it is noteworthy that revising the spleen of cat n°9 with a negative histological examination and positive ihc, only a mild increase in the perivascular number of macrophages was noticed multifocally, but was not considered as a typical lesion (figure 1a ). in light of the positivity to ihc (figure 1b) , these lesions could be considered as early lesions. this confirms that the presence of lesions potentially consistent but not absolutely diagnostic for fip should induce the pathologist to perform ihc since the combination of tests may be more informative than histology alone. our results demonstrated that the number of rt-npcr positivities exceeded those recorded in histology and ihc, due to the higher analytical sensitivity of this method [25] and to the possible occurrence of viremic episodes. moreover, the viral burden in fcov infected cats is reported to be very variable [26, 27] . therefore, some cats may have enough circulating viruses to be detected by molecular methods even when not affected by fip. the best concordance was found between histology and ihc, except for the lung, which showed the best concordance between ihc and rt-npcr. in non fip cats, the discordance between positive histology and negative ihc confirmed that lesions were due to causes other than fip, as hypothesized above. in fip cats, it may rather depend on the already reported variable distribution of lesions in different organs and viral antigens within lesions [21] . in these cases, however, other tissues from the same cat were positive at both histology and ihc, confirming that multiple tissues should be sampled to improve the probability to detect fip lesions and/or positive ihc since ihc might not be confirmatory if only one section is analyzed [12] . a substantial limitation of this study is that the sampling of organs post-mortem does not exactly represent what can be sampled in a clinical setting, our results demonstrated that the number of rt-npcr positivities exceeded those recorded in histology and ihc, due to the higher analytical sensitivity of this method [25] and to the possible occurrence of viremic episodes. moreover, the viral burden in fcov infected cats is reported to be very variable [26, 27] . therefore, some cats may have enough circulating viruses to be detected by molecular methods even when not affected by fip. the best concordance was found between histology and ihc, except for the lung, which showed the best concordance between ihc and rt-npcr. in non fip cats, the discordance between positive histology and negative ihc confirmed that lesions were due to causes other than fip, as hypothesized above. in fip cats, it may rather depend on the already reported variable distribution of lesions in different organs and viral antigens within lesions [21] . in these cases, however, other tissues from the same cat were positive at both histology and ihc, confirming that multiple tissues should be sampled to improve the probability to detect fip lesions and/or positive ihc since ihc might not be confirmatory if only one section is analyzed [12] . a substantial limitation of this study is that the sampling of organs post-mortem does not exactly represent what can be sampled in a clinical setting, and our results cannot be directly transferred to bioptic samples collected in vivo. moreover, tissues collected in vivo by bioptical means are reduced in size, and the possibilities of obtaining multiple sections from these kinds of samples, especially when they are sent to commercial laboratories, are low. unfortunately, the same approach adopted in this study would not have been possible in live pathogens 2020, 9, 852 9 of 15 animals. the ultrasound-guided collection of bioptic samples immediately after euthanasia would be an alternative future approach to reproduce the clinical setting. however, results of histology, immunohistochemistry, and/or rt-npcr may provide positive results even when evident gross lesions are not detected. therefore, knowing which tissues might be preferable to collect, even if not affected by macroscopic or sufficiently large lesions to be detected by imaging means, could possibly be useful. the low concordance between rt-npcr and histology or ihc was generally due to positive rt-npcr in tissues that were negative for histology and/or ihc. this is likely due to the above mentioned high analytical sensitivity of rt-npcr, coupled with the possible systemic spread of fcov in infected cats not affected by fip [6, 17, 28] . the single exception was represented by one lung sample from an fip cat, which had positive ihc but negative rt-npcr. different hypotheses could explain this discrepancy. first, lesions could be restricted to sections processed for ihc and not in those intended for rt-npcr, despite collected tissue sections always being adjacent to each other. second, the amount of viral rna in the sample intended for rt-npcr might not have been adequate to produce enough amplification products. third, the fcov genome within the lesion could have undergone mutation(s) that did not allow the binding of primers to target sequences. this latter hypothesis is unlikely, either because the 3 -utr's target of the rt-npcr is well conserved in fcovs [28] or because the kidney from the same cat was rt-npcr positive, although simultaneous infection by different mutated strains cannot be excluded. it is noteworthy that in non fip cats, positive results of rt-npcr may be misleading. positive results were found, especially in the mesenteric lymph node and spleen. these latter organs are thought to harbor high viral loads because of the migration of fecv infected monocytes through the vascular network [27] . conversely, the absence of rt-npcr positive results in the liver and lung of non fip cats is partly surprising and in disagreement with those studies which identified these organs as probable sites of fcov persistence in healthy cats, as the results recorded in the large intestine. again, this finding could be imputable to the low sample size of non fip cats [25] . however, in both fip and non fip cats, the rate of concordance between rt-npcr and ihc was very variable in the different tissues. again, this indicates that the probability of diagnosing fip, independent of the method used, increases when several organs are sampled. alternatively, it may be advisable to sample the liver, lung, and small intestine since these organs had the highest rate of positive results and may provide the same information independent of the diagnostic method used. indeed, the choice of tissue and test that more likely might provide diagnostic information should also be based on information about diagnostic accuracy. from this perspective, our results confirmed that, regardless of the examined organ, positive ihc is always diagnostic for fip since the specificity and positive predictive value reached 100%. on the contrary, negative ihc does not allow the exclusion of fip, based on the low sensitivity and negative predictive value and on the high negative lr. these results, in fact, indicate that, in the case of negative ihc, the chance that the cat is affected by fip is not null [29] . nevertheless, it should again be considered that performing ihc on a wide range of organs and lesions increases the probabilities to diagnose fip. additionally, considering results from cns tissues from cat n • 2, which were not included in the calculation of diagnostic performance of ihc, the diagnostic accuracy of ihc would have reached 100%. this additional information confirms that ihc performed on multiple tissues collected at necropsy, including cns, may be absolutely diagnostic for fip. however, this information would also deviate from the aims of this study, which is intended to recommend organs to sample in clinical practice on live animals, based on the frequency of positive results. from this standpoint, the higher specificity of ihc compared with histology, coupled with the low sensitivity of histology, suggest that a negative histological result alone cannot rule out fip and that in case of strong clinical suspicion, it is advisable to obtain serial sections of the same tissue to perform ihc, to increase the probability of finding the fcov antigen within histological lesions, which may not have been included in the first slide. contrary to ihc, rt-npcr showed an overall higher sensitivity but lower specificity, as already reported [17] , with rare exceptions. according to our results, positive rt-npcr cannot be considered diagnostic for fip, except for the liver, large intestine, and lung, in which this method exhibited absolute specificity. conversely, diagnostic accuracy on the kidney proved to be the highest, as demonstrated by the high positive likelihood ratio, that unlike predictive values, was not affected by the prevalence of the disease. in some cases, the sensitivity of rt-npcr was low, especially in the liver and large intestine. results regarding rt-npcr on the large intestine were quite surprising since samples from this latter organ are more likely expected to give false positive rt-npcr results due to the presence of fcov in the gastrointestinal tract of shedder cats. a possible explanation for this finding could be the action of fecal rnase on nucleic acids and their degradation. moreover, the high rate of negative rt-npcr results recorded in this study in the small and large intestine could depend on a decreased viral fecal excretion, which has been demonstrated to occur in fip affected cats, hypothetically due to the impaired tropism of mutated fcov for intestinal epithelial cells [30, 31] . on the other hand, non fip cats are less likely to shed the virus in the feces compared with fip cats [17] . nevertheless, the small sample size of the non fip group could also have affected the results, and also performing the rt-npcr on feces would have been useful to confirm if the cats were not shedding fcov at the moment of sampling. specificity was notably low in the mesenteric lymph node and spleen, suggesting that an rt-npcr positivity in these organs, which are frequently sampled in clinical routine for diagnostic purposes, cannot be considered as diagnostic for fip. this seems to be in contrast with the high sensitivity and specificity of quantitative rt-npcr on the mesenteric lymph node that has been recently reported [15] . overall, the low specificity of rt-npcr makes it mandatory to perform histology and ihc independently of the rt-npcr results, except in the liver and lung, where a positive result is absolutely specific for fip. tissue samples were collected post-mortem at the veterinary teaching hospital of milan from cats affected by fip or other systemic diseases or serious injuries, deceased or euthanized, and subjected to necropsy for diagnostic purposes. all the above methods were performed within routine diagnostic procedures and with the owner's written informed consent about the use of tissues and samples for research. therefore, according to the ethical committee of the university of milan (decision n • 2, 2016), residual aliquots of tissues were used for research purposes without any additional formal request of authorization to the ethical committee. the inclusion criteria were: • the possibility to collect and process tissues within six hours after death; • the availability of signalment, history, and clinical information (physical examination, clinical pathology, diagnostic imaging, depending on the clinical presentation). this information, along with necropsy findings and histology results, was used to classify the cats in the fip or non fip group [32] . samples were collected from the spleen, liver, mesenteric lymph node, kidney, large intestine, small intestine, and lung of the necropsied cats, paying particular attention to sample tissues with gross lesions, whenever present, and to always include the organs serosa (see table 1 for a detailed list of sampled organs). when possible, data regarding evident macroscopic alterations frequently seen in fip, namely: thickening of the serosa, focal or diffuse nodular lesions, organs increase in size, were recorded (see supplementary table s2 ). samples were then processed as described below. for diagnostic purposes, tissues from other organs showing macroscopic alterations, as well as organs not visibly affected but plausibly involved based on the patient's history (e.g., brain and cerebellum from cats with neurological signs), were also sampled during a necropsy to perform routine histology and immunohistochemistry. since rt-npcr was not performed on these tissues, the corresponding results were not included in comparison with rt-npcr but were used to achieve accurate classification of cases in the fip or non fip group as detailed below. from each organ, a sample was taken and sectioned with a sterile scalpel in two immediately adjacent halves of approximately one-centimeter diameter each. if evident nodular lesions were present, the two halves were obtained, sectioning the sample in the center of the lesion. one half was placed in plain tubes and immediately frozen at −20 • c for molecular biology, while the other half was collected into 10% neutral-buffered formalin to perform histology and immunohistochemistry. histopathology was performed by an experienced veterinary pathologist, blinded to both gross lesions as well as to the patient's signalment and history. formalin-fixed samples were sent to the department of comparative biomedicine and food science of the university of padua. sections (4 µm) obtained from formalin-fixed paraffin-embedded samples were stained with hematoxylin-eosin for histology with an automated stainer (autostainer xl, leica biosystems, wetzlar, germany). for immunohistochemistry (ihc), 4 µm paraffin sections were placed on surface-coated slides (superfrost plus, thermofisher scientific, milan, italy). immunostaining was performed with an automatic immunostainer (ventana benchmark xt, ventana medical system, tucson, az, usa). antigen retrieval was performed with standard cc1 (heat induced epitope retrieval, hier, in tris-edta buffer ph 7 at 95 • c for 44 min). as the primary antibody, a mouse monoclonal antibody against the feline coronavirus was used (dilution 1:500, clone fipv3-70 serotec, oxford, uk). after incubation at 37 • c for 30 min, a kit with a secondary antibody with horseradish peroxidase (hrp)-conjugated polymer that binds to mouse and rabbit primary antibodies (ultraviews universal dab, ventana medical system, tucson, az, usa) was used. all reagents were dispensed automatically except for the primary antibody, which was manually dispensed. fip positive tissues were used as internal positive controls. for negative controls, the antibody diluent (ventana medical systems) was applied instead of the primary monoclonal antibodies. from frozen-thawed samples, rna was obtained using a nucleospin rna kit (macherey-nagel, bethlehem, pa, usa). twenty milligrams of tissue were thinly shredded on sterile plates using sterile scalpels and vigorously vortexed in ra1 lysis buffer until completely dissolved. all the further steps were performed according to the manufacturer's instructions. rna samples were then frozen at −80 • c or immediately used for rt-npcr. rt-npcr targeting a 177 bp product of the highly conserved 3 untranslated region (3 utr) of the genome of both type i and type ii fcov was used [28] . rna pre-analytical quality control targeting vertebrate 12s rrna locus [33] was performed on randomly selected samples (results not shown). fcov rt-npcr positive rna was used as positive control and a fip-negative sample as negative control. rt-npcr products were visualized under uv transilluminator on a 1.5% agarose gel stained with ethidium bromide. cats included in this study were divided into two groups based on the following criteria. fip group: cats suspected to have fip based on history and/or clinical, hematological, biochemical, and serum protein electrophoretic alterations [11] on which diseases other than fip were excluded, showing gross and histological lesions consistent with fip in at least one tissue, as reported below, including tissues sampled for diagnostic purposes and not included in this study (e.g., brain or cerebellum). • non fip group: cats with or without a clinical presentation potentially consistent with fip, on which diseases other than fip were already diagnosed on a clinical basis (e.g., based on results of diagnostic imaging or cytology) and/or without any histological lesion consistent with fip. for each organ, lesions were considered consistent with fip if showing one or more of the following typical fip patterns ( figure 2) [9, 18] : pyogranulomas on one or more serosal surfaces; • granulomas with or without necrotic areas; • lymphocytic and plasmacytic infiltrates in specific sites (e.g., band-like infiltrate in serosal surfaces, perivascular infiltrate in meninges and cns); • granulomatous to necrotizing vasculitis and fibrinous serositis. pathogens 2020, 9, x for peer review 13 of 17 • non fip group: cats with or without a clinical presentation potentially consistent with fip, on which diseases other than fip were already diagnosed on a clinical basis (e.g., based on results of diagnostic imaging or cytology) and/or without any histological lesion consistent with fip. for each organ, lesions were considered consistent with fip if showing one or more of the following typical fip patterns ( figure 2) [9,18]: conversely, histological lesions were considered non-consistent with fip if changes consistent only with diseases other than fip or no lesions at all were present. histological lesions not diagnostic for fip as a standalone, but consistent with fip if associated with signalment, history, gross lesions, and other histological lesions (e.g., pyogranulomas) suggestive of fip, were considered potentially consistent with fip. these lesions (e.g., multifocal perivascular increase in macrophages) were categorized as positive or negative depending on the lesions recorded in other organs, as it would happen in a clinical setting. as previously described by pedersen and kipar, immunohistochemistry was considered positive if fcov antigen was detected within typical histological lesions above described [9, 18] . on the contrary, according to kipar and colleagues, the detection of fcov antigen in few scattered tissue macrophages (e.g., kupffer cells in the liver, pulmonary interstitial macrophages in the lungs as well as individual sinus macrophages in the lymph nodes) or epithelial cells (e.g., intestinal columnar cells, likely infected by fecv, figure 3) only, was not considered as a positive result [25] . • pyogranulomas on one or more serosal surfaces; • granulomas with or without necrotic areas; • lymphocytic and plasmacytic infiltrates in specific sites (e.g., band-like infiltrate in serosal surfaces, perivascular infiltrate in meninges and cns); • granulomatous to necrotizing vasculitis and fibrinous serositis. conversely, histological lesions were considered non-consistent with fip if changes consistent only with diseases other than fip or no lesions at all were present. histological lesions not diagnostic for fip as a standalone, but consistent with fip if associated with signalment, history, gross lesions, and other histological lesions (e.g., pyogranulomas) suggestive of fip, were considered potentially consistent with fip. these lesions (e.g., multifocal perivascular increase in macrophages) were categorized as positive or negative depending on the lesions recorded in other organs, as it would happen in a clinical setting. as previously described by pedersen and kipar, immunohistochemistry was considered positive if fcov antigen was detected within typical histological lesions above described [9, 18] . on the contrary, according to kipar and colleagues, the detection of fcov antigen in few scattered tissue macrophages (e.g., kupffer cells in the liver, pulmonary interstitial macrophages in the lungs as well as individual sinus macrophages in the lymph nodes) or epithelial cells (e.g., intestinal columnar cells, likely infected by fecv, figure 3) only, was not considered as a positive result [25] . rt-npcr was considered positive if it showed a 177bp band on agarose gel electrophoresis [28] . for histology, ihc and rt-npcr, true-positive (results consistent with fip in cats with fip) and false-positive results (results consistent with fip in cats without fip), as well as true-negative (results not consistent with fip in cats without fip) and false-negative results (results not consistent with fip in cats with fip) were recorded. sensitivity, specificity, accuracy, positive and negative predictive values, as well as positive and negative likelihood ratios (lr+ and lr−, respectively) were then calculated for histology, ihc, and rt-npcr. the concordance between histology, ihc and rt-npcr was assessed using cohen's k coefficient. concordance was classified as absent (k < 0); minimal (0 < k < 0.20); weak (0.21 < k < 0.40); moderate (0.41 < k < 0.60); strong (0.61 < k < 0.80), and almost perfect (0.81 < k < 1) [34] . rt-npcr was considered positive if it showed a 177bp band on agarose gel electrophoresis [28] . for histology, ihc and rt-npcr, true-positive (results consistent with fip in cats with fip) and false-positive results (results consistent with fip in cats without fip), as well as true-negative (results not consistent with fip in cats without fip) and false-negative results (results not consistent with fip in cats with fip) were recorded. sensitivity, specificity, accuracy, positive and negative predictive values, as well as positive and negative likelihood ratios (lr+ and lr−, respectively) were then calculated for histology, ihc, and rt-npcr. the concordance between histology, ihc and rt-npcr was assessed using cohen's k coefficient. concordance was classified as absent (k < 0); minimal (0 < k < 0.20); weak (0.21 < k < 0.40); moderate (0.41 < k < 0.60); strong (0.61 < k < 0.80), and almost perfect (0.81 < k < 1) [34] . in conclusion, results from this study prove that the frequency of positive results obtained with different methods (histology, ihc, and rt-npcr) on fip cats varies depending on the analyzed tissue. even if with a low frequency, some histological results consistent with fip were also recorded in non fip cats. conversely, ihc had a 100% specificity, and, therefore, it may be advisable to use ihc to confirm and exclude the disease in the presence of histological lesions consistent with fip. on the other hand, rt-npcr is not recommended as a first diagnostic approach, i.e., on fine-needle aspiration biopsy (fnab), due to its lower specificity and sensitivity. therefore, it is always advisable to confirm possible positive or negative rt-npcr results with ihc. however, data concerning concordance suggest that, when both tests cannot be performed, it would be preferable to examine either the lung or liver, on which the probability to obtain accurate information, independent of the analytical method, is good. instead, when the kidney or intestine are sampled, ihc should be preferred to rt-npcr as a confirmatory test since the concordance of the two methods is not sufficiently high, and the specificity is higher for ihc than for rt-npcr. supplementary materials: the following are available online at http://www.mdpi.com/2076-0817/9/10/852/ s1, supplementary table s1. details on histological lesions recorded for each sample included in the study. abbreviations. eo, eosinophilic, f, fibrinous; g, granulomatous; lp, lymphoplasmacytic; n, neutrophilic; m, macrophagic; pv, perivascular. lesions highly suggestive and classified as consistent with fip are reported in italic. a lesions compatible with fip, classified as negative (not consistent with fip). b lesions compatible with fip, later on, classified as positive (consistent with fip). supplementary table s2 . results of macroscopic and histological examination, immunohistochemistry, and rt-npcr obtained on each collected sample. negative (not consistent with fip) and positive (highly suggestive, therefore compatible with fip) results are listed. abbreviations: n, negative; n/a, not applicable (sample not collected); n/d, not done; p: positive. a tale of two viruses: the distinct spike glycoproteins of feline coronaviruses feline coronavirus in multicat environments an update on feline infectious peritonitis. companion anim feline coronaviruses: pathogenesis of feline infectious peritonitis replication of feline coronaviruses in peripheral blood monocytes amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis persistence and evolution of feline coronavirus in a closed cat-breeding colony the molecular dynamics of feline coronaviruses feline infectious peritonitis: still an enigma? clinical and laboratory features of cats with feline infectious peritonitis-a retrospective study of 231 confirmed cases (2000-2010) comparison of the performance of laboratory tests in the diagnosis of feline infectious peritonitis diagnosis of feline infectious peritonitis: update on evidence supporting available tests an update on feline infectious peritonitis: diagnostics and therapeutics performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases diagnosis of non-effusive feline infectious peritonitis by reverse transcriptase quantitative pcr from mesenteric lymph node fine-needle aspirates sensitivity of tru-cut and fine-needle aspiration biopsies of liver and kidney for diagnosis of feline infectious peritonitis limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis a review of feline infectious peritonitis virus infection: 1963-2008 feline infectious peritonitis diagnosis of feline infectious peritonitis: a review of the current literature cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis some aspects of humoral and cellular immunity in naturally occuring feline infectious peritonitis chronic kidney disease in aged cats cellular composition and interferon-γ expression of the local inflammatory response in feline infectious peritonitis (fip) sites of feline coronavirus persistence in healthy cats natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats high viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (fcov) type i and in naturally fcov-infected cats detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr on the use and computation of likelihood ratios in clinical chemistry feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene spike protein fusion peptide and feline coronavirus virulence feline coronavirus infections two universal primer sets for species identification among vertebrates the measurement of observer agreement for categorical data funding: this research received no external funding. the authors declare no conflict of interest. key: cord-283202-5fq1wxz8 authors: kent, marc title: the cat with neurological manifestations of systemic disease. key conditions impacting on the cns date: 2009-05-31 journal: journal of feline medicine & surgery doi: 10.1016/j.jfms.2009.03.007 sha: doc_id: 283202 cord_uid: 5fq1wxz8 practical relevance a number of systemic diseases are associated with neurological deficits. most systemic diseases that impact on the nervous system result in multifocal neurological signs; however, isolated deficits can also be observed. this article reviews the clinical signs, pathophysiology, diagnosis, treatment and prognosis of four important systemic diseases with neurological consequences: feline infectious peritonitis, toxoplasmosis, hypertension and hepatic encephalopathy. clinical challenges early recognition of systemic signs of illness in conjunction with neurological deficits will allow for prompt diagnosis and treatment. while neurological examination of the feline patient can undoubtedly be challenging, hopefully the accompanying articles in this special issue will enable the clinician to approach these cases with more confidence. evidence base the veterinary literature contains numerous reports detailing the impact of systemic disease on the nervous system. unfortunately, very few references provide detailed descriptions of large cohorts of affected cats. this review summarises the literature underpinning the four key diseases under discussion. knowledge of the epidemiology of fcov infection is important in order to understand the pathogenesis of fipv. feline coronavirus infection is virtually endemic, with studies revealing that: ✜ approximately 50% of cats in the united states and europe have antibodies against fcov; 3 ✜ in australia, the seroprevalence in owned cats is approximately 34%; 7 ✜ in the uk, 82% of show cats, 53% of breeding cats and 15% of single-cat homes have anti-fcov antibodies; 8 ✜ in italy, the seroprevalence is 82% in cats from breeding colonies, shelters and homes; 9 ✜ in switzerland, similarly 80% of breeding cats, and 50% of free-roaming cats, test positive for anti-fcov antibodies. 10 the significance of this worldwide distribution relates to the fact that fipv is the result of spontaneous mutation of fecv, which means that cats worldwide are susceptible to developing fip. 6 despite this, approximately 5% of cats in multicat homes and a smaller percentage of cats in single-cat homes develop fip. 3, 11, 12 notably, it is young cats and immunosuppressed cats that are most susceptible to developing fip. in addition, certain purebreed cats, specifically the birman, ragdoll, bengal, rex, abyssinian and himalayan breeds, have a greater risk of developing fip, which suggests a genetic influence on susceptibility. 13 transmission of fcov is through infected fecal material via the orofecal route, leading to enteric infection. enteric fcov infection typically results in inappetence and/or mild gastrointestinal signs such as vomiting and diarrhea. infected cats shed fecv for up to 10 months post infection; thereafter infected cats shed virus intermittently or continuously, serving as chronic carriers and thereby perpetuating reinfection of other individuals. 14 as with all coronaviruses, fecv undergoes a high rate of mutation. the degree of mutation, and therefore the development of a mutation leading to fip, appear greater in susceptible individuals as well as in individuals with a high viral load. 15, 16 in part, this may explain the fact that more than 50% of cats with fip are under a year of age. 3 the initial step in the pathogenesis of fip is the mutation of fcov, in the process of which the virus gains the ability to replicate within macrophages. once this occurs, the virus can be disseminated throughout the body. ultimately, fip is an immune complex disease that is a consequence of virus or viral antigens complexed with antiviral antibodies. 16 after distribution by macrophages, the virus may enter tissue and replicate, resulting in attrac-consisting primarily of lymphocytes, macrophages, and varying numbers of plasma cells forming perivascular cuffs. 18 subependymal periventricular inflammatory infiltrate may obstruct the mesencephalic aqueduct leading to obstructive hydrocephalus. similarly, obstruction of the central canal of the spinal cord may lead to hydromyelia. occasionally, infiltrate extends into the superficial neuropil and cranial nerve roots. 18 hematology in affected cats usually reveals a normocytic, normochromic, nonregenerative anemia, leukocytosis consisting of a neutrophilia, and lymphopenia. 22 approximately 50% of cats with the effusive form and 70% of cats with the dry form have increased serum proteins, primarily comprising a hyperglobulinemia. 23 protein electrophoresis discloses a polyclonal gammopathy, mainly involving the γ-globulins. 24 other biochemical changes may be observed depending on the severity of involvement of other organ systems including abnormal liver enzyme, bilirubin, blood urea nitrogen and creatinine levels. 25 although common clinicopathologic abnormalities in affected cats have been defined, changes in routine hematology and biochemical evaluations are often non-specific. consequently, establishing a definitive ante mortem diagnosis of fip is extremely challenging. 26 definitive diagnosis can only be achieved through histopathological identification of pyogranulomatous inflammation within tissue coupled with identification of the virus. 16 viral identification in tissue samples can be performed using immunohistochemistry or through pcr testing. however, there are several tests that may help support a presumptive ante mortem diagnosis. importantly, interpretation of results from such tests should be evaluated in conjunction with clinical signs and results of other diagnostics. taken outside the context of signs and other clinicopathologic data, most tests are unable to provide a definitive diagnosis. when present, effusions should be analyzed. typically, effusion from an affected cat should be consistent with a modified transudate. 3 cats with neurological signs should undergo magnetic resonance imaging (mri) of the brain. this may disclose hydrocephalus. 18 additionally, t2-weighted and t2weighted flair images may reveal periventricular hyperintensities consistent with periventriculitis. 18 although these findings are not pathognomonic, mri of the brain should be pursued in order to eliminate the potential that clinical signs may be a consequence of a disease process other than fipv. analysis of cerebrospinal fluid (csf) often reveals increased protein content (50-350 mg/dl) with a pleocytosis consisting of neutrophils, lymphocytes and macrophages. 20, 27, 28 the evaluation of fcov antibody titers (often erroneously referred to as an 'fip titer') in blood and other fluids has been extensively studied. however, despite years of investigation, caution should be exercised when interpreting fcov antibody titers in blood and effusions as high titers can be observed in healthy cats and low to absent titers in affected cats. 3 ✜ fipv is fatal and treatment is mainly palliative. since fip is an immune-mediated disease process, therapy has been directed at immunosuppression and/or immunomodulation with the goal of providing symptomatic care. immunosuppressive therapy using corticosteroids (prednisone at 2-4 mg/kg/day) may allow mildly affected cats to maintain an acceptable quality of life for weeks to months. in addition to corticosteroids, a wide array of drugs including chemotherapy agents (cyclophosphamide and melphalan), an antiviral (ribavrin), a thromboxane synthetase inihibitor (ozagrel hydrochloride) and a variety of immunomodulating drugs (promodulin, human interferon-α, propionibacterium acnes, and feline interferon-ω) have been investigated. 36 the interpretation of results from most studies has been hindered by a lack of control groups and the difficulty in establishing a definitive diagnosis of fip in treated cats. 36 still, most therapeutic trials have failed and, disappointingly, an effective treatment regime remains elusive. presence of neurological fipv. 18 cerebrospinal fluid fcov antibody titers correlate with serum fcov antibody titers but, most importantly, elevated csf fcov antibody titers may also be observed in cats affected by neurological diseases other than fip. 29 while pcr assays can be performed on blood and effusions in affected cats, they are unable to distinguish between the mutated fcov causing fip, and the non-pathogenic fcov. 30 in addition, healthy cats can be viremic with fcov. 31 therefore, pcr identification of virus in blood or effusions does not provide a definitive diagnosis. 32 while its application in csf has not been studied, pcr identification of virus in csf may allow a definitive diagnosis of fip. measurement of serum α1-acid glypoprotein (agp), an acute phase protein that increases during inflammation, has been used in the diagnosis of fip. 33, 34 in cats with signs and clinicopathologic data highly suggestive of fip, elevation in serum agp provides strong supportive evidence of fipv infection. 33 however, agp may also increase in other conditions associated with inflammation, such as feline immunodeficiency virus (fiv) infection, or in cats with a high viral load of fecv, which may limit its potential as a diagnostic tool for fip. 34, 35 prognosis unfortunately, the prognosis for cats with fip is grave as all affected cats succumb to the disease. to date, no therapy has been shown to alter the eventuality of humane euthanasia or death of affected cats. toxoplasmosis is caused by an obligate intracellular protozoan parasite, toxoplasma gondii. the definitive hosts are the domestic cat and other felidae. many mammals can become infected with t gondii and serve as intermediate hosts; however, fecal shedding of infective oocysts occurs only in cats. systemic and ocular toxoplasmosis have been well described in cats. the emphasis in the following discussion is on central nervous system (cns) toxoplasmosis. there are three methods of transmission of t gondii: fecal-oral, ingestion of tissue cysts, and congenital. reproduction of the organism can involve both a sexual and asexual phase. the asexual phase occurs in many mammals and birds, which serve as intermediate hosts. as the definitive host, the sexual phase of the life cycle can only occur in cats and it does so within the intestinal tract. as a result, unsporulated oocysts, which are non-infectious, are passed in the feces. these oocysts require 1-5 days for sporulation to occur, at which point they become infectious. ingestion of sporulated oocysts by another cat begins another cycle. toxoplasma gondii also displays an extraintestinal life cycle. after infectious oocysts have been ingested, the organism is capable of penetrating the wall of the intestinal tract and disseminating to multiple organs. within these other organs, asexual reproduction occurs, giving rise to tissue cysts -bradyzoites (so named given their slow replication) and tachyzoites (in which replication is rapid). ingestion of bradyzoites in tissue is probably responsible for the majority of infections. in cats it can result in intestinal replication, while ingestion of bradyzoites by other animals can only lead to extraintestinal infection. ingestion of infectious organism during gestation can also lead to congenital infection. 37 cysts that form as a result of extraintestinal infection are likely to persist for life. 38, 39 encysted bradyzoites are the most probable source of continual release of antigen and reactivation of infection. 38 reactivation of infection is thought to occur secondarily to immunosuppression. cats infected with fiv appear to be predisposed to the development of acute toxoplasmosis. 40 there appears to be a high seroprevalence of t gondii infection in cats co-infected with fiv. 41, 42 the significance of this relationship is unknown as the seroprevalence of t gondii infection in fiv-infected cats is similar to that in the general population. 39, 43 immunosuppression as a result of ciclosporin therapy has also led to acute toxoplasmosis. 44, 45 with the availability of renal transplantation in cats, the role of immunosuppression in reactivation of infection and the development of clinical disease has gained importance. [46] [47] [48] clinical signs clinical infection with t gondii is not common in cats. infections can be considered acute or chronic. 49 acute toxoplasmosis typically affects younger cats. the most common clinical signs are anorexia, lethargy, fever, dyspnea and sudden death. [49] [50] [51] [52] chronic toxoplasmosis typically affects older cats and manifests over weeks to months. signs are similar to acute infection and may include vomiting, diarrhea, anorexia, weight loss, fever and icterus. 42, [49] [50] [51] with the exception of finding t gondii in tissue, no single test provides a definitive diagnosis. serology for the detection of igg and igm anti-t gondii antibodies is widely used. 61 after experimental inoculation, an igm response is detected in 1-3 weeks and an igg response in 2-4 weeks. immunoglobulin m responses peak within 3 weeks and persist for 3-16 weeks. in cats co-infected with fiv, there is a delayed conversion from an igm to an igg response. 62 unfortunately, an igm response does not necessarily correlate with active disease as occasionally igm responses can be detected in clinically normal cats with chronic infection. likewise, a single high igg response does not predict active disease, as igg responses can last up to 6 years. 63 a rising titer is strongly suggestive of active disease, however, and maximal titers are reached within 2-3 weeks. 63 in practice, given the insidious nature of the disease many cats have reached maximal immune responses by the time they are examined by a veterinarian, making documentation of a rising titer difficult. 61 theoretically, identification of an immune response in the cns, an immunoprivileged site, would suggest infection. however, immunoglobulins may extravasate from the blood into the csf in other inflammatory diseases that disrupt the blood-brain barrier. defining a ratio between serum and csf igg responses may help eliminate the possibility of passive cross over of antibodies secondary to another disease that compromises the integrity of the blood-brain barrier. a serum:csf igg response > 1 suggests local cns production of immunoglobulin. in experimental oral inoculation, cats remain clinically normal yet develop a detectable igg response in the csf 4-12 weeks post inoculation and again 8-16 weeks after secondary exposure. 64 importantly, an igg response in the csf can occur after exposure to killed tachyzoites in previously infected cats. 65 therefore, observation of an igg response in csf does not necessarily document infection. 65 experimental inoculation does not result in an igm response in the csf. 64 potentially, therefore, detection of an igm response in the csf may be indicative of active disease, but this is yet to be confirmed. clinical signs reflecting organ involvement include lymphadenopathy, myocardial disease, pancreatitis, hepatitis, anterior uveitis and chorioretinitis. 41, 42, [49] [50] [51] 53, 54 the diagnosis of clinical toxoplasmosis can be challenging. hematologic findings are nonspecific, often consisting of non-regenerative anemia, neutrophilic leukocytosis, lymphocytosis and monocytosis. 41, 42, 50 biochemical abnormalities generally reflect organ involvement and include azotemia, elevation in liver enzymes, hyperbilirubinemia and hyperproteinemia. 41, 42, 50 thoracic radiographs may show a diffuse interstitial to bronchial pattern in which infiltrate may coalesce into areas of patchy alveolar patterns (fig 2) . [49] [50] [51] 60 neurological signs ✜ central nervous system involvement occurs in almost all clinically affected cats. 50 neurological signs typically reflect a multifocal distribution and include hypothermia, behavioral changes, seizures, ataxia, blindness, anisocoria, torticollis, vestibular disease, muscle hyperesthesia, and paresis/paralysis. 21 cerebrospinal fluid analysis typically reveals a mild lymphocytic pleocytosis predominantly, although other cell types may be observed; protein may be elevated to up to 149 mg/dl. 39 neutrophilic pleocytosis has also been reported. 58 with the exception of identifying t gondii in tissue, no single test provides a definitive diagnosis. a presumptive diagnosis is based on a combination of clinical signs, evidence of recent or active infection (gained via serology for immunoglobulins or immune complexes, or pcr), exclusion of other disease processes, and response to therapy. 39 hypertension has been defined as a sustained increase in systolic blood pressure ≥ 160-170 mmhg. 75 although the prevalence of hypertension in cats has not been accurately established, one study documented hypertension in 2% of healthy cats. 80 in cats referred for evaluation of disease associated with hypertension, or animals with clinical signs compatible with hypertension, a 30% prevalence was found. 79 hypertension can be divided into three categories: white coat, secondary and idiopathic. ✜ white coat hypertension is an artefactual increase in blood pressure that develops secondarily to excitement or anxiety, and is likely to be the result of activation of the sympathetic nervous system. 81 it is observed in cats, and results in a median increase in systolic blood pressure of 17.6 mmhg ± 1.5 mmhg. 81 although pcr assays have not been performed on csf for the detection of t gondii, pcr assays have been utilized in the aqueous humor, another immunoprivileged site. 66, 67 toxoplasma gondii can be identified in the aqueous humor of cats with uveitis using pcr; however, the organism can also be detected in the aqueous humor of clinically normal cats that have naturally been exposed to t gondii. 66 consequently, pcr detection of t gondii in aqueous humor does not provide definitive proof of active disease. a similar interpretation of pcr analysis of csf is likely. unfortunately, the prognosis is poor for cats displaying neurological signs or severe respiratory disease as most will succumb to the disease. 21, [56] [57] [58] 60, 73 despite this, cats with focal cns toxoplasmosis may achieve long term remission. 59 since the initial description of systemic hypertension in cats, 74 the impact of hypertension systemically and on the nervous system has become increasingly recognized. a testament to this is a recent consensus statement from the american college of veterinary internal medicine that has established guidelines for identification, evaluation and management of hypertension in dogs and cats. 75 in healthy cats, normal systemic blood pressure, which is often reported as a systolic measurement, is 118-162 mmhg. [75] [76] [77] the wide range in the reported normal values is likely to reflect a lack of standardization in technique and equipment used to measure blood pressure. 75 many factors affect blood pressure measurement including recording device, cuff size and operator skill, as well as patient factors such as size and demeanor of the cat. although increasing age was found to be associated with increased blood pressure in one study, other reports have found no effect of age on blood pressure. [77] [78] [79] the treatment of choice for cats with clinical toxoplasmosis is clindamycin hydrochloride at 12-25 mg/kg divided per day. 39, 42 clindamycin is almost completely absorbed after oral administration and achieves high concentrations in most tissues, including the lung. 68 concentrations in csf are low; 69,70 however, the concentration in the brain may be higher given the lipophilic nature of the drug. 71 clindamycin is well tolerated, with only minimal side effects (eg, vomiting and diarrhea) reported at dosages two and a half to four times the recommended dosage. 72 parenteral formulations can be used in animals unable to receive oral medication or those experiencing gastrointestinal toxicity. reports of successful treatment are rare, which may reflect the difficulty of establishing a definitive diagnosis. systemic clinical signs typically show improvement within 24-48 h of initiation of treatment. 39 cats with systemic or ocular disease treated with antibiotic therapy may achieve clinical remission; however, recurrence of signs is likely as antibiotic therapy is unlikely to eliminate the organism entirely. 42 chronic systemic hypertension has a variety of pathological consequences that collectively are referred to as end-organ or target organ damage. important target organ damage is observed in the kidneys, eyes, heart and nervous system. 90 in the kidney, this leads to an accelerated decline in renal function, proteinuria and death. hypertension can exist in animals at any stage of renal disease, and may be seen in nonazotemic animals. 75 in the eye, hypertension leads to hypertensive retinopathy and choroidopathy (fig 3) . exudative retinal detachment, retinal hemorrhage, multifocal retinal edema and tortuosity of the retinal vessels may be observed, and commonly result in blindness. 74, 82, 83, 89, [91] [92] [93] in the heart, hypertension may result in cardiomegaly and left ventricular hypertrophy. 74, 83, 89, 94 a systolic murmur, gallop rhythm and congestive heart failure may be observed. 82, 89 in the nervous system, hypertension may result in a hypertensive encephalopathy. 83, 85, 86, 89, 95 two studies have variously documented neurological signs in 29% and 46% of cats with hypertension. 83, 89 clinical signs since hypertension in most cats can be cat egorized as secondary, clinical signs typically reflect the underlying disease process. consequently, affected cats often demonstrate signs relating to renal disease or hyperthyroidism, given the high prevalence of hypertension with these disorders. although the pathophysiology underlying hypertensive encephalopathy remains unclear, it is thought to involve the development of vasogenic edema, which predominantly affects the white matter. 96, 97 with acute hypertension, the autoregulatory capacity of the brain vasculature may be exceeded, leading to hyperperfusion, breakdown of the blood-brain barrier, and cerebral edema. 96, 98 in experimental acute hypertension in cats, gross findings include coning of the vermis of the cerebellum, cerebel-lar herniation into the foramen magnum (fig 4) , rostral displacement of the colliculi, and widening and flattening of the cerebral gyri, all of which reflect raised intracranial pressure. 95 microscopically, the consequences of edema are observed such as marked pallor of the cerebral white matter, accentuation of the separation between axons and myelin sheaths, and widening of the perivascular space. 95 in chronic hypertension, brain vasculature may be chronically vasoconstricted leading to hypertrophy and hyperplasia of the smooth muscle. 89 as a result, fibrous changes develop, allowing leakage of plasma which ultimately causes degeneration of the vasculature predisposing to ruptures and microhemorrhages. 89 multifocal cerebral arteriosclerosis with hemorrhages has been observed in cats with spontaneous hypertension. 83 a presumptive diagnosis of hypertensive encephalopathy is relatively straightforward and requires the documentation of hypertension (systolic blood pressure ≥ 160-170 mmhg) with contemporaneous neurological signs. the gold standard for blood pressure measurement is invasive intra-arterial monitoring. however, this is often not feasible in clinical practice. 99 consequently, indirect blood pressure monitoring is used most commonly. 99 accurate and reliable indirect blood pressure measurements can be performed using doppler flow ultrasonography and oscillometry. 99 minimal, mild, moderate and severe risk categories for target organ damage have been defined based on blood pressure recordings (see below). 75 identification of hypertension should prompt investigation for an underlying disease process. a complete blood count, biochemistry profile and urinalysis should be performed in all hypertensive cats. in cats older than 5 years of age, serum thyroxine level should also be measured. when indicated, endocrinological testing for cushing's disease or diabetes mellitus should be performed. in cats with suspected or confirmed renal disease, quantification of a proteinuria should be performed. thoracic radiographs should be obtained to assess cardiovascular structures, and abdominal ultrasonography should be performed to assess renal structure and identify any concurrent disease. echocardi ography is warranted in cats with a murmur, gallop rhythm or other signs consistent with cardiac disease. in all cats with hypertension, echocardiography allows assessment of any secondary cardiac changes. in cats with severe neurological dysfunction mri may be warranted. in addition to assessing cns pathology related to hypertension, mri allows exclusion of other disease processes that can produce similar neurological signs. given the potential for raised intracranial pressure and brain herniation in hypertensive encephalopathy, caution should be exercised prior to advanced imaging; the requirement for general anesthesia can lead to deterioration or death in animals with severe raised intracranial pressure. in humans with hypertensive encephalopathy, mri of the 402 jfms clinical practice brain discloses hyperintensities in the white matter of the parietal and occipital lobes of the cerebrum on t2-weighted images. 96 less frequently, similar findings may be observed in the brainstem. 100 magnetic resonance imaging in hypertensive cats has not been studied; however, given the gross and microscopic changes observed in affected cats, similar findings would be expected. unfortunately, control of hypertension does not appear to have a significant effect on survival time. 82, 83, 108, 109 however, amlodipine does seem to reduce the degree of proteinuria in cats with renal disease, and a reduction in proteinuria appears to have a positive effect on survival time. 108, 109 unless animals are showing evidence of target organ damage, or are at severe risk of developing target organ damage (see box on page 401), there is no requirement for immediate therapeutic intervention. instead, repeated blood measurements over a period of time, combined with identification and treatment of any potential underlying disease process leading to hypertension, may be all that is needed to control blood pressure. in cats that remain hypertensive despite control of an underlying disease process, or those with idiopathic hypertension in the mild to moderate risk category for target organ damage, the decision to pursue hypertensive therapy requires a dedicated owner as treatment is generally lifelong and involves frequent re-evaluations. in animals displaying signs consistent with hypertensive encephalopathy, prompt intervention should be pursued. the treatment of choice for hypertension in cats is amlodipine besylate, a calcium channel blocker. 82,99,101-103 a dose of 0.625-1.25 mg/cat orally once to twice daily reliably reduces blood pressure with minimal risk of causing hypotension. 82,101-104 furthermore, treatment with amlodipine is not associated with increases in blood urea nitrogen and creatinine in cats with chronic renal failure. 103, 104 in cats in which amlodipine is ineffective at controlling hypertension, adjunctive therapy with the β1 selective β-blocker, atenolol, may be instituted at 6.25-12.5 mg/cat po q 12-24 h. 105 alternatively, the angiotensin-converting enzyme inhibitor, benazepril, at 0.25-0.5 mg/kg po q 12-24 h can be used. 106 however, benazepril therapy is associated with only a small but significant reduction in blood pressure in cats with chronic renal failure. 106 in acute hypertension, subcutaneous hydralazine (1.0-2.5 mg/cat) has been effective at reducing blood pressure without significant risk of hypotension. 84 while parenteral hypotensive medications may be preferable in the setting of severe hypertensive encephalopathy, the use of such medications requires continuous, direct arterial blood pressure measurement and is associated with a significant risk of hypotension. in cats with severe neurological dysfunction that do not respond to a reduction in blood pressure, treatment for raised intracranial pressure due to brain edema may be warranted. this entails diuretic therapy with mannitol (0.5 to 2 g/kg iv over 10-15 mins often combined with furosemide 0.7 mg/kg iv), or other hypertonic agents. 107 note, however, that diuretic therapy should not be used until blood pressure has normalized, as these agents can transiently increase blood pressure. 107 tre a t m e n t o f h y p e r t e n s i o n hepatic encephalopathy is the clinical syndrome of abnormal neurological function caused by portosystemic shunting, with or without intrinsic liver disease. 110 as a result, he can develop in cats with acquired or congenital liver disorders. by far the most common cause of he in cats is portosystemic shunting of blood secondary to a congenital vascular anomaly. 111 regardless of the cause of the underlying liver disease, the clinical signs of he are similar and can be divided into systemic and neurological signs. affected cats often display intermittent clinical signs that may be associated with eating. 112 cats with portosystemic shunts are generally small in stature, fail to thrive and grow, and lose weight. [112] [113] [114] [115] pytalism is a common clinical sign, occurring in approximately 75% of cats. 112, [116] [117] [118] [119] [120] other, less common clinical signs include gastrointestinal signs such as decreased appetite, anorexia, pica, vomiting, diarrhea or constipation. 112, 113, 117 cats may demonstrate polydipsia, and polyuria, pollakiuria and stranguria may occur as a consequence of cystic calculi. 112, 113, 117 affected cats may have copper-coloured irises. 121 although a complete understanding of the mechanisms underlying he remains elusive, it is clear that the pathophysiology involved is multifactorial. despite numerous potential factors, ammonia remains key in the development of he. 124 ammonia is produced by bacteria in the gastrointestinal tract, primarily the colon, as a consequence of protein metabolism. 125 it is also produced by the gastrointestinal cells as a result of metabolism of glutamine, the main cellular energy source for the epithelium. 126 a further source of ammonia is the kidneys, during states of hypokalemia or alkalosis. 126 normally, the liver efficiently removes ammonia from the portal vasculature, ultimately converting it to urea. in animals with hepatic failure or portosystemic shunts, hyperammonemia may develop. however, the severity of the neurological signs does not always correlate with the degree of hyperammonemia. 127 in fact, blood ammonia levels may be normal in cats with he. 127 this relates to a greater rate of uptake of ammonia in the cns in he, leading potentially to a high cns ammonia level in the face of a normal blood ammonia level. 128 although he is a syndrome of neuronal dysfunction, the neuropathological consequences of increased cns ammonia are played out in the astrocyte. 129 normally, cns ammonia undergoes energy-dependent metabolism to glutamine by the astrocytes. 130 with increased cns ammonia, astrocytes become overwhelmed, leading to energy depletion. 110 additionally, the increased concentration of glutamine in astrocytes may act as an osmotic stress, leading to cell swelling. 110 increased numbers of swollen astrocytes -referred to as alzeheimer type ii astrocytes -is the only structural change observed microscopically in the brain in he. 131 as a consequence of cellular edema, neurotransmitter processing in the astrocytes is affected, resulting in upregulation of neuronal benzodiazepine receptors and the production of neurosteroids which increase γ-aminobutyric acid (gaba) neurotransmission and thereby ultimately affect neuronal function. 110 ammonia may also have a direct toxic effect on neurons. 110 while ammonia remains a focal point in the pathogenesis of he, other factors are also involved. mercaptans are formed during the degradation of sulfur-containing amino acids. these substances exert a neurotoxic effect through inhibition of atpase activity, thereby potentiating the effect of ammonia. 132 short and medium chain fatty acids are derived from bacterial metabolism of carbohydrates or from incomplete β-oxidation of long chain fatty acids in the liver. 127 like mercaptans, these molecules may inhibit energy metabolism as well as inhibiting urea cycle enzymes in the liver. 126 a putative role for mercaptans, short and long chain fatty acids is unknown, but they may act synergistically with ammonia. 127 hepatic encephalopathy may develop through an imbalance of inhibitory (gaba) and excitatory (glutamate) neuro transmitters. 133 there is evidence to implicate excessive gabaergic tone in he, which would result in global inhibition of neurological function. 133 in humans with he, there are increased gaba concentrations in the cns leading to excessive gabaergic tone. 134 in addition, there may also be increased concentrations of endogenous benzodiazepines in the cns. 127, 133 benzodiazepines also bind to the gaba receptor, potentiating the effect of gaba. 126 false neurotransmitters may also play a role in he. 126 in liver disease, the production of branched chain amino acids is reduced. 127 branched and aromatic amino acids compete for the same transporter into the cns. 126 as a consequence of reduced branched chain amino acids, there may be a relative increase in the aromatic amino acids; these so-called false neurotransmitters may act like gaba and other inhibitory neurotransmitters. 135 ultimately, the clinical signs of he may be a result of global inhibition of neurotransmission. a complete blood count, biochemistry and urinalysis should be performed in all animals with clinical signs suggestive of he. on hema tol ogy, microcytosis may be present. 112 biochemical abnormalities may include low blood urea nitrogen, increased liver enzymes, decreased total protein and albumin concentrations, and hypocholesterolemia. 112 urinalysis may disclose hyposthenuria and ammonium urate crystals. 112 a presumptive diagnosis can be made by documenting altered liver function in the setting of clinical signs consistent with he. an elevated fasting blood ammonia level helps confirm the clinical suspicion. in order to provide accurate results, blood samples should be treatment should be directed at the underlying cause of he as well as controlling the clinical signs of he. one of the primary aims is to reduce blood ammonia levels. for animals with mild to moderately severe clinical signs, which are capable of taking oral medications, lactulose should be administered at a starting dose of 1 ml po q 8-12 h. 116 the dosage is adjusted based on stool consistency and response. in more severely affected cats, lactulose can also be administered per rectum. prior to rectal administration, warm water enemas should be performed to remove fecal material. lactulose is a non-aborbable disaccharide that undergoes extensive metabolism by colonic bacteria, first to constituent monosaccharides and then to volatile fatty acids. 140 ultimately, lactulose decreases the production/absorption of ammonia. 140 this is accomplished in several ways: namely, by decreasing the colonic luminal ph, leading to conversion of ammonia (nh 3 ) to ammonium (nh 4 + ), trapping it intraluminally; decreasing transit time through the osmotic cathartic effect of lactulose; and interfering with intestinal absorption of glutamine, thereby decreasing the production of ammonia. 141 antibiotic therapy is often combined with lactulose administration. antibiotics with activity against ureaseproducing bacteria are effective at reducing ammonia production. neomycin is an oral aminoglycoside antibiotic that undergoes minimal systemic absorption. it is administered at 20 mg/kg po q 8 h. 116 despite the limited systemic absorption, systemic concentrations capable of causing side effects are possible. 116 metronidazole is also effective at reducing urease-producing microbes, and is administered at 10 mg/kg po q 12 h. 116 reduced hepatic metabolism in animals with liver disease may result in an increased incidence of neurotoxicity. 142 alternatively, ampicillin or amoxicillin-clavulanate can be administered. in severely affected animals, antibiotics (ampicillin, amoxicillin or metronidazole) should be administered parenterally. there are several precipitating factors that can lead to he, and these should be identified and corrected in the individual animal. correction of dehydration, hypoglycemia and hypokalemia is imperative. animals with clinical signs suggestive of gastrointestinal bleeding, such as melena, anorexia and vomiting, should be treated with h 2 -blockers. in moderately to severely affected animals, food should be withheld until therapy results in significant improvements. once a clinically significant improvement is obtained, affected animals should be fed a diet restricted in protein, with limited aromatic amino acids and short chain fatty acids. 114 key points: hepatic encephalopathy placed in a heparinized tube and transferred to the laboratory on ice for immediate testing. red blood cells contain large amounts of ammonia; hence hemolysis may result in falsely elevated blood ammonia levels. alternatively, a presumptive diagnosis of he can be made by demonstrating altered liver function through fasting bile acid stimulation testing and on the basis of response to therapy. a suspected or confirmed diagnosis of he should prompt investigations for congenital or acquired portosystemic shunting. portosystemic shunts are most commonly diagnosed by ultrasonography or per rectal portal scintigraphy using 99m technetium pertechnetate. [136] [137] [138] [139] positive contrast portography also can be performed; however, this necessitates general anesthesia and laparotomy. 113 the prognosis for animals with he is dependent on the underlying liver disease. with treatment, most animals experience an improvement in the clinical signs related to he. severely affected animals may not respond to therapy, however. animals with increased blood ammonia tend to respond better to treatment than those with normal blood ammonia. feline infectious peritonitis, part 2 feline infectious peritonitis and feline enteric coronavirus infections. i. feline enteric coronaviruses clinical, cerebrospinal fluid, and histological data from twenty-seven cats with primary inflammatory disease of the central nervous system inflammation and changes in cytokine levels in neurological feline infectious peritonitis use of anti-coronavirus antibody testing of cerebrospinal fluid for diagnosis of feline infectious peritonitis involving the central 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mercaptans and ammonia or fatty acids in the production of coma: a possible role for mercaptans in the pathogenesis of hepatic coma experimental hepatic encephalopathy: changes in the binding of gamma-aminobutyric acid hyperammonaemia, plasma aminoacid imbalance, and blood-brain aminoacid transport: a unified theory of portal-systemic encephalopathy per rectal portal scintigraphy using 99m technetium pertechnetate to diagnose portosystemic shunts in dogs and cats diagnosis and treatment of portosystemic shunts in the cat ultrasonographic diagnosis of portosystemic shunting in dogs and cats transcolonic sodium pertechnetate tc 99m scintigraphy for diagnosis of macrovascular portosystemic shunts in dogs, cats, and potbellied pigs: 176 cases (1988-1992) the treatment of hepatic encephalopathy new mode of action for lactulose medical management of animals with portosystemic shunts available online at www.sciencedirect.com key: cord-264315-3hum7rqm authors: paltrinieri, s; grieco, v; comazzi, s; cammarata parodi, m title: laboratory profiles in cats with different pathological and immunohistochemical findings due to feline infectious peritonitis (fip) date: 2001-09-30 journal: journal of feline medicine & surgery doi: 10.1053/jfms.2001.0126 sha: doc_id: 264315 cord_uid: 3hum7rqm abstract blood was collected from 55 cats with feline infectious peritonitis (fip) and from 50 control cats in order to define whether differences in pathological findings and in distribution of feline coronaviruses (fcov) can be associated with changes in haemograms, serum protein electrophoresis, and antibody titres. compared to controls, the whole group of fip-affected cats had blood changes consistent with fip. based on the pathological findings or on the immunohistochemical distribution of viral antigen, fip-affected cats were divided in the following groups: subacute against acute lesions; low against strong intensity of positivity; intracellular against extracellular positivities; positive against negative lymph nodes. lymphopenia was more evident in cats with acute forms, strong intensity of positivity, extracellular antigen and negative lymph nodes. cats with positive lymph nodes had the most evident changes in the protein estimations. these results suggest that differences in pathological findings might depend on different reactive patterns to the fcovs. f eline infectious peritonitis (fip) is a fatal disease for wild and domestic felidae, caused by a feline coronavirus (fcov). the feline infectious peritonitis virus (fipv) is a mutated form of the enteric one (fecv) (vennema et al 1995 , poland et al 1996 . both these fcovs are able to pass from the intestine to the blood (herrewegh et al 1995) ; unlike fecv, fipv is able to replicate within the macrophages that phagocytose the virus in the lymph nodes and diffuse it through the body (pedersen 1995a) . the development of the disease depends on the balance between humoral and cellular immunity: antibodies could facilitate the viral uptake by macrophages (hodatsu et al 1993 , hodatsu et al 1994 and in the absence of cell-mediated immunity, antigen-antibody complexes are responsible for a type iii hypersensitivity reaction that leads to vasculitis and effusion (hayashi et al 1977 , pedersen, 1995a . weak cellular immunity leads to dry forms, characterised by type iv hypersensitivity reactions (pedersen 1987 , paltrinieri et al 1998a . in vivo the two types of immune reactions probably coexist (paltrinieri et al 1998b) . in experimental and in spontaneously occurring fip, fluctuating levels of circulating antibodies and immune complexes as well as discrepancies between -globulin levels and antibody titre have been reported (jacobse-geels et al 1982 , pedersen 1995a , paltrinieri et al 1998b . the severity of the disease has been associated with a decrease of circulating lymphocytes (ward et al 1974 , pedersen 1995a , paltrinieri et al 1998b . furthermore, the distribution and the histological pattern of the lesions, as well as the distribution of viral antigens within the lesions, show great variations among the cats and often among different organs of the same cat (weiss & scott 1981 , walter et al 1989 , tammer et al 1995 , kipar et al 1997 , kipar et al 1998 , paltrinieri et al 1998a . in order to define whether this variability is a casual finding or if it could be associated with different systemic reactive patterns, the relationship between the histological findings and the distribution of the virus on one hand, and haemograms, protein levels and antibody titres on the other were analysed in this study. fifty-five cats presenting clinical signs of fip were referred from private clinicians of the area of milan (italy). the characteristics of these cats, their pathological findings and the sampled organs are reported in table 1 . as a negative control, blood was taken from a group of 50 healthy cats, comparable with the group of cats with fip in terms of age, breed, sex and mode of life (pets, free roaming, and breeding cats). blood (3 ml) was withdrawn from the cephalic or from the jugular vein of each cat. one millilitre of blood was collected in edta-coated tubes and the remaining amount was put in tubes without anticoagulant. by mean of an automatic cell counter (hemat 8, seac, firenze, italy) leukocyte, platelet and erythrocyte numbers as well as haemoglobin (hb) concentration, hematocrit (ht) percentage, mean corpuscular volume (mcv), mean corpuscular haemoglobin (mch) and mean corpuscular haemoglobin concentration (mchc) were evaluated. the differential leukocyte count was microscopically evaluated on may grü nwald-giemsa (mgg) stained smears and the reticulocyte percentage on brillant cresyl blue stained smears as described by pasquinelli (1984) . the reticulocyte production index (rpi) was evaluated as suggested by jain (1993) . total proteins in serum were measured by a discrete analyser (abbott vp, abbott lab., irving, tx, usa) using the biuret method (abbott lab., abbott park, il, usa). serum protein electrophoresis was performed using the semimicromethod, with cellulose polyacetate strips (seac, firenze, italy) in a barbitone and tris buffer (helena lab. italia spa, assago, mi, italy). the strips were run for 40 min, 150 v and then stained for 15 min in red ponceau (0.5 g in 100 ml of 5% trichloroacetic acid), destained in 5% acetic acid and put in a diaphanising solution (helena lab. italia spa, assago, mi, italy). the gels were scanned in a densitometer (bt512, biotecnica instruments, roma, italy). antibody titres against fcovs were evaluated using a commercially available elisa kit (dyaset, portomaggiore, fe, italy) in 96 wells microtitre plates coated with fipv proteins and using anti-feline horseradish peroxidaseconjugated antibodies. the plates were read at 450 nm in an automatic elisa analyser (dasit multiskan, dasit spa, cornaredo, mi, italy) . serology for feline immunodeficiency virus (fiv) and for feline leukaemia virus (felv) was performed using commercially available elisa kits (snap, idexx lab, westbrook, ma, usa), according the procedure suggested by the manufacturer. a sample (approximately 1 cm 3 ) of the affected organs (see table 1 ) was taken from each dead cat, fixed in buffered 10% iso-osmotic formalin and embedded in paraffin. microtome sections (5 m) were used to confirm the diagnosis by hematoxylin-eosin stain and by immunohistochemistry using a monoclonal antibody against the fcov (kindly provided by prof n.c. pedersen, davis, usa). two to five sections of each sample and 5 to 10 serial sections from lymph nodes draining the lesions were immunohistochemically analysed. the avidin biotin complex (abc) method with a commercially available kit (vectastain elite, vector laboratories inc., burlingame, ca, usa) was used to detect the positive reaction, as described by hsu et al (1981) after inhibition of the endogenous peroxidase (h 2 o 2 1% in methanol) and antigen unmasking using microwave pretreatment (2 cycles of 5 min in citrate-buffered solution, 0.01 m, ph 6) (cattoretti et al 1993) and 3-amino-9-ethyl-carbazole as chromogen. some sections of each sample were used as negative controls, with the primary antibody substituted by an equal amount of normal mouse serum (dako a/s, glostrup, denmark). in each session of test a section of liver with a fibrinous perihepatitis and with intraparenchymatous pyogranulomatous foci from a cat with fip was used as positive control. histological and immunohistochemical sections were read by two different readers. since different histopathological patterns as well as different immunohistochemical characteristics were present in different lesions of the same animal and often in different foci of the same organ, for each cat the histological and immunohistochemical characteristics of each sample were recorded and groups were formed on the basis of the most prevalent finding in the same cat. the following groups were analysed and compared each other: acute vs subacute forms. the cases of fip on which the main histological finding was the presence of perivisceral fibrin and necrosis with scattered leucocytes were classified as acute (fig. 1) ; the cases of fip with histological signs of organisation of the perivisceral fibrin, with larger pyogranulomatous foci and/or with presence of intraparenchymatous pyogranulomatous foci were classified as subacute (fig. 2) . intensity of the positivity. on the basis of the number of positive cells within the foci two groups were formed: low intensity when less than 50% of the cells were positive (fig. 3) , and strong intensity when more than 50% of the cells were positive (fig. 4) . type of the positivity. two groups were formed according to the presence of intracellular (fig. 5) or extracellular (fig. 6 ) positivity: in the latter group, viral antigen was detectable both in the cells and in the extracellular spaces, where granular positivities were present. presence or absence of positivity in the lymph nodes draining the lesions. these groups were formed based on the presence or absence of positive cells that appeared as single cells with dendritic projections or large vescicular cells in germinal centres (fig. 7) , in absence of fip lesions such as pyogranulomatous lymphadenitis or of lymphoid depletion. haematological, serological and electrophoretic data of fip-affected cats were compared with those of controls using specific software (statsoft. inc, tulsa, ok, usa), by student's t-test. when the data did not have a normal distribution, the corresponding non-parametric test mann-whitney u test was used. the same tests were used to compare each of the results obtained in the different groups and to compare the different groups with controls. results from control cats (table 2 ) were in agreement with those reported in literature (jain 1993 , kaneko et al 1997 . furthermore all the cats from this group were fiv and felv negative. in contrast variable anti-fcov antibody titres were detected in this group. when considered as a whole and compared against controls (table 2) cats with fip had a normocytic, normochromic non-regenerative anaemia, neutrophilic leucocytosis with lymphopenia, eosinopenia and monocytosis, hypoalbuminaemia and hyperglobulinaemia with a decreased albumin/globulin (a : g) ratio, and increased 2 --and -globulin concentrations. also in this group no fiv or felv positives were present. the cats were then divided into groups according to their pathological and immunohistochemical findings. values related to erythrograms never exhibited any significant or evident difference among the groups. for this reason that these data are not reported in the following tables. acute against subacute forms (table 3) . thirty-five cats (63.6%) had acute forms, while in 20 cases (36.4%) histological findings were consistent with subacute forms. compared to controls, both groups had leucocytosis, neutrophilia with left shift, eosinopenia, monocytosis, hypoalbuminaemia, hyperglobulinaemia with inverted a : g ratio and increased 2-, -and -globulins. in contrast lymphopenia was present only in acute forms and hyperproteinaemia in the subacute ones. lymphocyte numbers and -globulins were significantly lower in cats with acute forms than in those with subacute forms. before analysing the results of groups formed according their immunohistochemical findings, it must be underlined that often foci with different characteristics of positivity were detectable in the same cat. nevertheless, the characteristics of positivity were similar in contiguous organs (eg intestine-omentum, liver-diaphragm, lungpericardium, etc.). the immunohistochemical findings that were more represented in the cats with fip were low intensity (31/55=56.4%), extracellular positivities (38/55=69.1%) and negative lymph nodes (30/55=54.5%) ( table 4 ). however immunohistochemical findings were not uniformly distributed among the different groups of cats: the main immunohistochemical finding in cats with acute lesions were negative lymph nodes and extracellular positivities, while cats with subacute lesions more frequently had low intensity of positivity; cats with intracellular positivities more often had positive lymph nodes, while those with extracellular positivities were mainly characterised by negative lymph nodes; finally, cats with negative lymph nodes showed a prevalence of low intensity of positivity. blood parameters were then analysed in the different groups of cats with the following results (table 5) : low against strong intensity of positivity. compared to controls, both groups had the above mentioned changes consistent with fip, except for hyperproteinemia that was present only in cats with low intensity of positivity. leucocytosis was significantly higher in cats with low intensity of positivity and lymphocyte counts were significantly lower in cats with strong intensity of positivity. intracellular against extracellular positivity. compared to controls, both the groups showed the changes consistent with fip, except for lymphopenia, that was present only in cats with extracellular positivity. these cats also had significantly lower eosinophil and lymphocyte counts than those with intracellular positivities. presence against absence of viral antigen in the lymph nodes. compared to controls, all the changes consistent with fip were detectable in both the groups, except for lymphopenia, that was present only in cats with negative lymph nodes, and hyperproteinaemia and hyper-globulinaemia, that were present only in cats with positive lymph nodes. lymphocyte counts and total and -globulin concentrations were significantly lower in absence than in presence of lymph node positivities. all the parameters in control cats were within the normal range (jain 1993 , kaneko et al 1997 . however, it must be underlined that the total proteins and protein fractions were characterised by marked individual variations, most likely due to the composition of the control group. in order to be comparable with cats with fip, in fact, the control group was composed of cats of different age, sex, and breed, as well as of cats living in different environments and it is well known that these variables can influence the protein levels af=acute forms; sf=subacute forms; li=low intensity of positivity; si=strong intensity of positivity; cp=cellular positivity; ep=extracellular positivity; nl= negative lymph node; pl=positive lymph node. li, low intensity of positivity; si, strong intensity of positivity; cp, cellular positivity; ep, extracellular positivity; nl, negative lymph node; pl, positive lymph node. (kristensen & barsanti 1977) . the finding of variable antibody titres in control cats is not surprising: anti-fcov positivities have already been reported in healthy cats (pedersen 1976 , barlough & stoddart 1990 , pedersen 1995a , paltrinieri et al 1998b . in the present sample cats from catteries were included. this increased further the possibility to have high anti-fcov antibody titres, since in multiple-cat environments the fcovs are often endemic, and seropositives are frequent (pedersen 1995b , richards 1995 . the haematological and serum protein profiles of cats with feline infectious peritonitis (fip) were in agreement with those reported in previous works (sparkes et al 1991 , pedersen 1995a , paltrinieri et al 1998b . after forming the groups and subsequently reducing the number of cats per group, variability among the cats increased further. by consequence, for some parameters statistical significance was rarely reached, despite strong differences in the mean values among the different groups. nevertheless, cats with acute lesions showed a significant decrease of lymphocytes compared to controls and to animals with subacute lesions. this suggests that the decrease of lymphocytes might exacerbate the disease, in agreement with the previous hypothesis on the role of lymphocytes in modulating the development of the disease (ward et al 1974 , pedersen 1995a , haagmans et al 1996 . -globulins and antibody titres were higher in subacute than in acute fip, suggesting an early involvement of immune system, although it cannot be excluded that the low antibody titre in acute fip depends on immunecomplexes formation. in experimental infections, an early increase of 2 -globulins was reported (stoddart et al 1988) , while -globulins and antibody titres increase just before the appearance of the symptoms either in effusive (stoddart et al 1988 , pedersen 1995b , gunn-moore et al 1998 or in non-effusive forms (pedersen, 1976) . in the present study it is not possible to find this correlation: in spontaneously occurring fip, in fact, it is not possible to assess the moment of infection nor to have an exhaustive follow-up, because the cats are usually euthanatised just after blood sampling. furthermore the development of the disease is heavily influenced by the serological status before infection (pedersen 1976 ). pedersen (1995b) suggested that cats that develop fip are almost always fcov seropositive, while other authors found that seropositive cats are less susceptible to the disease (addie et al 1995 , herrewegh et al 1997 . although it is not possible to assess whether our cats were seropositive or not, it cannot be excluded that the abovementioned increase of antibody titres and -globulins could have been present before the samplings. the distribution of the viral antigen and the characteristics of positivity in the different animals, as already reported (cammarata parodi et al 1993 , tammer et al 1995 , kipar et al 1998 , were very variable. for these reasons the prevalence of the different characteristics of positivity were recorded and used to form the groups. the finding of similar characteristics of positivity in contiguous organs, however, suggests the possibility of an early diffusion of the virus from the site of the first appearance of the lesions to the nearest tissues, as previously demonstrated in experimentally induced diseases (weiss & scott 1981) . in most of the cats, viral antigen was detectable in less than 50% of the cells within the lesions and it was also found as granular positivity in extracellular spaces. although this might be an artifact due to a different distribution of the virus within each lesion, it is most likely due to a different reactivity against the virus, probably related to a different immune status of the cats. cats with different susceptibility to fcov infections, due to a breed predisposition, have been already reported . in the present sample the distribution of viral antigen in persian cats was similar to that of cats from other breeds; however, the hypothesis of an individual predisposition is supported by the finding that cats with more acute lesions and with extracellular antigen, that has been interpreted as the release of virus from lysed cells (weiss & scott 1981 , paltrinieri et al 1998a mainly had negative lymph nodes. a further support on the hypothesis of a different reactivity in cats with different immunohistochemical findings, came from the observation that some haematological changes were associated with the increasing spread of viral antigen. in particular, lymphopenia was present in cats with stronger positivity and with extracellular antigen. the relationship between lymphopenia and the severity of the clinical symptoms has been already underlined (weiss & scott 1981) and the lack of protective cellular immunity is considered to play a central role in the development of effusive fip (pedersen 1987) . the association detected in this study between lymphopenia and the spread of the virus supports this hypothesis. however, the data presented here do not allow determination if viral spread can exacerbate the lymphopenia, as previously suggested by the finding of apoptotic lymphocytes close to fipv-infected macrophages (haagmans et al 1996) . the finding of viral antigen in the germinal centres of lymph nodes in the absence of typical fip lesions must be considered with particular attention. the positive cells were morphologically identifiable as interfollicular dendritic cells, that work as antigen presenting cells (steinmann 1991) and are thus involved in the activation of the immune system. cats with positive lymph nodes had -globulin levels significantly higher than those of the other group. this suggests that they have already mounted an humoral reaction against the virus. this is consistent with the lower amount of viral antigen found in these cats and it is in agreement with previous reports in which the ability to resist the infection has been associated with a follicular hyperplasia in the lymph nodes (kipar et al 1999) . lymphopenic cats, in contrast, are most likely unable to mount this response: their lymph nodes were negative and -globulins were lower than in the other cats. in conclusion the simultaneous analysis of pathological, immunohistochemical and blood findings in cats affected by fip allowed us to define different reactive patterns against the virus. ultimately, the results presented here further support the hypothesis of an association between lymphopenia and the severity of fipv infection. further efforts to investigate the relationship between fipv and the immune system are needed to help us understand the histopathogenesis of the lesions. the risk of feline infectious peritonitis in cats naturally infected with feline coronavirus feline infectious peritonitis using direct immunofluorescence to detect coronaviruses in peritoneal and pleural effusion antigen unmasking on 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immunologic aspects of feline infectious peritonitis virus infection an overview of feline enteric coronavirus and infectious peritonitis virus infections the history and interpretation of feline coronavirus serology two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus problems in the interpretation of feline coronavirus serology (specificity vs. sensitivity of test procedures) feline infectious peritonitis: a review of clinicopathological changes in 65 cases, and a critical assessment of their diagnostic value the dendritic cell system and its role in immunogenicity cats inoculated with feline infectious peritonitis virus exhibit a biphasic acute phase plasma protein response immunological demonstration of feline infectious peritonitis virus antigen in paraffin-embedded tissues using feline ascites or murine monoclonal antibodies a comparison of the genomes of fecvs and fipvs: what they tell us about relationship between feline coronaviruses and their evolution eine modification der abc-methode (avidin-biotin-peroxidase-complex) fü r den nachweis von viralen antigenen bei der infektion der katze durch ein coronavirus (fip) und der infektion des hundes durch das parvovirus-typ 2 feline infectious peritonitis: experimental evidence for its multiphasic nature pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence this work was supported by the grant ex-m.u.r.s.t. 60% (italy). the authors are grateful to dr giuseppe sironi and manuela teti for their technical assistance and to dr cristina crosta, emiliana monzani and to the other practicians that submitted their cases. key: cord-023121-hewbl5yu authors: parodi, m. cammarata; cammarata, g.; paltrinieri, s.; lavazza, a.; ape, f. title: using direct immunofluorescence to detect coronaviruses in peritoneal in peritoneal and pleural effusions date: 2008-04-10 journal: j small anim pract doi: 10.1111/j.1748-5827.1993.tb02591.x sha: doc_id: 23121 cord_uid: hewbl5yu twenty‐one cases of feline infectious peritonitis (fip) were diagnosed using a direct immunofluorescence test on cytocentrifuged pleural and peritoneal effusions from cats sampled in vivo (11 cases) and at necropsy (10 cases). a commercial fluorescent polyclonal antiserum of feline origin reacting with fipv and cross reacting with transmissible gastroenteritis virus and canine coronavirus was used. eleven cats with ascites of a different origin were used as negative controls. the direct immunofluorescence test was 97 per cent reliable (31 cases of 32) and can be used in routine diagnosis. ~ feline infectious peritonitis (fip) is a disease which affects domestic cats and several wild feline species (barlough and weiss 1983 , pedersen 1987a , scott 1987 , barlough and stoddart 1990 . it was described for the first time in the usa by wolfe and griesemer (1966) and is now reported worldwide. fip is caused by a virus of the coronaviridae family, antigenically related to other coronaviruses which are responsible for mild feline infections, such as feline enteric coronavirus (fecv), or asymptomatic infections, like transmissible gastroenteritis virus (tgev) or canine coronavirus (ccv) (reynolds and others 1977 , pedersen and others 1978 , holmes 1985 , mcintosh 1985 , fenner and others 1987 , tupper and others 1987 , pastoret and burtonboy 1991 . fip is not easy to diagnose in vivo, partly because of its subtle onset and partly because of the variety of signs and lesions which accompany the different clinical forms (doherty 1971 , wolfe and griesemer 1971 , montali and strandberg 1972 , pastoret and others 1974 , legendre and whitenack 1975 , hayashi and others 1977 , rosmini and simoni 1979 , weiss and scott 1981 , pedersen 1983b , barlough and summers 1984 , lutz and others 1985 , renzoni and others 1985a ,b, pedersen 1987a ,b, kelnerr and litschi 1989 . suspicion of the onset of peritoneal forms is often aroused by the appearance of ascites, accompanied by listlessness, mild jaundice, and fever, which does not respond to antibiotic treatment. diagnosis is much more difficult in effusive non-peritoneal forms and even more so in noneffusive forms (robison and others 1971 , lutz and others 1985 , pozza and avezza 1986 , pedersen 1987a ,b, wise and macy 1990 . the titration of circulating anti-fipv antibodies is not diagnostic, although reliable results are often obtained using the enzyme-linked immunosorbent assay (elisa) or competitive elisa (c-elisa) (faravelli and others 1991) . in fact fip signs appear only in a proportion of seropositive felines and in some cases antibody levels become undetectable during the course of the disease. false positives due to infections from serologically related coronaviruses are also reported (pedersen and others 1980 , weiss and scott 1980 , pedersen 1983a ,b, barlough 1985 , pedersen 1987b , barlough and stoddart 1990 , wise and macy 1990 . according to shelly and others (1988) , physicochemical examination of the intracavitary effusions can supply useful information, especially when the y-globulin concentration exceeds 32 per cent. at the moment it appears that there are in order to overcome these difficulties the authors tried to evaluate the results of direct immunofluorescence (dif) on cytocentrifuged cavitary effusions of affected animals in comparison with cryostatic sections of the related organs. the aim was to verify the possible application of this method to intravital diagnosis of fip. thirty-two cats were included in the investigation. they all showed signs of fip. in particular, signs of effusions in at least one serous cavity were present. approximately 2 ml of effusive fluid were sampled in vivo from the affected cavity of 22 cats. in the remaining 10 cats, sampling of effusive fluid was carried out at necropsy within two days of death. the information regarding sex, breed and age of the cats is shown in table 1 . within 15 hours of sampling, two slides were obtained from each sample through cytocentrifugation using the cytospin 2 (shandon) at 130 g for 10 minutes. one slide was stained with may grunwald-giemsa and the other submitted for dif. all the cats, including those sampled in vivo, were subjected to post mortem examination. the final diagnosis was based on the necropsy findings and histological examination. a commercial feline polyclonal fluoresceinconjugated antiserum (vmrd inc) was chosen for the dif test; this detects both fipv biotypes i and 11, and cross reacts with tgev and ccv. the test was applied on freshly prepared cytocentrifugates and cryostatic sections of organs with typical fip lesions (10 cases). the control sections came from organs with lesions from other diseases (11 cases). if immediate staining was not possible, it was carried out after the slide had been stored at -20°c for no more than seven days. the slides submitted for dif were fixed and dehydrated in acetone-methanol (75 to 25 per cent) for 20 minutes and incubated with 100 pl of labelled serum for 30 minutes at 37oc in a moist chamber. after washing four times for 10 minutes with a 25 per cent solution of carbonate buffer (ph g ) , the slides were mounted with buffered glycerol and examined under a fluorescent microscope at 250 to 400 x magnification. the technique was verified using a known positive cryostatic section. an attempt was made to show the presence of coronavirus by transmission electron microscopy on 10 samples of ascitic fluid chosen from those cats positive to the dif, and following ultracentrifugation with a beckman airfuge and negative staining with a 2 per cent sodium salt of phosphotungstic acid. a clear correlation was found between pathological findings and analysis of the intracavitary effusions by dif for all the cats examined, except one (table 1 ). in 11 of the 32 cats, the pathological picture and laboratory tests led to a diagnosis different to fip, referable to nocardiosis (three cases), intrathoracic neoplasms (three cases), hepatodystrophy (two cases), foreign body peritonitis (one case), septic pleurisy (one case), and chylothorax (one case). in all of these cases the cytocentrifugates of the intracavitary effusions were negative by the dif test. may griinwald-giemsa stain often supplied useful indications for the diagnosis, which were subsequently confirmed by histological and, or, microbiological examination. in the remaining 2 1 cats, the clinical diagnosis of fip was confirmed by pathological and histological findings and was also confirmed in 10 of these cases by a positive dif test carried out on cryostatic sections of affected organs. a marked disagreement between the result from the dif test on ascitic fluid and the final fip diagnosis was found in only one case (case 11; table 1) which at the age of four months showed clinical signs of thoracic effusions with fever. both the elisa for the detection of anti-fipv antibodies and dif test carried out on the effusions were negative. following antibioticcortisone treatment, the clinical signs partially subsided and general health improved, with the exception of persistent fever. after four months of treatment, there was a sudden serious deterioration in the clinical picture and concurrent appearance of ascites and serious cardiac failure. further elisas gave a positive result for fip whereas the dif test on peritoneal fluid remained negative. shortly before death, the in the positive exudates the examination of cytocentrifugates by ultraviolet microscope showed variable numbers of cells with a vivid green cytoplasmic fluorescence (fig 1) . granulocytes also showed a green cytoplasmic fluorescence, similar to that of the positive elements, although they were easily recognisable by the plurilobated nucleus. the autofluorescence of other cells was also different from that of positive cells because it was not so intense and the colour tended more towards yellow. the corresponding cytocentrifugates stained by the may griinwald-giemsa method showed pictures consistent with fip infiltrates: there was a polymorphous cell population mainly composed of macrophages, lymphocytes, mesothelial cells and occasionally granulocytes. the result of the dif test was verified on cryostatic sections prepared from affected organs. they showed cytoplasmic fluorescence in mononucleate round cells which infiltrated the necrotic areas. however, the dif-positive cells were represented in a different fashion both in mononuclear infiltrates and in various affected organs. so in the same animal it was possible to find organs with positive lesions and organs with negative lesions, and often the mononuclear infiltrates revealed the presence of coronavirus antigen in a limited number of macrophages only. on ultramicroscopic examination, the presence of coronavirus particles in the intracavitary effusions positive to the dif test was confirmed in five of the 10 samples examined. most of the viral particles appeared under the form of immunocomplexes, that is compact clumps of a it is well known that the clinical diagnosis of fip is frequently difficult (robison and others 1971 , pedersen 1983b , lutz and others 1985 , pozza and avezza 1986 , pedersen 1987a ,b, shelly and others 1988 ; this also emerged from the authors' experience and particularly from the regular occurrence of suspected cases, not confirmed at necropsy or by laboratory tests. this was occasionally observed even when the clinical history was suggestive and serological tests were positive. similar conclusions regarding the reliability of the serological test had already been drawn by other authors (weiss and scott 1980 , tupper and others 1987 , ingersoll and wylie 1988a ,b, barlough and stoddart 1990 , wise and macy 1990 . it is well known that on the basis of this test alone there are no differences between cats that are clinically ill with fip and those that are either only infected or have come into brief contact with the virus or have been infected with fecv. on the other hand, the absence of serum antibodies does not mean exclusion of infection, because the formation and deposition of immunocomplexes can cause temporary 'antibody eclipses' (pedersen and others 1978 , pedersen 1987b . the dif test that the present authors used on cytocentrifugates from intracavitary effusions is very suitable, giving a positive result in most of the cases of fip (20 out of 21) which were subsequently confirmed by necropsy and histopathological examinations and, or, by a dif test on cryostatic sections. the cases where pathological entities different to fip were identified and where the dif test had never been positive on either the samples of the effusions or the cryostatic sections of affected organs were useful negative controls. however, the single case of fip where the dif on the intracavitary effusion was negative should not be underestimated. therefore, these results seem to suggest that a positive dif test on the intracavitary fluids can be considered reliable for the diagnosis of fip, whereas the negative results are less reliable, due to false negatives, even though these are a rare occurrence. various authors scott 1980, barlough and stoddart 1990) state that the most reliable diagnostic method consists of a histological biopsy examination: by using the dif test on the ascitic liquid, biopsy could be limited to those cases where non-effusive fip disease is suspected. cats, coronaviruses and coronavirus antibody tests feline infectious peritonitis encephalitis due to feline infectious peritonitis virus in a twelve-week-old kitten viral diseases: feline infectious peritonitis ocular manifestation of feline infectious peritonitis ricerca degli anticorpi anti-fipv in gatti sani e malati mediante tecnica immuno-enzimatica competitiva (c-elisa) coronaviridae. in: veterinary virology systemic vascular lesions in feline infectious peritonitis replication of coronaviruses identification of viral antigens that induce antibody responses on exposure to coronaviruses comparison of serologic assays for measurement of antibody response to coronavirus in cats augenveranderungen bei der felinen infektiosen peritonitis feline infectious peritonitis with spinal cord involvement in two cats la peritonite infectieuse feline: etat actuel de connaisances using dlf to detect coronaviruses in peritoneal and pleural effusions mciniosh, k. (1985) coronaviruses. in: virology extraperitoneal lesions in feline infectious peritonitis le point sur la peritonite infectieuse feline description et etude experimentale de la peritonite infectieuse feline feline infectious peritonitis and feline enteric coronavirus infections. part i: feline enteric coronavirus feline infectious peritonitis and feline enteric coronavirus infections. part 11: feline infectious peritonitis coronavirus diseases (coronavirus enteritis, feline infectious peritonitis) in: diseases of the cat virologic and immunologic aspects of feline infectious peritonitis virus infection antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species feline infectious peritonitis osservazioni anatomo-istopatologiche in corso di peritonite infettiva del gatto e rilievi patogenetici ulteriori rilievi sulla peritonite infettiva del gatto (fip) detection of transmissible gastroenteritis virus neutralizing antibody in cats naturally occurring feline infectious peritonitis: signs and clinical diagnosis peritonite infettiva dei gatti: contributo anatomopatologico ed ultrastrutturale feline coronaviruses: which are really pathogenic? le point veterinaire 19 protein electrophoresis on effusion from cats as a diagnostic test for feline infectious peritonitis antigenic and biological diversity of feline coronaviruses: feline infectious peritonitis and feline enteritis virus laboratory diagnosis of feline infectious peritonitis pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence feline infectious peritonitis feline infectious peritonitis: review of gross and histopathologic lesions a 10-year-old spayed lhaso apso had a slowly enlarging mass on the right hindleg, extending from the distal femur to mid tibia. this had been present for three years. there was recent lameness in the affected limb. radiographs demonstrated soft tissue swelling with periosteal new bone on the tibia, and lytic lesions on the tarsus and tibia. aspirants of the mass were consistent with a diagnosis of lipoma. an infiltrative fatty tumour was found on surgical exploration, involving both soft tissue and bone and extending into, and throughout, the stifle joint. histological examination confirmed this to be a lipoma. no other treatment was undertaken and the mass continued to enlarge and was only mildly painful. a second biopsy confirmed the mass as remaining lipomatous. a four-year-old labrador bitch had a chronic vaginal prolapse, first noticed during oestrus. artificial insemination had been performed after the prolapse had been manually reduced. multiple prolapses, treated unsuccessfully by sutures, recurred in the ensuing weeks (up to 58 days post oestrus). radiographic examination failed to demonstrate any fetal skeletons and the bitch was deemed not to be pregnant. surgical correction by hysteropexy and surgical removal of the prolapse took place and recovery was uneventful. six days later, the bitch delivered a live, healthy male pup. serum progesterone and oestradiol concentrations were taken and were 1.8 ng/ml and 1-75 pg/ml, respectively. bitches with chronic vaginal prolapse should not be used for breeding. key: cord-285335-agm4zbcx authors: kennedy, melissa; boedeker, nancy; gibbs, pam; kania, stephen title: deletions in the 7a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis date: 2001-08-08 journal: vet microbiol doi: 10.1016/s0378-1135(01)00354-6 sha: doc_id: 285335 cord_uid: agm4zbcx a population of persian cats experienced an epidemic of feline infectious peritonitis (fip) over 2 years. twelve cases of fip occurred in litters born during this period. cats contracting fip were all genetically related through the sire. feline coronavirus (fcov) genomic rna was detected consistently in this study in biologic samples from adult cats, kittens suffering from fip, and their siblings. analysis of viral 7a/7b open reading frame (orfs) were analyzed and revealed two distinct virus variants circulating in the population, one with an intact 7a orf and one with two major deletions in the 7a orf. the 7b orfs were intact and similar among all virus isolates, although point mutations resulting in amino acid changes were present. the sire was determined to be infected with both variants, and was persistently virus-infected. we speculate the deletion variant arose from the non-deletion variant during viral replication in this population, possibly in the sire. feline infectious peritonitis (fip) is a serious disease of domestic and wild felidae. it is an important disease of cats in multi-cat households, catteries, and shelters (scott et al., 1992; wolf, 1995) . this disease can manifest as an effusive peritonitis and/or pleuritis with a short course ending in death; or it may present as a more insidious disease with veterinary microbiology 81 (2001) [227] [228] [229] [230] [231] [232] [233] [234] granulomatous lesions affecting multiple organs that also progresses to death (pedersen, 1987; hoskins, 1993; pedersen, 1995) . the etiologic agent of fip is feline coronavirus (fcov) . in environments where large numbers of cats are housed closely together, 75-100% of the animals may be seropositive to the virus (pedersen, 1995) . despite these numbers, fip occurs sporadically -fatal disease is an uncommon manifestation of infection with fcov. virus factors are important to disease development, as virus strains vary in virulence (pedersen, 1987) . virulent fcov is theorized to arise from mutation of the infecting fcov during replication in the intestinal tract of infected cats (poland et al., 1996; vennema et al., 1998) . the 7b open reading frame (orf), the 3 0 -most gene, has been speculated to have a role in virulence, as deletions in this region lead to decreased virulence (vennema et al., 1992; herrewegh et al., 1995; vennema et al., 1998) . despite this data, the specific virus and host factors involved in the production of lethal disease are not known. the occurrence of fip in feline populations is rarely higher than 5% (hoskins, 1993) . we investigated a colony of 15 adult persian cats that experienced 12 cases of fip in their kittens over 2 years. we detected fcov genetic material in the feces of the adult cats as well as tissue and fluid sample from the fip victims. the 7a/7b orf from the fcov in this population was characterized to determine if mutations in this region were occurring. a persian cattery consisting of 15 adult cats was selected for study. all animals in the population were negative for feline leukemia virus and feline lentivirus. they were routinely vaccinated for feline herpesvirus, calicivirus, panleukopenia virus, and rabies. twelve cases of fip occurred in six litters over 2 years (1997) (1998) (1999) . diagnosis was based on clinical signs, cbc and blood chemistry, coronavirus serology, histopathology, and pcr on plasma or effusion. this latter parameter was found to correlate with histopathology for diagnosis of fip (kennedy et al., 1998) . fecal samples, whole blood, tissue and ascites from two clinical cases were collected at the time of illness. in addition, fecal and whole blood samples were collected from seven adult cats and unaffected kittens in the household at the same time point. total rna was extracted from the specimens using trizol ls according to the manufacturer's directions for reverse transcription and nested polymerase chain reaction (gibco brl, baltimore, md). primers encompassed the 7a/7b orfs, the 3 0 -most orfs of the genome (kennedy et al., 1998) . reverse transcription was done with moloney murine leukemia virus reverse transcriptase according to the manufacturer's recommendations with the downstream external primer (gibco brl, baltimore, md). nested polymerase chain reaction was done using extaq polymerase (intergen, purchase, ny) as described previously (kennedy et al., 1998) . products were analyzed on a 1% agarose gel. amplification products were cloned into pcr 2.1 using the ta cloning system (invitrogen, carlsbad, ca). cloned cdna was sequenced by molecular biology resources service (university of tennessee, knoxville, tn) and analyzed using gcg software (university of wisconsin, madison, wi). a minimum of two clones from two separate pcr reactions was used for sequencing and analysis. we detected fcov in a purebred population of cats that had experienced a high rate of fip over a 2-year period, with 12 cases of fip in six litters. the fcov infecting the population was analyzed, focusing on the 7a/7b orf. this genomic region was chosen for analysis because of its speculated role in virulence (vennema et al., 1992; herrewegh et al., 1995; vennema et al., 1998) . amplification of fcov rna was successful in samples from nine cats. seven of these were resident adults and included the sire and queens of affected offspring. two were from kittens that died from fip. two distinct virus variants were found, one with an intact 7a orf and one with two deletions in the 7a orf (fig. 1) . the deletions encompassed nucleotides 20-120 and nucleotides 164-226 of the 7a gene. four additional nucleotides (tctt) were present in all of the deletion mutants at the position corresponding to nucleotide 227 of the undeleted virus. the protein predicted from the nucleotide sequence contains different amino acids in the 3 0 one-half of the predicted protein due to the altered reading frame caused by the second deletion (fig. 2) . it is not known if the 7a protein predicted from the nucleotide sequence is expressed in the deletion mutant virus. the remainder of the 7a/7b orf was highly conserved among all isolates, including the region encoding the 7b orf (fig. 3) . interestingly, amino acid residues 202 and 203 of the predicted 7b protein were histidine and lysine. this is contrary to the findings of vennema et al. (1998) that fip viruses contained tyrosine and lysine at these positions whereas putative avirulent fcov contained histidine and arginine. both virus variants were identified in one cat, the sire ''dan'', as sequence analysis of clones from a single pcr from this animal revealed the presence of the 7a deletion mutant as well as the intact isolate (dan 1 and 2 in fig. 2 ). this cat was pcr-positive for virus in multiple samples and may be persistently infected with fcov. he was also the sire of all the fip victims in the first 18 months of the study. litters resulting from the breeding of this sire to his daughters from previous litters experienced morbidity and mortality of 75-100% within the litter (n ¼ 4). during the last 6 months of the fip outbreak, a new sire was used and bred to the original sire's daughters. morbidity and mortality has since decreased to 0-25% within litters (n ¼ 4), with the last case occurring in spring of 1999. amplification of fcov 7a/7b orf from the most recent cases of fip (n ¼ 4) have not been successful. fig. 4 shows the phylogenic relationship among the virus isolates resulting from nucleotide sequence alignments. the two distinct variants are clearly delineated. the deletion mutants show a very close similarity to one another and form a closely related cluster. we have characterized the 7a/7b orf of fcov variants in a population experiencing an epidemic of fip. one variant had an intact 7a/7b orf, while another had two major deletions in the 7a orf. the latter group appears to be very closely related, and probably arose from a single mutant strain. the first deletion resulted in no change in the reading frame, but the second deletion led to an alteration of the reading frame by one nucleotide leading to an altered amino acid sequence in the predicted protein. we speculate that these deletions arose from ''looping out'' of rna regions due to the predicted secondary structure of the single stranded rna genome in this region (data not shown). this may result in the viral rna polymerase ''skipping'' certain regions during transcription. it is not known if the 7a protein is expressed in the mutant virus. the first 18 nucleotides of the cdna sequence, which corresponded to nucleotides 1-18 of the 7a gene and included the start codon, corresponded to the upstream internal primer. some of these nucleotides must be present in the viral template in order for hybridization of primer to template to occur but it is not known how many of nucleotides 1-18 are present in the virus. thus, it is unclear if this start codon is present. if it is, translation of the 7a orf in the deletion mutant would result in a predicted protein that is nearly half the size of the native protein (58 amino acids in deletion mutant versus 108 amino acids residues in the native virus). the sire of the majority of fip kittens was dually infected with both virus variants as revealed by sequence analysis of cloned 7a/7b genes from this cat. other cats in this study may have also been dually infected with both virus variants, however, only one virus variant was identified by pcr in each of the remaining cats tested. this may be due to a quantitative difference in the amount of each virus variant in each cat tested. both variants were circulating as every cat tested was infected with one of the virus variants. some infected with the intact 7a/7b orf variant were ill and some infected with the deletion mutant were ill. however, as both variants were circulating and thus, every cat was exposed to both variants, there may be a causal relationship between the mutation that occurred and the increased incidence of disease seen in this population. the variation in disease may be related to host factors, such as generation of an effective immune response, rather than solely to the virus itself. alternatively, mutations in other genomic regions may be responsible for the variation in virulence observed with these isolates. host factors may play an important role in the virulence of fcov. increased incidence of fip in purebred cats, as well as in cheetahs, which are relatively genetically homologous, is known to occur (o'brien et al., 1985; foley and pedersen, 1996) . in this investigation, all of the fip victims were genetically related through the sire. those related to him through both the queen and sire had the highest morbidity while those related to him through the queen or sire only had lower morbidity. this would support the belief that susceptibility to fip following infection with fcov has a host genetic component. genetic detection targeting the 7a/7b orf of fcov was not successful in all fip victims. the deletions we have characterized in the 7a orf occurred near or within the binding site of the internal upstream primer. as the virus has persisted in this population, we postulate that this primer-binding site may have been lost due to the occurrence of additional deletions in the 7a viral gene. mutation of fcov in some cats may lead to changes in virulence ultimately resulting in fip. point mutations, recombination, and deletions have been observed (poland et al., 1996; herrewegh et al., 1995) . mutations are more likely to occur if virus is not cleared from a host population and viral replication continues at a significant level . it is not known if two virus variants entered this population or if one variant is a mutant of the other. the latter may be more likely, as the cats in this population have remained virus-infected for an extensive period increasing the likelihood of genetic mutation. deletions have been noted in the 7b orf from previous studies (vennema et al., 1995) . this is the first report of a deletion occurring in the 7a orf in a natural infection of a cat population. other virus mutations in addition to those which we have characterized may have occurred in the fcov from this population and may correlate directly with virulence. further analysis of additional genomic regions of these fcovs is required to completely explain the increased occurrence of fip in this population. the inheritance of susceptibility to feline infectious peritonitis in purebred catteries the molecular genetics of feline coronavirus: comparative sequence analysis of the orf 7a/7b transcription unit of different biotypes perspectives on feline coronavirus evolution veterinary clinics of north america: small animal practice correlation of genomic detection of feline coronavirus with various diagnostic assays for feline infectious peritonitis genetic basis for species vulnerability in the cheetah virologic and immunologic aspects of feline infectious peritonitis virus infection an overview of feline enteric coronavirus and infectious peritonitis virus infections two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus evaluation of the safety and efficacy of primucell-fip vaccine genomic organization and expression of the 3 0 -end of the canine and feline enteric coronaviruses feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses the impact of feline infectious peritonitis on catteries we gratefully acknowledge and thank morris animal foundation for its financial, technical, and administrative assistance in funding and managing the research through which this information was discovered. we would also like to thank dr. michael kiningham for his assistance in this investigation. key: cord-022203-t2f0vr1w authors: dowers, kristy l; lappin, michael r title: the pyrexic cat date: 2009-05-15 journal: problem-based feline medicine doi: 10.1016/b978-0-7020-2488-7.50024-7 sha: doc_id: 22203 cord_uid: t2f0vr1w nan • temperature > 39.2˚c (102.5˚f). • true fever results from a cascade of events, which starts with activation of leukocytes. pyrogenic factors released from the leukocytes increase the thermoregulatory set point in the hypothalamus. signs that may be associated with fever include: • elevated body temperature. • reluctance to move. • anorexia. • depression. • hyperpnea. • muscle or joint stiffness/discomfort. • shivering. • inflammation anywhere in the body can result in elevation of core body temperature above 39.2˚c (102.5˚f). • the most common etiology for fever in the cat is percutaneous cellulitis or abscess. viral diseases such as fiv, felv and fip are important diseases to consider. conjunctivitis is the predominant sign and is often initially unilateral and becomes bilateral. ocular discharge is serous initially then mucopurulent, but is usually mild. fever, anorexia and lethargy may occur. true fever must be differentiated from hyperthermia, which can be caused by increased muscle activity, increased environmental temperature and stress. true fever results from activation of leukocytes that release factors (pyrogens) such as interleukin-1 and tumor necrosis factor. • these factors cross the blood-brain barrier and increase the thermoregulatory set point in the hypothalamus. • leukocytes are activated by a multitude of infectious agents, neoplasia, tissue necrosis and immune-mediated diseases. fever is defined as systemic elevation of core body temperature above 39.2˚c (102.5˚f). the most accurate measurement of core body temperature is obtained rectally. aural temperature is approximately −17.2˚c (0.5˚f) lower than the rectal temperature. fever is a general clinical sign that can be associated with many different diseases. the most common disease causing fever in the cat is percutaneous cellulites or abscess. many viral and bacterial diseases cause fever because leukocytes are recruited and activated as part of the general immune response. organ inflammation, such as pancreatitis, cholangiohepatitis and myocarditis, can be associated with an elevated temperature even when an infectious agent is not present. classical signs • fever. • anorexia (partial or complete). • reluctance to move, lethargy and depression. • pain, heat or swelling at site of abscess or cellulitis. cellulitis usually precedes an abscess, and if treated appropriately, the abscess may not even form. cellulitis may be the only evidence of a previous abscess. an abscess may rupture spontaneously, and the owner may notice foul-smelling, purulent discharge on the fur. • some abscesses resolve on their own with or without rupture, if they have been present long enough. regional lymphadenopathy may occur near the affected site. cellulitis spreads rapidly with the development of multiple fistulae and a febrile response. • lameness from septic arthritis is a common sequelae to infection with l forms. joints are affected by the hematogenous route and may be distant to the initial site. lower limbs (tarsus and carpus) are most commonly affected. the joints often ulcerate with a grayish mucinous exudate. infection remains confined to subcutaneous tissues and joints without systemic spread to internal organs. history supports access to outdoors or conflict with other cats indoors. palpation reveals a tender area or fluctuant swelling, with or without evidence of puncture wounds. microscopic examination of a fine-needle aspirate of the abscess reveals a heterogeneous population of bacteria, numerous degenerate neutrophils and intracellular bacteria. a complete blood count will generally show neutrophilia. l forms are not visible in tissue samples even with special stains, nor do they grow on culture. on electromicroscopy, organisms are visible intracellularly within phagocytes. diagnosis is often made by response to tetracyclines in a therapeutic trial (doxycycline 10 mg/kg po, q 24 h). response is rapid and evident within 48 h. non-healing abscesses should have histopathology and culture of tissue. causes include nocardia, fungi, mycobacteria, and tumors. see page 1081, the cat with non-healing wounds. in plague-endemic regions, yersinia pestis (plague) must be considered, if the swelling is predominately in the neck region and the cat's fever is in the region of 40.5˚c (105˚f). cautionary measures such as gloves, masks and isolation of the suspect cat should be taken until diagnosis established. (see below for discussion of y. pestis infections). fracture. ligament/tendon injury. neoplasia. clip area looking for evidence of puncture wounds. drainage of the purulent material is the key to treatment. surgical drainage can be done under sedation or general anesthesia with a #15 blade. make a 1/4-1/2" incision over the dependent area, or the area most likely to allow for continued drainage. flush the wound thoroughly with sterile saline or a saline/betadine mixture. explore the wound with a sterile cotton swab or hemostats to assess the extent of dead-space and to look for a possible foreign body. leave the wound open to allow drainage of further purulent material. do not suture incision closed, as this will only allow the abscess to reform. a penrose drain may be placed for 2-3 days to allow maximum drainage for abscesses that close too early. antibiotic therapy for 7-10 days directed against anaerobes: penicillins, cephalosporins, clindamycin and metronidazole are reasonable choices. most abscesses respond extremely well to drainage and amoxicillin at 10-20 mg/kg po q 12 hours for 7 days or amoxicillin/clavulonic acid (12.5 mg/kg po q 12 hours). l-forms and mycoplasma spp. respond to doxycycline or tetracycline within 48 hours, but not other antibiotics. if the wound is not healing well, or the cat has had recurrent abscesses, felv/fiv testing is recommended to rule out an underlying immunodeficiency. further considerations are inappropriate antibiotics (consider culture and sensitivity testing) or the presence of an undetected foreign body (consider surgical exploration of the area) or involvement of underlying bone (osteomyelitis). prognosis is good unless there is an underlying immunodeficiency. restrict the cat to an indoor environment only; although less effective, confine cat indoors at least from dusk to dawn. neuter male intact animals to decrease territorial behavior. felv and fiv serology should be repeated 2-4 months following bite wounds. classical signs acute onset of sneezing followed by oculonasal discharge. discharge progresses from serous to mucoid to mucopurulent. severe conjunctivitis with tearing, photophobia and chemosis. hypersalivation may occur as an initial sign before the classic signs of upper respiratory tract appear. punctate corneal ulcers that may coalesce to larger ulcers or perforation. fever of 1-2 days duration, anorexia and depression. retching or coughing may occur. cats with anterior uveitis have occasionally have herpesvirus 1 in the aqueous humor. presumptive diagnosis can be made on the basis of history and clinical signs because treatment for feline herpes virus-1 and calicivirus are similar. ocular ulcerations and chemosis are more suggestive of fhv-1. definitive diagnosis is by direct ifa of cells obtained from conjunctival or nasal scrapings, or by viral isolation or polymerase chain reaction assays from oropharyngeal or nasal swabs. sudden onset of serous ocular discharge and mild conjunctivitis; these signs may begin unilaterally, but often progress bilaterally. initial signs are rapidly followed by sneezing, which are not paroxysmal and are less prominent than in herpesvirus. nasal discharge is primarily serous to mucoid and rarely progresses to purulent. oral ulcerations are common, especially on the tongue, and may be associated with drooling or hypersalivation. ulcers may also occur at the mucocutaneous junction, hard palate and nose. fever generally spikes initially after infection prior to onset of signs, and returns with onset of clinical signs. viral pneumonia occurs occasionally with certain strains, and may produce significant mortality. death is often sudden and preceded by laboured respiration. a rare variant strain (fcv-ari) reported from the united states, produces a high fever, facial and paw edema (50% of cats), ocular and nasal discharge, conjunctivitis and ulcerative stomatitis (50% of cats), hemorrhage from the nose, git, etc. (30-40% of cats), icterus (20% of cats) and rapid death. mortality is high (30-50%). presumptive diagnosis can be made on basis of history and clinical signs because treatment for feline herpes virus-1 and calicivirus are similar. oral ulcerations or clinical signs of pneumonia are more suggestive of calicivirus. definitive diagnosis is by viral isolation or reverse transcriptase polymerase chain reaction assays from swabs taken from the oropharynx, ideally in the first week of illness. demonstration of increasing serum antibody titers to feline calicivirus in paired samples is also useful, whereas measurement of a single titer is not useful because many cats have titers from vaccination. identification of fcv-ari is based on the clinical syndrome, pathology and culture of virus from blood, nasal or ocular discharge, spleen or lungs. clinical signs are often non-specific and include fever, anorexia and weight loss. dyspnea and harsh lung sounds without coughing is common. peripheral and visceral lymphadenopathies are frequently present. pale mucous membranes, icterus, hepatomegaly or splenomegaly may be evident. ocular signs are uncommon, but can occur. gastrointestinal signs are uncommon in cats compared to dogs, and include chronic diarrhea, mesenteric lymphadenopathy and anorexia. osseous lesions produce soft tissue swelling and lameness. diagnosis is by demonstration of the organism in lymph nodes, draining tracts, bone lesions or vitreous humor. the organism has a thin capsule and is intracellular within macrophages. no reliable serologic test available. genetic predisposition appears to play a role. fip is most common in catteries and multi-cat households. there are two clinical forms of fip, effusive or wet form and non-effusive or dry form. both are characterized by a fluctuating fever unresponsive to antibiotics, anorexia, lethargy and weight loss. typical age of onset is 6 months to 2 years, but any age can be affected. the effusive form may have any of the following signs: • abdominal effusion that is non-painful but progressive. the amount of effusion varies from volumes causing abdominal enlargement, to amounts only detectable by abdominocentesis. fluid is straw-colored and highly viscous, like egg white. • pleural effusion resulting in dyspnea occurs in 30% of cats with the effusive form. pericardial fluid may be evident on ultrasound. usually it not associated with clinical signs, but occasionally can produce cardiac tamponade. • male cats may present with scrotal swelling. the non-effusive form may have any of the following signs: • ocular signs result from pyogranulomatous inflammation of the iris and ciliary body. they include bilateral uveitis, perivascular exudates (cuffing), retinal hemorrhage, retinal detachment. • neurologic signs include cerebral and cerebellarvestibular signs such as seizures, personality changes, nystagmus, head tilt, circling, head tremor and hyperesthesia. • dysfunction of any organ system may result from granuloma formation within the tissue of that organ, e.g., liver, kidney, spleen, intestines, lungs, etc., however, organ failure producing clinical signs only rarely occurs, and most dysfunction is only detected on biochemical tests. • granulomatous masses may be palpable in abdominal viscera especially mesentery, mesenteric lymph nodes and omentum as tender, irregular masses. occasionally vomiting or diarrhea results from extensive lesions on the bowel wall. jaundice may occur with either form of the disease. histopathology of affected tissues provides the only definitive antemortem diagnosis. the classic fip lesion is pyogranulomatous infiltration around venules. the following are typical abnormalities associated with fip. all asterisked items must be present for a high likelihood of fip; if any one parameter is not present, fip is unlikely. a negative coronavirus ("fip") titer suggests fip is not the cause of the fever, although a few cats with the effusive form of the disease are titer negative. lymphopenia (< 1.5 × 10 3 cells/μl).* occurs in many cats with fip, and many cats without fip. except where the classical effusive fluid is present, definitive diagnosis of fip requires organ biopsy and demonstration of classical histopathological lesions. various non-specific abnormalities may be evident on laboratory tests, including increased total white cell count, mild to moderate anemia, and increased concentrations of bilirubin, liver enzymes, bun, creatinine, fibrinogen, globulin and mild proteinuria. csf typically has increased protein (> 2 g/l) and cell counts (>100 cells/ml) which are predominantly nonlytic neutrophils. ocular signs: toxoplasmosis, fungal agents. neurologic signs: toxoplasmosis, neoplasia (e.g., lymphoma), trauma, congenital abnormalities in young cats. other clinical signs: rule out other diseases associated with the apparent organ dysfunction. lymphocytic, plasmocytic cholangiohepatitis occasionally produces a high protein abdominal fluid similar to that of effusive fip. fip is a fatal disease with no known treatments. the therapies listed below have been used in an attempt to slow progression and/or to improve quality of life. glucocorticoids at immunosuppressive doses (prednisolone 4 mg/kg/day). cyclophosphamide (200-300 mg/m 2 q 2-3 weeks or 2.2 mg/kg daily for 4 days each week) or chlorambucil (20 mg/m 2 q 2-3 weeks). +/− broad-spectrum antibiotics to control secondary bacterial infections while the cat is immunosuppressed. prognosis is poor. the mortality is > 95%. fecal-oral transmission is most likely; transplacental transmission is rare. fomites, e.g., food bowls and litter trays, may be an important mode of transmission, as some strains of fcov survive in dried secretions for several weeks. a seronegative cat introduced into a household where coronavirus is endemic has a 1 in 6 chance of developing fip; a seropositive cat under the same conditions has a 1 in 12 chance. both young and old animals seem to be most susceptible due to vulnerable immune systems. maternal antibodies that protect kittens wane at approximately 5-6 weeks of age. reduce fecal-oral contamination by providing one litterbox for every 1-2 cats, cleaning litterboxes daily, and placing litterboxes away from feeding areas. minimize stress, especially crowding in catteries. do not introduce fcov-positive cats into a multi-cat household. wean kittens at 5 weeks and remove from the queen's environment if she is seropositive. an intranasal vaccine is available for use in seronegative cats. however, efficacy has not yet been demonstrated against wild strains. classical signs see main reference on page 540 for details (the anemic cat). onset of illnesses occurs over an extended period of time (months to years), although young kittens can become acutely ill. chronic, opportunistic infections occur that do not respond to appropriate antibiotic therapy and are primarily due to immunosuppression. fever may occur in any age cat but is primarily seen initially in the viremic stage or later in response to neoplastic, inflammatory or immunosuppressive effects. chronic fever occurs in later stages of disease. weight-loss/cachexia. non-regenerative anemia. thrombocytopenia. lymphoma is associated with felv-positive cats, especially thymic and multicentric forms. history and clinical signs may be suggestive. complete blood count showing anemia, thrombocytopenia, leukemias, increased mcv and leukopenia are supportive. bone marrow aspirate may show myeloproliferation and arrested erythroid differentiation. a positive felv antigen test (viral core antigen p27) on whole blood using an ifa (can also be done on bone marrow sample) or an elisa test (also on serum, plasma, saliva, tears). see page 543 for interpretation. polymerase chain reaction is available from some laboratories. • pale mucous membranes. see main reference on page 530 for details (the anemic cat). classical signs are pale mucous membranes and/or icterus primarily from extravascular hemolysis due to complement binding of infected erythrocytes. severe, regenerative hemolytic anemia may ensue. anorexia and depression are typical. fever occurs in 50% of cats in the acute phase, and may occur intermittently in chronic infections. history and clinical signs are suggestive, especially if an immunosuppressive disorder is present concurrently. diagnosis is via demonstration of the organism on the surface of erythrocytes. use a marginated blood sample for diagnosis, e.g., ear vein. multiple blood smears over a number of days may be required as most of the organisms are removed from circulation by the time clinical signs are apparent. infected cats may be coomb's positive. a polymerase chain reaction test is available in some laboratories for diagnosis. classical signs gastrointestinal signs, primarily abdominal discomfort and small bowel diarrhea, are due most likely to replication of the organism (tachyzoites) in enteroepithelial cells resulting in necrosis. clinical signs in the acute, fatal form of extraintestinal disease are caused primarily by tissue damage from the rapidly dividing tachyzoites. tachyzoites begin to disappear from tissues approximately 3 weeks after infection. the organism may persist in tissues as tissue cysts containing bradyzoites. chronic disease may be a result of delayed hypersensitivity reactions and tissue reaction to antibody-antigen complex deposition. concomitant illness, such as felv, fiv and immunosuppression with glucocorticoids, has been reported in some cases. gastrointestinal disease. • mild, self-limiting small bowel diarrhea may occur in the definitive host (cats), but only after ingestion of tissue cysts, oocysts or sporulated oocysts. • young kittens are more likely to have gastrointestinal signs, although mild clinical disease has been reported in adult cats as well. all newborn kittens experimentally infected developed severe diarrhea 5-6 days later. • fatal extraintestinal disease is most likely to occur in transplacentally infected kittens. • kittens may be stillborn or exhibit signs that are severe and rapidly progressive and reflect involvement of the lungs, liver and cns tissues. these signs may also be observed in postnatally infected kittens and include: -a distended abdomen from an enlarged liver and/or ascites. -icterus from hepatitis or cholangiohepatitis. -dyspnea is present in most kittens and cats with signs of acute infection. -neurologic deficits; continuous vocalization; excessive sleeping. -fever, anorexia, depression often accompanies the tissue-specific signs. • cats may have a moderate fever, lethargy and depression that waxes and wanes. • hyperesthesia and stiff painful joints or shifting lameness may be evident, presumably due to an immune-mediated process. • unilateral or bilateral anterior or posterior uveitis may occur with possible sequelae of lens luxation, glaucoma or retinal detachment. • seizures and ataxia may be present if cns tissues are involved. • rarely, a toxoplasma granuloma (tissue cyst) forms in the gastrointestinal tract or pancreas causing chronic vomiting. clinical signs consistent with toxoplasmosis are suggestive, especially when other causes of the signs have been ruled out. igm titers > 1:64 and a four-fold increase in igg:igm titers within 2 weeks correlate best with clinical toxoplasmosis. however, some cats do not develop detectable igm titers, and in other cats, positive igm titers can persist for months to years after infection. elevated ocular and csf titers relative to serum titers in cats with ocular or neurologic signs, respectively, are very suggestive. coefficient values > 1.0 are highly suspect and > 8.0 strongly suggest local production of t. gondii antibodies. response to therapy for toxoplasmosis is a useful indicator of infection. definitive diagnosis requires demonstration of the organism in inflamed tissues by histology, immunohistochemistry or polymerase chain reaction assay. rule out diseases associated with affected organs, e.g., fip for neurologic and ocular signs. clindamycin at 10-12 mg/kg orally q 12 hours for 4 weeks is usually effective. • cats should respond within several days of treatment. • if no response is evident after 3 weeks of antibiotic therapy, reconsider the diagnosis. • the chronic form may recur even after successful treatment, as drugs tend to suppress replication rather than kill the parasite. other systemic drugs with potential efficacy include the trimethoprim sulfas combination, doxycycline, minocycline, azithromycin and clarythromycin. cats with ocular lesions should also be treated with corticosteroids, either topically (e.g. topical 0.5% prednisolone acetate drops applied q 6-12 h) or systemically to control inflammation and its sequelae (glaucoma, lens luxation). gastrointestinal disease has a good prognosis, although it may lead to inflammatory bowel disease in rare cases. acute extraintestinal disease has a guarded to poor prognosis. chronic extraintestinal disease has a fair to good. • the placenta or milk with tachyzoites. • ingestion of meat infected with tissue bradyzoites, e.g., rodents. • ingestion of sporulated oocysts in food or water. t. gondii has a zoonotic potential. infection of humans can occur via: • ingestion of undercooked meat containing tissue bradyzoites (most common mode of transmission). • ingestion of sporulated oocysts from the environment. • transplacentally, if first-time exposure to the organism occurs during pregnancy. only cats host the sexual replication that results in oocysts in the feces. • oocysts are shed for 1-2 weeks. • most seropositive cats do not shed oocysts on repeat exposure. oocysts must sporulate to be infectious: • sporulation occurs 1-5 days after environmental exposure, thus handling individual cats rarely results in infection of humans. transplacental transmission occurs in cats and people after primary exposure. discourage cats from going outdoors and hunting behavior. do not feed cats undercooked meat. • cook meat at 80˚c (176˚f) for 15 minutes. • use gloves when gardening or changing the litterbox, and wash hands well. • change litterboxes daily. use litterbox liners or clean with scalding water. • lethargy and anorexia. see main reference on page 272 for details (the cat with depression, anorexia or dehydration). the classical signs are not as well-defined for cats as for dogs for the following reasons: • cats tend to have intermittent bouts of chronic pancreatitis. • diagnostic tests for pancreatitis are not as reliable in cats. • there is poor correlation of biochemical parameters with pancreatitis in the cat. lethargy and anorexia is variable depending on chronicity. vomiting only occurred in 35% of cases in one study. dehydration occurred in 50% of cases in the same study. abdominal pain is quite variable. fever is variably present, and generally mild. in severe acute pancreatitis it may progress to hypothermia, which is a poor prognostic sign. diagnosis is unreliable based on a biochemistry panel. lipase may be increased or normal in pancreatitis. • hyperbilirubinemia and elevated liver enzymes may be present. • hypocalcemia occurs in 40% (total serum calcium) or 60% (plasma ionized calcium concentration) of cats due to soponification of fat. cats with a plasma ionized calcium concentration < 1.00 mmol/l (< 4.00 mg/dl) have a grave prognosis (77% mortality) and aggressive medical treatment is indicated. pancreatic lipase immunoreactivity is probably a more sensitive diagnostic tool for confirming pancreatitis in cats than measurement of plasma lipase concentration or trypsin like immunoreactivity. a feline-specific assay must be used. abdominal ultrasound to visualize an enlarged pancreas or heterogeneous echogenicity in the area of the pancreas is considered by many to be most sensitive. demonstration of higher lipase levels in abdominal fluid compared to those of the serum is suggestive. diagnostic peritoneal lavage may be necessary to obtain a fluid sample. clinical signs may be acute, chronic or intermittent. typically, there is anorexia and depression together with icterus or increased bilirubin and liver enzymes on a biochemistry panel. vomiting and dehydration may be present. fever, especially in the suppurative form occurs in approximately 38% of the cases. chronic cholangiohepatitis may lead to end-stage liver disease and the cat may present with ascites and hepatic encephalopathy. multiple causes include bacterial, protozoal (t. gondii) and immune-mediated disease. complete blood count may show neutrophilia with a left shift, and mild non-regenerative anemia. biochemistry panel shows hyperbilirubinemia, elevated liver enzyme activities (alp, alt, ggt), +/− elevated serum bile acids. • signs of late-stage liver disease are occasionally present, such as decreased bun, glucose and albumin concentrations. abdominal ultrasound should be performed to evaluate the gall bladder and bile duct for cholelithiasis, bile sludging and cholecystitis. liver aspirates/biopsy allows for differentiation of suppurative from non-suppurative forms of cholangiohepatitis. clinical signs are primarily due to immunosuppression, i.e., chronic recurring infections that do not respond to appropriate therapy. gingivitis, stomatitis and peridontitis are more common findings in fiv infections than in felv, although one study suggests that these signs may be to an effect of age, rather than a consequence of fiv infection. fever is chronic and is related to production of tumor necrosis factor and/or il-1 in infected cats. weight loss/cachexia are common in the late stages of fiv, as in human hiv infections. diarrhea resembles a panleukopenia-type syndrome that may be due to actual enterocyte infection by the virus or secondary to inflammation. cats are often thin and scruffy with an unkempt haircoat, and may have miliary dermatitis. diagnosis may be suspected based on history and clinical signs, but requires antibody or antigen tests for confirmation. virus isolation and polymerase chain reaction for virus detection is available at some research facilities. cats infected with fiv can be co-infected with felv. chronic nasal discharge can be unilateral or bilateral and is generally serosanguineous. sneezing and stertorous breathing is often present. facial deformity may occur due to invasion of the surrounding bone. chronic low-grade fever may be present. depression, anorexia and weight-loss are signs of disseminated disease. neurologic signs occur via hematogenous spread or invasion into the cns through the cribiform plate but are uncommon. signs include seizures, blindness, depression and ataxia. the skin form typically produces nodules which often ulcerate. diagnosis is by demonstration of narrow-based budding yeast with a very thick capsule from affected tissue or by culture of affected tissue or csf. demonstration of cryptococcus antigen in serum, urine or csf is also diagnostic. occurs in cats of all ages, with and without outdoor access. progressive clinical signs occur over a period of 1-4 weeks. according to one study, non-supportive meningoencephalitis may be the most common cause of seizures in cats. systemic signs, which are not present in all cats, include fever, anorexia, lethargy, vomiting, diarrhea and lymphadenopathy. the condition, however, does not appear to be contagious to other cats. csf tap can be very useful to rule out other causes of cns signs, specifically toxoplasmosis and fip; csf analysis reveals a normal or mild protein elevation (typically < 1 g/l) and/or an increased white cell count (< 50 cells/μl). complete blood count findings are non-specific and may include leukopenia or leuko-cytosis, eosinophilia and anemia. • uniphasic or biphasic fever. • depression. • lethargy. • mild generalized lymphadenopathy. • +/-signs of cardiac failure. • viral, e.g., fip has been shown to cause cardiac infection. • trypanosoma cruzi, which causes chagas' disease in humans. • streptococcus and borrelia (lyme's disease) in certain geographic areas. no single agent has been identified, and the disease may be multifactorial. fever is biphasic in 50% of the cats; if biphasic: • first fever occurs approximately 10 days after exposure, lasts 1-3 days and peaks at 39.3-40.7˚c (102.7-105.2˚f). • second fever occurs 1-2 weeks after the first fever (at 3-4 weeks post-exposure), lasts 5 days and peaks at 39.9-40.9˚c (103.8-105.6˚f). appetite is mildly decreased in some cats, but most continue to eat and drink. some animals exhibit mild generalized lymphadenopathy. irritable disposition and hyperesthesia may occur, and are most likely due to fever and malaise. in a few case reports, cats have died from peracute cardiac failure, but this outcome is not common. myocarditis/diaphragmitis is a diagnosis of exclusion. biochemistry and complete blood counts are unremarkable, except for a mild to moderate increase in ck in less than 50% of experimentally infected cats. definitive diagnosis can only be made at necropsy. histopathology shows a neutrophilic infiltrate with a foci of myonecrosis in myocardium and diaphragm. any other causes of fever should be ruled out including infectious, inflammatory, immune-mediated, drugs, neoplasia and metabolic. other causes of cardiac failure that should be ruled out include congenital deformities, hypertrophic cardiomyopathy, restrictive cardiomyopathy and dilatative cardiomyopathy. supportive therapy is indicated if dehydration or cardiac disease are present. broad-spectrum antibiotics are indicated if complete blood count supports an infectious cause. fever and depression resolve spontaneously in the majority of cats. prognosis is poor if peracute cardiac failure is present with systemic signs of fever and depression. although an infectious agent is suspected, no single etiologic agent has been identified, making recommendations for prevention difficult. • cardiovascular or respiratory compromise. • external signs of injury. cardiovascular compromise may result in tachycardia, hypovolemia or hypotension. respiratory compromise may produce dyspnea/ tachypnea due to pneumothorax, hemothorax or pyothorax. internal injuries may result in abdominal pain from organ rupture, bone/joint pain or focal swelling. diagnosis is based on clinical signs and history. radiographs of the chest, abdomen and/or limbs may be required to characterize the injury. complete blood count and biochemistry panel is indicated to rule out specific organ injury and primary infection. • alert, febrile cat being treated with antibiotics or antifungal agents. history of treatment with antibiotics or antifungal agents. fever does not correspond to clinical appearance of animal. cats are bright, alert and responsive, despite a temperature in the range of 39.4-40˚c (103-104˚f). onset of fever is idiosyncratic and variable, but the fever is generally present for the duration of the drug treatment. tetracycline is the most common antibiotic cause of drug-induced fever in cats. amphotericin b can cause fever by disrupting cell membranes and releasing pyrogens into circulation. be aware that other drugs (griseofulvin, chloramphenicol and chemotherapeutic drugs) can cause bone marrow suppression leading to a cat with fever, neutropenia and secondary bacterial infection. these cats are obviously sick, whereas the drug-induced fever animals are bright and alert in comparison. • history is of treatment with fever-inducing drugs, especially tetracycline and amphotericin b. • clinical signs are inappropriate, that is, the cat appears bright and alert although febrile. temperature normalizes after drug is discontinued. classical signs • sneezing. • conjunctivitis and ocular discharge. see main reference on page 13 for details (the cat with acute sneezing or nasal discharge). marked conjunctivitis is the predominant sign, which often starts unilaterally, but usually progresses to both eyes. classic triad of upper respiratory infection signs including oculonasal discharge and sneezing. serous ocular discharge accompanied by blepharospasm, chemosis and conjunctival hyperemia are initial signs. discharge becomes mucopurulent over the course of the disease. mild to moderate fever can be seen in the acute phase. pneumonia is rarely associated with this infection. history and clinical signs are highly suggestive. cytology of conjunctival scrapings reveal dark blue inclusion bodies (giemsa stain). immunofluorescent antibody staining or polymerase chain reaction assay to demonstrate the organism in conjunctival scrapings is available from some laboratories. • acute onset of depression. • acute onset of vomiting. rapid onset of depression, anorexia, and vomiting especially in peracute and acute disease. fetid diarrhea (may be hemorrhagic) typically follows 1-2 days after initial onset of signs. severe dehydration and electrolyte abnormalities. initial fever followed by hypothermia as the disease progresses. high mortality rate when signs are severe. the disease should be suspected in cats less than one year of age with no history of vaccination and a rapid clinical course. panleukopenia evident on hematology. parvoviral antigen can be detected in feces using the canine parvoviral antigen tests or electron microscopy. histopathologic changes include denuded intestinal crypts and blunted villi (often a post-mortem diagnosis). classical signs francisella tularensis is a gram-negative coccobacillus. clinical signs are associated with gram-negative endotoxins and bacteremia. there are two main strains of the organism, both of which have been isolated from cats. • associated with tick-rabbit cycle. • found only in north america. • highly virulent for laboratory rabbits. • associated with more severe disease in humans. • associated with a more complex cycle involving rodents, ticks, mosquitoes, mud and water. • found throughout the northern hemisphere. • avirulent for laboratory rabbits. history of contact with rabbits, especially if the cat is a hunter. any age of cat can be infected, but younger cats are more susceptible to developing septicemia. the spectrum of illness varies from severely affected to asymptomatic. fever is generally > 40˚c (104˚f). marked depression, anorexia and lethargy, with or without vomiting are typical. on physical examination, peripheral lymphadenopathy, icterus and palpable splenomegaly and hepatomegaly are reported. oral, lingual or pharyngeal ulcers may be present. clinical signs together with a history of exposure to wild rabbits is highly suggestive. hematologic and serum biochemical abnormalities may include panleukopenia, with severe toxic changes in neutrophils, high band neutrophil count, thrombocyto-penia and hyperbilirubinemia. definitive diagnosis is via identification of the bacterial agent by ifa or bacterial culture, but should only be performed in a qualified laboratory. • samples can be obtained from affected lymph nodes, bone marrow, urine or blood. serum antibody titers > 1:120 or a four-fold increase in serum antibodies in samples collected during acute and convalescent phases (10-14 days) are considered diagnostic. fip, fiv, panleukopenia. toxoplasmosis. multicentric lymphoma. antimicrobial efficacy studies have not been done in the cat, therefore therapy is derived from case reports and/or human therapy regimens. enrofloxacin (5 mg/kg q 12 hours iv or po). tetracycline and chloramphenicol may be effective, but because they are bacteriostatic for f. tularensis, relapses can occur. in humans, the drugs of choice are streptomycin and gentamycin. prognosis is poor to fair as mortality rate varies across case reports. f. tularensis has a serious zoonotic potential if there is contact with infected animal tissue. bites from infected ticks, deer flies or mosquitoes are the most common method of transmission. infection can also occur via ingestion of infected meat. • this is the most common method of transmission to humans in cat-associated cases. • the infected cat may have no obvious signs of illness, but have a history of hunting wild animals, especially rabbits. inhalation of aerosolized organisms may also transmit the disease. care should be taken by veterinary and laboratory personnel handling suspected animals or samples being prepared for ifa or culture. discourage hunting behavior in cats. ectoparasite control, especially tick control. onset of illness occurs 24-72 hours after exposure to the organism. transmission to cats is either via ingestion of infected rodents or a fleabite from infected fleas. rapid multiplication of organism causes tissue damage and necrosis. the host immune response contributes to pathology. three forms of the plague exist: bubonic (local infection), bacteremic/septicemic and pneumonic. bacteremia occurs in many cases, resulting in the septicemic or pneumonic form of plague. endemic regions of the world include the western usa, south america, africa, asia, eastern europe. history of hunting rodents, especially in known endemic areas. current flea infestation is evident. acute onset of fever, anorexia, depression over a period of 2-6 days. the clinical course may last 6-20 days. submandibular or cervical swelling associated with lymph nodes (can be unilateral or bilateral). the inflamed, swollen lymph node is referred to as a bubo. subcutaneous abscessation may occur and appear similar to a cat bite abscess. in the pneumonic form (~10% of cases), upper and lower respiratory signs may be present, including sneezing, nasal discharge, coughing, dyspnea/tachypnea. initially, microscopic examination of a lymph node aspirate, especially a markedly swollen lymph node (bubo) should reveal a homogeneous population of bipolar-staining coccobacilli. • blood should be examined in cats with the bacteremic/septicemic form. fluorescent antibody testing of sample provides a definitive diagnosis. culture of organism should be performed by a qualified laboratory only. a four-fold rise in antibody titers (taken 10-14 days apart) is suggestive of plague. these results must be interpreted carefully, as high titers can persist for up to one year after infection. chest radiographs may reveal patchy, nodular lesions if the pneumonic form is present. be aware that the risk of exposing other staff members to the disease should be weighed against the benefit of the diagnostic test. reactive lymph nodes from a percutaneous abscess or tooth-root abscess. • aspirates of cat bite abscesses contain a mixed bacterial population compared to y. pestis, which is homogeneous. neoplasia, although it is less common in the us for cats with lymphoma to have peripheral lymphadenopathy. respiratory signs may be due to other upper respiratory infections (calicivirus, herpesvirus, chlamydophila) or lower respiratory disease (parenchymal lung disease, pleural disease). other diseases which cause high fever (tularemia, toxoplasmosis, fip, etc.). absolute caution must be practiced in all suspect plague cases. cautionary measures include gloves, mask, isolation of animal and limited exposure to other staff members. doxycycline/tetracycline: (1) doxycycline at 5 mg/kg q 12 hours po for 14-21 days or (2) tetracycline 25 mg/kg q 8 hours po. • begin treatment immediately after samples for diagnosis have been collected. • doxycycline is preferred as tetracycline has been associated with relapse. consider aminoglycosides or enrofloxacin (5 mg/kg im q 8 hours) for the first 3 days to avoid placing hands into the cat's mouth (see transmission section below). prognosis for bubonic plague is fair to good. prognosis for the pneumonic form is guarded to fair. prognosis for septicemic form is guarded. persistent fever > 40˚c (104˚f) despite treatment is associated with a poor prognosis. y. pestis has a serious zoonotic potential, and great care should be taken in suspect cats to prevent transmission to humans and other cats. • infected cats are no longer a zoonotic risk after 3 days of antibiotic therapy. infection can also occur via inhalation of aerosolized organism, either from aspirates of infected tissue or nasal discharge/sneezing of cats with pneumonic form. discourage hunting behavior especially during the peak flea season (april to october). provide effective flea control to prevent flea bites. • anorexia and weight loss. see main reference on page 547 for details (the anemic cat). this disease is uncommonly reported in cats and is difficult to diagnose because of its vague and variable clinical signs. age range of cats with documented disease was 2-10 years of age, with no breed or sex predilection reported. infection has a variable effect on appetite, from mild inappetence to anorexia and mild to moderate weight loss. chronic intermittent fever in the moderate range is common. lymphadenopathy was reported in three of 23 cats. hyperesthesia, joint pain or irritable disposition is common. complete blood counts may show a non-regenerative anemia with a leukopenia or a leukocytosis; thrombocytopenia is present in about 25% of the cats. biochemistry abnormalities are uncommon, except for hyperglobulinemia in about 33% of documented cases. a complete blood count and biochemistry panel consistent with chronic ehrlichia spp. infection is suggestive. diagnosis is by demonstrating e. canis and/or anaplasma phagocytophilum serum antibody titers or a positive ifa test. demonstration of morulae in mononuclear cells, neutrophils or eosinophils (rare) is diagnostic. pcr assays can be positive. • anorexia, lethargy, weight loss. • ± fever. • signs depend on tumor type and organ system involved. anorexia, lethargy and weight loss. poorly groomed coat. some cats have a fever associated with neoplasia, which is generally a secondary neoplastic syndrome. tumors which destroy the bone marrow and result in neutro-penia are classically associated with fever. fever may occur with other tumors via other mechanisms, including antibody stimulation from tumor antigens, and tissue necrosis which activates leukocytes to release pyrogenic factors. signs are specific to the organ system involved. lymphoma (mediastinal, gi, renal) , mammary adenocarcinoma, squamous cell carcinoma (nasal, oral) and mast cell tumor are the most common tumors in cats. hematology, biochemistry panel, radiology, ultrasound and/or bone marrow aspirates may be necessary to provide evidence that a tumor is present, especially if it involves the hematopoietic system (leukemia) or is located internally (splenic mast cell tumor). identification of the tumor type is via fine-needle aspirates and/or biopsies. organ dysfunction due to infectious or degenerative disease process. felv/fiv or other immunosuppressive illness. benign masses (granulomas, abscesses, reactive lymph nodes, benign tumors). treatment involves surgical excision of identifiable masses +/− regional lymph nodes, especially for mammary adenocarcinoma, nasal squamous cell carcinoma, splenic mast cell tumor, etc. chemotherapy may be effective and needs to be based on tumor type, e.g., cop (cyclophosphamide, vincristine, prednisolone) protocol in lymphoma cases. radiation therapy is used for local disease only, and response to radiation therapy is tumor dependent (e.g., squamous cell carcinoma). • radiation therapy is most effective after surgical debulking of the primary mass. the effectiveness of radiation therapy may be enhanced with concurrent chemotherapy. classical signs acute onset of fever and malaise are initial clinical signs. vomiting, diarrhea and abdominal pain may occur, however, approximately 50% do not have gastrointestinal signs. dehydration. shock may occur if septicemia/bacteremia develops. mortality rate approaches 10% and may be higher if the cat is concurrently immunosuppressed. typically, the cat is an outdoor cat with a history of hunting behavior, especially of birds. complete blood count and biochemistry panel supports infectious diarrhea or septicemia, e.g., neutropenia with a left shift, bacterial rods in blood leukocytes if overwhelming sepsis present, hypoglycemia, hypoproteinemia, pre-renal azotemia. blood cultures provide the best definitive diagnosis if positive. three separate samples over a 4-6 hour period should be taken during febrile episodes using aseptic techniques. fecal cultures may isolate salmonella organisms, but because many animals are subclinical carriers, positive culture does not prove that the organism is the cause of the clinical signs. • skin lesions. • respiratory signs. • ocular lesions. • fever, anorexia, depression. the geographical distribution includes south-west usa, central america and south america in areas that have sandy soil with low rainfall and high temperatures. soil is the reservoir for infection, and the highest frequency of cases occur when the soil is dry and dusty, and organisms are disseminated in the wind. most humans and animals in endemic areas become infected, but the majority of infections are subclinical or cause only mild, transient clinical signs. cats are more resistant to infection and signs are less common than in dogs. infection is contracted via inhalation, and only a few organisms are required to produce signs, which occur after 1-3 weeks. initial infection is confined to the respiratory tract, but dissemination may occur resulting in chronic disease over months or years with signs referable to bones, eyes, central nervous system and abdominal organs. localized infection following a penetrating skin wound appears to be rare. cats appear to be resistant to clinical disease. skin lesions are the most frequent types of infection in cats and were reported in 56% of cats in one study. • lesions begin as small bumps and progress to abscesses, ulcers or draining tracts. • in cats, underlying bone involvement is uncommon. systemic signs such as fever, anorexia and depression are commonly reported (44% of cats) and can be seen with skin lesions. respiratory signs such as coughing and wheezing are less common in cats and occur in approximately 25% of cases. musculoskeletal signs such as lameness, with or without painful bone swelling, were reported in 19% of cats. ocular lesions are seen infrequently and include chorioretinitis and anterior uveitis. ocular or cns signs were reported in 19% of cats. most cats have clinical signs for less than 4 weeks prior to diagnosis. hyperproteinemia is present in approximately 50% of cats. definitive diagnosis is by identification of the organism via biopsy of lesions. antibody detection is available using latex agglutination (igm), agid (igm) or elisa (igm or igg). tube precipitin (tp) for igm and complement fixation (cp) for igg were previously thought to be less reliable in cats, but have been subsequently demonstrated to detect feline infections. itraconazole (10 mg/kg po if possible, q 24 h or 5 mg/kg q 12 h) is the treatment of choice. treatment is required for 4-6 months and must be continued for at least 2 months after all signs have resolved. • some cats develop anorexia, and less commonly vomiting or diarrhea. stop the drug for a few days until the cat is eating, and then restart at 1/2 the dose for 7-10 days, before increasing back to the full dose, which is usually then tolerated. amphoteracin b is also effective (0.25 mg/kg in 30 ml dextrose 5% iv over 15 minutes q 48 h or given subcutaneously) -see page 26, for cyptococcosis in the cat with signs of chronic nasal disease. continue amphotericin b therapy until a cumulative dose of 4 mg/kg is given or until bun > 17.9 mmol/l (50 mg/dl). amphotericin has the disadvantage of requiring frequent parenteral or subcutaneous administration and causes significant nephrotoxicity. • because of its quick onset of action, amphoteracin b in combination with itraconazole is useful in cats with severe pulmonary signs that are rapidly deteriorating. if the cat survives, after a few weeks treatment can be continued with itraconazole alone. • lipid-complexed amphoteracin formulations allow higher dosages with less toxicity, and should be used in cats with severe pulmonary signs, although the cost is higher. dose at 2-3 mg/kg iv 3 days per week for a total of 9-12 treatments (cumulative dose of 24-27 mg). dilute to a concentration of 1 mg/ml in dextrose 5% and infuse over 1-2 hours. if the titer has decreased four-fold and there is a similar improvement in physical and radiographic signs, treatment can be stopped after 4-6 months. antibodies may persist for long periods and obtaining a zero titer is not a useful treatment goal. classical signs • fever. • respiratory signs. • ocular signs. • lymphadenopathy. the geographical distribution includes north america, central america and africa. soil is believed to be the reservoir for infection, and living near a lake or river increases the risk of infection in dogs. signs are more common in dogs than in the cats. disseminated disease is primarily contracted via inhalation. respiratory signs include coughing, dyspnea and harsh lung sounds. ocular disease, such as uveitis, glaucoma and retinal detachment, is a frequent finding. fever, anorexia, depression, weight loss and lymphadenopathy are systemic signs associated with disseminated disease. draining skin lesions may occur and are usually a manifestation of systemic disease rather than local disease. neurological signs are associated with cns involvement of the brain or spine and include circling, disorientation, anisocoria, paresis, decreased conscious proprioception, or upper motor neuron signs, hyperesthesia and extensor rigidity. definitive diagnosis is by demonstration of an extracellular, broad-based budding yeast in aspirates or biopsies from lymph nodes, draining tracts, bone lesions or vitreous humor. an antibody detection test is available, but may be negative. itraconazole (10 mg/kg po if possible, q 24 h or 5 mg/kg q 12 h) is the treatment of choice. treatment is required for 4-6 months and must be continued for at least 2 months after all signs have resolved. • some cats develop anorexia, and less commonly vomiting or diarrhea. stop the drug for a few days until the cat is eating, and then restart at 1/2 the dose for 7-10 days, before increasing back to the full dose, which is usually then tolerated. amphoteracin b is also effective (0.25 mg/kg in 30 ml dextrose 5% iv over 15 minutes q 48 h or given subcutaneously) -see page 26, for cyptococcosis in the cat with signs of chronic nasal disease. continue amphotericin b therapy until a cumulative dose of 4 mg/kg is given or until bun > 17.9 mmol/l (50 mg/dl). amphotericin has the disadvantages of requiring frequent parenteral or subcutaneous administration and causing significant nephrotoxicity. • because of its quick onset of action, amphoteracin b in combination with itraconazole is useful in cats with severe pulmonary signs that are rapidly deteriorating. if the cat survives, after a few weeks treatment can be continued with itraconazole alone. • lipid-complexed amphoteracin formulations allow higher dosages with less toxicity, and should be used in cats with severe pulmonary signs, although the cost is higher. dose at 2-3 mg/kg iv 3 days per week for a total of 9-12 treatments (cumulative dose of 24-27 mg). dilute to a concentration of 1 mg/ml in dextrose 5% and infuse over 1-2 hours. classical signs see main reference on page 534 for details (the anemic cat). primarily found in the south-central and southeast united states. the north american bobcat is the natural host. there is usually a history of exposure to ticks in the previous 5-20 days (incubation period is 5-20 days). the clinical course of disease is approximately 1 week and often ends in death. clinical signs are the result of an overwhelming hemolytic crisis. rapid onset of fever, dyspnea, anorexia, pale mucous membranes, icterus and dark-colored urine are typical. collapse and death occur 2-3 days after the fever peak. hypothermia occurs in the terminal stages. there appear to be non-pathogenic strains as well. a complete blood count reveals regenerative anemia, hemoglobinemia and neutrophilia or neutropenia. the biochemistry panel commonly has hyperbilirubinemia. urinalysis may show evidence of hemoglobin and bilirubin. demonstration of the organism in erythrocytes (merozoite stage) is possible only relatively late in the disease, approximately 1-3 days before death. parasitemic cats usually have only 1-2% of rbcs affected, and up to 50% of cats have parasitemias that are very low or undetectable. demonstration of the organism in macrophages from bone marrow, spleen, liver or lymph node aspirates may be possible even when organisms are not evident in blood. serum antibody levels and direct fa test for detection of tissue phase are available through some labs. weight loss in spite of normal to increased appetite. polyuria/polydipsia. behavioral changes which often include hyperactivity and aggression. unkempt, rough hair coat and sometimes overgrown nails. tachycardia accompanied by a "gallop" rhythm and/or a systolic murmur. mild fever which may be intermittent in nature and reflect the increased metabolic rate in this disease. these cats are easily stressed and may present dyspneic and tachycardic with a mildly elevated temperature, usually not greater than 39.4˚c (103.0˚f). enlarged thyroid glands are often evident on palpation of the neck. diagnosis is based on clinical signs and history and confirmed by demonstrating increased thyroid hormone concentrations (total t4, free t4). thyroid glands can be palpated in approximately 80% of cats with hyperthyroidism, and are unilaterally or bilaterally enlarged. • enlarged thyroid glands may not be palpable if the abnormal thyroid tissue is within the thoracic inlet. complete blood count and a biochemistry panel are required to rule out diseases such as diabetes mellitus, renal disease, etc. a trh stimulation test may be necessary when clinical signs are highly suggestive and total and free t4 are in the upper region of the reference range for normal cats. thyroid radionuclide uptake and imaging with pertechnetate ( 99m tc) is also available at some institutions. response to therapy with anti-rickettsial drugs (tetracycline or doxycycline) is highly suggestive. • subclinical or mild fever and occasional ocular signs. bartonella henselae is an intracellular bacterium within erythrocytes. bacteremia is present in many healthy cats in the population, and cats are reservoirs for infection. b. henselae is an important pathogen because of its zoonotic potential in immunocompromised humans. • humans may develop fever, malaise, lymphadenopathy and skin eruptions following cat scratches or bites. • b. henselae causes bacillary angiomatosis, bacillary peliosis and encephalitis in human aids patients. naturally infected cats usually only develop subclinical infection. mild, self-limiting fever lasting 48-72 hours has been documented in some experimentally infected cats. anterior uveitis and fever were documented in naturally exposed cats. lymphadenopathy. atypical seizures occur in some cats. antibody titers are prevalent in healthy cats, but there is a poor correlation with blood culture and pcr assay results. intermittent bacteremia may occur for longer than one year following infection, with 25-41% of healthy cats bacteremic for up to 22 months. the organism is present within erythrocytes, therefore, hemolyzing red blood cells increases the sensitivity of the culture. other causes of mild transient fever need to be considered, such as mild cellulitis following a cat fight. other infectious causes of anterior uveitis need to be ruled out, such as toxoplasmosis, fungal diseases, felv, fiv, cuterebra or dirofilaria. antimicrobial efficacy has not been clearly demonstrated. clinical signs of disease have resolved when the cats are administered doxycycline at 25-50 mg po q 12 h for 28 days. azithromycin is used in humans and is a safe alternative in cats when administered at 10 mg/kg po q 24 h for 28 days fluoroquinolones also may be effective. while clinical signs resolve, bacteremia is usually only temporarily suppressed. b. henselae has very low pathogenicity in cats. once cleared of infection, cats are resistant to re-infection by innoculation, but are still susceptible if transmitted via blood transfusion. transmission is via arthropod vectors. in endemic areas, cats infested with fleas and/or ear mites are more likely to be seropositive. the organism survives in flea feces for at least 9 days. because of the frequency of bacteremia in healthy cats, blood transfusions are a likely route of infection. primary immune-mediated disease is extremely rare in cats. stimulation from primary infectious disease antigens is the most common cause of immune-mediated disease in cats, and is most often associated with hemobartonella (mycoplasma) and calicivirus. systemic lupus erythematosus is rare in cats. a multitude of signs may occur including fever, weight loss and cutaneous lesions. immune-mediated hemolytic anemia is most commonly associated with hemobartonellosis. signs include anemia, icterus, fever and anorexia. cats with immunosuppressive disorders such as felv may be more susceptible. immune-mediated thrombocytopenia is rarely reported in cats. felv-positive cats, however, may have thrombocytopenia that is thought to be the result of an immune-mediated response. immune-mediated polyarthritis is uncommon in cats, but has been documented in kittens and adult cats with post-calicivirus vaccination. 19 -the pyrexic cat 391 a bacteriologic investigation of subcutaneous abscesses in cats clinical, clinicopathologic, and pathologic features of plague in cats: 119 cases (1977-1988) bacterial diseases; fungal diseases; and protozoal diseases feline toxoplasmosis: interpretation of diagnostic test results bartonella spp. antibodies and dna in aqueous humor of cats feline toxoplasmosis and the importance of the toxoplasma gondii oocyst feline infectious peritonitis. part i. etiology and diagnosis feline infectious peritonitis. part ii. treatment and prevention consensus statement of ehrlichial disease of small animals from the infectious disease study group of the acvim feline infectious myocarditis/diaphragmitis diagnostic approach and medical treatment of seizure disorders an appraisal of the value of laboratory tests in the diagnosis of feline infectious peritonitis tularemia in two cats key: cord-302161-ytr7ds8i authors: lutz, mirjam; steiner, aline r.; cattori, valentino; hofmann-lehmann, regina; lutz, hans; kipar, anja; meli, marina l. title: fcov viral sequences of systemically infected healthy cats lack gene mutations previously linked to the development of fip date: 2020-07-24 journal: pathogens doi: 10.3390/pathogens9080603 sha: doc_id: 302161 cord_uid: ytr7ds8i feline infectious peritonitis (fip)—the deadliest infectious disease of young cats in shelters or catteries—is induced by highly virulent feline coronaviruses (fcovs) emerging in infected hosts after mutations of less virulent fcovs. previous studies have shown that some mutations in the open reading frames (orf) 3c and 7b and the spike (s) gene have implications for the development of fip, but mainly indirectly, likely also due to their association with systemic spread. the aim of the present study was to determine whether fcov detected in organs of experimentally fcov infected healthy cats carry some of these mutations. viral rna isolated from different tissues of seven asymptomatic cats infected with the field strains fcov zu1 or fcov zu3 was sequenced. deletions in the 3c gene and mutations in the 7b and s genes that have been shown to have implications for the development of fip were not detected, suggesting that these are not essential for systemic viral dissemination. however, deletions and single nucleotide polymorphisms leading to truncations were detected in all nonstructural proteins. these were found across all analyzed orfs, but with significantly higher frequency in orf 7b than orf 3a. additionally, a previously unknown homologous recombination site was detected in fcov zu1. feline coronaviruses (fcovs) are endemic in the domestic cat population and are found worldwide with high seroprevalences. fcovs are positive-stranded enveloped rna viruses with a non-segmented genome of~29 kilobases (kb) encoding for a polymerase (replicase complex 1a and 1b), four structural proteins (spike (s), membrane/matrix (m), envelope (e), and nucleocapsid (n)), and five nonstructural/accessory proteins (nsp) 3abc and 7ab [1] . due to infidelity of the rna polymerase, fcovs show high mutation rates upon replication. this leads to the formation of quasispecies, a cloud-like appearance of multiple genetic virus variants that are linked by mutations [2, 3] . virus variants that acquire high virulence can lead to feline infectious peritonitis (fip), an immune-mediated disease that is currently the most frequent fatal infectious disease in young pedigree cats and cats in shelters [4, 5] . however, the pathogenesis of fip is still not fully understood [6] . fcovs occur as two pathotypes, the low virulence so-called feline enteric coronaviruses (fecv) and the virulent fip viruses (fipv). the former dominates since fcov infection is via the fecal-oral route and primarily targets the intestine; it does, if at all, only induce mild enteritis. however, 4-5% of adult cats and 5-10% of kittens develop fip at some point after infection [7, 8] as a consequence of de novo occurrence of highly virulent fcovs that arise from low virulent fcovs by mutations in the individual infected cat [9] ; these are generally not horizontally transmitted. it was initially thought that the essential process for the development of fip would be a broadening of the viral target cell spectrum from enterocytes to also include monocytes/macrophages, which would be mediated by specific viral mutations and would allow systemic infection, leading to fip [10] . in vitro studies subsequently revealed that it is effective and sustainable replication in macrophages rather than the capacity to enter the cells that confers virulence of fcovs [11, 12] . also, it was soon shown that healthy carrier cats become systemically infected and carry low amounts of virus in different organs [13, 14] . irrespective of the pathotype, fcovs can be classified into two serotypes based on their neutralization reactivity with s protein-specific monoclonal antibodies [15, 16] . serotype ii fcovs have arisen from a double rna recombination event between canine coronavirus (ccov) and serotype i fcovs [17] . only type ii fcov can be efficiently propagated in cell cultures [5] . however, viruses of both serotypes can cause fip. previous studies found deletions in open reading frame (orf) 3c and mutations in orf 7b in fcovs from organs of cats that succumbed to fip [9, 18] . these were not found in fecal fcov isolates from healthy carrier cats and were hence considered to be linked to the fip pathotype and, consequently, to the development of fip. in fact, an intact orf 3c was shown to be critical for replication in enterocytes and thus, shedding of the low virulent pathotype fcovs [19, 20] . the fact that the deletions and mutations were unique to each cat supported the de novo mutation theory [21] . fcovs found in fip often exhibit mutations in orf 3c that lead to truncation of the protein; however, a significant proportion (up to 40%) of fipv still have an intact orf 3c [9, 18, 20, 22, 23] . hence, a virulence switch cannot be explained with orf 3c deletions alone [18, 20] . nevertheless, mutations that affect the expression of the accessory proteins might contribute to increased viral replication in monocytes/macrophages [24, 25] and, thereby, to the development of fip. conflicting evidence exists regarding the role of orf 7a and b. while some studies found deletions in orf7 to be associated with reduced in vivo virulence or in vitro replication [24] [25] [26] [27] , others did not detect such links [28] [29] [30] . a high n protein diversity was identified as evidence of quasispecies formation, but not as a marker of pathotypes [31] . mutations in the e or m genes were not found to be critical, neither in vivo [21] nor for in vitro replication [30] . the fcov s glycoprotein mediates viral cell entry through receptor binding and is thus responsible for cell tropism [32] . early in vitro studies found that macrophage tropism can be promoted by introducing the s protein of a highly virulent type ii fcov strain into its low virulent counterpart, suggesting that this protein plays a role in virulence. however, the study did not associate a distinct mutation with the new features of the recombinant strain [30] . specific s gene mutations were only identified in a more recent full genome sequencing approach, analyzing fcovs of both pathotypes. the majority of fipv pathotype fcovs were found to carry a mutation in a nucleotide position that was associated with an amino acid change in the putative fusion peptide of the s protein [33] . the same mutation plus a substitution leading to an amino acid change in the heptad repeat 1 (hr1) region, possibly leading to an altered fusogenic activity of the s protein and thus an altered cellular tropism, was found in another study [34] . however, it was subsequently shown that the amino acid change described by chang and coworkers [33] correlates with systemic spread of the virus and not directly with fip [35] . another study examined a furin cleavage site between the receptor binding and fusion domain of the s gene. the site was found to be conserved in less virulent fcovs, whereas fip pathotype fcovs exhibited a higher variability in the core motif and surrounding residues. these mutations alter cleavability of the s protein by furin. depending on the exact position and the exchanged nucleotide, cleavage was either enhanced or suppressed [36] . again, the study design did not consider systemically infected healthy carrier cats in this context. reverse genetics approaches with recombinant chimeric viruses in which genes of an avirulent (fcov black) strain were successively substituted by genes from a highly virulent serotype ii fipv strain (79-1146) led to seroconversion and systemic infection, but did pathogens 2020, 9, 603 3 of 20 not induce fip [37, 38] . despite the fact that many comparative studies have described mutations only present in fip pathotype fcovs, their relevance for the development of fip remains unclear. based on the hypothesis that certain mutations are essential for the capacity of fcovs to spread systemically, the present study investigated a cohort of systemically infected healthy carrier cats at different time points post experimental infection for the presence of a range of mutations in the genes encoding for the s protein, nsp 3abc, and nsp 7b, which have been shown to have implications for the development of fip. it also assessed the changes in the swarm composition of the challenge stock virus present in the organs to determine if its complexity would increase or rather decrease with time. to track viral sequence mutations in organs of healthy fcov carrier cats, we investigated fcov sequences detected in the colon, liver, and thymus, as well as feces of seven experimentally fcov infected cats. in two animals, the thymus was found to be negative by fcov reverse transcription quantitative real-time pcr (rt-qpcr) and lung and tonsil were included as alternatives (table 1) . fcov rt-qpcr was performed from undiluted and 10-fold diluted rna samples to detect any potential pcr inhibition and the rna dilutions with the lower cycle threshold (ct) value, corresponding to the higher viral load, were selected for analysis by one-step rt-pcr (table 1) for further cloning and sequencing. due to low tissue viral loads and/or lack of primer hybridization due to possible sequence mismatches, in five of the 28 samples, the analysis of mutations in either orfs 3abc or 7b was not possible (cat 3257b: amplicon nsp7ab from liver and tonsil; cat d4: amplicon nsp7ab from the liver; cats y1 and 3138b: amplicons s-e and s-m from the thymus, respectively). in total, 51 sequences were obtained. the plan was to sequence three colonies from each amplicon. due to the often low concentrations of eluted viral cdna, this was not possible for all samples. however, sequences for 129 of the expected 168 amplification reactions were obtained. we determined the sequence of the putative fusion region of the s protein from at least one amplicon from each organ of five cats (3132b, 3138b, 3312b, d4, y1). the one-step rt-pcr for the s gene already yielded an amplicon of the expected length (615 bp) from the colon of five cats (3132b, 3138b, 3312b, d4, y1) and the liver of cat 3138b, and after the nested pcr, amplicons with the expected length (134 bp) were obtained from the liver of four cats (3132b, 3312b, y1, 3257b), the thymus of four cats (3132b, 3138b, 3312b, y1), and the tonsil of cat 3257b. from one cat (y2), no amplicon was obtained. to determine how a virus swarm changes in its composition and viral sequences during in vivo infection and with systemic spread, the genomic sequences of fcovs in the tissue and fecal samples were compared to those of challenge stock virus. the number of sequences identical to the consensus was always higher in the latter than in the tissue/fecal counterparts of the same strain and orf (table 2 ). the number of single nucleotide polymorphisms (snps) per nucleotides analyzed did not differ significantly between challenge stock virus and tissue/fecal viruses. however, a tendency towards higher mutation frequencies was observed for orf7b in the tissue/fecal viruses compared to the challenge stock virus (table 2, figure 1 ). interestingly, zu3 orf3 showed higher mutation frequencies in the stock virus than in the tissue/fecal viruses. to determine whether there was a higher selective pressure on a certain gene after in vivo infection, we compared the mutation frequencies and resulting amino acid changes of genes 3a, 3b, 3c, and 7b ( table 3 ). the variability per gene of the tissue/fecal fcovs of each cat and in the challenge to determine whether there was a higher selective pressure on a certain gene after in vivo infection, we compared the mutation frequencies and resulting amino acid changes of genes 3a, 3b, 3c, and 7b ( table 3 ). the variability per gene of the tissue/fecal fcovs of each cat and in the challenge stock virus was small and ranged from 0.051 to 0.553%. while the 3a, 3b, and 3c genes seemed to have equal variability, the 7b gene exhibited a significantly higher mutation frequency than the 3a gene ( figure 2 ). to determine whether there was a higher selective pressure on a certain gene after in vivo infection, we compared the mutation frequencies and resulting amino acid changes of genes 3a, 3b, 3c, and 7b ( table 3 ). the variability per gene of the tissue/fecal fcovs of each cat and in the challenge stock virus was small and ranged from 0.051 to 0.553%. while the 3a, 3b, and 3c genes seemed to have equal variability, the 7b gene exhibited a significantly higher mutation frequency than the 3a gene ( figure 2 ). we wanted to determine whether the length of infection had an effect on mutation frequencies. the overall comparison of fcov gene sequences from the different cats euthanized at different time points after infection did not reveal any significant differences (table 3) . nevertheless, when the cats previous studies have provided evidence of a link between truncated 3c proteins and the development of fip [9, 18] . therefore, we investigated the effect of the mutations and deletions found in the present study on the encoded proteins. table 4 shows all deletions and snps detected in viral sequences from tissue and fecal samples and the challenge stock virus that led to a major alteration, i.e., leading to more than just one amino acid change, of the encoded protein. deletions were detected in five of the 129 viral gene tissue/fecal sequences. most mutations of viral sequences led to truncated 7b proteins. two deletions and one snp led to truncated 3a and 3c proteins. two snps and one deletion caused the start codon or the stop codon to disappear. the challenge virus only contained one truncated 3b protein. to determine the serotype of the strains fcov zu1 and fcov zu3 and their tissue/fecal derivate strains, we constructed bootstrap phylogenetic trees with fcov and ccov sequences from the ncbi database (genbank). consensus sequences of virus sequences from the same cat and origin and the previous studies have provided evidence of a link between truncated 3c proteins and the development of fip [9, 18] . therefore, we investigated the effect of the mutations and deletions found in the present study on the encoded proteins. table 4 shows all deletions and snps detected in viral sequences from tissue and fecal samples and the challenge stock virus that led to a major alteration, i.e., leading to more than just one amino acid change, of the encoded protein. deletions were detected in five of the 129 viral gene tissue/fecal sequences. most mutations of viral sequences led to truncated 7b proteins. two deletions and one snp led to truncated 3a and 3c proteins. two snps and one deletion caused the start codon or the stop codon to disappear. the challenge virus only contained one truncated 3b protein. to determine the serotype of the strains fcov zu1 and fcov zu3 and their tissue/fecal derivate strains, we constructed bootstrap phylogenetic trees with fcov and ccov sequences from the ncbi database (genbank). consensus sequences of virus sequences from the same cat and origin and the consensus sequence of the challenge stock virus were aligned to the reference sequences from the genbank for genes 3a, 3b, 3c, and 7b (figure 4a-d, respectively) . fcov zu3 and consensus sequences obtained from cat 3138b inoculated with fcov zu3 clustered with type i fcovs in all genes, whereas in tissue and fecal samples of cats inoculated with the fcov zu1 strain, only viral consensus sequences encoding the 3a gene clustered with type i fcovs. sequences related to fcov zu1 encoding genes 3c and 7b clearly clustered with type ii fcovs. while sequences of the 3c and 7b genes could undoubtedly be assigned to serotype ii, because they clustered with the sequences of the corresponding serotype, this was not the case for the 3a and 3b gene. here, sequences clustered together but could not be unquestionably assigned to a given serotype. therefore, we speculate that the 3a and/or 3b gene of the challenge virus fcov zu1 harbors a new recombination site. pathogens 2020, 9, x 8 of 21 consensus sequence of the challenge stock virus were aligned to the reference sequences from the genbank for genes 3a, 3b, 3c, and 7b (figure 4a-d, respectively) . fcov zu3 and consensus sequences obtained from cat 3138b inoculated with fcov zu3 clustered with type i fcovs in all genes, whereas in tissue and fecal samples of cats inoculated with the fcov zu1 strain, only viral consensus sequences encoding the 3a gene clustered with type i fcovs. sequences related to fcov zu1 encoding genes 3c and 7b clearly clustered with type ii fcovs. while sequences of the 3c and 7b genes could undoubtedly be assigned to serotype ii, because they clustered with the sequences of the corresponding serotype, this was not the case for the 3a and 3b gene. here, sequences clustered together but could not be unquestionably assigned to a given serotype. therefore, we speculate that the 3a and/or 3b gene of the challenge virus fcov zu1 harbors a new recombination site. based on sequence alignments with defined serotype i (fecv-ucd3 (fj943761)) and serotype ii (fipv 79-1146 (dq010921)) sequences, the recombination site could be tentatively mapped to the overlapping region between orf3a and orf3b ( figure s1 ). to evaluate the sequence evolution patterns of the fcov zu1 and zu3 challenge stock viruses for both the 3abc and 7b genes in each cat (i.e., at different time points post infection), bootstrap phylogenetic trees were constructed, with each available sequence obtained from different colonies in relation to the respective challenge stock virus consensus sequence (supplementary figures s2-s5) . in cats 3132b, 3138b, 3312b ( figures s2 and s3) , and d4 ( figures s4 and s5 ), the variability between the different sequences was very small and no subtree resulted from the analysis of either gene. the virus variation seemed to be evenly distributed across tissue and fecal samples independent of the time interval between infection and euthanasia (14, 28, or 48 days) . in cat y2, the gene 3abc based analysis revealed an equal distribution of virus variability in all examined tissue and fecal samples ( figure s4 ). however, for the 7b gene, the analysis revealed a higher variability, with viral sequences from different origin grouping to a subtree. the evolutionary pressure on the 7b gene seemed to be similar in all samples of this cat ( figure s5 ). phylogenetic analysis of cat y1 sequences based on genes 3abc and 7b showed subtrees in both genes. the sequence variability in the 3abc genes was smaller than in the 7b gene ( figures s4 and s5 ). analysis based on the 7b gene led to three different subtrees where viral sequences of the same tissue and feces grouped together and were clearly distinct from each other. also, in cat 3257b, the variability in the 7b gene was slightly higher than in the 3abc genes ( figures s4 and s5 ). trees constructed with both genes formed subtrees. two colonies sequenced from the colon of this cat grouped together when the analysis was based on the 3abc genes. analysis of the 7b gene yielded two subtrees, one consisting of two sequences from the feces and one of each feces and a colon sequence. the genetic difference of the viral sequences identified in the colon of cat 3257b to fcov zu1 (figures s4 and s5 ) was the highest of all sequences analyzed, potentially reflecting the longer time span after infection (80 days) in this animal. to assess if the fcov zu1 strain evolved divergently in different sites (tissues and/or feces) of individual cats, bootstrap phylogenetic trees were constructed with all sequences from feces and tissues. figures 5 and 6 show the analysis based on 3abc and 7b genes, with all viral sequences of the same origin. also, these trees revealed a higher variability in the viral 7b genes ( figure 6 ) compared to the 3abc genes ( figure 5 ). with the exception of the tree based on the analysis of the 3abc genes from thymus, all trees generated subtrees (figure 5: colon; feces; liver). subtrees consisted of sequences originating from the same cat, except for one subtree for liver sequences where two sequences of cat y1 and y2 grouped together ( figure 5 : liver). sequences that diverged in the analysis based on genes 3abc did not originate from the same animals and/or colonies as those diverging in the analysis based on gene 7b. sequences with mutations that led to truncated proteins did not cluster together but were rather dispersed and the majority were not represented in the subtrees ( figures 5 and 6: red dots) . when the analysis was based on genes 3abc of the colon, feces, and liver of cat 3132b, i.e., the cat that was sacrificed after the shortest time post infection (14 days), the sequences always formed a subtree that was clearly distinct from the challenge virus fcov zu1 and sequences found in other cats infected with the same virus (figure 5: colon; feces; liver). this was not seen in the 7b gene analysis (figure 6 : colon; feces; liver). the trees based on the 7b gene indicated that in cats y1, y2, d4, and 3257b, the 7b genes were generally more distant from parent fcov zu1 than from those of the other cats. orf 7b colon orf 7b feces orf 7b liver orf 7b thymus figure 6 . phylogenetic analysis based on the sequences encoding for the nonstructural protein 7b of all sequences of colon, feces, liver, or thymus, respectively (red dot, sequence carries a deletion that leads to a premature stop codon (3312b c3, y1c2, d4 c1); blue dot, snp causes the disappearance of a start codon (y1 c2, y1 c1); bar, mean number of differences per 1000 sites). unfortunately, none of the nested pcr products could be sequenced, neither by direct sequencing nor after cloning. however, sequences were obtained for five of the six samples that were positive after the first rt-pcr step. four of these sequences derived from the colon and one from the liver. all sequences had an atg codon at position 1058, resulting in a methionine residue (figure 7) . the fipv c1je strain exhibits a ttg codon at this position, resulting in a leucine residue [39] . at position 1060, all sequences had a tct codon. none of our samples exhibited the serine to alanine mutation described in a minority of fipvs at this position [33] . other snps detected in the immediate surrounding of position 1058 and position 1060 in our samples did not alter the amino acid figure 6 . phylogenetic analysis based on the sequences encoding for the nonstructural protein 7b of all sequences of colon, feces, liver, or thymus, respectively (red dot, sequence carries a deletion that leads to a premature stop codon (3312b c3, y1c2, d4 c1); blue dot, snp causes the disappearance of a start codon (y1 c2, y1 c1); bar, mean number of differences per 1000 sites). unfortunately, none of the nested pcr products could be sequenced, neither by direct sequencing nor after cloning. however, sequences were obtained for five of the six samples that were positive after the first rt-pcr step. four of these sequences derived from the colon and one from the liver. all sequences had an atg codon at position 1058, resulting in a methionine residue (figure 7) . the fipv c1je strain exhibits a ttg codon at this position, resulting in a leucine residue [39] . at position 1060, all sequences had a tct codon. none of our samples exhibited the serine to alanine mutation described in a minority of fipvs at this position [33] . other snps detected in the immediate surrounding of position 1058 and position 1060 in our samples did not alter the amino acid composition. overall, none of the mutations identified by chang and coauthors in the putative fusion region of the s protein were present [33] . composition. overall, none of the mutations identified by chang and coauthors in the putative fusion region of the s protein were present [33] . during the last years, many studies have been performed with the aim to identify mutations responsible for the virulence switch in fcovs towards the fipv pathotype. there has been evidence that mutations in accessory genes and the s gene of fcovs are associated with fip development. so far, most of these studies compared less virulent fcovs from healthy cats with fcovs in animals suffering from fip [18, 20, 22, [40] [41] [42] [43] . in the present study, we investigated the orfs 3abc and 7b and the s gene mutations of viral sequences identified in different organs and feces of healthy cats that had been experimentally infected with different fcov field strains and had developed a systemic fcov infection [14] . in the original study, the challenge virus used for experimental infection had been isolated from the feces of naturally infected cats. since fcovs show high mutation rates, due to the infidelity of their rna-dependent rna polymerase and homologous recombination events [44] , various fcov variants are transmitted in natural fcov infections [45] ; therefore, due to our experimental setup, we hypothesized a similar scenario. such virus swarms are called quasispecies, defined by a dominant nucleotide sequence and its mutant spectrum. evolutionary selection acts on the entire quasispecies rather than on single viral mutants [46] . in our study, we compared the swarm composition of the challenge stock viruses used for infection to the viruses detected in organs and feces at different time points after infection, focusing on the sequences of accessory genes. in parallel, we investigated the s genes for the presence of mutations described to have an impact on the development of fip [33] . the analysis of the accessory genes of more than 20 colonies of the challenge stock viruses confirmed the existence of one dominant sequence and several mutated derivates in each orf and strain (data not shown); this observation aligns with the quasispecies theory. in the viral genomes detected in the tissue and fecal samples of the infected cats, the extent of sequences identical to the consensus sequence was lower than in the challenge stock virus. additionally, viral sequences identified in cats that were euthanized at a later time point after infection overall showed significantly more variation than those from cats that were euthanized earlier, although due to the small number of cats analyzed, this significance could subsequently not be assigned to a specific accessory gene. a limitation for the interpretation of the results is the fact that only few cats (n = 7) were included in this study, of which just one was infected with the fcov zu3 strain. furthermore, two of these animals had been subcutaneously vaccinated with the inactivated fcov zu1 strain before challenge with the homologous strain. these cats were included in the study to add a further time point to monitor mutation frequencies over time. as the vaccine was inactivated and unable to replicate, we did not expect to find it in the tissues and feces. the animals seroconverted and started to shed virus in the feces only upon challenge and at the same time, with no differences in titers between vaccinated during the last years, many studies have been performed with the aim to identify mutations responsible for the virulence switch in fcovs towards the fipv pathotype. there has been evidence that mutations in accessory genes and the s gene of fcovs are associated with fip development. so far, most of these studies compared less virulent fcovs from healthy cats with fcovs in animals suffering from fip [18, 20, 22, [40] [41] [42] [43] . in the present study, we investigated the orfs 3abc and 7b and the s gene mutations of viral sequences identified in different organs and feces of healthy cats that had been experimentally infected with different fcov field strains and had developed a systemic fcov infection [14] . in the original study, the challenge virus used for experimental infection had been isolated from the feces of naturally infected cats. since fcovs show high mutation rates, due to the infidelity of their rna-dependent rna polymerase and homologous recombination events [44] , various fcov variants are transmitted in natural fcov infections [45] ; therefore, due to our experimental setup, we hypothesized a similar scenario. such virus swarms are called quasispecies, defined by a dominant nucleotide sequence and its mutant spectrum. evolutionary selection acts on the entire quasispecies rather than on single viral mutants [46] . in our study, we compared the swarm composition of the challenge stock viruses used for infection to the viruses detected in organs and feces at different time points after infection, focusing on the sequences of accessory genes. in parallel, we investigated the s genes for the presence of mutations described to have an impact on the development of fip [33] . the analysis of the accessory genes of more than 20 colonies of the challenge stock viruses confirmed the existence of one dominant sequence and several mutated derivates in each orf and strain (data not shown); this observation aligns with the quasispecies theory. in the viral genomes detected in the tissue and fecal samples of the infected cats, the extent of sequences identical to the consensus sequence was lower than in the challenge stock virus. additionally, viral sequences identified in cats that were euthanized at a later time point after infection overall showed significantly more variation than those from cats that were euthanized earlier, although due to the small number of cats analyzed, this significance could subsequently not be assigned to a specific accessory gene. a limitation for the interpretation of the results is the fact that only few cats (n = 7) were included in this study, of which just one was infected with the fcov zu3 strain. furthermore, two of these animals had been subcutaneously vaccinated with the inactivated fcov zu1 strain before challenge with the homologous strain. these cats were included in the study to add a further time point to monitor mutation frequencies over time. as the vaccine was inactivated and unable to replicate, we did not expect to find it in the tissues and feces. the animals seroconverted and started to shed virus in the feces only upon challenge and at the same time, with no differences in titers between vaccinated and non-vaccinated animals. for these reasons, we did not expect to see a difference in mutation frequencies due to vaccination, but we also cannot completely exclude it. only three colonies per amplicon of each tissue/fecal sample were sequenced. this sequencing approach was most likely picking the most represented sequences, which might not be enough to appropriately characterize the quasispecies swarm. a targeted high throughput sequencing study would allow for much greater sensitivity for detecting sequence variations in the virus population. nevertheless, taken together, the results point towards an expansion of the viral swarm with increasing infection time. yet, stabilization of the virus swarm over a longer period might subsequently occur. this has been shown in a previous study, which also provided evidence that persistently infected cats are likely no source of novel virus variants, since they carry highly conserved fcov swarms [45] . single nucleotide polymorphisms (snp) were found randomly scattered across all accessory genes without defined patterns (hot spots) both in the viral rna from tissues/feces and the challenge viruses. this suggests that they are a consequence of the infidelity of the viral rna-dependent rna polymerase. the present study found a generally higher mutation rate in the 7b gene than in the 3abc genes; the difference was significant for orf 3a. this confirms previous findings suggesting that the amino acid sequence of the nsp 7b is less conserved than that of other nsps [27, 47] . a bias could have been introduced since a non-proofreading taq polymerase was used for pcr amplification to increase the efficiency of the one-step rt-pcr because of the low viral loads in the tissues. but as sequencing errors are supposed to be equally randomly distributed across the genes, this would not impact on the difference of the mutation frequencies. interestingly, we found deleterious mutations and snps that led to truncated nsp 7b proteins in four viral sequences from the feces and colon of different cats infected with the fcov zu1 strain. truncated 7b proteins were previously shown to correlate with attenuated virulence: four well-characterized fcov strains (fecv 79-1683, fipv tn406-hp, fipv ucd2, and fipv df2) were found to be avirulent after they had acquired orf 7b gene deletions during in vitro passage in cell culture. in contrast, field fcov isolates consistently carried an intact 7b gene regardless of their pathotype [21] . thus, it was postulated that an intact 7b gene confers a selective advantage in natural infection but is not necessary for in vitro growth [27] . our study detected truncated 7b proteins solely in either feces or the colon, i.e., the main site of replication and persistence of less virulent fcovs, possibly representing virus variants that were indeed of low virulence and potentially even unable to infect monocytes/macrophages and to spread systemically. this would support the hypothesis that orf 7b mutations are not involved in the development of fip [29] . we also identified each two sequences each with truncated 3a, 3b, and 3c proteins, respectively. these results differ from those of a previous study which sequenced the structural and accessory genes of fcov from feces and organs of fip cats and mainly found truncated 3c proteins in the diseased tissue of fip cats [21] . the same study also claimed that orfs 3a, 3b, and 7a show the least variability among the other accessory and structural genes. the present study does not support this assumption, but rather indicates that the variability of the orf 3 genes is equally distributed at least in systemic infections with fcovs of low virulence. at the same time, the absence of orf 3c deletions and orf 7b mutations that have previously been linked to the development of fip in fcovs identified in the organs of our cats provides indirect support for the previous hypothesis that these might indeed be a feature of fipvs [19] . however, since healthy carrier cats also harbor virus in different organs, they are likely not a prerequisite of systemic spread in an infected animal. the orfs 3abc and 7b based phylogenetic analysis showed clustering of fcov zu3 with serotype i fcovs, whereas fcov zu1 sequences clustered with serotype ii fcovs. this was remarkable since both viruses were identified as type i fcovs after parts of the s gene had been sequenced in 2004 (dq256137 and dq256139) [14] . serotype definition is commonly based on the reactivity of antibodies to the s protein, hence it is generally accepted that fcov type i and ii can be distinguished based on the s gene sequences [15, 48] . the rna-dependent rna polymerase of covs is well known to be error-prone and to incorporate one mutation per 10 kb [49] ; this allows for three mutations per replication cycle of the 30 kb fcov genome and can lead to deleterious mutations that yield non-viable viruses. to overcome this problem, the viruses might rely on homologous recombination. the latter mostly occurs at specific hotspots in the genome, where secondary rna structures are formed that cause the polymerase to pause. four such hotspots, leading to double recombination events that result in type ii fcovs, have previously been identified; upstream ones in the polymerase sequence and downstream ones in genes e and m, respectively [17] . according to this configuration, after a double recombination event, the spike and nsp 3 genes should belong to type ii, whereas the nsp 7 should belong to type i. however, the exact locations of these recombination sites vary in the different strains, indicating that serotype ii fcovs continuously arise through independent recombination events [17, 50, 51] . therefore, homologous recombination can also generate new variants as long as they do not have evolutionary disadvantages [52] . this might also hold true for fcov zu1 where a recombination event in the 3a/3b gene resulted in a genome with type i fcov s gene, whereas the adjacent 3c gene as well as the 7b gene are those of a type ii fcov. controversial evidence exists concerning a set of defined s gene mutations first described in 2012 [33] in fipvs for which a role in the development of fip was suggested. two years later, two studies investigated these mutations in more detail. one study compared fecal samples of healthy fcov carriers with fecal and/or ascites samples of natural fip cases by sequencing the accessory and the s genes. this revealed a conserved methionine residue at amino acid position 1,058 in 9/10 fcovs from healthy carriers, whereas the m1058l mutation was found at this position in 5/6 ascites samples of fip cats whose feces generally displayed both genotypes. the authors also investigated the animals for orf 3c mutations/truncations and concluded that these together with mutations at amino acid position 1,058, account for the pathotype switch [22] . another study, published in the same year, concluded that the spike mutations in question are relevant for systemic spread and are not associated with fip. the authors found fcovs that carried the m1058l mutations in the majority of extra-intestinal tissue samples obtained from both cats with fip and healthy cats, whereas the majority of fcovs derived from fecal samples had a methionine residue. percentages of mutated versus conserved sequences in the different types of samples did not significantly differ between fip cats and controls [35] . we were also interested in the potential presence of these mutations in fcovs in our cohort of healthy carrier cats. sequence information covering the region of interest was obtained from four cats and two different organs: colon (four sequences, one per cat) and liver (one sequence). interestingly, none of these samples exhibited one or both mutations in question (m1058l and s1060a) [33] . these results could indicate that the above-mentioned mutations are not even markers of systemic spread per se [35] , but rather of another relevant aspect in the development of fip that, like systemic spread, is mediated by and/or occurs in monocytes/macrophages. however, our results are based on a single successfully sequenced extra-intestinal sample and are therefore of very limited value. mutations in the s gene are presumed to be associated with infection of monocytes and macrophages [53] , however unfortunately only few samples (2 out of 75) showed a monocyte-associated viremia [14] and due to the rna degradation, could not be further analyzed. at present, the relevance of the above-mentioned viral mutations for fip is still not clear. complementary transparent studies using clearly defined animal populations to obtain sufficient data for a definitive conclusion are clearly lacking and before these are available, no given mutation should be propagated as a diagnostic marker. a real-time rt-pcr has been marketed since august 2014 as a confirmatory diagnostic test for fip (fip virus realpcr™ test; idexx reference laboratories). a detailed description of the investigations on which the test validation was based seems to be lacking; test sensitivity (98.7%) and specificity (100%) were generated in a population of 186 cats "who were either healthy or had confirmed fip based on biopsy". it is questionable whether healthy cats are adequate controls for fip cases. after all, porter and coworkers found fcovs with the mutation described by chang and coworkers [33] not only in 91% of the tissue samples from cats with fip, but also in 89% of the tissue samples from fcov positive cats without fip [35] . therefore, with the current knowledge, we recommend considering the detection of the s gene mutations in question solely as further support of a diagnosis of fip, but not as definite proof. the exact role of the fcov spike protein in the pathogenesis of fip is still unknown. it is most likely the key determinant of cell tropism and is crucial for viral host cell entry [32] . substitution by recombination of the s2 region of fipv 79-1146 into fecv 79-1683 was found to be sufficient to enable the hybrid virus to effectively infect macrophages in vitro [30] . thus, it is likely not the receptor binding (via s1) but the fusion process that is critical in this context. also, for type i fcovs, which, unlike type ii fcovs, hardly grow in cell culture, the receptor is not yet known. interestingly, a cell culture adapted type i fcov strain (ucd 1) was found to carry a mutation in the furin cleavage site that renders the s protein susceptible to cleavage by heparan sulfate [54] , leading to the speculation that the amino acid composition of the furin cleavage site is critical for cell culture adaption and thus cell tropism. indeed, a strong correlation was later found between conservation of the furin cleavage site and fcov pathotype [36] . unfortunately, attempts to target the furin cleavage site at the boundary of s1 and s2 [36] did not yield conclusive results in our study (data not shown). fip pathogenesis includes several key processes, i.e., the establishment of systemic fcov infection; effective and sustainable viral replication in monocytes/macrophages; and activation of infected monocytes [6] . efficient entry into monocytes/macrophages is an essential prerequisite for all these, but there are most likely additional s protein-unrelated factors that allow the increase in viral replication in monocytes/macrophages and their subsequent activation. therefore, s gene mutations likely only represent contributing factors among multiple events, ultimately resulting in fip. in conclusion, despite some limitations mentioned above, using known field viruses and a controlled experimental setting, we were able to establish that fcovs in the tissue and fecal samples of healthy fcov infected cats do not carry the orf 3abc, 7b, and s gene mutations that have previously been linked to the development of fip. these findings support the hypothesis that these alterations are linked to fipv pathotype viruses. however, like all other studies so far, they do not answer the question whether their occurrence does directly cause fip. interestingly, the viruses detected in the tissues also lacked mutations seen in association with systemic spread of fcovs [35] , which indicates that they are not essential for this key prerequisite of fip. furthermore, we detected a homologous recombination site in the strain fcov zu1 that has not been described before. further comparative studies, and in particular, those using a ngs approach, are required to ultimately assess whether the development of fip can be attributed to virus characteristics alone or whether it represents an interplay between fipv pathotype viruses and a host prone to a specific ultimately detrimental immune response. the tissue and fecal samples used in this study originated from a study performed between 2000 and 2003 [14] . the study was approved by the swiss ethics committee (tvb66-2000). specific pathogen-free (spf) cats aged between 8 and 16 weeks were orally infected with different fcov type i strains (fcov zu1 [dq256137.1] or fcov zu3 [dq256139.1]) isolated from feces of healthy field cats or from the intestines of cats that had been experimentally infected with the same virus strains. cats d4, y1, and y2 originated from an unpublished study where the cats had been subcutaneously vaccinated with the inactivated homologous fcov strain 10, 7, and 4 weeks before challenge. cats d4 and y1 had been vaccinated with the inactivated strain mixed 1:1 with the adjuvant diluvac forte (intervet ltd., uk). for cat y1, a cpg motif, as described in a previous vaccine study [55] , had been added together with the adjuvant. cat y2 had been mock vaccinated. the challenge virus (1 ml) was administered twice, as previously described [14] . all infected cats had remained clinically healthy until euthanasia at 14, 28, 48, or 80 days after infection (table 1) . a full postmortem examination was carried out. the gross and histological examination did not reveal any pathological changes apart from lymphatic hyperplasia [14] . challenge stock virus samples of the fcov zu1 and zu3 strains originated from the same material originally prepared and aliquoted for the experimental challenge. all samples used in the current study had been stored at −80 • c since 2004. feces, colon, liver, and thymus or, when the thymus was not fcov positive by rt-qpcr, tonsils or lung, were analyzed. viral rna was isolated with the rneasy mini kit (qiagen, hombrechtikon, switzerland). approximately 25 mg of tissue was taken from the frozen samples and placed in a 2 ml eppendorf tube containing 600 µl of qiagen lysis buffer rlt, 3.5 µl β-mercaptoethanol, and a 3 mm steel bead (schieritz & hauenstein ag, arlesheim, switzerland). samples were homogenized and lysed by mixing at 30 hz for 1 min in a mixer mill 300 device (qiagen). after mixing, samples were centrifuged shortly to reduce the foam that had formed during the homogenization step. the homogenate was loaded on a qiashredder spin-column (qiagen) and centrifuged at 14,000 rounds per min (rpm) for 2 min; 350 µl of 70% ethanol were added to the flow-through and mixed by pipetting. samples were transferred to rneasy spin columns placed in 2 ml collection tubes. downstream operations were performed according to the manufacturer's instructions and rna was eluted with 50 µl nuclease free h 2 o and centrifugation at 10,000 rpm for 1 min. fcov loads were determined by a rt-qpcr assay that detects a 102 bp long amplicon of the 7b gene, modified from previously described protocols [56] . briefly, 25 µl reactions contained 12.5 µl 2× reaction buffer (one step rt qpcr mastermix plus low rox, rt-qprt-032xlr, eurogentec, seraing, belgium), 0.375 µl each of forward and reverse primers (20 µm) (microsynth, balgach, switzerland), 0.75 µl probe (microsynth), 0.125 µl euroscript reverse transcriptase & rnase inhibitor (eurogentec), 5 µl template rna, and rnase free water to 25 µl. the reaction underwent reverse transcription (rt) at 48 • c for 30 min, denaturation for 10 min at 95 • c, and 45 cycles of 95 • c for 15 s and 60 • c for 1 min. to test for any inhibition, each rna sample was tested neat and after 10-fold dilution in rnase free water. all qpcr assays were performed using an abi 7500 fast sequence detection system (applied biosystems, thermofisher scientific, reinach, switzerland). positive and negative controls were included in each rt-qpcr run. synthesis of cdna and pcr amplification were performed with the superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen, basel, switzerland) using specific primers. viral rna isolated from a cell culture supernatant infected with the fcov wellcome strain or from the fcov zu1 and fcov zu3 gut homogenates used for infection of the cats was always included as positive controls. also, two negative controls were included, of which one was kept open throughout the manipulation to control for airborne contaminations. pcr reactions (25 µl) contained 12.5 µl 2× reaction mix (provided in kit), 1 µl superscript iii rt/platinum taq mix, 0.5 µl each of forward and reverse primers (10 µm) (microsynth), 4 µl template rna, and autoclaved distilled water to 25 µl. three novel assays were developed. one amplified parts of the s gene, the 3abc genes, the e gene, and parts of the m gene of the strain fcov zu1. primers used for this amplicon (s-m, 1845 bp) were fcov_se_f (5 -tgc tgt tta act act ggt tgt tgt gga-3 ) and fcov_sm_r (5 -gca ccc gct ata cta agg ccg-a 3 ). the second assay amplified parts of the s gene, the 3abc genes, and parts of the e gene of the strain fcov zu3. primers used for this amplicon (s-e, 1348 bp) were fcov_snsp3b.f (5 -ctt ggt atg tgt ggc tac taa ttg g-3 ) and fcov_se_r (5 -atc aac agg agc cag aag aag aca ct-3 ). the third assay amplified parts of the 7a and the whole 7b gene of both strains. for this amplicon (nsp7ab, 855 bp), already described primers were used, namely 7a-f1 (5 -ctg cga gtg atc ttt cta g-3 ) [29] and fcov1229r (5 -aac aat cac tag atc cag acg tta gct-3 ) [56] . all assays were run on a biometra tpersonal thermocycler (labgene, chatel-st-denis, switzerland). cycling conditions were 55 • c for 30 min, 94 • c for 2 min; 40 cycles of 94 • c for 15 s, 60 • c for 30 s, and 68 • c for 2 min; 5 min at 68 • c and then cooling to 10 • c for the first assay (s-m); and 55 • c for 30 min, 94 • c for 2 min; 40 cycles of 94 • c for 15 s, 55 • c for 30 s, and 68 • c for 1 min; 5 min at 68 • c and then cooling to 10 • c for the second and third assay (s-e and nsp7ab). for the amplification of the s gene targeting the mutations m1058l and s1060a, a modified previously published nested rt-pcr assay was used [33] . the fcov-ucd1-s.3022 (5 -caa tat tac aat ggc ata atg g-3 ) forward and fcov-ucd1-s.3636 (5 -ccc tcg agt ccc gca gaa acc phylogenetic and molecular evolutionary analyses were conducted using geneious prime (biomatters ltd.). nucleotide sequences were edited, assembled, and aligned. alignments were manually adjusted when necessary. bootstrap phylogenetic trees were constructed using the neighbor-joining algorithm and the tamura-nei genetic distance model [57] . four bootstrap phylogenetic trees based on the 3a, 3b, 3c, or 7b genes were constructed with the consensus sequence of all viral rna sequences from the same tissue or feces of each cat and different fipv, fcov, and ccov strains retrieved from the genbank. additional neighbour-joining trees were constructed with either all 3abc or 7b sequences of the same cat or with all sequences from the same sample. the consensus sequences of fcov zu1 and fcov zu3 served as outgroups. variable sites and deletions of genes 3a, 3b, 3c, and 7b were analyzed with graph pad prism version 8.4.2 (san diego, ca, usa). data were tested for normal distribution with the kolmogorov-smirnov test. statistical significance was determined using one-way anova analysis of variance with bonferroni correction to compare the mutational frequency of the different genes. mean mutation frequencies in challenge stock viruses and viral sequences from tissues/feces for orf 3abc and orf 7b of fcov zu1 and zu3 were evaluated using the wilcoxon signed-rank test. the time-dependent mutation frequency between genes was analyzed using kruskal-wallis with dunn's multiple comparison posttest. data were expressed as median with boxes and whiskers showing the maximum and minimum values within a group or as means in a bar chart. a p-value < 0.05 was considered statistically significant in all cases. supplementary materials: the following are available online at http://www.mdpi.com/2076-0817/9/8/603/s1, figure s1 : nucleotide alignment of the orf3abc region between fcov zu1 and defined serotype 1 fecv-ucd3 (fj943761)) and serotype ii (fipv 79-1146 (dq010921)) viral sequences. sequences were aligned using the geneious prime software (www.geneious.com, biomatters ltd.) and the muscle method. dots depict identical nucleotides. the grey bars indicating the different orf3abc locations are based on the sequence of fcov zu1. figure s2 : phylogenetic analysis based on the sequences encoding for nonstructural proteins 3abc for cats 3132b, 3138b, and 3312b. (*, day p.i. of euthanasia; yellow dot, sequence carries snp that leads to a premature stop codon; bar, mean number of differences per 1000 sites), figure s3 : phylogenetic analysis based on the sequences encoding for nonstructural protein 7b for cats 3132b, 3138b, and 3312b. (*, day p.i. of euthanasia; red dot, sequence carries a deletion that leads to a premature stop codon; bar, mean number of differences per 1000 sites). figure s4 : phylogenetic analysis based on the sequences encoding for nonstructural proteins 3abc for cats d4, y1, y2, and 3257b. (*, day p.i. of euthanasia; red dot, sequence carries a deletion that leads to a premature stop codon (y1) or a shortened protein without frameshift (y2); green dot, snp causes disappearance of a stop codon; bar, mean number of differences per 1000 sites). figure s5 : phylogenetic analysis based on the sequences encoding for nonstructural protein 7b for cats d4, y1, y2, and 3257b. (*, day p.i. of euthanasia; red dot, sequence carries a deletion that leads to a premature stop codon; blue dot, snp causes disappearance of a start codon; bar, mean number of differences per 1000 sites). cycling conditions were 55 • c for 30 min, 94 • c for 2 min; 40 cycles of 94 • c for 15 s, 47 • c for 30 s, and 68 • c for 2 min; 5 min at 68 • c and then cooling to 10 • c. for the second nested pcr step, if needed, two newly designed primer pairs amplifying the region of interest (amplicon 134 bp), fcov-ucd1-s.3027f (5 -aat ggt gct tcc tgg ggt tg-3 ) and fcov-ucd1-s.3160r (5 -gca cct gca tag caa aag gc-3 ), and the phusion hot start ii high-fidelity dna polymerase (thermo scientific scientific) were used. then, 5 µl of one-step rt-pcr cycling conditions were 98 • c for 3 min; 40 cycles of 98 • c for 10 s, 59 • c for 30 s, and 72 • c for 2 min or alternatively, a gene ruler dna ladder mix (thermo scientific) or a 10-kilobase-pair dna ladder (eurogentec), was used for molecular size 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distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to acknowledge enikö gönczi and andrea spiri for their excellent technical support. the laboratory work was performed using the logistics of the center for clinical studies at the vetsuisse faculty of the university of zurich. this work was mainly performed by mirjam lutz and aline r. steiner as partial fulfillment of their masters theses. the authors declare no conflict of interest.pathogens 2020, 9, 603 key: cord-273424-iz1vat9p authors: ceciliani, fabrizio; grossi, claudia; giordano, alessia; pocacqua, vanessa; paltrinieri, saverio title: decreased sialylation of the acute phase protein α1-acid glycoprotein in feline infectious peritonitis (fip) date: 2004-04-12 journal: vet immunol immunopathol doi: 10.1016/j.vetimm.2004.02.003 sha: doc_id: 273424 cord_uid: iz1vat9p feline infectious peritonitis (fip) is an immune-mediated disease of domestic and exotic felides infected with feline coronavirus. fip is characterized by the overexpression of an acute phase protein, the α1-acid glycoprotein (agp). in humans, agp is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. we studied the changes in agp glycosylation in the course of fip. specifically, we focussed our attention on the degree of sialylation, fucosylation and branching. this study presents a purification method for feline agp (fagp) from serum, using an ion exchange chromatography strategy. the glycosylation pattern was analyzed in detail by means of interaction of purified fagp with specific lectins. in particular, sambucus nigra agglutinin i and maackia amurensis agglutinin lectins were used to detect sialic acid residues, aleuria aurantia lectin was used to detect l-fucose residues and concanavalin a was used to evaluate the branching degree. by this method we showed that fagp did not present any l-fucose residues on its surface, and that its branching degree was very low, both in normal and in pathological conditions. in contrast, during fip disease, fagp underwent several modifications in the sialic acid content, including decreased expression of both α(2–6)-linked and α(2–3)-linked sialic acid (76 and 44%, respectively when compared to non-pathological feline agp). decreased sialylation of the acute phase protein a1-acid glycoprotein in feline infectious peritonitis (fip) feline infectious peritonitis (fip) is an immunomediated disease of domestic and exotic felides infected with feline coronavirus (fcov) (pedersen, 1995) . fip develops when cats are exposed to variants of fcov that replicate within macrophages, thereby spreading the virus into target organs. the pathogenesis of fip is complex and still not well understood. two positive acute phase proteins, a1-acid glycoprotein (agp) and haptoglobin (hp), have been reported to increase during fip (duthie et al., 1997) . hp concentrations in fip are approximately twice the maximum value of healthy cats. more interesting, however, is that levels of agp increase from 0-1.5 to 4-8 g/l. the biological significance of agp overexpression during fip and its correlation with the veterinary immunology and immunopathology 99 (2004) [229] [230] [231] [232] [233] [234] [235] [236] abbreviations: fip, feline infectious peritonitis; agp, a1-acid glycoprotein; fagp, feline a1-acid glycoprotein; fcov, feline coronavirus; hp, haptoglobin; slex, sialyl lewis x; hplc, high pressure liquid chromatography; snai, sambucus nigra agglutinin; maa, maackia amurensis agglutinin; aal, aleuria aurantia lectin * corresponding author. tel.: þ39-02-50318100; fax: þ39-02-50318095. e-mail address: fabrizio.ceciliani@unimi.it (f. ceciliani). pathogenesis of the disease are still unknown. agp is a glycoprotein with a molecular weight ranging from 40 to 44 kda, depending on the species. one of the most interesting characteristics of agp is the extent of its carbohydrate moiety, which accounts for approximately the 45% of its molecular weight (fournier et al., 2000) . glycans are n-linked in the form of five, or six in some species, complex-types, that could, theoretically, increase to more than 10 5 different glycoforms. so far no more than 12-20 glycoforms of agp have been detected in non-pathological human serum. human agp is overexpressed during systemic inflammatory responses in the course of several pathologies (hochepied et al., 2003 ) but its precise function is unknown: all the data reported so far would suggest an immunomodulatory activity. specifically, agp inhibits the neutrophils chemotaxis and activation (vasson et al., 1994) and may be responsible for an increase in il1 receptor antagonist secretion (tilg et al., 1993) . in vivo experiments further suggest that agp may be involved in the induction of non-specific resistance to infection, for example by blocking the binding of hiv to the chemokine receptor ccr5 of human macrophages (atemezem et al., 2001) . the oligosaccharides micro-heterogeneity of agp is profoundly affected by pathologic conditions and different glycoforms can appear in plasma during systemic inflammation or diseases (de graaf et al., 1993; van dijk et al., 1995) . the alterations of the glycan moiety is believed to be important for several agp immunomodulatory activities. for example, a reduction in the degree of branching of attached oligosaccharides has been correlated to a reduction of il1 activity (bories et al., 1990) . a change in the amount of fucose residues was found to increase in some human inflammatory diseases, and fucosylated agp that expresses the sialyl lewis x (slex) epitope could counteract the extravasation of leukocytes, slex being the ligand of endothelial selectins (brinkman-van der linden et al., 1996; de graaf et al., 1993) . finally, several defensive functions proposed for agp can be associated to the sialic acid content: sialic acid is a very important component of membrane glycoproteins, and agp may therefore act as a competitor for cell surface receptors, blocking the binding and the invasion of infectious agents (friedman, 1983; hochepied et al., 2000) . therefore, agp carbohydrate moiety modifications may represent a very important mechanism in the strategy of the inflammatory response. high levels of agp have been described in cat serum during fip (duthie et al., 1997) . due to the importance of the glycan moiety on the immunomodulatory activity of agp, we targeted our attention to the post-translational processing of that protein during fip infection. in the present study we used the lectin binding specificity for carbohydrates in order to gain insight into some major (branching) and minor (sialic acid content) glycan microheterogeneity of feline agp (fagp) purified from fip affected cats. in particular, the aim of this study was to (a) setup a protocol for the purification of fagp from cat serum, (b) evaluate the normal and pathological expression on the surface of fagp of sialic acid and fucose and (c) evaluate the possible changing of the degree of branching of oligosaccharides chains. serum samples (0.5-1 ml) were obtained from 16 clinically healthy cats (10 males and 6 females) used as controls for fagp purification and for the determination of the non-pathological glycosylation pattern of fagp. since analysis of the glycosylation patterns of the individual non-pathological fagp samples revealed no differences, fagp from 16 healthy cats were pooled and used throughout the experiment as the control. samples were then collected from 24 cats affected by fip (14 males and 10 females), as diagnosed following post mortem examination and immunohistochemical detection of fcov within the lesions, and from 8 fcovexposed cats (4 males and 4 females), i.e. cats that cohabited with fip affected cats but did not develop the disease. all the cats used in these experiments were negative for feline immunodeficiency virus and for feline leukemia virus. fagp was isolated by conventional hplc ion exchange chromatography avoiding desialylation and structural degradation. cat serum (0.5-1 ml of each sample) was dialyzed overnight against the buffer used for the initial chromatography (10 mm citrate-phosphate buffer, ph 4.0). the serum was centrifuged at 13,000 â g for 5 min, to remove proteins not soluble at ph 4.0, and the supernatant was applied to an hitrap q sepharose strong anionic exchange column (amersham biosciences), equilibrated with the starting buffer. the column was washed from the starting buffer until no absorbance was recorded at 280 nm, and the protein was eluted in 100 mm citrate-phosphate buffer, ph 4.0. the fraction containing fagp (approximately 2 ml) was concentrated with centricon 10 (millipore) to 400 ml, and directly loaded onto an hitrap sp sepharose strong cationic exchange column (amersham biosciences), equilibrated in 100 mm citrate-phosphate buffer, ph 4.0. in these conditions fagp is not retained by the column and is therefore eluted in void volume. purified fagp (2 mg each lane) was subjected to sds-page in a discontinuous ph system (laemmli, 1970) using a miniprotean ii apparatus (bio-rad) with a 4% stacking gel and a 12% separating gel. gel were stained with coomassie blue, or electroblotted onto nitrocellulose. total proteins were visualized by staining the gels for 1 h according to the bio-safe 1 coomassie method (bio-rad). western blotting was performed with a mini trans blot electrophoresis cell (bio-rad) onto nitrocellulose. fagp was detected with an anti-fagp polyclonal antibody raised in sheep that was generously provided by dr. duthie (university of glasgow) and an alkaline phosphatase-conjugated goat anti-sheep secondary antibody. the blots were developed using the amplified ap immun-blot kit (bio-rad). four lectins were used in this study. the digoxigenin-conjugated lectins sambucus nigra agglutinin (snai) (roche) and maackia amurensis agglutinin (maa) (roche) bind sialic acid residues in specific linkages. snai is specific for sialic acid a(2-6)-linked to galactose, and maa recognizes sialic acid a(2-3)-linked to galactose. these two lectins were used at a concentration of 2 and 5 mg/ml, respectively. the biotin-conjugated lectin from aleuria aurantia (aal) (vector laboratories) shows affinity for a(1-6)-, a(1-2)and a(1-3)-linked fucose. concanavalin a (vector laboratories) binds to the diantennary glycans, and with lower affinity to tri-and tetraantennary glycans. these two lectins have also been widely utilized to evaluate the presence of fucosylation and slex (aal) and the degree of branching (cona) on glycoproteins surface. these lectins were used at concentrations of 8 mg/ml (aal) and 1 mg/ml (cona). glycan analysis of fagp that reacts with digoxigenin-conjugated lectins was performed following the glycan differentiation kit (roche), according to the method of haselbeck et al. (1990) . digoxigeninlabeled lectins were identified with an alkaline phosphatase conjugated sheep anti-digoxigenin immunoglobulin. glycan analysis of fagp that reacts with biotinconjugated lectins was performed following a peroxidase reaction using the vectastain abc kit (vector laboratories) and developed using hrp immun-blot assay kit (bio-rad). the protein content was determined by direct spectophotometric measurement at 280 nm and by the coomassie blue dye binding method (bradford, 1976) . densitometric analysis was performed using the imagemaster 1d software (amersham biosciences). the membranes were processed with a scanner and subjected to computerized image analysis. on each membrane image, profile plots were obtained displaying the fagp peak, and the volume of each peak was calculated by the software. fig. 1 reports the chromatographic procedure set up to purify fagp. the fraction containing fagp (fig. 1a , fraction 2) from q-sepharose chromatography was loaded onto a sp-sepharose chromatography column. the eluted fraction (fig. 1b, fraction 4) contains the purified fagp. the purity was determined by sds-page ( fig. 1 insert, lane 4) , which showed a single band on the gel after coomassie blue staining. the identification of the homogeneous band as fagp was determined by western blotting using a polyclonal anti-fagp antibody as primary antibody (fig. 1 insert, wb, lanes 2 and 4). fig. 2a shows a representative glycosylation pattern of non-pathological fagp. this glycosylation pattern was identical among all 16 control cats. in particular, the degree of sialylation was determined by the lectinbinding of fagp with snai (sialic acid a(2-6)-linked to galactose) and maa (sialic acid a(2-3)-linked to galactose), in lanes 1 and 2, respectively, the degree of fucosylation with aal (lane 3) and the degree of branching with cona (lane 4). fagp strongly reacts with snai and, with minor affinity, with maa. the reactivity of fagp with snai and maa has been presented as a density plot in fig. 2b . the dendrogram displaying the average volume of density profile plots of non-pathological fagp after reaction with snai and maa is shown in fig. 2c . the figure shows that the variability in non-infected cats is very low. on the contrary, fagp glycoforms from healthy cats did not bind to the lectins aal and cona. the non-reactivity of fagp with these two lectins therefore suggests that in non-pathological conditions the protein does not present any fucose residue on its surface, and that the glycoforms with one or more diantennary glycans are limited. fig. 3 reports the glycosylation patterns of fagp purified from fip affected cats: lane np represents pooled non-patological fagp, while pathological fagp from the 24 fip affected cats were loaded onto lanes 1-24. in all 24 cases, pathological fagp did not react with aal (fig. 3c ) or with cona (fig. 3d) . this result suggested that fagp expressed during fip does not present any fucose residues on its surface, and its branching degree is not increased during disease. fig. 3a shows that there are several differences in the a(2-6) sialylation of fagp among fip affected cats. several proteins, for example those in lanes 2, 3, 4, and 10, share an evident desialylation, that can be quantified by densitometry in 44, 42, 52 and 60%, respectively (data not reported) when compared to controls. on the contrary other proteins (lanes 7 and 8) exhibit an increase in the degree of sialylation (123 and 122%, respectively, data not reported). in other cats (lanes 5, 6, 12 and 14) the a(2-6) sialylation of fagp is not modified during fip. histograms in fig. 4 present the semi-quantitative evaluation to the peaks corresponding to fagp profiles shown in fig. 3 . using the lectin snai, the sialic acid a(2-6)-linked content of fagp from fip affected cats decreases to an average of 76% when compared to non-pathological fagp. we can therefore conclude that the a(2-6) sialylation of fagp is reduced during fip. on the contrary, the reaction with maa lectin (fig. 3b) , that preferentially binds a(2-3)-linked sialic acid residues, showed a marked difference in the binding between non-pathological and pathological fagp. all except one (fagp in lane 8) of the pathological fagp purified in this experiment clearly showed a marked reduction in the reaction with maa lectin and the decrease was very strong in several cats. some proteins, for example those in lanes 2, 3, and 6, showed clear desialylation, quantified by densitometry in 11, 7, and 20%, respectively (data not reported) when compared with nonpathologic fagp. this result strongly supports the hypothesis that there is a marked decrease of the sialic acid residues linked to galactose in position a(2-3). using the lectin maa, the sialic acid a(2-3)-linked content of pathological fagp decreases to an average value of 44% when compared to non-pathological fagp. fig. 3 . analysis of the glycosylation pattern of pathological fagp. detection of oligosaccharide chains has been performed after western blotting using the interaction with (a) snai, (b) maa, (c) aal and (d) cona. np: 2 mg pooled non-pathologic fagp, lanes 1-24: 2 mg each of fagp from fip affected cats. fagp were stained as described in section 2. the arrows on the right indicate the position of fagp. we further examined whether any differences between non-pathological fagp and fagp from fip-exposed cats could be observed (data not shown). as in the previous experiments, aal and cona did not show any reaction with agp. on the contrary, snai and maa exhibited strong reactions with fagp. however, very few differences between the non-pathological and the pathological forms were observed (fig. 4b) . moreover, the strong similarity of the electrophoretic mobility of all these proteins (data not presented) may suggest that fagp from fip exposed cats share the same oligosaccharide moiety with fagp from healthy cats. it has been reported that plasma acute phase protein agp concentrations increase by two to five times during fip (duthie et al., 1997) . in this study we have demonstrated several post-translational modifications of fagp in the course of fip. specifically, the glycan moiety was analyzed by binding with specific lectins. the most evident post-translational modification during fip is a decrease in the degree of sialylation. the function of fagp has not been clearly defined, however most reported data suggest an anti-inflammatory and an immunomodulatory role. the heavy glycosylation level of agp (45% of total mw) strongly suggests the involvement of the glycan moiety in these agp functions. fig. 4 . histograms displaying the average volume of density profile plots of non-pathological fagp compared with fip affected cats (a) and with fip exposed cats (b), after reaction with snai and maa. value are expressed in relative pixel intensity. the fagp glycosylation pattern in fip is surprisingly original, if compared to that reported in literature for other diseases. in human diseases, including rheumatoid arthritis and hiv, serum agp exhibits several features, including an increase in the fucosylation levels and in the degree of branching level (elliot et al., 1997) . we found that the branching degree and the fucosylation level of fagp are not increased in fip. it has been reported in mice that fucose epitopes are not exposed during prolonged inflammatory reaction, due to the absence of a3-fucosyltranferase in mouse liver (havenaar et al., 1998) . it is possible that cats may also lack this enzyme. furthermore, our results clearly indicate that fip affected cats exhibit a marked modification of sialic acid content such as a reduction (76%) of the sialic acid a(2-6)-linked to galactose, that was particularly marked in several subjects (4 out of 24). in other cats the number of epitopes of a(2-6) sialic acid were unchanged or increased. we therefore may conclude that the a(2-6) desialylation occurs on an individual basis and it is likely correlated with the disease. we also observed a strong reduction (44% if compared to controls) of sialic acid a(2-3)-linked to galactose, from the surface of purified fagp. this desialylation occurred, with varying degrees, in 22 out of 24 cats. fcov exposed cats apparently do not present any sialic acid, l-fucose or glycan branching modification. our results suggest that it is probably the fip, and not the simple infection with fcov, that may cause the major glycan modification on the surface of agp. at present, few data are available regarding biological differences between a(2-6) and a(2-3) linkage of sialic acid to galactose. due to their negative electric charges and the terminal localization, sialic acid residues play a very important role in mediating intercellular recognition events. it is also thought that the sialylation of a glycoprotein exerts an antiproteolytic activity, de facto prolonging the life of the protein. desialylation causes the molecule to be recognized by galactose specific lectins: this recognition targets the desialylated serum glycoproteins to hepatocytes for their removal from circulation (lamari and karamanos, 2002) . moreover, sialic acid is a component of receptors for cytokines, selectins and siglecs (sialic acid-binding immunoglobulin superfamily lectins) (traving and schauer, 1998) . the data presented here are not sufficient to suggest a role of fagp in the pathogenesis of fip. at present we can only speculate on the physiological and pathological role of the desialylation in the immunomodulatory function of agp. is the desialylation of agp connected with a reduction of the half-life of agp itself and, therefore, with a reduction of its immunomodulatory properties? the agp interacts with monocytes, inducing an increase of the secretion of il-1 receptor antagonist, and modulates the lpsinduced cytokine expression (fournier et al., 2000) . does the modification of the glycan moiety alter the binding of pathological agp to monocytes, thus increasing or decreasing the activation capability of these cells? fip develops when cats are exposed to mutated fcov that acquire the capability to replicate into macrophages (stoddart and scott, 1989) , and therefore it is conceivable that the interaction of different agp glycoforms may elicit different macrophage reactions. in conclusion, we have reported in this study that a change in glycosylation of fagp occurs during fip. since no information is currently available concerning glycosylation of fagp in other feline pathologies (and very few are indeed available in humans and mice), it is at the moment impossible to propose fagp glycosylation pattern as an additional specific marker for the diagnosis of this pathology. however, further studies of the carbohydrate moiety of fagp in other disease states, and the evaluation of whether this protein is overexpressed or not, are warranted. human a 1 -acid glycoprotein binds to ccr5 expressed on the plasma membrane of human primary macrophages prevalence of tri-and tetraantennary glycans of human alpha 1-acid glycoprotein in release of macrophage inhibitor of interleukin-1 activity a rapid and sensitive method for the quantitation of microgram quantities or protein utilizing the principle of protein-dye binding glycosylation of alpha 1-acid glycoprotein in septic shock: changes in degree of branching and in expression of sialyl lewis(x) groups inflammation-induced expression of sialyl lewis xcontaining glycan structures on alpha 1-acid glycoprotein (orosomucoid) in human sera value of a1-acid glycoprotein in the diagnosis of feline infectious peritonitis investigation into the concanavalin a reactivity, fucosylation, and oligosaccharide miscroheterogeneity of a 1 -acid glycoprotein expressed in the sera of patients with rheumatoid arthritis alpha-1-acid glycoprotein control of malaria virulence by alpha 1-acid glycoprotein (orosomucoid), an acute-phase (inflammatory) reactant structural characterization of glycoprotein carbohydrate chains by using digoxigenin-labeled lectins on blots sialyl lewisx epitopes do not occur on acute phase proteins in mice: relationship to the absence of a 3-fucosyltransferase in the liver involvement of the acute phase protein a 1 -acid glycoprotein in nonspecific resistance to a lethal gram-negative infection 1 -acid glycoprotein: an acute phase protein with inflammatory and immunomodulating properties cleavage of structural proteins during assembly of the head of bacteriophage t4 separation methods for sialic acids and critical evaluation of their biologic relevance an overview of feline enteric coronavirus and infectious peritonitis virus infection intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence antiinflammatory properties of hepatic acute phase proteins: preferential induction of interleukin 1 (il-1) receptor antagonist over il-1 beta synthesis by human peripheral blood mononuclear cells structure, function and metabolism of sialic acids alpha 1-acid glycoprotein (orosomucoid): pathophysiological changes in glycosylation in relation to its function effects of a 1 -acid glycoprotein on human polymorphonuclear neutrophils: influence of glycan microheterogeneity we thank dr. paol roccabianca and dr. laura kramer for the very helpful support in the critical reading of this paper. thanks also go to dr. scarafoni and dr. barbirolli for the help in the densitometric analysis with the imagemaster 1d software (amersham biosciences). we acknowledge the very important gift of the antibody anti-fagp from dr. duthie, university of glasgow. this work was financed by grant first/2002 funded to prof. paltrinieri. key: cord-317411-6lc0wpoo authors: giori, l.; giordano, a.; giudice, c.; grieco, v.; paltrinieri, s. title: performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases date: 2011-02-21 journal: j small anim pract doi: 10.1111/j.1748-5827.2011.01042.x sha: doc_id: 317411 cord_uid: 6lc0wpoo objectives: feline infectious peritonitis (fip) can be difficult to diagnose. histopathology is considered the gold standard test but immunohistochemistry (ihc) is mandatory to confirm/exclude the disease. this study aimed to assess the performances of tests carried out in vivo or at postmortem examination in challenging cases in which fip was confirmed or excluded based on ihc or on adequate follow‐up. methods: twelve cases (four without fip, eight with fip) were retrospectively studied. clinical findings, serum protein electrophoresis (spe), analysis of the effusions (ae), antifeline coronavirus serology, serum concentration of α1‐acid glycoprotein (agp) and histopathology were classified as consistent, doubtful or non‐consistent with fip. sensitivity, specificity and concordance (κ) with the final diagnosis were calculated. results: concordance was absent for serology (κ=−0·08) and ae (κ=−0·52), poor for histopathology (κ=0·09), fair for spe (κ=0·25) and perfect for agp (κ=1·00). sensitivity was high for agp (100%) and low for ae (50%), spe (37·5%) and histopathology (37·5%). specificity was high for agp or histopathology (100%) and low for spe (50%) and ae (0%). clinical significance: ihc must always be performed to confirm fip. if this is not possible, when histopathology is controversial, elevated agp concentrations may support the diagnosis of fip. feline coronavirus (fcov) causes feline infectious peritonitis (fip), a lethal disease characterised by vasculitis and/or pyogranulomatous lesions in different organs (pedersen 2009 ). the diagnosis of fip is difficult, especially in non-effusive (dry) cases (kipar and others 1999 , hartmann 2005 , pedersen 2009 ). a diagnosis cannot be based solely on serology or polymerase chain reaction (pcr), which detects the antibody response or the virus but does not provide information about the relationship between infection and clinical disease (pedersen 1976 , herrewegh and others 1995 , pedersen 1995 , meli and others 2004 . a diagnosis should be based on a combination of history (cats younger than 3 years or older than 10 years, especially living in crowded conditions), clinical signs and clinico-pathological or pathological changes (sparkes and others 1991 , 1994 , foley and others 1998 , andrew 2000 , addie and others 2004 , hartmann 2005 , pedersen 2009 ). common abnormalities include lymphopenia and changes in serum protein electrophoresis (spe) such as hyperproteinaemia with inverted albumin/globulin (a/g) ratio and increased serum α 2 -and γ-globulin concentrations (sparkes and others 1991 , 1994 , paltrinieri and others 1998 , addie and others 2004 . this electrophoretic pattern is also evident in effusions when present (shelly and others 1988, paltrinieri and others 1998) . the macroscopic, physico-chemical and cytological pattern of the effusion is also typical and has a high positive predictive value for fip (paltrinieri and others 1999) . it is described as a yellowish, transparent to cloudy, sticky fluid, often with fibrin clots and characterised by high protein content, high specific gravity and variable amounts of cells, mostly composed of non-degenerated neutrophils, macrophages and lymphocytes in a granular proteinaceous eosinophilic background. recently, it has been proposed that the serum concentration of α 1 -acid glycoprotein (agp) is used as a diagnostic tool for fip (paltrinieri and others 2007, pedersen 2009 ). agp is the major performance of diagnostic tests for fip taken for postmortem examination and histopathology and ihc were performed as described below. the details of the tests included in this study are as follows. analysis of the effusions: approximately 2 ml of fluid was placed in an edta-coated tube. cells were counted using an impedance counter (hemat 8, seac, calenzano, firenze, italy) . then 50 to 100 μl of fluid was cytocentrifuged (cytospin 2, shandon scientific, runcorn, cheshire, uk) at 130g for 10 minutes, and the remaining effusion was centrifuged (500g for 8 minutes), to obtain the cell-free supernatant used to evaluate the total protein by the biuret method with an automated analyser (cobas mira classic, roche diagnostics basel, switzerland), to perform electrophoresis using the same method described below for serum and to estimate the specific gravity using a portable refractometer (clinical refractometer 5711-2020, schüco, tokyo, japan). cytocentrifuged slides were stained with may grünwald-giemsa, cover slipped and examined under a light microscope. serum protein electrophoresis: this was performed using an automated system (genio, interlab srl, rome, italy). cellulose acetate electrophoresis was run at room temperature in a buffer solution (tris buffer >5%, sodium 5,5-diethylbarbiturate <1%, ph ≥8) for 16 minutes at 140 v. strips were stained with ponceau red, destained, diaphanised in appropriate solutions provided by the manufacturer and tested by densitometry to obtain electrophoretograms and the percentage area under each peak. agp: agp concentrations were measured using a radial immunodiffusion (srid) kit (tridelta development ltd, maynooth, kildare, ireland). five microlitres of each serum sample or of standard solutions containing 0·5 and 2·0 mg/ml of feline agp, respectively, were put in each well of a multi-well srid plate. after incubation (48 hours at room temperature), the diameter of precipitation rings was measured. values of the two standard solutions were used to design a standard curve. values from the case samples were then plotted to extrapolate absolute agp levels, expressed as mg/ml. serology: the presence of anti-fcov antibodies was assessed using an indirect immunofluorescence test performed on 10 multi-well slides produced at the university of zurich according to osterhaus and others (1977) , by coating each well with 4·5×10³ pd-5 cells, half of which were infected with swine transmissible gastroenteritis virus (serologically cross-reacting with fcovs). twofold dilutions (1:25 to 1:400) of each serum sample were prepared and 20 µl of each dilution was applied to the wells. after incubation (30 minutes, 37°c in a moist chamber), slides were washed with phosphate-buffered saline (pbs), dried and 15 µl of fluorescein isothiocyanate-conjugated rabbit-anticat immunoglobulin (nordic immunological laboratories, tilburg, the netherlands) was added to each well. after incubation (30 minutes, 37°c in a moist chamber), slides were washed, dried, cover-slipped with pbs and kaiser's glycerin (1:3 v/v) and observed on a fluorescence microscope. when the dilutions showed a clear fluorescent signal in about half of the cells, they were judged as positive. samples that were still positive at a 1:400 dilution were further diluted on a twofold basis until they were negative. histopathology/ihc: tissue samples were fixed in buffered 10% iso-osmotic formalin and embedded in paraffin. microthomic acute phase protein in cats and it increases in several inflammatory and non-inflammatory conditions (ceron and others 2005, paltrinieri 2008 ). nevertheless, the most prominent increases in serum agp concentration have been recorded in cats with fip (duthie and others 1997, giordano and others 2004) and agp could also be measured in effusions (bence and others 2005) . however, even moderate increases in serum concentration of agp can be used to support a diagnosis in cats with a strong clinical suspicion of fip (paltrinieri and others 2007) . histopathology is considered the only method for a conclusive diagnosis (sparkes and others 1991 , 1994 , pedersen 2009 ). however, the low yield of histopathological lesions in tru-cut sections limits the potential application of this approach for antemortem diagnosis of fip (giordano and others 2005) . in the authors' experience, postmortem test results and routine histopathology are occasionally inconclusive, especially in cases with an atypical clinical presentation. this difficulty has also been highlighted elsewhere, with reports of fip lesions misdiagnosed as tumours or vice versa others 1999, pedersen 2009 ). anti-fcov immunohistochemistry (ihc) is mandatory to confirm/exclude the disease in doubtful cases (addie and others 2004 , hartmann 2005 , pedersen 2009 ). the purpose of this study was to retrospectively assess the results of tests recorded in vivo and at postmortem examination in doubtful cases where fip was clinically suspected and definitely confirmed or excluded by ihc or based on complete recovery and to evaluate which test had the best sensitivity, specificity and concordance for fip. the database in our department was retrospectively analysed to select cases with the following criteria: (1) presence of complete information about in vivo and postmortem tests and/or a minimum follow-up of approximately 2 years; (2) inclusion of fip among the differential diagnoses based on the presence of clinical or laboratory abnormalities supportive of fip and also the presence of one or more clinical, laboratory, gross or histopathological features not considered consistent with fip or suggestive of a disease other than fip; (3) final confirmation/exclusion of fip based on the following criteria: cats were classified as not affected by fip (without fip) based either on gross or histopathological findings inconsistent with fip with negative ihc for intralesional fcovs or on the in vivo identification of diseases other than fip and/or based on complete remission of clinical signs after appropriate therapy. conversely, cats were classified as affected by fip based on positive ihc despite gross or histopathological lesions not completely consistent with fip. at admission, all the selected cats underwent a complete clinicopathological screening, except for pcr that, when performed, was requested by the referring veterinarians at their own respective service laboratories. in the case of death, cats were immediately following reasons: cat 1 recovered after local therapies and cat 2 had a diaphragmatic hernia. lesions consistent with fip or intralesional fcovs were not found in tissues from the remaining two cats and in cat 3, the "effusion" actually was the fluid content of a hepatic cyst. the cat recovered completely after surgical removal of the cyst, cat 4 had a focally extensive hepatic nodular lesion, histologically diagnosed as hepatocarcinoma and negative for fcov on ihc. final diagnosis in the eight cats with fip three of the cats with fip had atypical clinical presentations, including a mediastinal mass cytologically consistent with lymphoma (cat 6), a haemorrhagic syndrome with abdominal effusion classified as "transudate" (cells and proteins were virtually absent; cat 7) and absence of other signs except stunted growth (cat 9). no effusions were detected in vivo in this latter cat but a small amount of abdominal fluid was collected at postmortem examination (see electrophoretical analysis in fig 1) . five cats with fip had in vivo tests partially consistent with fip, including the presence of effusions, but postmortem examination findings were atypical due to the presence of the following: mesenteric fibrotising lymphadenopathy without additional lesions (cats 5 and 12), intestinal pyogranulomatous lesions containing ziehl-neelsen positive rods and periodic acid-schiff (pas)-positive fungal hyphae (cat 8), segmental thickening of the intestinal wall histologically characterised by severe fibrosis (cat 10, fig 2) intralesional fcovs were detected by ihc in all the eight cats belonging to this group (fig 4) . clinical findings and results of clinicopathological or postmortem tests are summarised in table 2 , where the results have been categorised according to the criteria reported in table 1 . to sections (5 µm) were stained by haematoxylin-eosin and by ihc using a monoclonal antibody (mab) against the fcov (kindly provided by prof. n.c. pedersen, davis, usa). the avidin biotin complex (abc) method with a commercially available kit (vectastain elite, vector laboratories inc., burlingame, ca, usa) was used to detect the positive reaction (paltrinieri and others 2003) , after inhibition of the endogenous peroxidase (h 2 o 2 1% in methanol) and antigen unmasking using microwave pretreatment (two cycles of 5 minutes each in citrate-buffered solution, 0·01 m, ph 6·2). 3-amino-9-ethyl-carbazole or diaminobenzidine served as chromogen for the reaction and then the slides were counterstained with mayer's haematoxylin. some sections of each sample were used as negative controls, with the primary antibody replaced by normal mouse serum (dako a/s, glostrup, denmark). data regarding clinical, clinicopathological and postmortem examination results recorded in each case were classified as consistent, doubtful or non-consistent with fip as described in table 1 . based on the final diagnosis, data were used to classify test results as true positive (tp), false positive (fp), true negative (tn) and false negative (fn). based on these values, sensitivity [sens=(tp/tp+fn)×100] and specificity [spec=(tn/ tn+fp)×100] were calculated (stockham and scott 2008) . the concordance between each single test interpretation and the final diagnosis was assessed by determining the cohen's κ coefficient and graded as suggested by landis and koch (1977) as poor (κ: <0), slight (κ: 0·0 to 0·20), fair (κ: 0·21 to 0·40), moderate (κ: 0·41 to 0·60), substantial (κ: 0·61 to 0·80) and almost perfect (κ: 0·81 to 1·00). final diagnosis in the four cats without fip these four cats had symptoms consistent with fip such as uveitis (cat 1) or effusions (cats 2, 3 and 4), but fip was excluded for the summarise, in cats without fip, the clinical features and intracavitary effusions, when present, were always consistent with fip. serology and spe were occasionally consistent with fip. postmortem examination or histopathology, when performed, and serum agp concentration were never consistent with fip (i.e. agp > 1·5 mg/ml), although in two cases, agp values [cat 2 (0·74 mg/ml) and cat 4 (1·33 mg/ml)] were higher than the upper reference limit of the laboratory (0·56 mg/ml). in cats with fip, the tests that were not consistent with or doubtful of fip included clinical signs (3 of 8 cases), analysis of effusion (ae; 3 of 6), spe (5 of 8), serology or pcr (4 of 5) and postmortem examination/histology (5 of 8). conversely, all these cats had agp values higher than the cut-off established as consistent with fip (1·5 mg/ml). the serum concentration recorded in these cats ranged from 2·04 to 14 mg/ml (median value of 2·89 mg/ml). data regarding sensitivity, specificity and concordance with ihc are reported in table 3 . the diagnostic concordance was maximal only for agp, which had 100% specificity and sensitivity, and low for histopathology, which had high specificity but low sensitivity, and poor or slight for the remaining tests. the ae was not specific but had a moderate sensitivity. these results confirm that the diagnosis of fip can be difficult in vivo. in this study, both the history and clinical presentation could be misleading even if they were highly consistent or inconsistent with fip. likewise, macroscopic and cytological aspects of the effusion were often not considered pathognomonic, as it is commonly accepted that the diagnosis of fip should be based on histopathological features (fibrinous polyserositis, infiltration of mononuclear cells and/or pyogranulomatous parenchymal foci; kipar and others 1998 , addie and others 2004 , pedersen 2009 ). in this study, however, histopathology proved highly specific for fip but was not a sensitive diagnostic test as it failed to confirm fip in most affected cases and it had a poor concordance with ihc, which is considered to have the highest specificity for diagnosing fip (pedersen 2009 ). as such, the detection of lesions clearly consistent with fip is intrinsically conclusive, but when the interpretation of lesions is not clear, fip cannot be excluded. there is limited information available on how to approach these cases when ihc is not available. although our study is limited by the small number of cases, the measurement of serum agp concentration appeared to be most helpful as it was the only diagnostic test in complete concordance with ihc. however, it must be pointed out that agp is an acute phase protein and, by definition, its serum concentration increases in any inflammatory/infectious condition (kajikawa and others 1999 , ceron and others 2005 , paltrinieri 2008 or even in non-inflammatory disease such as lymphoma (correa and others 2001) or other tumours (selting and others 2000) . the excellent performance of agp in this study could thus be biased because of the lack of cases with other severe inflammatory diseases. nevertheless, two factors must be they were also observed in inflammatory conditions other than fip. cytology of effusions is therefore not specific and cannot be used to support a diagnosis of fip when histopathology is doubtful. nonetheless, the cytological evaluation of effusions is always mandatory in the investigation as it can identify diseases other than fip (e.g. tumours or septic effusions) that were not included in this study. direct staining of fcovs within macrophages by immunofluorescence in cytocentrifuged effusions is considered the most specific tool to confirm fip (hartmann 2005) . however, it could not be applied in non-effusive forms or in poorly cellular samples as those detected in some cats in this study. in addition, although this test was first established by our group years ago (cammarata parodi and others 1993), it is not routinely performed in our laboratory, as it is time consuming and other tests, such as spe or agp, can provide information that strongly support the diagnosis of fip in cats with a high pretest probability of disease (paltrinieri and others 2007) . performance of diagnostic tests for fip taken into account in evaluating agp concentrations to support a clinical diagnosis of fip: the magnitude of the increase and the pretest probability of fip. the cut-off employed in this study was as suggested by duthie and others (1997) who noted that, although agp can increase in any inflammatory condition, the increase is more pronounced in fip than in other diseases. this has been confirmed by other studies others 2004, paltrinieri and others 2007) and ultimately also by the results of this study. in a recent study (paltrinieri and others 2007) , it was demonstrated that agp can be used to support a clinical diagnosis of fip according to the bayesian approach. when the pretest probability of fip is high (when most of the in vivo or postmortem tests are consistent with but not absolutely conclusive of fip), a "positive test" (a high agp value) can increase the posttest probability of disease. in other words, although agp alone cannot be used to confirm a clinical suspicion of fip, it could be confirmatory when fip is probable but not definitely diagnosed based on other clinicopathological findings or on postmortem examination results. in conclusion, the detection of lesions consistent with fip is a confirmatory test in cases with atypical clinical presentation. in the absence of lesions clearly consistent with fip or potentially consistent with other diseases, ihc should always be performed. if this is not possible, the presence of typical spe patterns or of effusions cytologically consistent with fip must not induce the pathologist to interpret ambiguous histological results as "consistent with fip." conversely, the presence of high concentrations of agp may support the diagnosis of fip in those cases in which results of other clinicopathological tests or histopathology are inconclusive. none of the authors of this article has a financial or personal relationship with other people 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peritonitis: a review of clinico-pathological changes in 65 cases, and a critical assessment of their diagnostic value coronavirus serology in healthy pedigree cats introductory concepts uncommon mediastinal cyst-like manifestation of feline infectious peritonitis key: cord-276617-chgjpg0v authors: takano, tomomi; azuma, natsuko; hashida, yoshikiyo; satoh, ryoichi; hohdatsu, tsutomu title: b-cell activation in cats with feline infectious peritonitis (fip) by fip-virus-induced b-cell differentiation/survival factors date: 2008-11-30 journal: arch virol doi: 10.1007/s00705-008-0265-9 sha: doc_id: 276617 cord_uid: chgjpg0v it has been suggested that antibody overproduction plays a role in the pathogenesis of feline infectious peritonitis (fip). however, only a few studies on the b-cell activation mechanism after fip virus (fipv) infection have been reported. the present study shows that: (1) the ratio of peripheral blood sig(+) cd21(−) b-cells was higher in cats with fip than in spf cats, (2) the albumin-to-globulin ratio has negative correlation with the ratio of peripheral blood sig(+) cd21(−) b-cell, (3) cells strongly expressing mrna of the plasma cell master gene, b-lymphocyte-induced maturation protein 1 (blimp-1), were increased in peripheral blood in cats with fip, (4) mrna expression of b-cell differentiation/survival factors, il-6, cd40 ligand, and b-cell-activating factor belonging to the tumor necrosis factor family (baff), was enhanced in macrophages in cats with fip, and (5) mrnas of these b-cell differentiation/survival factors were overexpressed in antibody-dependent enhancement (ade)-induced macrophages. these data suggest that virus-infected macrophages overproduce b-cell differentiation/survival factors, and these factors act on b-cells and promote b-cell differentiation into plasma cells in fipv-infected cats. feline coronavirus (fcov) belongs to group 1 of the family coronaviridae. fcov is classified into two biotypes based on differences in pathogenicity: feline enteric coronavirus (fecv) and feline infectious peritonitis virus (fipv). fecv infection is asymptomatic in cats. in contrast, fipv infection causes a fatal disease, feline infectious peritonitis (fip). the difference in pathogenicity between fecv and fipv in cats is considered to be associated with macrophage tropism of the viruses [27, 29] . it has been proposed that fipv arises from fecv by mutation [11, 26, 37] , but the exact mutation and inducing factors have not yet been clarified. when anti-fcov antibody-positive cats are inoculated with fipv, the onset time of fip is earlier than that in antibody-negative cats, and symptoms are severer [25] . this phenomenon is known as antibody-dependent enhancement (ade) of fipv infection. a similar phenomenon has been observed with other virus infections, such as dengue and human immunodeficiency virus infections [10, 33, 35] . ade of fipv infection has been reported to be induced by antibodies against fipv spike (s) protein [5, 12, 23] . in ade-induced macrophages (ade macrophages), tnf-alpha production increases with viral replication, and this tnf-alpha acts on macrophages and promotes virus receptor (feline aminopeptidase n; fapn) expression [31] . there are several events suggested to involve anti-fipv antibodies in fip development, other than ade. for example, antibodies produced due to fipv infection bind to the virus and form immune-complexes [1, 24] . these complexes are deposited in micro blood vessels. complement binding to the deposit injures the vascular tissue and causes vasculitis (immune-complex-mediated vasculitis). hyperproteinemia also develops with an increase of gamma-globulin in fip cats [1] . moreover, increased levels of interleukin (il)-6, a cytokine involved in the survival of b-cells and their differentiation into plasma cells, in ascites t. takano á n. azuma á y. hashida á r. satoh á t. hohdatsu (&) laboratory of veterinary infectious disease, school of veterinary medicine, kitasato university, towada, aomori 034-8628, japan e-mail: hohdatsu@vmas.kitasato-u.ac.jp and culture supernatant of peritoneal exudative cells (pec) from fip cats have been reported [8] . this suggests a close involvement of antibodies in fip pathogenesis. however, the mechanism leading to the alteration of b-cells into plasma cells has not been investigated. overproduction of the virus and tnf-alpha also occurs in ade macrophages in fipv infection [31] , but it is not clear whether the production of b-cell differentiation/survival factors, such as il-6, is involved in the induction of ade. in this study, we collected peripheral blood mononuclear cells (pbmcs) from fip cats and analyzed the b-cell surface antigens as well as measured the mrna expression level of the plasma cell master gene, b-lymphocyteinduced maturation protein 1 (blimp-1). we also collected macrophages from fip cats and measured the expression levels of the viral rna and mrna of b-cell differentiation/survival factors: il-6, cd40 ligand (cd40l), and bcell-activating factor belonging to the tumor necrosis factor family (baff). furthermore, we investigated the relationship between fipv infection-induced ade activity of macrophages and il-6, cd40l, and baff mrna expression levels. type-ii fipv strain 79-1146 (10 4 tcid 50 /ml) was administered orally to 6-to 8-month-old, specific-pathogen-free (spf) cats. thirteen cats that developed fip symptoms (fip cats), such as fever, weight loss, peritoneal or pleural effusion, dyspnea, ocular lesions, and neural symptoms, and thirteen 6-to 8-month-old spf cats as controls were used in this study. fip diagnosis was confirmed upon postmortem examination, revealing peritoneal and pleural effusions and pyogranuloma in major organs. all experiments were performed in accordance with the guidelines for animal experiments of kitasato university. felis catus whole fetus-4 (fcwf-4) cells was grown in eagles' minimum essential medium containing 50% l-15 medium, 5% fetal calf serum (fcs), 100 u/ml penicillin and 100 lg/ml streptomycin. feline alveolar macrophages and pbmcs were maintained in rpmi 1640 growth medium supplemented with 10% fcs, 100 u/ml penicillin, 100 lg/ml streptomycin, 50 lm 2-mercaptoethanol, and 2 lg/ml of polybrene. type-ii fipv strain 79-1146 was grown in fcwf-4 cells at 37°c. fipv strain 79-1146 was supplied by dr. m. c. horzinek of state university utrecht, the netherlands. phycoerythrin (pe)-conjugated anti-cd21 monoclonal antibody (mab) (anti-canine cd21 homologue of feline cd21 and human cd21: mab cd2.1d6) and fluorescein isothiocyanate (fitc)-conjugated igg f(ab 0 )2 fragments of rabbit anti-feline igg(anti-feline sig ab) were used to stain feline b-cells. pe-conjugated mab cd2.1d6 was obtained from serotec ltd (uk). fitc-conjugate anti-feline sig ab was obtained from rockland inc. (usa). mab 6-4-2 (igg2a) used in the present study recognizes s protein of type-ii fipv, as demonstrated by immunoblotting [13] . it has been reported that mab 6-4-2 exhibits a neutralizing activity in fcwf-4 and crfk cells, but exhibits an enhancing activity in feline macrophages, depending on the reaction conditions [14] . heparinized blood (10 ml) was diluted twofold with phosphate-buffered saline (pbs) and subjected to ficoll-hypaque density gradient centrifugation at 1,700 rpm for 20 min. the pbmc layer was collected, washed twice with pbs, and resuspended at 2 9 10 6 cells/ml. a total of 2 9 10 6 cells were incubated with pe-conjugated mab cd2.1d6 or fitc-conjugated anti-feline sig ab at 4°c for 45 min. after the cells were washed three times with pbs containing 0.1% nan 3 , the number of stained cells was determined by counting 5,000 cells on a facs 440 (becton dickinson, usa). albumin-to-globulin ratio for plasma samples, blood was collected from cats using a heparinized syringe and centrifuged at 3,000 rpm for 10 min, and the supernatant was collected. the albumin and globulin levels were determined in plasma samples of spf cats and in fip cats, using an automatic analyzer. the albumin-to-globulin ratio was calculated by dividing the albumin levels by the globulin levels. feline alveolar macrophages were obtained from spf and fip cats by broncho-alveolar lavage with hank's balanced salt solution (hbss), as previously described by hohdatsu et al. [12] . 28 t. takano et al. rna isolation and cdna preparation rna isolation and cdna preparation were performed by the method of takano et al. [30, 32] . determination of levels of feline gapdh mrna, il-6 mrna, cd40l mrna, baff mrna, blimp-1 mrna and fcov n gene expression cdna was amplified by pcr using specific primers for feline gapdh mrna, il-6 mrna, cd40l mrna, baff mrna, blimp-1 mrna, and the fcov n genes. the primer sequences are shown in table 1 . pcr was performed by the method of takano et al. [30, 32] . band density was quantified under appropriate uv exposure by video densitometry using scion image software (scion corporation, usa). il-6 mrna, cd40l mrna, baff mrna, blimp-1 mrna, and the fcov n genes were quantitatively analyzed in terms of the relative density value to the mrna for the housekeeping gene gapdh. sequencing and analysis of blimp-1 and baff cdna pcr products (21 ll) were electrophoresed with dna markers on a 1.5% agarose gel. singlet bands were excised and transferred to microtubes, and dna was purified using a qiaquick gel extraction kit (qiagen gmbh, germany). the purified dna was sent to shimadzu corporation (japan) for sequencing, and the partial base sequences of blimp-1 cdna and baff cdna were determined. the sequences determined were then analyzed with the genetyx computer program (software development, japan). equal volumes a viral suspension (fipv strain 79-1146, 2 9 10 3 tcid 50 /0.1 ml) and a mab 6-4-2 solution were mixed and reacted at 4°c for 1 h, and 0.1 ml of this reaction solution was used to inoculate feline alveolar macrophages (2 9 10 6 cells) cultured in each well of 24well multi-plates. as controls, medium alone, virus suspension alone, or mab 6-4-2 solution alone was added to feline alveolar macrophages. after virus adsorption at 37°c for 1 h, the cells were washed with hbss and 1 ml of growth medium. the cells and culture supernatant were collected every 24 h thereafter. the cells were used for measurement of the fcov n genes, il-6 mrna, cd40l mrna, and baff mrna, and the culture supernatant was used for measurement of the virus titer. confluent fcwf-4 cell monolayers in 24-well multi-plates were inoculated with 100 ll of the sample dilutions. after virus adsorption at 37°c, the cells were washed with hbss, and 1 ml of growth medium containing 1.5% carboxymethyl cellulose was added to each well. the cultures were incubated at 37°c for 2 days, fixed in 10% buffered formalin, and stained with 1% crystal violet. data were analyzed by student's t test. the data in fig. 1a and b were also analyzed by the mann-whitney test. p values .05 were considered to indicate a significant difference between compared groups. fig. 1b . the ratio of sig ? cd21 ? b-cells in pbmcs was not significantly different between spf and fip cats. in contrast, the ratio of sig ? cd21 -b-cells was significantly higher in fip than in spf cats. figure 1c shows (table 1) . using these primers, rt-pcr was performed on pbmcs from fip cats, and a 186-bp band was specifically amplified. this amplified dna was sequenced, and the base sequence was partially identified (partial base sequence) (data not shown). the amino acid sequence was also deduced from the partial base sequence. homologies among the partial base sequences of feline, canine, bovine, mouse, and human blimp-1 mrna and the deduced amino acid sequences are shown in table 2 . the blimp-1 mrna expression level in pbmcs was measured in spf and fip cats. rt-pcr was performed using feline blimp-1-specific primers, and the blimp-1 mrna expression level in pbmcs was compared between fip and spf cats. the blimp-1 mrna expression level was significantly elevated in pbmc from fip cats, compared to that in spf cats (fig. 3) . partial base sequence analysis of baff cdna, deduction of amino acid sequence, and comparison with other animal species il-6, cd40l, and baff are known as b-cell differentiation/survival factors. we investigated whether these factors in macrophages were increased in fip cats by measuring mrna expression. since the nucleotide sequence of feline baff mrna has not yet been determined, we partially sequenced the cdna of baff mrna. the sequences of mouse (genbank accession number: nm_033622), human (nm_006573), and bovine (xm_864542) baff mrna have been determined. primers corresponding to a highly conserved region were prepared for the detection of feline baff mrna (table 1) . using these primers, rt-pcr of pbmcs from fip cats was performed, and a 164-bp band was specifically amplified. this amplified dna was partially sequenced (data not shown), and the amino acid sequence was deduced from this. homologies among the partial nucleotide sequences of feline, canine, bovine, mouse, and human baff mrna and their deduced amino acid sequences are shown in table 2 . measurement of fcov n gene expression and il-6, cd40l, and baff mrna expression in alveolar macrophages of spf and fip cats the production of b-cell differentiation/survival factors in fip cats was investigated. macrophages, one of the target cells of fipv, were collected from fip cats. the il-6, cd40l, and baff mrna expression levels in these cells were measured by rt-pcr and compared to those in spf cats. fig. 3 blimp-1 mrna expression levels in pbmcs of fip and spf cats. pbmcs (2 9 10 6 cells) were collected from fip and spf cats, and the blimp-1 mrna was detected by rt-pcr. blimp-1 mrna was quantitatively analyzed in terms of their relative density compared to that of the mrna for the housekeeping gene gapdh virus replication in macrophages was investigated in fip cats by measuring fipv negative-strand rna expression. in all fip cats investigated, fipv negative-strand rna was expressed in alveolar macrophages (fig. 4a) . when the mrna expression levels of b-cell differentiation/survival factors were measured, expression of il-6, cd40l, and baff mrna was significantly enhanced in fip cats compared to spf cats (fig. 4b) . relationship between fipv replication and il-6, cd40l, and baff mrna expression levels in alveolar macrophages to investigate the relationship between fipv replication and b-cell differentiation/survival factor production in macrophages, alveolar macrophages from spf cats were inoculated with fipv alone or a mixture of fipv and anti-fipv s mab (mab 6-4-2). the culture supernatant and cells were collected every 24 h for 3 days, and fipv replication and b-cell differentiation/survival factor production were measured. in the measurement of fipv replication, the virus titer in the macrophage culture supernatant was measured by the plaque method, and intracellular virus production was measured using fipv negative-strand rna expression as an index. to assess bcell differentiation/survival factor production, the il-6, cd40l, and baff mrna expression levels in the cells were measured. fipv negative-strand rna was strongly expressed in macrophages inoculated with a mixture of fipv and anti-fipv s mab compared to that in macrophages inoculated with the virus alone, and the virus titer in the culture supernatant was also significantly higher ( fig. 5a and b) . the mrna expression levels of all b-cell differentiation/survival factors, il-6, cd40l, and baff, were also significantly increased in macrophages inoculated with the mixture (fig. 5c) , suggesting that virus production increased in ade macrophages, in which b-cell differentiation/survival factor production also increased. in fip cats, the gamma-globulin level increases, and immune-complex-mediated vasculitis develops, suggesting that overproduced antibodies are closely involved in the pathogenesis of fip. however, only a few studies on b-cell characteristics and their activation mechanism after fipv infection in fip cats have been reported. here, we show that the ratio of sig-positive, cd21negative cells increases in pbmcs of fip cats, and correlates with the albumin-to-globulin ratio in plasma samples. we also show that the blimp-1 mrna expression level is significantly elevated in pbmcs from fip cats. tedder et al. [34] reported that cd21 molecules disappeared from the cell surface, when b-cells differentiated into antibodyproducing cells. shapiro-shelef and calame [28] reported that blimp-1 is the master gene that causes the final differentiation of b-cells into plasma cells. thus, the number of plasma cells may increase in the peripheral blood of fip cats. plasma cells are mainly present in the bone marrow, spleen, and lymph nodes, but not in peripheral blood. an increase in plasma cells in peripheral blood may be evidence of enhanced b-cell activation. b-cells are stimulated by antigen-presenting cells (dendritic cells and macrophages) in lymphatic tissues, and start to differentiate into plasma cells. for b-cells to differentiate into plasma cells, various factors (cytokines, antigens, etc.) are necessary. we focused on il-6, cd40l, and baff as b-cell differentiation/survival factors. il-6 is involved in the differentiation of b-cells into plasma cells, and signal transmission via the il-6 receptor inhibits b-cell apoptosis [7, 18, 36] . cd40l inhibits the induction of bcell apoptosis by binding to cd40 expressed on the b-cell surface, activating b-cells [3, 16, 17, 20] . baff induces an apoptosis-inhibitory gene, bcl-2, by binding to the baff receptor expressed on b-cells, inhibiting b-cell apoptosis [6, 22] . we clarified that il-6, cd40l, and baff mrna expression was increased in macrophages in fip cats. the il-6, cd40l, and baff mrna expression levels were also increased with virus replication in macrophages inoculated with fipv, suggesting that fipv replication a b fig. 4 fcov n gene, il-6, cd40l and baff mrna expression levels in alveolar macrophages of fip and spf cats. a alveolar macrophages (2 9 10 6 cells) were collected from fip and spf cats, and the fipv negative-strand rna was detected by rt-pcr. b alveolar macrophages (2 9 10 6 cells) were collected from fip and spf cats, and il-6, cd40l and baff mrna was detected by rt-pcr. il-6, cd40l and baff mrna were quantitatively analyzed in terms of their relative density compared to that of the mrna for the housekeeping gene gapdh induced baff, il-6, and cd40l production in macrophages of fip cats, and these factors promoted b-cell differentiation into plasma cells. virus production and il-6, cd40l, and baff mrna expression were markedly enhanced in ade macrophages in vitro compared to macrophages inoculated with the virus alone. we reported previously that when the fipv antigen was detected by the fluorescent antibody method, 10-20% of cells were positive when spf cat-derived alveolar macrophages were inoculated with fipv ? anti-fipv s mab, whereas 1-2% of cells were positive when alveolar macrophages were inoculated with fipv [12, 15] . it seems that the increased number of virus-infected macrophages leads to the over-expression of b-cell differentiation/survival factor mrna in macrophages. however, there is no evidence that this hypothesis is correct. a continued examination of the mechanism of ade activity in fipv infection would strengthen this hypothesis. we reported previously that ade activity in fipv infection of feline macrophages caused overproduction of tnf-alpha, which may lead to serious fip symptoms [31] . the increased il-6, cd40l, and baff mrna expression levels in ade macrophages may also be closely involved in aggravation of pathogenesis, as well as tnf-alpha. that is, our data suggest that fipv re-infection induces ade and advances fip development in cats. however, addie et al. [2] reported that fcov re-infection of anti-fcov antibody-positive domestic cats might not result in the development of ade. although the details are unclear, differences in the immunological condition of fcovinfected cats may be the reason why the phenomenon noted in ade does not occur in domestic cats. it is necessary to analyze mechanism of ade in detail. of the b-cell differentiation/survival factors measured, significant elevation of the ascitic and serum il-6 levels in fip cats has been reported [8] . regarding cd40l, tissue deposition of immune-complexes, similar to that in fip, is involved in the pathogenesis of systemic lupus erythematosus, rheumatoid arthritis, and sjogren's disease, in which a significantly increased serum cd40l level has been reported [9] . similarly, increased levels of immune-complexes in the blood of mice induced to overexpress baff have been reported [21] . therefore, it is easily predicted that il-6, cd40l, and baff are involved in the development of 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(fipv)-induced macrophages, upregulates expression of type ii fipv receptor feline aminopeptidase n in feline macrophages analysis of the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection: aminopeptidase n is not important and a process of acidification of the endosome is necessary two receptors are required for antibody-dependent enhancement of human immunodeficiency virus type 1 infection: cd4 and fc gamma r expression of c3d receptors during human b cell differentiation: immunofluorescence analysis with the hb-5 monoclonal antibody antibody-dependent enhancement of virus infection and disease il-6 rescues the hyporesponsiveness of c-rel deficient b cells independent of bcl-xl, mcl-1, and bcl-2 feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses key: cord-254375-otj044by authors: paltrinieri, s; cammarata, m.parodi; cammarata, g; comazzi, s title: some aspects of humoral and cellular immunity in naturally occuring feline infectious peritonitis date: 1998-10-23 journal: vet immunol immunopathol doi: 10.1016/s0165-2427(98)00155-x sha: doc_id: 254375 cord_uid: otj044by haematology, antibody titers and serum protein electrophoresis from 48 cats (34 effusive and 14 noneffusive forms) affected with feline infectious peritonitis (fip) were studied and compared with those of 20 healthy cats. in the effusive form, antibody titers and protein electrophoresis in the effusions were analyzed. the distribution of the immune cells and of the virus in fip lesions were also investigated immunohistochemically with the avidin–biotin complex (abc) method, using antibodies against the fip virus (fipv), myelomonocytic (mac387) and lymphoid (cd3, cd4 and cd8 for t-cells and igm and igg for b-cells) antigens. seropositive animals (antibody titer>1:100) were present among both the fip infected cats (73%) and the healthy cats (70%). cats with effusive fip had neutrophilic leukocytosis (p>0.05), lymphopenia (p<0.01) and eosinopenia (p<0.001). in both effusive and noneffusive forms decreased albumin/globulin ratio (p<0.001) with hypoalbuminemia (p<0.001), hyperglobulinemia (p<0.001) and increased α(2)(p<0.05), β(p<0.05) and γ-globulins (p<0.001) were found. hypergammaglobulinemia was not related to the antibody titers, suggesting the presence of other proteins with γ-motility (e.g. complement fractions). the electrophoretic pattern of the effusions was always similar to that of the corresponding serum. antibody titers higher than those of the corresponding serum were often detected in the effusions. immunohistochemical findings were not related to the antibody titers, but they were related to the histological aspect of the lesions. in cellular foci of fip lesions many virus-infected macrophages and few lymphocytes, mainly cd4(+), were found. extracellular viral and myelomonocytic antigens were also detectable in the foci with intercellular necrosis. only few fipv-infected cells were present at the periphery of the larger necrotic foci: in these lesions mac387(+) cells were mainly neutrophils, with many mac387(−) macrophages, probably due to their activated state; a small number of lymphocytes, with an increasing percentage of cd8(+) cells was present. lymphocytes were more abundant when cellular foci and fip-infected macrophages were centered around neoformed vessels. igm and igg exposing b-cells were always few and scattered. in conclusion the simultaneous analysis of body fluids and of the cellular composition of the lesions showed a complex immune status, on which type iii and type iv hypersensitivity could coexist. feline infectious peritonitis (fip) is a disease of felids due to coronaviruses. the fip virus (fipv) originates from the less pathogenic feline enteric coronavirus (fecv) by a minor mutation (pedersen and floyd, 1985) , and acquire the ability to pass through the epithelium into the lymph and to replicate in the cells of the monocytic±macrophagic line (stoddart and scott, 1989) . within the monocytes, fipv spreads throughout the body to target organs. the two feline coronaviruses (fcov) have slight and poorly understood differences in their genoma (vennema et al., 1995) , but they are morphologically and antigenically identical (boyle et al., 1984) . this leads to some problems in both diagnosis and understanding of the pathogenesis of the disease. the appearance of the disease and the different clinical forms (effusive or noneffusive) depends on the cellular immune efficiency: cats with a strong cellular immunity may not develop the disease, while those with a weak cellular immunity have the noneffusive fip and those without a cellular reaction have the effusive form (pedersen and black, 1983; pedersen, 1995a pedersen, , 1987 humoral immunity, in contrast, seems to increase the probability of getting the disease. even if antibody-enhanced infection has been recently questioned (olsen et al., 1992 (olsen et al., , 1993 addie et al., 1995) , many experimental results demonstrate that anti-fcov antibodies facilitate the uptake of the virus by the macrophages (hayashi et al., 1983; stoddart and scott, 1989 ; barlough and stoddart, 1990; hohdatsu et al., 1994; pedersen, 1995a) , and that immunocomplexes lead to a type iii hypersensitivity reaction with disseminated intravascular coagulation and fibrinoid necrosis of the vessel's walls, responsible for the effusions (hayashi et al., 1977 (hayashi et al., , 1978 pedersen and boyle, 1980; weiss et al., 1980; jacobse-geels et al., 1980; weiss and scott, 1981; fenner, 1987; pastoret and bourtonboy, 1991; pedersen, 1995a) . this hypothesized immune-mediated pathogenesis is also supported by epidemiologic considerations (pedersen and floyd, 1985; addie et al., 1995) and by other immunopathological findings, such as the fluctuations of antibody titers and complement fractions during the course of the infection (pedersen and boyle, 1980; jacobse-geels et al., 1982) . the morphology of the lesions, similar to those of other immunological granulomas, such as the tubercular ones (pedersen, 1987; paltrinieri et al., 1998) and the detection of a cutaneous fipv-induced delayed-type hypersensitivity-like reaction in cats (weiss and cox, 1988) , suggests that type iv hypersensitivity reactions could be involved in the pathogenesis of focal lesions (pedersen, 1987) . to further understand the pathogenesis of the disease, parameters indicative of the involvement of humoral immunity (total and fractioned proteins and antibody titers in serum and in effusions), and the distribution of viral antigen and immune cells in the lesions were studied in cats with spontaneous fip. forty eight cats of different sex, age and breed and with clinical signs of spontaneous fip submitted by clinicians were examined: 34 cats had the effusive form, while the remaining 14 had noneffusive form of the disease. the clinical diagnosis was confirmed by necropsy, histology and by the immunohistochemical detection of the fip antigen in the lesions. a blood sample of 2 ml was taken from the cephalic or jugular vein of each cat: 1 ml was put in a edta-coated tube for hematology and 1 ml in a tube without anticoagulant for serum. from the cats with effusive fip, 1.0 ml of ascitic fluid or thoracic effusion was also withdrawn by paracentesis before the cats were euthanatized or immediately after death and collected in edta-coated tubes. hundred ml of each effusion were cytocentrifuged in a multiwell cytocentrifuge (bioprobe). the slides were routinely stained with may gru ènwald±giemsa. the remaining amount of the effusions was centrifuged and the supernatants separated to perform both protein analysis and serology. the sera or the effusions were aliquoted and stored at à208c. a complete hemogram in an automatic counter (seac hemat 8) and a differential leukocyte count on a may gru ènwald±giemsa stained smear were carried out. the percentages of reticulocytes were evaluated in cresyl brillant blue-stained smears. total proteins in serum and effusions were measured by a discrete autoanalyzer (abbott vp) using the colorimetric biuret method (abbott). serum protein electrophoresis was carried out by the semimicro-method, using cellulose polyacetate strips (gelman) in a barbitone and tris buffer (helena lab.). undiluted serum or effusions were applied on the strips and run for 40 min, 150 v in the same buffer. then the strips were stained for 15 min in red ponceau (0.5 g in 100 ml of 5% trichloroacetic acid), destained in a 5% acetic acid and cleared (helena lab.) the gels were scanned in a densitometer (gelman dcd16). antibody titers were evaluated using a commercially available elisa kit (diagnostica 2000) . serial 2-fold dilutions in phosphate buffered solutions (pbs) were prepared and 100 ml of each dilution were put in a microtiter plate coated with polyclonal fip proteins. after incubation (60 min at 378c) 100 ml of anti-feline horseradish peroxidaseconjugated antibody were added. after 60 min of incubation at 378c, 100 ml of substrate buffer solution were added for 20 min. the reaction was stopped with 50 ml of stop solution. the multiwell microtiter plates were then read at 450 nm in an automatic elisa analyzer (dasit multiskan). negative and positive controls were included in each plate. from each affected organ, including the central nervous system (cns), when nervous symptoms were present, and kidney, even without any lesions, two samples (approximately 1 cm 3 ) were taken, one was fixed in buffered 10% iso-osmotic formalin and embedded in paraffin, the other was frozen. one of the cryostatic sections and one of the formalin-fixed, paraffin-embedded sections were used to confirm the diagnosis by hematoxylin±eosin stain. the histological lesions were recorded and a comparison with the results of body fluid analysis was carried out. a monoclonal antibody against the fip (kindly provided by prof. n.c. pedersen, davis, usa) was used to detect the virus in lesions. myeloid cells were identified using the monoclonal antibody mac387 (dako). a polyclonal antibody against cd3 antigen (dako) and monoclonal antibodies against cd4 and cd8 antigens (both of these kindly provided by dr. willet, glasgow, uk), were used to identify t-lymphocytes and their subpopulations. polyclonal antibodies were employed to detect igg-and igm-expressing b-lymphocytes. cd3 and mac387 antibodies were tested in formalin-fixed and paraffin-embedded sections after antigen unmasking by microwave pre-treatment (cattoretti et al., 1993) ; igg and igm expressing b-lymphocytes were investigated in formalin-fixed and paraffinembedded sections after antigen unmasking using a proteolytic pretreatment (protease xxiv, sigma); cd4 and cd8 antibodies were detected in frozen sections. immunohistochemistry was performed with the avidin biotin complex (abc) method with a commercially available kit (vectastain elite, vector laboratories, ca, usa), as previously described (hsu et al., 1981) . briefly, the formalin-fixed and paraffinembedded sections were deparaffinated and rehydrated in xylene and ethanol, endogenous peroxidase were inhibited with h 2 o 2 (1%) in methanol and antigen unmasking was carried out using the protease xxiv (0.5% in prewarmed tris-buffered solution, ph 7.7, for 15 min at 378c) or microwave pretreatment (2 cycles of 5 min in citrate-buffered solution, 0.01 m, ph 6). in cryostatic sections, endogenous peroxidase was inhibited with h 2 o 2 (1%) in 0.1% sodium azide. the blocking sera (20 min at room temperature) were obtained from goats (for cd3), from rabbits (for igg and igm) or from horses (for monoclonal antibodies). then the primary antibodies were applied overnight at 48c. after three washes in tris-buffered solution (ph 7.7) the biotinylated secondary antibodies (30 min at room temperature), and finally the abc complex (30 min at room temperature) were added. diaminobenzidine tetrahydrochloride or 3-amino-9-ethyl-carbazole served as chromogen for the reaction. the reaction was blocked by washing in running tap water and the slides were counterstained with mayer's haematoxylin and then the coverslips were mounted. some sections of each sample were used as negative controls, with the primary antibody substituted by tris-buffered solution. the results obtained from hematology and protein analysis in effusive and noneffusive fip-infected cats were compared with those obtained from the controls using an one-way anova test. the nonparametric spearman correlation test was employed to plot the antibody titers against total and fractioned protein concentrations. protein analysis of the serum were compared with those of the corresponding effusions by a t-test for dependent samples. the nonparametric wilcoxon matched paired test was employed to compare the antibody titers in serum and in corresponding effusions. all 48 cats had pathological features of fip, consistent with those described in literature (wolfe and griesemer, 1971; montali and strandberg, 1972) : 34 cases had a yellowish, dense, and fibrinous effusion in the abdomen (79.4%), in the chest (17.6%) or in both the cavities (2.9%). perivisceral fibrin was present on the omentum and on all the peritoneal or pleural surfaces; small nodular foci were detectable under the intestinal serosa, and in the liver and kidneys. larger nodular foci were often detectable in the kidneys. the involvement of the central nervous system was characterized by a fibrinous meningitis. histology of the lesions showed the presence of perivisceral fibrin with few scattered inflammatory cells or with subperitoneal or subpleural cellular foci: these larger foci contained central necrosis with peripheral fibrosis. a lymphohistiocytic perivasculitis was often detectable, as well as intraparenchymatous pyogranulomatous hypercellular lesions with necrotic centers. fibrinous and lymphohistiocytic meningitis was found in the cns. the remaining 14 cases were the noneffusive form, with nodular lesions (diameter: 0.3±1 cm) in the kidneys or in the lymph nodes or, less frequently, in other organs. these lesions were characterized by a central necrosis surrounded by a pyogranulomatous reaction similar to those detectable in the larger and intraparenchymatous lesions of the effusive forms. means and standard deviations observed in controls and in fip-infected cats are reported in table 1 . all the fip cats were anemic, with a decreased number of erythrocytes (p<0.001), haemoglobin concentration (p<0.001) and pcv (p<0.01). no change in reticulocyte number was detected between the groups. leukocytosis (p<0.05) with increased number of segmented (p<0.001) and band (p<0.05) neutrophils and decreased number of eosinophils (p<0.001) and lymphocytes (p<0.05) were present in cats with effusive fip. the total protein concentration (table 2) in cats with effusive fip (73.5ae18.1) and in those with noneffusive fip (80.2ae14.5) was similar to those observed in controls (70.1ae9.4 g/l). the fip cats demonstrated a decrease of albumin/globulin (a/g) ratio (p<0.001), due to decreased albumin (p<0.001) and increased globulin (p<0.001) concentrations. hypoalbuminemia was greater in the effusive than in the noneffusive form (p<0.001). all globulin fractions increased in fip cats, but the increase was lower in -(p<0.05) than in -(p<0.01) and in g-(p>0.001) globulins; the increase of -globulins was due mainly to an increase in the 2 fraction (p<0.05). antibody titers higher than 1:100 were present in healthy cats (70%) and fip-infected cats (73%) (fig. 1) . the prevalence of seropositivity was higher in the effusive (79%) than in the noneffusive form (57%) (fig. 2) . no correlations were found between the antibody titers and total or fractioned protein concentrations. thirty of the 34 examined effusions (88.2%) had cytologic findings consistent with the clinical diagnosis of fip, according to cowell et al. (1989) , with neutrophils, mesothelial cells and a proteinaceous background and without any bacteria or noninflammatory cells. most (32 of 34) effusions (94.1%) had a protein content higher than 35 g/l, a value considered diagnostic for fip (sparkes et al., 1991) . the mean protein concentration (61.7ae16.1 g/l) was lower than those observed in the serum (p<0.001) and the mean ratio between protein content in the effusions and sera was 0.86ae0.17 (table 3 ). the electrophoretic pattern of the effusions was similar to those of the corresponding serum. no differences were found between the albumin concentrations in serum and in effusions, with an effusion/serum ratio of 0.97ae0.30. the concentrations of total and -globulins were higher in the serum (p>0.001 for both parameters). these differences were higher for 1 -than 2 -globulins (p<0.001 and p<0.01, respectively). all of these parameters had the lowest effusion/serum ratio (see table 3 ). mean values of -and g-globulins concentrations were similar in serum and in the corresponding effusions, may be due to the high variability, with many cats that had higher values of these compounds in the effusions than in the serum (effusion/serum ratio 0.99ae0.68 and 1.39ae2.93, respectively). the percentage of animals in which the effusions had antibody titers higher than 1:100 (79.4%) was similar to those observed in the serum (fig. 3) . no correlation was found between the titers observed in serum and in the corresponding effusions. 47% of the animals had higher titers in the effusions than in serum. each examined cat had fip viral antigen in the lesions. the amount and the localization of viral antigen was very variable between the lesions, without any relationship to the clinical form, antibody titer or the size of the foci. in contrast, the distribution of the fip in each focus seemed to be related to the pattern of the lesion: in cellular foci, many of the histiocytic cells were fipv , at the periphery of necrotic foci. in some cases, viral antigen was detectable in dendritic-like cells into germinal centers of lymph nodes draining the affected organs. antigen was never detected where immune complexes may be deposited (vessel's walls or the glomeruli). the number of mac387 myeloid cells was high within cellular lesions. many of these cells near necrotic foci were granulocytes. no differences in the distribution of the mac387 and mac387 à myeloid cells were found between cats with different antibody titers or protein concentrations. cd3 lymphocytes were randomly distributed within fibrin and small cell foci; their numbers increased in the larger organized lesions where they were concentrated around the periphery of necrotic foci. cd3 à cells were also present, mainly in more organized lesions. no differences in the localization and in the number of cd3 lymphocytes were found between cats with different antibody titers or protein concentrations. igg and igm lymphocytes and plasma cells were detectable in fip lesions, but their number was always low and their distribution very variable among the cases. no relationship with the histological pattern of the lesions, or with the humoral findings of the cats has been detected. the number of cd4 and cd8 lymphocytes increased with the age/size of the fip lesion. in the small foci, cd4 cells predominates, whereas in the larger necrotic foci, the number of cd8 cells were increased. the cd4 lymphocytes were uniformly distributed throughout the lesions, whereas the cd8 lymphocytes were at the periphery of the foci in each stage of the granuloma. all these findings were detectable both in the cats with high antibody titers and in those with low antibody titers. no changes indicative of immune system dysfunction were detectable by the hemograms: nonregenerative anemia, neutrophilic leukocytosis and lymphopenia are well-recognized hematologic findings in fip (jain, 1986 (jain, , 1993 pedersen, 1995a) . in other immune-mediated diseases such as leishmaniasis (carvalho et al., 1992; bourdoiseau et al., 1997) , canine blastomycosis (legendre and becker, 1982) and feline immunodeficiency virus (lin et al., 1990) or feline leukemia virus infections (hoover et al., 1987) , lymphopenia has been interpreted as a sign of reduced cell-mediated immunity. this conclusion was based upon a reduced lymphocyte blastogenesis (legendre and becker, 1982; lin et al., 1990; bishop et al., 1992; carvalho et al., 1992) or a low cd4/cd8 ratio (bishop et al., 1992; lawrence et al., 1995; hoffmann-fezer et al., 1996) . this study gives only a quantitative evaluation of lymphocytes, and no information about blood lymphocyte subpopulations or functions in fip are available in literature. thus, although an impairment of lymphocyte functions can not be excluded, the neutrophilia and eosinopenia only in the cats with the acute effusive form suggest a stress leukogram (jain, 1986 (jain, , 1993 . hyperproteinemia has been reported in fip (sparkes et al., 1991 (sparkes et al., , 1994 pedersen, 1995a) , but was not observed in spite of the increase of total globulins, because of the decrease in serum albumin levels. hypoalbuminemia was more evident in the effusive form perhaps due to the loss of albumin in the effusions. in fact no differences were found between albumin concentration in serum and in effusions, with high effusion/serum ratios. the electrophoretic pattern of fip cats was consistent with those reported in literature (sparkes et al., 1991 (sparkes et al., , 1994 pedersen, 1995a) . the changes in -and -globulins are well documented in experimental infections by stoddart et al. (1988) and characterized by a biphasic acute phase plasma protein response with increases of 1 -orosomucoid, 2haptoglobin and 2 -transferrin levels. the same author stated that g-globulins increases as the antibody titers begin to mount. in the spontaneous disease the early phases of antibody production are not detectable: however the occurrence of animals with high antibody titers without symptoms and of symptomatic cats with low antibody titers have been reported (pedersen, 1976 (pedersen, , 1977 (pedersen, , 1995a reynolds et al., 1977; vanden bossche, 1990; sparkes et al., 1992a, b) . based upon the pattern of antibody and g-globulin levels, four groups of cats were identified (fig. 4) : 1. cats with low antibody titers and low g-globulin concentrations: these cats were mainly those of the control group; 2. cats with high antibody titers and low g-globulin concentrations: these were cats with effusive fip or control cats with fcov antibodies: the latter occurs in fecv-endemic environments (pedersen, 1995b) ; 3. cats with low antibody titers and high g-globulin concentrations: all of these cats were fip affected, with either the effusive or noneffusive forms. on these animals, the low antibody titers result from the formation of immune complexes (pedersen, 1987) ; 4. cats with high antibody titers and high g-globulin concentrations: they were symptomatic cats with effusive or noneffusive fip. furthermore, cats with the same antibody titer but with different -, and g-globulin levels were detected (fig. 5) , suggesting that some of the detected proteins are not immunoglobulins. many of complement fractions or immune complexes have similar motility. the c1q subunit, involved in immune complex disease, migrates with theglobulins (yamada and hirayama, 1983) . a c3 activating factor (c3af) (arroyave et al., 1976) and the c1r subunit (ziccardi and cooper, 1976 ) have a g-globulin motility (roberts et al., 1981) . the changes in the level of these proteins support the previous studies on the fluctuation of complement fractions (jacobse-geels et al., 1982) and further confirm the role of type iii hypersensitivity in fip. these changes were present in fig. 4 . percentage of animals with different antibody titers and g-globulin concentrations (alow titer, low gglobulins; blow titer, high g-globulins; chigh titer, low g-globulins; dhigh titer, high g-globulins) in the controls and in cats with effusive and noneffusive fip fig. 5 . serum protein electrophoresis in cats with the same antibody titers: dashed linecontrol cat, titer 1:25, gglobulins 12.7 g l; solid linefip cat, titer 1:25, g-globulins 75.9 g l. both the effusive and noneffusive forms, suggesting a similar involvement of the immune system. the observed protein concentrations and the electrophoretic pattern agree with those reported in literature (jain, 1986; shelly et al., 1988; sparkes et al., 1991 sparkes et al., , 1994 . in the effusive form, -and g-globulin concentrations in the effusions were often higher than those of the corresponding serum. in immune complex disease, low antibody titers are expected. in this study, the effusions often had antibody titers higher than the corresponding serum (jacobse-geels et al., 1982) . despite the variability in total and fractioned protein concentrations and antibody titers, no differences were found between the forms in the distribution of the lesions. fibrinous perivisceral lesions were not seen in the noneffusive cases. the histologic pattern of the foci was similar in effusive and in noneffusive forms: three different patterns of the lesions were identified: (a) cellular infiltrates without necrosis; (b) cellular infiltrates with scattered necrotic intercellular debris; (c) large foci with a central necrosis. the distribution of the virus and of the immune cells were not influenced by the antibody titers or by the total and fractioned protein concentration. the distribution of antigen and of immune cells varied depending upon the type of lesion identified: in the first many fipv cells were distributed across the lesions, with many mac387 macrophages, lymphocytes cd3 , many of which cd4 and rare cd8 , igg and igm lymphocytes were detectable. these foci were considered as acute lesions. cd3 and cd4 lymphocytes were often detectable near vessels. the mac387 macrophages have recently been identified as blood-origin macrophages (poston and hussain, 1993) typical of perivascular acute inflammations (bhardwaj et al., 1992) . expression of the mrp14 protein, the target antigen of this antibody (goebeler et al., 1994) , is largely absent in activated tissue macrophages (lagasse and weissman, 1992) . in the foci with scattered necrosis, viral and myeloid antigen was present in the necrotic debris, most likely due to the lysis of virus-infected macrophages. many of the mac387 cells in these lesions were granulocytes. a moderate number of cd3 and cd4 lymphocytes, were diffusely distributed across the lesions, while the distribution and the number of cd8 , igg and igm lymphocytes were similar to those of the cellular foci. when a central necrosis was present, necrotic debris was diffusely and weakly positive for myeloid antigen. viral antigen was detectable only in the cells and in the intercellular necrotic debris around the necrotic center. in these lesions mac387 cells were mainly granulocytes, and mac387 à activated macrophages (bhardwaj et al., 1992; lagasse and weissman, 1992) . the latter are likely involved in immune-cell down-regulation, as demonstrated in other granulomas, such as those of human tuberculosis and sarcoidosis, or in hypersensitivity reactions, such as contact hypersensitivity (roitt et al., 1996) . cd3 /cd4 , igg and igm lymphocytes were scattered over the cells around the necrosis while the number of peripherally distributed cd8 lymphocytes appeared to be greater than those observed in the foci without necrosis. these results agree with those reported in previous studies on effusive fip lesions (paltrinieri et al., 1998) and with those on other immunological granulomas, in which tlymphocytes (mainly cd4 ) and macrophages, with few igm-exposing b-lymphocytes are present in each stage of the lesion and fewer numbers of cd8 cells was detectable at the periphery of the larger lesions (modlin et al., 1984; momotani et al., 1989; scanziani et al., 1990) . the results of this study strongly support the role of type iii hypersensitivity (hayashi et al., 1977 (hayashi et al., , 1978 pedersen and boyle, 1980; weiss et al., 1980; weiss and scott, 1981; jacobse-geels et al., 1980; fenner, 1987; pastoret and bourtonboy, 1991; pedersen, 1995a) . in particular, the differences between globulin fractions and antibody titers suggest either the presence of immune complexes or the presence of complement fractions in fip affected cats. these changes occur in both the effusive and in noneffusive forms. in contrast, the results regarding the distribution of the virus and of the immune cells in lesions support the role of type iv hypersensitivity in their pathogenesis as suggested by others (pedersen, 1987 (pedersen, , 1995a paltrinieri et al., 1998) . in particular advanced lesions, with central necrosis, activated macrophages, few and scattered igg and igm positive blymphocytes and peripherally distributed cd8 t-lymphocytes, were very similar to those observed in other granulomatous diseases (scanziani et al., 1990) . the concurrent detection of humoral and cellular findings consistent with both the pathogenetic mechanisms indicates a complex involvement of the immune system. a better understanding of the pathogenesis of this disease may derive from the study of others and more sensitive indicators of the immune status such as the cytokines. the risk of typical and antibody enhanced feline infectious peritonitis among cats from feline coronavirus endemic households serum factors activating the alternative complement pathway in autoimmune 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title: oral mutian®x stopped faecal feline coronavirus shedding by naturally infected cats date: 2020-06-30 journal: research in veterinary science doi: 10.1016/j.rvsc.2020.02.012 sha: doc_id: 266155 cord_uid: hf3retap abstract feline coronavirus (fcov) is common among cats living indoors in groups. in about 10% of infected cats, a potentially lethal disease, feline infectious peritonitis (fip) occurs. virus transmission is faecal-oral. mutian® xraphconn (mutian x) is a product marketed to treat cats with fip but is also being used to stop virus shedding, although no clear guidelines exist for its use for this purpose. the aim of this study was to establish the minimum dose and treatment duration required to ensure viral clearance from the faeces of asymptomatic virus-shedding cats. in five multicat households, 29 cats naturally infected with fcov and actively shedding virus in the faeces were given mutian x pills. virus shedding was monitored using reverse-transcription quantitative polymerase chain reaction (rt-qpcr) controlled for faecal inhibitors to ensure sensitivity. mutian x given orally cleared the virus in 29 cats; although four cats required a repeated course to finally stop virus shedding. a dose of 4 mg/kg q24 h for four days was found to be the optimal treatment protocol: 2 mg/kg cleared only 80% of cats. post-treatment using a sensitive rt-qpcr test was essential to ensure that virus clearance had been achieved, since failure to clear even one cat can result in re-infection of the others. records of virus shedding by cats before treatment provided a retrospective control: significantly more cats stopped shedding virus after mutian x than recovered from infection during the control period (p < .00001). this is the first report of the successful elimination of faecal fcov shedding in chronically infected cats. feline coronavirus (fcov) is a positive strand rna virus ubiquitous in multi-cat environments such as breeding catteries, rescue shelters, cat sanctuaries and large cats in zoos, but rare in stray and feral cats. (addie et al., 2012; cave et al., 2004) . two fcov genotypes are currently known: fcov type i (fcov-i), which is predominant in the field, (addie et al., 2003; hohdatsu et al., 1992; li et al., 2019) and fcov type ii (fcov-ii), which derives from recombination between fcov-i and canine coronavirus (decaro and buonavoglia, 2008; herrewegh et al., 1998; terada et al., 2014) . fcov is primarily a pathogen of the gastrointestinal tract of domestic cats, replicating in the intestines and is spread by faecal-oral transmission from cats that are either persistently or transiently several products, some specifically described as gs-441524, and others with proprietary names, are being sold through the internet to cat guardians who treat their own cats suffering from fip. fcov persists in multicat households by infection and re-infection of healthy cats by the same, or another, strain of virus and by the development of a persistently infected carrier status in 13% of fcov-infected cats which act as reservoirs of infection in a feline population without developing fip (addie et al., 1995; addie and jarrett, 2001; addie et al., 2003; herrewegh et al., 1997) . there is an urgent need of methods to prevent cats in high density environments such as breeding and rescue catteries becoming infected with fcov. an fip vaccine is licensed in some countries, but the first dose is administered at 16 weeks (gerber et al., 1990) which is too late to protect kittens, that are usually infected when maternally derived antibody wanes at between five and seven weeks of age (addie and jarrett, 1992; pedersen et al., 1981) . at present, therefore, the major way to prevent fip is to prevent exposure to the virus by quarantine and rigorous hygiene. fcov transmission is faecal-oral; sharing litter trays with a fcov shedder and fomite transmission are the major risk factors for uninfected cats. in dry indoor environments, fcov can survive up to seven weeks in fomites (scott, 1988) . another method of control would be to stop the excretion of virus in the faeces of infected cats by antiviral therapy. some cat guardians have considered whether the drugs shown to cure fip might also stop asymptomatic fcov-infected cats shedding virus, thereby preventing them acting as a source of infection and providing a means to establish households of cats that are free of the virus. mutian® x (nantong mutian biotechnology co. ltd. china) is one of those products, it is formulated in capsules containing nicotinamide mononucleotide, crocin i, s-adenosylmethionine, silymarin and mutian x, which is a novel synthetic adenosine analogue (patent pending in china), exhibiting broad-spectrum activity against rna viruses (tony xue, ceo of nantong, personal communication). nucleoside analogues function by replacing adenosine, thus terminating replication of the rna virus genome. mutian x is efficacious by the oral route unlike previously described anti-coronavirus drugs, which must be given by painful injection, often causing severe inflammatory responses (pedersen et al., 2019) , which is of concern for the development of feline injection site sarcoma. although recombinant feline interferon omega (feifnω: virbagen omega, virbac, france) was previously shown to reduce fcov shedding, this is the first report to document an anti-viral that stopped the excretion of fcov in the faeces of naturally infected cats. fcov testing was implemented because the households had experienced fip among their cats. a summary of the cats and the reasons for screening for fcov shedding is shown in table 1 . thirty-four of 50 cats in five multicat households (a-e) were naturally infected with fcov and were treated with mutian x. results from five cats from household e could not be included because the intervals between faecal tests left the possibility that the cats might have spontaneously stopped shedding virus, rather than mutian x having stopped virus shedding: thus they were excluded from both treatment and control groups (table 1) . therefore, there remained data of 29 cats treated with mutian x. household c was a ragdoll breeding cattery with a history of endemic fcov for at least four years despite testing for fcov antibodies and shedding, and rigorous hygiene. in 2015 this cattery experienced eight fip deaths. keeping cats in small groups or singly to attempt to prevent fcov transmission resulted in clearance of virus in eight of 24 cats. prior to the observational study we report here, the cattery owner (sc) discovered she could reduce coronavirus shedding in some cats using mutian x tablets: we worked with her to optimise dose and duration of treatment for stopping virus shedding. faecal samples were mailed to the veterinary diagnostic laboratory (vdl) at the university of glasgow, uk. the samples were mailed using royal mail or fedex: they were not on ice, nor in rna-preserving buffer. cat guardians were advised to try to submit faecal samples with no cat litter attached, since cat litter can inhibit the pcr reaction. fcov rna was detected by semi-quantitative reverse-transcriptase polymerase chain reaction (rt-qpcr) as previously described (dunbar et al., 2019) , except that to control for false negative results gapdh was replaced by a control for pcr inhibitors in faecal samples, the exact nature of this control cannot be revealed because the laboratory deems it a commercial secret. testing was performed in duplicate. those performing the fcov rt-qpcr tests were blinded to which treatments the cats were receiving. threshold cycle (c t ) number was used as the measure of viral load. the lower the c t , the more virus present in the sample. samples with no signal at c t 40 were considered negative. cat guardians purchased mutian x online. mutian x was available in either tablet format, containing 4 mg of the anti-viral mutian x ingredient, or capsules (mutian x 200) containing 10 mg. the exact nature of mutian x is a commercial secret, prior to chinese patent being granted: the manufacturer describes it as an adenosine nucleoside analogue (tony xue, personal communication). cat guardians were using 4 mg/kg q24 hours per os for seven days as per advice from social media groups for stopping fcov shedding. the owner of household c (sc) was experimenting with different doses and protocols with the aim of minimising dose and duration of treatment to stop faecal virus shedding: we report the outcome of using 2 mg/kg and 4 mg/kg doses. the owners of household e (fb and bc) set up a camera and alarm system which enabled the recording of the exact number of hours post onset of treatment that a faecal sample was passed, and to ensure the samples were not contaminated or degraded. prior to the use of mutian x, cat guardians were attempting to prevent fcov transmission within their households by quarantining fip cats (households a and d), keeping cats in as small groups as possible given their facilities (households c and e), and using dr. elsey cat attract cat litter or other bentonite-based cat litter (households a and e respectively). some households also tried to reduce fcov shedding using probiotics, recombinant feifnω, and a 21 day course of itraconazole (households a and d) which has been shown to have in vitro activity against type i fcov (takano et al., 2019) . these various treatments were grouped into a single control period for each household (figs. 1-3), since numbers of cats for each of these parameters were too small to be statistically significant, and also because combinations of prevention strategies were used. this was an observational study so that there was no opportunity to conduct a placebo-controlled trial. however, fcov shedding records prior to treatment with mutian x, where available, were used as a retrospective control period. cats acted as their own controls, which meant that variables such as housing, breed, and age, which affect fcov shedding, were constant. fisher's exact test (two-tailed) and correlation coefficient were carried out using the statistics package in excel (microsoft office 2007). details of the five households of cats which were cleared of feline coronavirus using mutian x. (herrewegh et al., 1995; longstaff et al., 2017; lorusso et al., 2019) . cat a1 ceased virus shedding following treatment with feifnω, itraconazole, and gc376 protease inhibitor injections (pedersen et al., 2018) . cat a1 was kept isolated from the other three cats and they were tested for fcov shedding prior to re-uniting them, in order not to re-infect cat a1. cat a2 stopped shedding virus subsequent to seven injections of gc376 given because fip was suspected, but his condition was a urinary tract infection which resolved with antibiotics. thus mutian x was only required for cats a3 and a4. dr. elsey cat attract cat litter was used and cat a2 remained uninfected despite continued sharing of litter trays with cats a3 and a4. all four cats tested fcov negative five months after their first negative faecal tests. b1 -b2 turkish van, indoor pet cat 1 1 cat b1 was in contact with cat b2 who was treated for fip using mutian ii injections and mutian x tablets prior to this study. cat b1 was shedding fcov. ragdoll breeding cattery 17 8 cats were routinely tested because this was a breeding cattery wishing to eliminate fcov, and therefore fip. eight fip deaths had occurred prior to testing beginning. at the start of the mutian x treatment, 14 of 24 cats were shedding fcov; one fcov shedding cat (c10) was introduced during the study period (the breeder having confidence in her ability to stop cat c10 shedding virus using mutian x). cats were kept in small groups of up to three cats. sawdust based cat litter was used. two of ten negative cats became infected during the study period, bringing the total treated to 17. siberian pet cats 1 1 cat d2 had effusive fip diagnosed by a positive fcov rt-qpcr test on ascites but was not shedding virus in the faeces. d1 was the housemate cat found to be shedding fcov and was treated to avoid re-infecting cat d2. a 21 day itraconazole course had failed to stop cat d1 shedding virus. domestic shorthair rescue cats 13 4 one cat died of histopathologically suspected fip prior to this study (immunohistochemistry was not performed to confirm diagnosis). fip was suspected in a second cat (e14) and gs-441524 treatment was begun; but he had multicentric lymphoma, so the anti-viral drug was stopped. cats were divided into three groups of up to eight cats. ten cats became infected during the study period despite clumping bentonite-based cat litter being used. results of five infected cats (e6, e8, e10, e11 and e13), could not be counted as having stopped shedding due to mutian x because of the absence of a faecal sample immediately prior to mutian x treatment: it is therefore possible that they had ceased shedding virus prior to onset of treatment. however, it is also possible that they had stopped shedding virus within 24 hours of treatment. the results are summarised in table 2 . prior to this study, the owner of household c had attempted various mutian x treatment regimes in her cats. a single dose treatment was attempted on two cats (c10 and c12) and while virus load reduced, it failed to clear infection and virus load increased again in cat c10. cat c1 was given a single injection of mutian three days prior to oral treatment which had reduced her virus shedding from c t 20 to 32. thus a single dose was ineffective at clearing infection and may have risked the emergence of resistant viruses. ten cats received a full course at 2 mg/kg and two other cats began at 2 mg/kg then were increased to 4 mg/kg. eight of ten cats stopped shedding fcov using mutian x tablets at a dose of 2 mg/kg sid ( fig. 2a) for seven or four days. two probable fcov carrier cats (c1 and c2) that had shed virus at levels of c t 18-20 (c1) and c t 35 (c2) for over a year prior to treatment were treated for seven days at a dose of 2 mg/kg after which their faeces were negative, c1 and c2 remained negative when tested five and seven days later. the remaining eight cats were treated for four days as shown in fig. 2a and table 2 . daily samples were available for five cats; two (c3 and c5) cleared the virus within 24 hours and three under 72 h (c4, c6 and c7). the other cats were not tested daily, so an accurate time of cessation of virus shedding could not be determined. cats c8 and c10 failed to totally clear the virus using the 2 mg/kg dose for four days; therefore they were treated again at 4 mg/kg, after which they became negative, as shown in fig. 2b and table 2 . cat c6 was in the same group as c8 and c10, and began shedding virus again within nine days, requiring a second course of treatment (figs. 2a and b) . two more cats (e1 and e2) started treatment at 2 mg/kg sid but were changed to the higher dose within 48 hours due to the results from household c (fig. 3) : these cats were therefore included in the 4 mg/kg rather than the 2 mg/kg category. in summary, a mutian x dose of 2 mg/kg was considered inadequate: it had cleared the virus in eight cats and reduced, but not abrogated, virus shedding in two cats ( fig. 2a) . thereafter, the dose was increased to 4 mg/kg. a dose of 4 mg/kg reliably cleared virus within seven days in 21 of 22 (95%) cats (two cats from household a, one from household b, ten of eleven from household c and eight from household e) (figs.2b and 3). cat c18 was the only cat who failed to clear the virus after a six day course of 4 mg/kg dose, but she vomited some of the capsules and she required a repeat course for three more days after which she was negative. ten cats received a full course of mutian x at 2 mg/kg: eight cats (80%) cleared infection, but cats c8 and c10 did not, and cat c6 who was housed with them, began shedding virus again. therefore at the end of that part of the study seven cats were clear of virus, and three required to be re-treated at 4 mg/kg. in this graph, the records of faecal fcov rna detection-rt-qpcr c t -is shown on the y axis and the time in days on the x axis for three cats a3, a4 and b1. each time point indicates a faecal test. this graph is normalised to time zero for test period for mutian x start. mutian x was given for seven days from days 0 to cats a3, a4, and b1 after which their faecal samples remained negative for 155, 157 and 51 days (x axis only shows up to 60 days). the graph shows that no intermittent virus shedding or re-infection occurred. faecal fcov shedding before and after treatment with mutian x at a dose of 2 mg/kg and 4 mg/kg q24 h in households c and d. faecal fcov rna detection-rt-qpcr c t -is shown on the y axis and the time in days on the x axis for ten cats treated with mutian x at an oral dose of 2 mg/ kg sid ( fig. 2a) , and for eleven cats at a dose of 4 mg/kg sid (fig. 2b) . each time point indicates a faecal test. these graphs are normalised to time zero for the start of the mutian x treatment and cut off at day 60 and 400 prior to testing: the exact extent of the no treatment control period is indicated in table 2 . two cats (c8 and c10) failed to clear the infection at 2 mg/kg dose ( fig. 2a) and were treated again at 4 mg/kg (fig. 2b) . cat c6 was kept in a group with cats c8 and c10, became re-infected, and required re-treatment, which is why this cat also features in both figures. twenty-two cats received a course of mutian x at a dose of 4 mg/kg and 21 cats (95%) became negative. cat c18, who had vomited some of the capsules, required re-treatment, after which she too, became negative. thus all 29 cats were cleared of infection, but four cats had required a second course of treatment. to determine whether cats that were shedding a higher amount of virus required more days of anti-viral treatment than cats with a lower virus quantity, starting fcov rna c t was plotted against days to become negative. serial accurately timed daily faecal samples were available for 14 cats (fig. 4) and all 14 cleared virus within 75 h of starting treatment. no correlation between starting fcov rna c t and time to become negative was found (r = 0.0048. pre-treatment virus shedding records were used as a retrospective control in this observational study: fcov virus shedding results prior to mutian x administration were available for 25 treated cats (shown in table 2 ). two additional cats in household c spontaneously stopped shedding virus prior to the start of treatment period (data not shown): one cat was kept in isolation and one was allowed outdoors. therefore two of 27 (7.4%) cats stopped shedding virus during the control period: the difference between the number of cats stopping shedding virus during the control period and subsequent to mutian x at 4 mg/kg was statistically significant (p < .00001). repeat negative samples were available from six cats dosed at 2 mg/ kg and 12 cats dosed at 4 mg/kg (table 2) . cats dosed at 2 mg/kg and 4 mg/kg remained negative for at least three to 18 days and one to 157 days respectively from their first negative result, showing that the cats remained negative post-treatment and did not start spontaneously shedding virus again. fcov antibody titres were available for cats a3 and a4 five months post-treatment: they had reduced from 1280 to 80 and from over 1280 to 320 respectively. mutian x pills stopped faecal fcov shedding in 29 naturally infected cats; however, four of the 29 cats required a second course of treatment before virus was eliminated. since successful treatment of fip using anti-viral drugs was first reported (pedersen et al., 2018; pedersen et al., 2019) cat guardians have been able to source various anti-fcov drugs, including mutian x, via social media. our view is that it is preferable that if they propose to use such medications, even though the products are unlicensed, they should do so under proper veterinary guidance, rather than following advice from social media group moderators, however knowledgeable. professor pedersen has provided a lucid view of the current situation in relation to the treatment of cats with fip with 'black market' drugs, and the subsequent dilemma for veterinary surgeons. (https://ccah.vetmed. ucdavis.edu/sites/g/files/dgvnsk4586/files/inline-files/black %20market%20production%20and%20sale%20of%20gs_0.pdf). to paraphrase professor niels pedersen, we would prefer that these drugs be approved, licensed, and made available in the normal manner, rather than being sold on the black market, but we are willing to advise veterinary surgeons and their clients. advice from those with fip experience is likely to reduce unnecessary use of anti-coronavirus drugs: in two of the five households reported here fip therapy levels of antiviral treatment was begun in two cats which did not have fip, and ceased when a proper diagnosis was obtained: anti-viral treatment has been stopped in nine other mis-diagnosed cats (da personal observation). we present an observational study of cat guardians who were using mutian x to stop their cats from shedding virus, and have defined a protocol which optimises and minimises its use. furthermore, we hope that by stopping cats shedding fcov, especially in purebred cats, the prevalence of fip will reduce, which will further reduce the use of unlicensed drugs. a four day course of mutian x at a dose of 2 mg/kg cleared coronavirus in eight cats, but failed to clear infection in cats c8 and c10. some opinion leaders have expressed concern that use of mutian x to stop virus shedding in cats without fip will lead to drug resistance and failure of the drug to treat fip, although the ability of mutian x to treat fip has not actually yet been documented, so far as we are aware. we ask those holding such views to consider the following: first that this drug is already being used for the purpose of virus elimination by cat guardians and surely it is better that a proper protocol has been developed, rather than they are left to do trial and error themselves. second, we have found that an intensive four day course stopped virus shedding while cats being treated for fip have higher virus loads (kipar et al., 2006) and require a longer course of treatment, up to 12 weeks (pedersen et al., 2019) , which surely is more likely to allow resistant virus strains to develop? indeed, virus resistance to a nucleoside analogue was documented in a cat being treated for fip (pedersen et al., 2019) . anti-virals are used prophylactically in other viral infections: there is precedent for this approach. we found no evidence for the development of drug resistance in asymptomatic cats at a dose of 4 mg/kg. in our view treating breeding queens for four days to stop fcov shedding is less likely to induce drug resistance than treating at least one in ten of their kittens for fip, requiring 12 weeks of medication. however, treatment of virus shedding cats must be done properly: one concern is that cat guardians may be tempted to reduce cost and use an insufficient dose or duration of treatment (sc and dda, personal observations), which might result in selection of viruses which are resistant to mutian x. another concern is that a blunderbuss approach may be taken: treating all the cats in a household without first confirming that they are virus shedders. no correlation was found between fcov rna c t prior to treatment and time to become negative, therefore cats shedding more virus did not require a longer treatment course than cats shedding a lower amount of virus and a four day course is likely to suffice, rather than seven days. fig. 3 . faecal fcov shedding before and after treatment with mutian x in household e. faecal fcov rna detection-rt-qpcr c t -is shown on the y axis and the time in days on the x axis for eight cats in household e treated with mutian x at an oral dose of 4 mg/kg sid for four days (cats e1 and e2 were started on a dose of 2 mg/kg, but increased to 4 mg/kg). each time point indicates a faecal test. cat results are normalised to time zero for the start of the mutian x treatment. since the study was observational rather than prospective, there was no formal placebo group. the lack of a placebo group in this study was problematic: it could be argued that the cats spontaneously stopped shedding fcov, co-incidental with the onset of mutian x therapy. cats do spontaneously stop shedding fcov some months post-infection: fcov-i is shed in the faeces for months to years (addie et al., 1995; addie and jarrett, 2001; addie et al., 2003) . little is known about fcov-ii shedding, in one experimental infection of laboratory cats virus was shed for up to 15 days (stoddart et al., 1988) ; and no persistently fcov-ii infected cat has been reported (addie and jarrett, 2001; addie et al., 2003) . that said, it is unlikely that 29 cats would all cease virus shedding co-incidentally within days of beginning mutian x treatment, we did retrospectively compare the results prior to mutian x, when virus eradication was attempted by quarantine and hygiene, and/or itraconazole, and feifnω with and without probiotics. another argument might be that the cats stopped shedding virus as a delayed result of the treatments given to some cats prior to mutian x: this argument could not apply to households b, c, and e, where the cats did not receive any other treatments. however, in household a, virus load did decrease in two cats on probiotics (fortiflora, purina pro plan, usa) and feifnω, but remained at a constant level in the third cat. therefore, a combination of interferon and probiotics may have reduced fcov shedding. feifnω has previously been shown to reduce, but not stop, fcov shedding (gil et al., 2013) . probiotics are routinely used to treat children with viral diarrhoea (szajewska et al., 2019) , but the interaction of the gut microbiome and fcov has not been extensively explored (meazzi et al., 2019) : further investigation into the possibility that probiotics might reduce coronavirus load is warranted. cats in households a and d also received itraconazole which has been shown to have in-vitro activity against fcov-i (takano et al., 2019) . however, a 21 day course of treatment failed to clear these three cats of fcov, although viral rna quantity did decrease in cat a3 while she was on itraconazole, but bounced back as soon as it was stopped. another possibility was that the mutian x could be excreted in the faeces and be inhibiting the fcov rt-qpcr assay, but this was unlikely because, as shown in figs. 2 and 3, fcov rna was being detected in cats during mutian x treatment, and also because faecal samples remained negative after the cats had ceased mutian x. while a four-day course of mutian x tablets at 4 mg/kg appeared to be effective in stopping fcov shedding, daily monitoring of faecal fcov a cat c12 was treated for one day, missed a day, one day of 2 mg/kg then 2 days of 4 mg/kg -low positive at c t 38. then 3 days of 4 mg/kg then negative. b cat c18 faecal sample was c t 38 at day 6 and so the cat was re-treated for 3 days and subsequently became negative. this cat had vomited some of the capsules. shedding using a laboratory which rapidly reports virus quantity is necessary to tailor treatment duration according to the circumstances of each individual cat. the virus is highly contagious, and if cats are sharing litter trays, they will be rapidly re-infected, as was the experience in households c and e, with large numbers of cats, even though they were kept in small groups. in household c, re-infection of cat c6 occurred in less than nine days: she was housed with one cat who had failed to clear the virus on a 2 mg/kg dose of mutian x; the cat litter used was sawdust based. it could be argued that cat c6 was shedding virus intermittently, but c6 was housed in a group who had failed to clear virus at the 2 mg/kg dose, thus was far more likely to have been re-infected than latently infected. intermittent virus shedding was not seen in any cat in this trial, nor in previous studies of sequential tests on hundreds of cats using the modern, sensitive fcov rt-qpcr technology (addie, unpublished observation) , and controlling for false negative results due to faecal pcr inhibitors (dye et al., 2008) . in our experience, cats either shed virus continuously or they stop shedding virus: the appearance of intermittent virus shedding could always be traced back to exposure to the faeces of an infected cat; or where virus quantity was so low that it was on the borderline of the ability of the assay to detect it, or by the presence of excessive cat litter on the sample: sodium bentonite based cat litters bind organic material. no side effects of mutian x were observed by us other than one cat having initially vomited some capsules, which might have been an adverse reaction, although that cat didn't vomit subsequent capsules. however, that does not mean that mutian x is without side effects: elevation of liver enzymes has been seen in cats with fip that are being treated for many weeks (addie, unpublished observation) . use of a laboratory that has an adequately sensitive fcov rt-qpcr test is absolutely essential for the detection and eradication of fcov from a household. false negative results will doom the effort to failure. six of eight samples sent to one highly reputable university veterinary laboratory were falsely reported as negative (data not shown). a major problem identified during our study was that the delay in reporting fcov rt-qpcr results-usually of at least one week from the date of sample submission-delayed separation of negative and positive cats, and allowed re-infection of cats that had cleared infection. this study showed that the oral administration of mutian x tablets, at a dose of 4 mg/kg for four days, stopped shedding of fcov from naturally infected cats. this treatment would be a useful adjunct for establishing fcov-free households of cats, in addition to current measures of hygiene, housing cats in small groups and the use of virusinhibiting cat litter . further observations of cats which stopped shedding virus should determine whether mutian x can prevent the development of fip. also, if the serum anti-fcov titres of treated cats decline over the coming year, it will indicate that the virus has been cleared systemically, as well as from the gastrointestinal tract. cats' guardians funded the treatment and blood testing of their pets. we are indebted to www.catvirus.com angelica trust donors who funded much of the fcov rt-qpcr testing. dda is hugely grateful to catvirus.com subscribers for supporting her through the writing of this paper. there is no conflict of interest of any authors in relation to the submission. fig. 4 . lower fcov rna c t did not correlate with requirement for a longer duration of treatment. the aim of this scatter plot was to determine whether cats that were shedding a higher amount of virus required more days of anti-viral treatment than cats with a lower virus quantity. daily monitoring of faeces was available for 14 cats (there are 4 results at c t 23, two are overlaid which gives an appearance of 13 samples). although there was a slight incline in the trendline, there was no correlation between starting fcov rna c t and days to a negative result (r = 0.0048). a study of naturally occurring feline coronavirus infection in kittens use of a reverse-transcriptase polymerase chain reaction for monitoring feline coronavirus shedding by healthy cats the risk of feline infectious peritonitis in cats naturally infected with feline coronavirus persistence and transmission of natural type i feline coronavirus infection quarantine protects falkland islands (malvinas) cats from feline coronavirus infection effect of cat litters on feline coronavirus infection of cell culture and cats risk factors for feline coronavirus seropositivity in cats relinquished to a uk rescue charity an update on canine coronaviruses: viral evolution and pathobiology diagnosis of non-effusive feline infectious peritonitis by reverse transcriptase quantitative polymerase chain reaction from mesenteric lymph node fine needle 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infection in cats natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats circulation and genetic diversity of feline coronavirus type i and ii from clinically healthy and fip-suspected cats in china feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis feline gut microbiota composition in association with feline coronavirus infection: a pilot study infection studies in kittens, using feline infectious peritonitis virus propagated in cell culture efficacy of a 3c-like protease inhibitor in treating various forms of acquired feline infectious peritonitis efficacy and safety of the nucleoside analog gs-441524 for treatment of cats with naturally occurring feline infectious peritonitis update on fip virus shedding and immune responses in cats inoculated with cell culture-adapted feline infectious peritonitis virus systematic review with meta-analysis: lactobacillus rhamnosus gg for treating acute gastroenteritis in children -a 2019 update antiviral activity of itraconazole against type i feline coronavirus infection emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses we sincerely thank the guardians of the cats for allowing their data to be used to help other cats, with special mention to eric and carrie, for their commitment to saving their cats.we are immensely grateful to os jarrett for editorial help and scientific advice in the writing of this paper.sc thanks bethany victoria smith for helpful discussions.we are grateful to the office, technical and veterinary staff of glasgow university veterinary diagnostic laboratories for their support.we thank dr. tony xue and niki ng of the nantong mutian biotechnology for the donation of pills for the treatment of cats in households c and e. key: cord-313439-cadyykks authors: felten, sandra; hartmann, katrin title: diagnosis of feline infectious peritonitis: a review of the current literature date: 2019-11-15 journal: viruses doi: 10.3390/v11111068 sha: doc_id: 313439 cord_uid: cadyykks feline infectious peritonitis (fip) is a fatal disease that poses several challenges for veterinarians: clinical signs and laboratory changes are non-specific, and there are two pathotypes of the etiologic agent feline coronavirus (fcov), sometimes referred to as feline enteric coronavirus (fecv) and feline infectious peritonitis virus (fipv) that vary fundamentally in their virulence, but are indistinguishable by a number of diagnostic methods. this review focuses on all important steps every veterinary practitioner has to deal with and new diagnostic tests that can be considered when encountering a cat with suspected fip with the aim to establish a definitive diagnosis. it gives an overview on all available direct and indirect diagnostic tests and their sensitivity and specificity reported in the literature in different sample material. by providing summarized data for sensitivity and specificity of each diagnostic test and each sample material, which can easily be accessed in tables, this review can help to facilitate the interpretation of different diagnostic tests and raise awareness of their advantages and limitations. additionally, diagnostic trees depict recommended diagnostic steps that should be performed in cats suspected of having fip based on their clinical signs or clinicopathologic abnormalities. these steps can easily be followed in clinical practice. feline infectious peritonitis (fip) is a fatal disease that occurs in domestic and wild felids worldwide. the etiologic agent, the feline coronavirus (fcov), occurs in two distinct pathotypes that can be distinguished by their biological behavior, but not by their morphology. some authors use different names for these two pathotypes, although they both belong to the same virus species. the feline enteric coronavirus (fecv) is highly prevalent in multi-cat environments and highly contagious-nearly 100% of cats that get in contact with fecv from feces of shedding cats become infected. however, infection is mostly asymptomatic or only causes mild and transient diarrhea [1] [2] [3] . the feline infectious peritonitis virus (fipv), in contrast, is not infectious via the fecal-oral route, but arises by mutation from the avirulent fecv within a small percentage of infected cats and then causes the fatal disease feline infectious peritonitis (fip) [4] [5] [6] . it is still unknown which exact genes harbor the mutation(s) leading to fipv development. since promising results using new drugs for treating cats with fip have been published recently, definitive ante mortem diagnosis is crucial in order to correctly identify the population of cats which could benefit from such antiviral treatment. at the same time, definitive diagnosis is challenging, since most existing diagnostic tests cannot differentiate between fecv and fipv, and especially in cats without body cavity effusions, it is often difficult to reach a definitive diagnosis ante mortem. in contrast to what was believed earlier, it is now well-accepted that antibody tests cannot differentiate between antibodies against fecv and fipv, and therefore, even high antibody titers in blood are not a specific indicator for fip [6, 68] . additionally, there is evidence of cross-reactivity between fcov and other coronaviruses, such as tgev and canine coronavirus (ccv) [68, 69] . a large proportion of the cat population (up to 80% and more, especially in multi-cat households) has serum antibodies against fcov, but most of these cats never develop fip [54, 60, [70] [71] [72] [73] [74] . moreover, antibodies can be detected in the serum of cats that were vaccinated against fcov [75] [76] [77] . the significance of the presence of antibodies for diagnosing fip in an individual cat therefore is very limited [11, 16, 19] . opinions on the value of antibody measurement for the diagnosis of fip vary, but based on the results of numerous studies, which are shown in the following paragraphs, it is the authors' opinion that antibody measurement is of no use in a cat suspected of having fip. if antibodies are measured in a cat suspected of having fip, then a titer should be determined in any case. especially low and medium titers are of zero diagnostic value for the diagnosis of fip [16, 78] . although a rising titer can sometimes be detected during progression of the disease [55, 60] , this can also be seen in conjunction with fecv reinfection, and thus is not an indicator for fip [70] . high and rising titers can also be found in healthy fecv-infected cats [70, 73] and should never be used to confirm a suspicion of fip. likewise, a negative antibody test result cannot exclude fip [79, 80] . in end-stage fip, especially with fulminant effusions, declining antibody titers are possible and sometimes antibody concentrations can even drop below the limit of detection [60, 68] . approximately 10% of cats with fip do not have serum antibodies [16] . most likely, the immune-mediated vasculitis in these cats leads to extravasation of blood components including antibodies into the effusion, and as a consequence, they can no longer be detected in the blood by antibody assays. additionally, a large number of antibodies is most likely bound by the high virus load in end-stage fip [81] [82] [83] , and thus, cats with fip without effusions can have negative serum anti-fcov antibody titers [84] . moreover, it has been demonstrated in longitudinal studies that in healthy cats originating from environments endemic for fip, antibody titers can vary greatly over time [85, 86] . therefore, measurement of a single antibody titer at a single random time point does not provide any diagnostic information. the results of studies evaluating the performance of antibody tests for diagnosing fip are summarized in table 1 . sensitivity and specificity partially vary depending on the antibody cut-off titer used. most studies reported the diagnostic value of antibody measurement compared to histopathology [16, 19, 78] . one study included cats with fip in which the diagnosis was established upon a combination of tests performed ante mortem and histopathology [79] . another study included cats in which fip was suspected clinically, but the diagnosis was not confirmed [87] . the studies included control groups consisting of either healthy cats [79] and/or cats with diseases other than fip [19, 79] ; in most studies, fip was an important differential diagnosis for all control cats [16, 19, 78] . table 1 . studies evaluating sensitivity and specificity of the detection of serum antibodies in comparison to either histopathology, a combination of diagnostic tests or clinical suspicion of feline infectious peritonitis (fip). control groups either consisted of healthy cats or cats with diseases other than fip (with or without clinical signs consistent with fip). as stated before, the detection of antibodies in plasma or serum (no matter which titer) is not proof for the presence of fip. in an attempt to increase specificity, a few years ago, an antibody assay was developed that only detected antibodies against the fcov 7b protein. this test was designed based on the erroneous assumption that fipv, but not fecv, contains an intact 7b gene. however, the test did not result in a better diagnostic performance than other antibody assays, as both cats with fip and fecv-infected cats (healthy or suffering from other diseases) had anti-7b antibodies [79] . nevertheless, measurement of serum antibodies is useful in guiding preventative measures and can be used for fcov control in multi-cat households. as such, antibody detection can be performed to screen cats that are about to be newly integrated into a group, it can help to confirm successful elimination of fcov in groups of cats and can guide separation of infected and non-infected cats [61] . cats without detectable antibodies most likely do not shed fcov with their feces [62, 70, 85] . however, fecal shedding has been observed in experimentally infected cats despite the absence of serum antibodies [54] . the diagnostic utility of anti-fcov antibody detection in effusion has been examined in a few studies as well [16, 87] . in cats with fip confirmed by histopathology, antibody detection in effusion had a sensitivity of 86% and a specificity of 85% [16] . although these numbers sound more promising than what is reported for the antibody detection in serum, it has to be emphasized that the diagnostic value of antibody measurement in both serum and effusion is similarly low and bears the same limitations. additionally, it has to be mentioned that many studies on antibody detection in effusion suffer from some limitations, such as inclusion of cats without a definitive diagnosis of fip. for example, a comparison of antibody detection in serum and effusion in cats with a clinical suspicion of fip revealed good concordance of test results in both body fluids; the antibody titer measured in effusion was lower than that determined in serum in 23% of cats. however, fip was not definitively confirmed in any of the cats included [87] . the same was true for a study in which fip was suspected in 61 cats based on different effusion features (cases had to fulfill all or most of the criteria for fip diagnosis given by the european advisory board of cat diseases recommendations of 2009 [88] ), but the diagnosis was not definitively confirmed in any of the cats. detection of anti-fcov antibodies had a sensitivity of 84%. the fact that 18 cats without detectable antibodies were positive for fcov rna in their effusion demonstrated that anti-fcov antibody detection alone has only limited diagnostic value [89] . one study even found an inverse correlation between fcov antibody titers and fcov viral load; therefore, false negative antibody results can occur in cats with high viral rna loads [81] . in conclusion, antibody detection in effusion is not more useful for the diagnosis of fip than antibody detection in serum (table 2) . while the detection of anti-fcov antibodies in the csf of cats with neurologic fip was first regarded as useful for the diagnosis [45] , subsequent investigations including larger and more appropriate control groups showed that the detection of antibodies in the csf, as in serum and effusion, is not suitable for a definitive ante mortem diagnosis of fip [46] . sensitivity and specificity were calculated with histopathology as reference standard; the control groups either consisted of only cats with any kind of neurologic disease [45] or of a combination of cats with neurologic and non-neurologic disorders [46] (table 3) . a recent study included cats with neurological signs suggested to be caused by fip, although the diagnosis was not confirmed by histopathology. in this study, csf from a large number of cats was analyzed and revealed a low sensitivity of the detection of fcov antibodies for the diagnosis of fip. however, the authors hypothesized that the detection of fcov rna in csf would confirm fip and, as they found fcov rna in all cats with a csf antibody titer of 1:640 or higher, based on these results concluded that a csf antibody titer of 1:640 or higher might be confirmatory for fip. however, the results are of limited value, as control cats were not included in the study and the diagnosis of fip was not confirmed [90] . due to the correlation of serum and csf antibodies and the fact that antibody titers measured in serum were always higher than those measured in csf, extravasation of antibodies from serum into the csf across an impaired blood-brain barrier is discussed as the most likely explanation for the detection of anti-fcov antibodies in the csf of cats without fip. another possibility seems to be local antibody production by migrating b lymphocytes in cats with fecv infection [46] , whereas there is no evidence for specific antibody production by intrathecal b lymphocytes [44] . the production of non-protective antibodies and formation of immune complexes are discussed as key features for the development of typical immune-mediated lesions in cats with fip [56, [91] [92] [93] [94] [95] [96] [97] , and circulating immune complexes could be detected in serum and effusion of fcov-infected cats [53, 54, 95, [98] [99] [100] . compared to histopathology as reference standard, the detection of immune complexes in serum via competitive enzyme-linked immunosorbent assay (elisa) had a sensitivity of 48% and a specificity of 91% for diagnosing fip (table 4 ). control cats in that study were suspected of having fip, but another disease was diagnosed [16] . other investigations confirmed the presence of fcov-specific immune complexes in healthy cats without fip [53, 54] . it has been suggested that also in fecv-infected cats, immune complexes seem to circulate, although for a limited period of time only [99] . in conclusion, detection of immune complexes is not useful for the diagnosis of fip [16] . monocytes/macrophages are the target cells for fipv replication [91, [101] [102] [103] [104] [105] . both fecv and fipv can replicate within monocytes/macrophages and lead to monocyte-associated viremia [54, [106] [107] [108] [109] . for a long time, it was believed that only fipv can replicate in a sufficient way that allows intracellular antigen detection via immunologic methods [88] , in which antibodies bind to fcov antigen within its target cells and enzymatic reactions or fluorescent conjugates result in coloring of the antigen. however, this assumption has been questioned recently, as a number of studies, which will be shown in the following paragraphs, have detected positive immunostaining also in cats not suffering from fip. the nature of these false positive results remains unclear; it is possible that not only fipv, but also fecv can be detected intracellularly in macrophages via immunologic staining methods. immunohistochemical staining of fcov antigen within characteristic histopathological tissue lesions is still considered gold standard for the diagnosis of fip [11] [12] [13] 102, 110] . when first reported in 1989, a study described the detection of fcov antigen in epithelial cells of a cat's nictitating membrane by immunofluorescence staining of impression smears [111] . a different study used an avidin-biotin-complex (abc) method to detect fcov antigen in tissue samples of various organs by enzymatic color change [112] . since then, successful immunostaining of antigen has been demonstrated several times in different organs from cats with fip-either by indirect immunofluorescence [113, 114] or by immunohistochemistry (ihc) within tissue macrophages in histopathologically evident tissue lesions caused by fip [102, 115, 116] . ihc was shown to have excellent sensitivity of 97-100% in cats with histopathologically confirmed fip [115, 116] and also specificity of up to 100% to exclude fip in control cats with other diseases diagnosed by histopathology [115] . an overview of studies reporting the detection of fcov antigen in tissue samples is shown in table 5 . in many cases, ihc can only be performed post mortem, because ante mortem tissue sample collection via laparotomy or laparoscopy is an invasive procedure that bears several risks for the sick cat. many different organs have to be sampled due to the fact that fcov antigen is variably distributed within the fip-induced tissue lesions [102] . unfortunately, the use of liver, kidney or lymph node samples obtained by minimally invasive ultrasound-guided fine-needle aspirates (fna) or tru-cut biopsies (tcb) for immunocytochemical or immunohistochemical staining has been shown to result in low sensitivities [117, 118] . a possible explanation for this could be inadequate material obtained for analysis due to cellular damage [119] . additionally, a false positive immunocytochemical result was found in a mesenteric lymph node sample, resulting in a specificity of only 91% [118] . therefore, immunocytochemistry (icc) or ihc on samples obtained by fna or tcb cannot reliably confirm or exclude fip as a sole diagnostic test [117, 118] . besides its diagnostic use, ihc has also been applied to detect fcov antigen in macrophages in unusual tissues or non-domestic felids. as such, fcov antigen was detected in the skin of two cats with atypical skin lesions caused by fip [120, 121] , in the penile tissue of a male cat [122] , in different tissues obtained from a mountain lion [123] and in ocular tissues from a lion [124] . most often, immunofluorescence staining has been performed to detect fcov antigen in effusions. for this, fluorescein-conjugated antibodies are used to detect fcov antigen within the cytoplasm of macrophages [125] . in earlier studies, this test was reported to have an excellent specificity of 100%. therefore, it was believed that a positive result would in all cases allow a definitive diagnosis of fip [16, 78, 125, 126] . however, more recent investigations also showed positive results of immunofluorescence or icc in cats with effusions caused by other diseases than fip. possible explanations for false positive results include binding of the antibody to cellular structures within macrophages (non-specific staining) or systemic spread and subsequent detection of fecv in cats without fip [127, 128] . sensitivity of immunostaining in effusion is not absolute; thus, a negative test result cannot exclude fip [16, 78, [125] [126] [127] . low numbers of macrophages in effusion samples or competitive binding of fcov antigen by circulating antibodies in the effusion are possible reasons for false negative results. one study demonstrated that all cats with false negative test results had anti-fcov antibodies in serum or effusion [78] . only one study reported a sensitivity of 100% for the detection of fcov antigen in effusion by immunofluorescence staining [128] . in contrast to other studies, which examined effusion samples obtained both ante and post mortem [125] [126] [127] , this study only included samples collected ante mortem [128] . additionally, a monoclonal biotinylated antibody was used, whereas previous studies applied a polyclonal fluorescein-conjugated antiserum in their staining protocols [16, 125, 126] . finally, samples were analyzed within 24 hours after collection [128] and longer intervals between sampling and analysis could result in decreasing sensitivities. a decreasing sensitivity could be observed with increasing time interval between sample collection and analysis, although fcov antigen could still be detected for at least two days in samples stored at 4 â�¢ c or room temperature [128] . table 6 shows all studies investigating the detection of fcov antigen in effusion. reference standard was either histopathology [16, 78, 125, 128] and/or ihc [126, 127] . control groups consisted of cats with clinical signs consistent with fip but a definitive diagnosis of another disease [16, 78, [125] [126] [127] [128] . it is also possible to detect fcov antigen in the csf of cats with fip (with and without neurological signs) [129, 130] . the only prospective study to date reported a sensitivity of 85% and a specificity of 83% for the immunocytochemical demonstration of fcov antigen in macrophages in the csf of cats with and without neurological signs (table 7) . in this study, ihc was the reference standard for the diagnosis of fip; control cats had clinical signs typical for fip, but other diseases were diagnosed histopathologically and ihc was negative in all cats. if only cats with neurological signs were evaluated, sensitivity surprisingly decreased to 78%, whereas specificity slightly increased to 88%. if only cats without neurological signs were evaluated, sensitivity was 91% and specificity only 50% [130] . table 7 . sensitivity and specificity from a study evaluating immunostaining for the detection of feline coronavirus (fcov) antigen in cerebrospinal fluid (csf) samples. feline infectious peritonitis (fip) was confirmed by histopathology and immunohistochemistry (ihc) of tissue samples. the control group consisted of cats with diseases other than fip with clinical signs (neurological or non-neurological) consistent with fip. in cats without fip that do not present with effusion, uveitis is common and ocular signs (with or without involvement of the central nervous system) can be noticed in 60% of the affected cats [12, 131] . secondary to the inflammation of ocular structures and breakdown of the blood-ocular barrier, fcov-bearing macrophages are present in the eye and fcov antigen can be detected immunocytochemically within macrophages in the aqueous humor [47, 132] . the results of the only prospective study that evaluated icc in aqueous humor in cats with immunohistochemically confirmed fip and control cats with similar clinical signs but histopathologically diagnosed other diseases are depicted in table 8 [132] . the technique only has a moderate sensitivity and specificity for the diagnosis of fip, and therefore, it cannot be used as a single confirming diagnostic test. since its first application for the detection of fcov [133] , rt-pcr is frequently used to amplify fcov rna in different materials and to diagnose fip. according to the long-existing hypothesis that only fipv but not fecv is able to spread systemically, it was initially believed that detection of fcov rna via rt-pcr in blood, effusion or any other body fluid or tissue except for the gastrointestinal tract indicates presence of the virulent pathotype fipv [6, 101, 104, 134] . this hypothesis has, however, been refuted by several studies (tables 5-9 ) and it is important to keep in mind that fcov rna can also be amplified outside of the gastrointestinal tract in cats without fip. nevertheless, it is well known that cats with fip exhibit much higher viral loads than healthy fecv-infected cats [54, [135] [136] [137] . therefore, an ideal rt-pcr assay should be able to quantify the viral load in order to facilitate diagnosis. not only are tissue samples from cats with fip more likely to be fcov-positive, but also viral loads are significantly higher in cats with fip compared to healthy fecv-infected cats [135, 137] . as an example, in a study analyzing a large number of tissue and fluid samples, 90% of the tissue samples and 78% of the fluid samples from cats with fip were positive, whereas only 8% of the tissue samples and 2% of the fluid samples from control cats were positive for fcov rna. additionally, relative fcov copy number (a ct value of 40 in rt-pcr was assigned to a relative copy number of 1, and a relative copy number was then calculated for each rt-pcr-positive sample using the following equation: 1.96 ) was significantly higher in tissue samples from cats with fip than from fcov-infected cats with other diseases or healthy fcov-infected cats (median 8.3 ã� 10 3 vs. median 25) [135] . thus, a positive rt-pcr result with a high viral load is at least very suggestive for fip [138] . in cats with fip, large quantities of fcov rna can only be found in tissues with inflammatory changes. tissues which are not involved in the disease process contain only small amounts or no viral rna at all. among the organs with highest viral loads are the omentum, mesenteric lymph nodes and spleen, so these tissues are most useful for analysis by rt-pcr. the kidneys, liver, lung, myocardium and popliteal lymph nodes, in contrast, contain little or no viral rna [57] . studies using rt-pcr to target fcov rna in tissues are summarized in table 9 . however, in veterinary practice, rt-pcr is rarely applied to tissue samples to diagnose fip, as this would usually require invasive tissue sample collection via laparotomy or laparoscopy or has to be done post mortem. therefore, the studies cited here were mainly performed for research purposes and did not have the aim of evaluating rt-pcr in tissue samples as a diagnostic test for fip. when an early study applied rt-pcr to tissue samples (liver, kidney and/or spleen), fcov rna was identified in 88% cats with clinically suspected fip; 100% of experimentally infected cats tested positive for fcov by rt-pcr. however, fcov rna was also detected in the tissues from 61% of cats without fip [133] , indicating that the detection of fcov rna in tissue is not specific for fip. especially in hemolymphatic tissues (spleen, mesenteric lymph nodes, bone marrow), fcov rna could be detected in all cats with fip but also in 85% of clinically healthy fecv-infected cats. in cats with fip, real-time rt-pcr was positive in the spleen in 60% of cases, in mesenteric lymph nodes in 87% and in the bone marrow in 67% of cases. in healthy fecv-infected cats, fcov rna was found in the spleen in 38%, in mesenteric lymph nodes in 33% and in the bone marrow in 46% of cases [107] . as stated before, however, cats with fip usually exhibit higher viral copy numbers in tissues than healthy fecv-infected cats [107, 135, 137] . in order to develop a rt-pcr assay which is more specific for fip, one study evaluated a real-time rt-pcr detecting messenger rna (mrna) (and thus, replicating virus). it was hypothesized that this rt-pcr would only detect fipv from cats with fip but not fecv from healthy fcov-infected cats. indeed, in this study, tissues from healthy fecv-infected cats tested negative for fcov mrna in all cases [139] , whereas fcov mrna was found in the tissues of 71% of cats with immunohistochemically confirmed fip. organs with the highest frequency of harboring fcov mrna in these cats were the mesenteric lymph nodes (100%), spleen (88%), lung (86%), liver (75%), bronchial lymph nodes (67%), kidneys (63%) and intestine (60%). recent studies evaluated the use of a quantitative rt-pcr (rt-qpcr) to detect fcov rna in fna samples of the mesenteric lymph nodes and other abnormal tissues of clinical cases [119, 140] and hypothesized that this technique would be a useful tool to diagnose fip for veterinary practitioners, especially in cats without effusion. the first study included control cats that were either fcov antibody-negative or antibody-positive but without signs indicative of fip or with a confirmation of another cause of illness/death. additionally, cats with non-effusive fip, that was confirmed either by histopathology or a diagnostic algorithm, were included. in all of the cats, the mesenteric lymph nodes were sampled by minimally invasive ultrasound-guided fna and examined by rt-qpcr. it was found that the detection of fcov rna in mesenteric lymph node fna can be a sensitive and specific method to diagnose fip (even when analyzed after shipping without any preservatives), although fcov rna was also detected in the mesenteric lymph nodes of a small proportion of cats without fip, and therefore, was not completely specific for fip [119] . the second study retrospectively included eleven cats with fip without effusions and successfully amplified fcov rna from fna samples of various organs in all of the cats. since no control group was included in that study, specificity could not be evaluated [140] . the same finding was made in a study in which different samples (fna or incisional biopsies (ib)) were obtained from different tissues (mesenteric and popliteal lymph nodes, liver, kidney, spleen, omentum) of 20 cats with immunohistochemically confirmed fip in order to determine the prevalence of fcov rna in different tissues and to potentially evaluate the diagnostic performance of rt-pcr on those tissue samples for diagnosing fip. sensitivity was high in most tissues except for the popliteal lymph nodes and sensitivities did not greatly differ whether fna or ib were used. since no control group was included, specificity could not be determined [141] . based on the erroneous assumption that only fipv but not fecv is able to infect monocytes/macrophages and disseminate systemically, it was thought that the detection of fcov rna in blood was proof of an infection with fipv, and thus, fip. therefore, rt-pcr was initially developed using primers for the highly conserved 3'-untranslated region (3'-utr) of the fcov genome in order to detect all known fcov isolates. as a result, the rt-pcr could neither differentiate between fecv and fipv, nor could it distinguish fcov from ccv or tgev. nevertheless, as stated before, it was assumed that only fipv would be detectable in blood. however, over the years, it has been shown that fcov rna could also be detected in the blood of asymptomatic cats and cats with diseases other than fip [143, 144] . several studies evaluating the use of rt-pcr in serum, plasma or whole blood confirmed this finding of possible fcov viremia in cats without fip [16, 80, 106, [145] [146] [147] . additionally, a recent study demonstrated that fcov viremia does not even predict the development of fip, as none of the healthy cats that tested positive for genomic or replicating fcov developed fip or clinical illness within six months of testing [147] . all of the studies using rt-pcr on blood samples are summarized in table 10 . if different blood fractions were examined, sensitivity and specificity are presented as a range. most studies used histopathology as reference standard [16, 80, 106, 143, 145, 148, 149] ; one study also used the detection of fcov antigen in macrophages in effusion [149] . some studies had the limitation of including cats in which fip was suspected clinically, but the diagnosis was not confirmed [148, 150, 151] . the control groups either consisted of healthy cats [106, 143, 145, 147, 148, 150, 151] and/or of cats with other diseases than fip, sometimes showing clinical signs consistent with fip [16, 80, 143, 145, 148, 149] . one additional study worth mentioning is not shown in table 10 . this study included 63 serum samples from 62 cats with abdominal clinical signs. since no definitive diagnosis was established in these cats, sensitivity and specificity could not be determined, and therefore, the study was not mentioned in table 10 . nevertheless, it is worth noting that in this study, 18 cats had a positive result in a nested rt-pcr (rt-npcr) assay. one of these cats survived for almost three years after examination; four additional cats lived for at least 70 months. since median survival time for cats with fip is very short (unless new antiviral drugs are applied) [152] , it is likely that these cats did not have fip and that the rt-npcr was false positive [146] . since fcov viremia can be detected by rt-pcr in up to 80-90% of fecv-infected cats without fip [106] , detection of fcov rna in blood is not specific for fip and rt-pcr in blood samples should not be used to confirm a diagnosis of fip. additionally, fcov viral load is low in any blood fraction in experimental fip [57] and this is most likely also the case in natural infection, leading to a rather low sensitivity of rt-pcr in blood. rt-pcr had better sensitivity when peripheral blood mononuclear cells (pbmc) were used compared to serum [149] ; however, other studies came to differing results regarding sensitivity in the different blood fractions and most studies are not well comparable [80, 106, 141, 143] . sensitivity was poor in most of the studies; therefore, due to a rather low virus load and resulting low sensitivities, blood samples are not recommended for analysis by rt-pcr. although fecv can infect monocytes and macrophages, it is not capable of efficiently replicating within them [101, 147, 153, 154] . in an attempt to increase specificity of rna detection in blood, a rt-pcr specifically amplifying fcov mrna has been developed in order to detect only actively replicating virus in blood. in an initial study, this rt-pcr assay really did not detect fcov mrna in any of the control cats with clinical signs indicative of fip but other confirmed diseases, whereas fcov mrna was found in 93% of cats with histopathologically confirmed fip [148] . nevertheless, subsequent studies also identified fcov mrna, and thus, replicating fcov in 0.5-52% blood samples from healthy cats [147, 148, 150, 151] and the detection of replicating virus did not predispose to the development of fip [147] . therefore, this special rt-pcr assay also is not adequately specific for fip. in general, however, cats with fip exhibit higher viral loads and copy numbers than healthy fecv-infected cats [107, 137] and higher viral copy numbers are found in the feces of healthy fecv-infected cats when compared to their blood [147] . therefore, an rt-pcr that allows simultaneous amplification and quantification of viral mrna could potentially distinguish fecv (with low viral copy numbers) from fipv (with high viral copy numbers). a real-time rt-pcr based on primer-probe energy transfer detecting and quantifying subgenomic fcov mrna showed a very good sensitivity and specificity in one study, but was so far only applied on very small numbers of samples and not used in the field [139] . loop-mediated isothermal amplification (lamp) is a new molecular method with the advantage of delivering rapid in-house test results. reverse transcriptase lamp (rt-lamp) has been used to amplify fcov rna in blood, effusion, lymph nodes and feces of cats with suspected fip in a recent study, in which rt-npcr was used as reference standard. specificity of rt-lamp was 100% in all materials; sensitivity, however, was only low to moderate [155] . in cats with fip that have effusion, virus load is much higher in the effusion than in blood [57] . sensitivities and specificities of rt-pcr assays for the diagnosis of fip in effusion are listed in table 11 . as shown, sample numbers are relatively low in some and especially in the older studies. most studies used histopathology as reference standard [16, 22, 149, 158, 159] ; in some studies, the detection of fcov antigen in effusion [149, 159] or tissue [159, 160] or laboratory fluid analysis [158] was the reference standard. one study included 27 cats with a suspicion of fip, of which only 13 had histopathologic confirmation of the disease and 8/13 cats had effusion [80] . another study examined 61 effusion samples from cats with suspected fip based on effusion feature criteria established by the european advisory board on cat diseases in 2009 [88] . the diagnosis was, however, not confirmed by reference standard methods in any of the cats [89] . the control groups consisted of cats with a clinical suspicion of fip but different diagnoses [16, 149, [158] [159] [160] . one study examined a large number of effusion samples from 854 cats with clinically suspected fip; the disease was, however, not confirmed. it could also not be determined whether cats with negative rt-pcr results had diseases other than fip. therefore, sensitivity and specificity could not be calculated, but fcov rna was detected in the effusion of 377/854 cats (44%) [161] . in another study, plasma and/or ascites from 42 cats with histopathologically confirmed fip and 141 controls (healthy or with fip-typical clinical signs but other diseases) were examined by rt-npcr. fcov rna was detected in the plasma of 70% of cats with fip. five additional cats that tested negative in plasma had a positive rt-npcr result in effusion. however, it was not stated how many effusion samples were analyzed in total [145] ; thus, the study was not included in table 11 . although specificity of rt-pcr in effusion was high in many studies and a positive rt-pcr result can add to a suspicion of fip, it has to be kept in mind that rt-pcr already detects small amounts of viral rna, which can potentially leak from blood into effusion in the face of inflammation (even without fip) in cats with circulating fecv [110] . especially in recent studies, fcov rna could also be detected in the effusion of cats without fip [15, 135, 157, 159] . all studies evaluating rt-pcr for the detection of fcov rna in csf have reported specificities of 100%, meaning that fcov rna could only be detected in the csf of cats with fip (with and without neurological signs) but not control cats (table 12) . reference standards used in these studies was either histopathology [45, 162] and/or the detection of fcov antigen in effusion [162] or tissue [135] . the control groups consisted of cats with neurologic diseases only [45] or also included cats with non-neurologic diseases but clinical signs consistent with fip [135, 162] . although the blood-brain barrier has not specifically been examined in cats with fip yet, it can be assumed that it is impaired secondary to the inflammation caused by fip [163] . in general, various infectious and inflammatory diseases of the central nervous system lead to the release of cytokines, adhesion molecules, metalloproteases and other mediators, which can damage tight junctions, basal membranes and cerebrovascular endothelium. this enables the leakage of cells and pathogens into the csf across the blood-brain barrier [163] . therefore, although false positive rt-pcr results have not been reported in csf thus far, it seems theoretically possible that fecv could pass the blood-brain barrier in cats with neurologic diseases other than fip, as it is also suspected for anti-fcov antibodies [46] . one study detected a correlation between high anti-fcov antibodies and positive rt-pcr in the csf. in that study, fcov rna was found in only 15% of samples from cats with low, but in 100% of samples from cats with high csf antibody titers. in contrast, fcov rna was not amplified in any of the antibody-negative cats. however, fip was not confirmed in the cats in this study [90] . another type of sample material that can be examined for fcov rna by rt-pcr is the aqueous humor. to date, there have been very few studies that evaluated this sample material for analysis by rt-pcr in a small number of cats with fip (table 13) . additionally, a case report demonstrated positive rt-pcr in the aqueous humor of 3/10 cats with uveitis; however, there were no clear-defined fip and control groups [131] . fcov replication, like replication of all rna viruses, is prone to error [165] . multiple individual mutations occur during each cycle of viral replication [12, 58, 166, 167] . it is hypothesized that specific mutations or a combination of mutations then lead to the development of the virulent pathotype fipv and trigger the tropism switch from enterocytes to macrophages as a key event in fip pathogenesis [4, 153, 168] . several different fcov genes have been analyzed and suggested to harbor the mutation responsible for the fcov pathotype switch. open reading frame (orf) 3abc, which is coding for the accessory proteins 3a, 3b and 3c, has been sequenced and compared in fcov from cats with fip and healthy cats and differences were detected. for instance, it was found that fipv from tissue or effusion of cats with fip contained a truncated protein 3c, whereas fecv from feces of healthy cats most often contained an intact protein 3c [108, 167, [169] [170] [171] [172] [173] . the 3a and 3b genes could play a role in fip pathogenesis as well [174] [175] [176] . orf 7ab, coding for accessory proteins 7a and 7b, has also been subject of studies looking into fip pathogenesis, but somewhat contradictory results were found [77, 153, 169, 170, [176] [177] [178] [179] [180] [181] . the fcov spike (s) protein is responsible for viral cell entry. it includes a subunit for receptor binding (s1) and a subunit mediating membrane fusion (s2) [182] [183] [184] . therefore, mutations in the fcov s gene, more specific mutations in the s2 region and corresponding amino acid substitutions in the s protein, have been suggested to be responsible for the change of viral cell tropism [153, 170, [185] [186] [187] . as a result, only fipv but not fecv are capable of efficient and sustained replication in macrophages, producing large amounts of viral particles and spreading the infection to adjacent cells [153, 154] . therefore, the s gene is of particular interest with regard to determining which mutations are responsible for the transition from fecv to fipv. recently, commercial diagnostic testing was introduced for fcov s gene mutations. when the s genes of a large number of fcov derived from the feces of healthy cats and from tissues or ascites of cats with fip were sequenced, two single nucleotide polymorphisms (snp) were found that only were present in the fcov from cats with fip but not in the fcov from healthy cats. these snp were found at nucleotide position 23531 and 23537, which lie in close proximity within the s gene and the detection of one of the snp allowed differentiation between fcov from cats with fip and healthy cats in 96% of the examined fcov. in all of the sequenced fecal fcov from healthy cats, adenine was found at nucleotide position 23531, whereas thymine or cytosine was detected in 92% of the fcov from ascites or tissues of cats with fip. both snp led to the substitution of methionine by leucine at amino acid position 1058 within the putative fusion peptide of the s protein (m1058l). in all of the sequenced fecal fcov from healthy cats, thymine was detected at the second nucleotide position 23537, whereas guanine was found in 4% of the fcov from ascites or tissues of cats with fip. this snp led to the substitution of serine to alanine at position 1060 of the s protein (s1060a) [185] . after that first study provided evidence of an association of the two s gene mutations with fip [185] , a number of studies subsequently investigated the prevalence of the mutations in tissue samples from cats with and without fip (table 14) . table 14 . sensitivity and specificity from different studies evaluating the detection of feline coronavirus (fcov) spike (s) gene mutations in tissue samples. feline infectious peritonitis (fip) was confirmed by histopathology alone or in combination with immunohistochemistry (ihc), control cats were either healthy cats or cats with diseases other than fip (with or without clinical signs consistent with fip). opinions vary substantially among researchers, especially on the specificity of s gene mutations for the fip phenotype. for example, one recent study corroborated the initial findings by detecting m1058l in three tissue samples (mesentery, colonic lymph node and omentum) from cats with fip; moreover, the mutation fully discriminated the fcov found in these tissue samples from fcov found in fecal samples from healthy cats [187] . in contrast, fcov with substitution m1058l was not specific for the fip phenotype in a different study, which also found the mutation in tissue samples from cats without fip. not only the majority of fcov from tissue samples from cats with fip (39/43; 91%), but also the majority of those from tissue samples from control cats without fip (8/9; 89%) had a leucine (and thus, a mutated sequence) at position 1058 in that study. on the other hand, the majority of fcov from fecal samples from cats with (10/13; 77%) and without fip (6/6; 100%) had a methionine (and thus, a non-mutated sequence) at this position. additionally, a number of tissue samples from cats with fip (4/43; 9%) also exhibited a methionine at position 1058, which means that they did not contain a mutation despite being from cats with fip. therefore, the authors concluded that substitution m1058l was indicative of systemic spread of fcov rather than a marker for fip [137] . in this study, five tissue samples that did not contain the substitution m1058l were analyzed for substitution s1060a (the less common s gene mutation) by sanger sequencing, and s1060a was then detected in 1/4 samples from cats with fip but not in the one remaining sample from a control cat [137] . since the first description, pcr assays detecting mutations m1058l and s1060a frequently have been used for diagnostic purposes and a number of studies have evaluated sensitivity and specificity or diagnostic accuracy of the detection of the mutations in the diagnosis of fip (table 14) . however, largely contrasting results were also found in these studies, especially in terms of specificity, possibly as a result of different sequencing assays that were used. one study, for example, examined a large number of tissue, fluid and fecal samples from cats with immunohistochemically confirmed fip and controls in which fip was excluded and performed rt-qpcr followed by pyrosequencing of an s gene amplicon in the rt-qpcr-positive samples. in total, 260 tissue samples from cats with fip were analyzed. however, since histopathological data was only available for 225 of the tissue samples, only these numbers were included in the calculation of sensitivity and specificity (table 14) . of these 225 samples, 202 were rt-qpcr-positive and s gene mutations were detected in 182/202 samples (m1058l being much more common than s1060a; mixed mutated and non-mutated fcov or fcov with both m1058l and s1060a also present). of the 258 tissue samples from control cats, 19 were rt-qpcr-positive and 14/19 were positive for mutation m1058l. when comparing sensitivity and specificity of rt-qpcr alone with that of rt-qpcr plus s gene sequencing, specificity slightly increased from 93% to 95%, whereas sensitivity decreased from 90% to 81%. therefore, it was concluded that the detection of fcov s gene mutations did not improve the ability to diagnose fip. mutations were also found in cats without fip, which supported the hypothesis of the mutations being a marker for systemic spread of fcov and not for fip [135] . a somewhat similar result was obtained a year later when a study compared different laboratory tests in tissue samples (mesenteric lymph nodes, spleen, small intestine, lung) from cats with fip (positive ihc in at least one tissue) and control cats (histopathological diagnosis of another disease plus negative ihc). all samples were examined by rt-npcr targeting the highly conserved 3'-utr and sequencing was performed to detect s gene mutations. again, the detection of fcov s gene mutations improved specificity of the rt-npcr from 50% to 88%, but sensitivity decreased from 91% to 70%, and an s gene mutation (type of mutation not shown) was also detected in a cat without fip [15] . however, contrasting results were obtained when a real-time rt-pcr was developed and evaluated subsequently, which uses specific fluorescent hydrolysis probes to detect either one of the two snp or the wildtype sequence. in that study, none of the 30 cats without fip (with histopathological diagnosis plus negative ihc) tested positive for one of the s gene mutations in their tissues. of the 34 cats with immunohistochemically confirmed fip, 24 tested positive for one of the s gene mutations in at least one of their tissues (23/24 m1058l, one mixed genotype). in this study, sensitivity was 71% and as such comparable to the previous studies; however, specificity was excellent (100%) [142] . finally, a recent study evaluated the same real-time rt-pcr detecting s gene mutations in 20 cats with immunohistochemically confirmed fip. fna and ib samples were obtained from the mesenteric and popliteal lymph nodes, spleen, liver, kidney and omentum and additionally from different body fluids. samples were first examined by rt-pcr detecting the fcov 7b gene (detecting all fcov) and rt-pcr-positive samples were subsequently analyzed by real-time rt-pcr detecting s gene mutations. interestingly, fcov with s gene mutations was present in every cat, but the location of mutated fcov varied from cat to cat. sensitivity of the rt-pcr detecting all fcov was good for all tissues except for the popliteal lymph nodes, but sensitivity decreased when s gene mutations were evaluated. since sensitivity of fna and ib did not significantly differ, sampling via minimally invasive fna seems possible in order to obtain adequate samples for rt-pcr testing. specificity was not determined in this study, since a control group was not included in the study protocol. diagnostic accuracy of the detection of fcov s gene mutations has also been evaluated in blood samples (table 15 ). rt-npcr and subsequent s gene sequencing of serum and plasma samples from cats with fip (diagnosed by histopathology â± ihc or by positive immunofluorescence in effusion) and control cats (diagnosed with another disease either ante or post mortem) revealed a sensitivity of only 7%, which confirms the very low virus load in blood. rt-npcr was negative in all of the blood samples from cats without fip, and thus, specificity of the sequencing step could not be determined [156] . subsequent studies evaluated the aforementioned real-time rt-pcr detecting s gene mutations by specific fluorescent hydrolysis probes in blood samples (plasma, serum, buffy coat, whole blood) from cats with fip [141] and/or controls [157] (reference standard histopathology and ihc) and either did not detect mutated fcov in any of the blood samples or only found very low sensitivity. in some cats with fip, fcov rna was detected, but viral concentrations were too low to allow pathotyping [157] . this implicates that blood cannot be recommended as sample material. nevertheless, a study comparing different pcr tests including 3'-utr rt-npcr and a combined approach with rt-pcr followed by s gene sequencing reported a sensitivity of 75% for the rt-npcr and 43% for combined rt-pcr and sequencing when whole blood was used. potentially, whole blood can give better results than plasma or serum. although the authors state that specificity of both rt-npcr and combined approach were 100%, specificity of the sequencing step should not be calculated from that study, since none of the blood samples from cats without fip tested positive for fcov rna at all [15] . an association of substitutions m1058l and s1060a with fip was shown by a study which detected amino acid substitution m1058l in 83% fcov extracted from ascites of cats with fip (reference standard for the diagnosis not reported) and a non-mutated sequence (a methionine codon) in 90% fcov extracted from feces of healthy cats and 57% fcov extracted from feces of cats with fip [170] . however, contrasting results of pcr assays detecting fcov s gene mutations were again, as in tissue and blood, found subsequently when analyzing effusion and other fluid samples for diagnostic purposes (table 16) . one study including cats with fip confirmed by ihc and control cats classified as non-fip by confirmation of other diseases based on either histopathology and/or the definitive diagnosis of another disease ante mortem showed that rt-qpcr alone had a sensitivity of 85% and a specificity of 100%. of the 17 rt-qpcr-positive cats, 11 had a mutated fcov with substitution m1058l in their effusion detected by sequencing. one cat exhibited mutation s1060a. this resulted in a sensitivity of 60% for the detection of s gene mutations. since none of the control cats were rt-qpcr positive, specificity of the sequencing step could not be determined. thus, the majority of effusion samples that generated sequence data contained a mutated fcov, although in that study population, detection of s gene mutations did not improve specificity [160] . when an rt-npcr and subsequent s gene sequencing was used on effusion samples from cats with fip (diagnosed by histopathology â± ihc or by positive immunofluorescence in effusion) and control cats (diagnosed with another disease either ante or post mortem), a similarly moderate sensitivity of 65% was detected. of the 50 samples from cats with fip, 36 were rt-npcr-positive, and the majority of those (32/36) contained s gene mutations (m1058l n = 30, s1060a n = 2). rt-npcr was negative in all of effusion samples from cats without fip, and thus, specificity of the sequencing step again could not be determined [156] . evaluations of a real-time rt-pcr detecting the s gene mutations with specific fluorescent hydrolysis probes in effusion from cats with fip and controls (reference standard histopathology and ihc) detected similar sensitivities of 64-69% for the detection of s gene mutations [141, 157] . of the 35 samples from cats with fip in one study, 34 were rt-pcr-positive. mutated fcov containing m1058l was detected in the majority of samples (22/34) . additionally, mixed fcov (with and without s gene mutations) were detected in two cats. in 10 cats, rt-pcr was positive, but rt-pcr detecting s gene mutations was not successful, either because of a low virus load (n = 7) or despite a high virus load (n = 3). the reason for the latter could be the presence of unknown sequence variations or serotype ii fcov. rt-pcr was also positive in 3/24 effusion samples from cats without fip and m1058l was detected in one ascites sample from a cat with chronic kidney disease [157] . finally, one recent study collected different body fluids (csf, aqueous humor, ascites, pleural or pericardial effusion) from cats with fip (confirmed by ihc) and cats in which histopathological signs of fip were absent. of the 51 samples from cats with fip, 40 were rt-qpcr-positive. in a subsequent pyrosequencing step, 30 of these 50 (one sample was lost from s gene analysis) contained mutated fcov (m1058l n = 25, s1060a n = 1, mixed mutated and non-mutated fcov without differentiation of mutation n = 4). of the 47 fluid samples from cats without fip, one was rt-qpcr-positive and contained substitution m1058l (an abdominal fluid sample from a cat with necrotizing pneumonia). since individual results for effusion, csf and aqueous humor are not shown in that study, sensitivity and specificity cannot be calculated individually, but only for all fluid samples together (table 16) . as was demonstrated for tissue samples, the detection of s gene mutations in this study led to a decrease in sensitivity (78% for rt-qpcr alone compared with 60% for combined approach) and an s gene mutation was also detected in a cat without fip [135] . the detection of fcov s gene mutations in csf was evaluated in two studies presented as conference abstracts thus far (table 17) . one study obtained 16 csf samples from cats with fip (confirmed by ihc) and reported a sensitivity of 44%. control cats were not included in that study [141] . the other study looked at 31 cats with immunohistochemically confirmed fip (six with neurological signs) and 29 control cats with clinical signs indicative of fip (10 with neurological signs) but definitively diagnosed other diseases, and calculated a sensitivity of 10%. sensitivity increased to 17% if only cats with neurological signs were included. since fcov rna was not detected in any of the control cats, specificity of the detection of s gene mutations could not be calculated [188] . ante mortem diagnosis of fip cannot be made based on results of one single diagnostic test. it is important to consider signalment, history, clinical signs and standard clinicopathological abnormalities in every cat that is presented with a suspicion of fip. additional tests, especially tests for direct virus detection, should be utilized depending on the clinical picture. measurement of fcov antibodies is not useful for the diagnosis at all. routine hematology, serum biochemistry and, if present, analysis of effusion should be performed in every cat. in cats with neurological clinical signs, csf analysis should be done as well. diagnostic trees depicting relevant additional diagnostic steps that are recommended depending on a cat's clinical presentation are shown in figure 1 . in cats with effusion, laboratory analysis of the fluid including rivalta's test should be performed. if this test is negative, fip is rather unlikely, especially if the pre-test probability of fip is low. if rivalta's test is positive, however, further diagnostic steps should follow. rt-pcr on effusion can help in establishing a diagnosis, especially if viral copy numbers are high. a negative rt-pcr on effusion makes fip unlikely, unless suspicion is high based on clinical findings and other laboratory test results. high fcov load or detection of s gene mutations in an rt-pcr-positive effusion sample can substantiate a suspicion of fip. additionally, in order to increase sensitivity of the detection of s gene mutations, other sample material, such as fna of mesenteric lymph nodes, spleen and liver and whole blood can be included in the analysis. if, however, s gene mutations cannot be detected in an rt-pcr-positive sample and the virus load is low, but still the suspicion of fip is high in a case, then more invasive diagnostic procedures, such as histopathology and ihc on tissue samples obtained in laparotomy/laparoscopy should be considered in order to confirm or exclude fip. in cats presenting without significant effusion, analysis of different sample types should be combined. ultrasound-guided fna of different organs including the mesenteric lymph nodes, spleen and liver can provide sample material for general rt-pcr, which should be the first diagnostic test in such a case. in cats with neurological clinical signs, diagnostic imaging and csf sampling is an important step in reaching a diagnosis anyway and csf should be submitted for rt-pcr as well. the same is true for cats with uveitis in which the aqueous humor should be integrated in a set of different samples for analysis by rt-pcr. by testing several materials, sensitivity of any given test can be increased. if general rt-pcr is positive in a cat without effusion, then again, the presence of fcov s gene mutations or a high fcov load might aid in cases of uncertainty-if mutations can be detected in an 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comparative in vivo analysis of recombinant type ii feline coronaviruses with truncated and completed orf3 region the role of accessory proteins in the replication of feline infectious peritonitis virus in peripheral blood monocytes the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf7a/7b transcription unit of different biotypes mutation of neutralizing/antibody-dependent enhancing epitope on spike protein and 7b gene of feline infectious peritonitis virus: influences of viral replication in monocytes/macrophages and virulence in cats orf7-encoded accessory protein 7a of feline infectious peritonitis virus as a counteragent against ifn-alpha-induced antiviral response deletions in the 7a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis field strain feline coronaviruses with small deletions in orf7b associated with both enteric infection and feline infectious peritonitis ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex feline coronavirus: insights into viral pathogenesis based on the spike protein structure and function spike protein fusion peptide and feline coronavirus virulence mutation in spike protein cleavage site and pathogenesis of feline coronavirus genotyping coronaviruses associated with feline infectious peritonitis evaluation of a discriminative realtime rt-pcr in cerebrospinal fluid for the diagnosis of feline infectious peritonitis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license [188] csf fip (n = 31) controls (n = 29) only two studies that were presented as conference abstracts have evaluated sensitivity and specificity of the detection of fcov s gene mutations in aqueous humor (table 18 ). these studies included cats with fip confirmed by ihc and reported sensitivities of only 10-17%. specificity, however, could not be calculated in either one of the studies, either because no control cats were included or because none of the control cats were fcov-positive at all [141, 164] . key: cord-284963-p0y5rrpb authors: kipar, anja; meli, marina l.; failing, klaus; euler, tatjana; gomes-keller, maria a.; schwartz, dirk; lutz, hans; reinacher, manfred title: natural feline coronavirus infection: differences in cytokine patterns in association with the outcome of infection date: 2006-08-15 journal: vet immunol immunopathol doi: 10.1016/j.vetimm.2006.02.004 sha: doc_id: 284963 cord_uid: p0y5rrpb natural and experimental feline coronavirus (fcov) infection leads to systemic viral spread via monocyte-associated viraemia and induces systemic proliferation of monocytes/macrophages. in the majority of naturally infected animals, fcov infection remains subclinical and is associated with generalised b and t cell hyperplasia, but no other pathological findings. a minority of cats, however, develop feline infectious peritonitis (fip), a fatal systemic granulomatous disease. this is generally accompanied by b and t cell depletion. the obvious functional differences of lymphatic tissues in fcov-infected cats with and without fip suggest that they contribute to the outcome of fcov infection. this study attempted to evaluate the functional changes in haemolymphatic tissues after natural fcov infection, with special emphasis on the magnitude, phenotype and function of the monocyte/macrophage population. the spleen, mesenteric lymph nodes and bone marrow from naturally fcov-infected cats with and without fip and specific pathogen-free (spf) control cats were examined for the quantity and activation state of monocytes/macrophages both by immunohistology and by quantitative real time pcr for the transcription of interleukin (il)-1β, il-6, il-10, il-12 p40, tumour necrosis factor (tnf), granulocyte colony stimulating factor (g-csf), macrophage-csf (m-csf) and gm-csf. compared to cats with fip, fcov-infected cats without fip exhibited significantly higher il-10 levels in the spleen and significantly lower levels of il-6, gand m-csf in mesenteric lymph nodes. in cats with fip, however, il-12 p40 levels were significantly lower in lymphatic tissues in comparison to both spf cats and fcov-infected cats without fip. in comparison to spf cats, fip cats had significantly higher il-1β levels and lower tnf levels in mesenteric lymph nodes and lower m-csf levels in the spleen. findings indicate that fcov-infected cats which do not develop fip are able to mount an effective fcov-specific immune response and can avoid excessive macrophage activation and fip, possibly by upregulation of il-10 production. development of fip, however, might be due to a lack of il-12 which inhibits an effective cellular immune response and allows for monocyte/macrophage activation and the development of fip. feline infectious peritonitis (fip) is a well-known and widely distributed coronavirus (fcov)-induced systemic disease in cats, characterised by fibrinousgranulomatous serositis with protein-rich effusions into body cavities, granulomatous-necrotising phlebitis and periphlebitis and granulomatous inflammatory lesions in several organs (hayashi et al., 1977; weiss and scott, 1981; kipar et al., 1998 kipar et al., , 2005 . fcov is transmitted via the faecal-oral route and primarily infects enterocytes (pedersen, 1995) , but subsequently spreads systemically via monocyteassociated viraemia (gunnmoore et al., 1998; kipar et al., 1999; meli et al., 2004) . however, fcovinfected circulating monocytes are not only responsible for viral dissemination, but also, in an activated state, for the development of vasculitis (weiss and scott, 1981; jacobse-geels et al., 1982; pedersen, 1995; kipar et al., 2005) . despite the generally high prevalence of fcov infection in the cat population, fip morbidity is low and rarely surpasses 5% (addie et al., 1995; pedersen, 1995; gunn-moore et al., 1998) . this is due to the fact that virulent fcov are predominantly generated within the individual infected host (vennema et al., 1998) . in virulent fcov, deletions in genes encoding non-structural proteins of yet unknown function, which develop during replication, have been observed (vennema et al., 1998; kennedy et al., 2001) . previous studies revealed major differences in the composition and functional state of lymphatic tissues of fcov-infected cats with and without fip (kipar et al., 1999 (kipar et al., , 2001a . in cats with fip, t and b cell depletion is consistently observed (weiss and scott, 1981; kipar et al., 2001a) . in experimentally infected animals with fip, enhanced lymphocyte apoptosis in b and t cell zones of the spleen and mesenteric lymph nodes was also described (haagmans et al., 1996; dean et al., 2003) . prior to lymphocyte depletion, however, most animals appear to develop a specific, systemic b cell response with formation of secondary follicles (kipar et al., 2001a) . in addition, lymphatic tissues contain increased numbers of monocytes/ macrophages, some of which are proliferating (kipar et al., 1999 (kipar et al., , 2001a . in contrast, fcov-infected cats without fip exhibit generalised b and t cell hyperplasia with a high rate of lymphocyte proliferation (kipar et al., 1999 (kipar et al., , 2001a . regardless of the development of fip, fcov infection induces a specific systemic immune response with fcov antibodies, circulating fcov-specific immune complexes and the presence of plasma cells positive for fcov-specific antibodies in lymphatic tissues (osterhaus et al., 1977; kipar et al., 1999; meli et al., 2004) . in fip lesions, b cells are the dominant lymphocyte subtype; they gradually replace the macrophages and seem to develop into plasma cells positive for fcov-specific antibodies (kipar et al., 1998 (kipar et al., , 1999 . these findings suggest that animals susceptible to fip fail to avoid the potential detrimental sequelae of fcov infection and therefore develop both fip and lymphatic depletion (kipar et al., 2001a) . based on morphological and limited functional studies, the pathogenesis of fip lesions is now relatively well understood (hasegawa and hasegawa, 1991; foley et al., 2003; berg et al., 2005; kipar et al., 2005) . however, data on the immunological processes associated with the development of the disease are limited and mainly restricted to experimentally infected animals (haagmans et al., 1996; dean et al., 2003) . in the present study, an attempt was made to evaluate the potential contribution of haemolymphatic tissues to the outcome of natural fcov infection in the individual cat. examinations focused on the assessment of the functional state of the spleen, mesenteric lymph nodes and bone marrow in cats with fip, in comparison to fcov-infected cats without fip and uninfected specific pathogen-free (spf) cats, with special emphasis on the magnitude, phenotype and function of the monocyte/macrophage population. it included quantitative real time pcr for feline interleukin (il)-1b, il-6, il-10, il-12 p40, tumour necrosis factor (tnf), granulocyte colony stimulating factor (g-csf), macrophage-csf (m-csf) and gm-csf. the study was performed on 15 necropsied cats which had died or been euthanased with lesions of fip (group 1). animals ranged from 5 months to 4 years of age (table 1) . group 2 consisted of 13 apparently healthy spf cats, aged 1 year, which had been housed for 30 weeks with cats developing fip (kipar et al., 1999) . all group 2 cats had shown positive results in tests for cov antibodies, circulating fcov-specific immune complexes and monocyte-associated fcov viraemia (kipar et al., 1999) . the heamolymphatic tissues from group 1 and group 2 cats had also tested positive for fcov rna by rt-pcr (kipar et al., 2006) . nine spf cats aged 9.5 months and five spf cats aged 15 months served as uninfected controls (group 3). all animals were necropsied at times ranging from a few minutes to 2 h after death, and samples from the spleen, mesenteric lymph nodes and bone marrow were collected and frozen at à80 8c for rna extraction. the tissue samples from the spleen and mesenteric lymph nodes were taken from areas without macroscopic fip lesions. additional haemolymphatic tissue and organ samples were fixed in 10% buffered formalin and routinely embedded in paraffin. sections (5 mm) were stained with haematoxylin and eosin (he) or by immunohistology (ih). in haemolymphatic tissues monocytes, recently blood-derived macrophages and myelomonocytic cells were demonstrated by ih, using a cross-reacting mouse monoclonal antibody against the human myeloid/histiocyte antigen (clone mac387, dakocytomation gmbh, ely, uk; kipar et al., 1998 kipar et al., , 2001a kipar et al., ,b, 2005 . expression of the cd18 component of b 2 -integrin adhesion molecules was shown by a mouse monoclonal antibody against feline cd18 (clone fe3.9f2, leukocyte antigen biology lab., university of califonia, davis, usa; kipar et al., 2005) . both haemolymphatic tissues and organ specimens with histologically confirmed fip lesions were examined for the presence of fcov antigen (clone fcv3-70, custom monoclonals int., west sacramento, usa; kipar et al., 1998 kipar et al., , 2005 . the percentage of cells labelled for myeloid/ histiocyte antigen and cd18 in relation to the total cell population in the different compartments (e.g. the splenic red pulp) was blindly evaluated semiquantitatively (i.e. by counting the immunolabelled cells and the total number of nucleated cells in several high power fields) by light microscopy. the scoring was made in 25% steps. using a previously described evaluation protocol on he-stained sections (kipar et al., 2001a) , activity and composition of haemolymphatic tissues in spf cats was considered as normal (controls). in spf cats, splenic white pulp was represented by small primary and/or secondary follicles and few, cell-rich periarterial lymphocyte layers in t cell zones; splenic red pulp exhibited moderate numbers of mononuclear cells. mesenteric lymph nodes generally exhibited cell-rich secondary follicles, broad, cell-rich t cell zones and mild histiocytosis of the medullary sinuses. lymphocyte depletion and hyperplasia, both in follicles and t cell zones were graded as mild (+), moderate (++) or severe (+++), based on the degree of reduction or increase in cellularity of the tissue compartments (kipar et al., 2001a) . grading as +/++ or ++/+++ was used when variability between different areas of the organ was observed. bone marrow activity was graded as low (+), moderate (++), moderate to high (++/+++) or high (+++), based on the cellularity of a cross section of the femoral bone marrow cylinder. in spf cats, bone marrow activity was mostly high and occasionally moderate to high. for rna extraction, approximately 100 mg of tissue was homogenised with a tissue homogeniser (pcr tissue homogenizing kit, süd-laborbedarf gmbh, gauting, germany) and lysed with 700 ml lysis buffer. total rna was extracted with a commercially available kit (rneasy mini kit; qia-gen, hilden, germany) according to the manufacturer's protocol (kipar et al., 2001b) . rna was dissolved in 30 ml rnase-free water and digested with rnase-free dnase i (promega gmbh, mannheim, germany) and subjected to the synthesis of cdna using avian myoblastosis virus (amv) reverse transcriptase (promega) according to published protocols (leutenegger et al., 1999; kipar et al., 2001b) . taqman-pcr for feline il-1b, il-6, il-10, il-12 p40, tnf, g-csf, m-csf and gm-csf quantitative real time taqman pcr, using an automated fluorometer (abi prism 7700 sequence detection system) was used for the relative quantification of feline (f) il-1b, fil-6, fil-10, fil-12 p40, ftnf, fg-csf, fm-csf and fgm-csf transcription (leutenegger et al., 1999; kipar et al., 2001b) . feline gapdh levels served as internal controls. for each target gene, two primers and an internal oligonucleotide as a probe (fam (6-carboxyfluores-cein)-labelled at the 5 0 end (reporter dye), tamra (6carboxytetramethylrhodamine)-labelled at the 3 0 end (quencher dye), phosphate-blocked at the 3 0 end to prevent extension by amplitaq gold dna polymerase) were used. primer (f, r) and probe (p) sequences were as previously published for the following systems: il-1b (76 bp pcr product; kipar et al., 2001b) , il-6 (110 bp pcr product; kipar et al., 2001a,b) , il-10 (76 bp pcr product; leutenegger et al., 1999) , il-12 p40 (81 bp pcr product; leutenegger et al., 1999) , tnf (74 bp pcr product; kipar et al., 2001b) and gapdh (82 bp pcr product; leutenegger et al., 1999) . primer and probe sequences for the csfs were as follows: g-csf (ncbi-accession no. y08558; 117 bp pcr product): fgcsf.410f from every cdna sample, parallel reactions were performed in duplicate in separate tubes for the detection of fgapdh and all cytokines. amplification conditions and assay compositions were identical for all reactions and followed previously published protocols (kipar et al., 2001b) . primer and probe concentrations were 400 nm and 80 nm, respectively, for fgapdh, fil-1b, fil-6, fil-10, fil-12 p40 and ftnf, 300 nm and 75 nm for fgm-csf primers and probe, and 300 nm and 200 nm for fg-csf and fm-csf primers and probes. the specificity of the taqman pcr systems for feline gapdh, il-1b, il-6, il-10, il-12 p40 and tnf was demonstrated previously (leutenegger et al., 1999; kipar et al., 2001b) . taqman pcr products for feline g-csf, m-csf and gm-csf were run on a 2% agarose gel. fragments were cloned, using a topo ta cloning 1 pcr 1 2.1-topo 1 kit (invitrogen bv, groningen, the netherlands), propagated in e. coli (top10f'one shot 1 e. coli) vector, and sequenced with a fluorescence-based automated sequencing system (abi 377 dna sequencer; seqlab, sequence laboratories göttingen gmbh, göttingen, germany) to confirm the specificity. relative quantification of cytokine signals was done by the comparative c t method and was reported as relative transcription or the n-fold differences relative to the calibrator cdna (fgapdh) (leutenegger et al., 1999; kipar et al., 2001b) . for each sample, differences between the target and internal control c t were calculated and served to normalize for differences in the amount of total nucleic acid added to each reaction and the efficiency of the reverse transcriptase step as previously described (kipar et al., 2001b) . statistical analysis was performed, using the statistical programme package bmdp (dixon, 1993) . the kruskal-wallis test, followed by the nemenyi comparison, was applied to compare cytokine transcription levels in organs of all three groups of cats. additionally, the wilcoxon mann whitney test was used to compare cytokine transcription levels in organs of cats with fip and spf cats. lesions typical for fip were represented by a fibrinous to granulomatous peritonitis and pleuritis, often associated with effusion, and/or by granulomatous lesions in various organs as well as lymph nodes and the central nervous system (table 1 ). brain and spinal cord involvement was characterised by a granulomatous leptomeningitis, often with granulomatous phlebitis and periphlebitis. the latter was also observed occasionally in eyes, renal cortices and lungs. immunohistologically, fcov antigen was demonstrated within macrophages in the lesions. cats with fip exhibited mild to severe follicular depletion in spleen (13/15) and mesenteric lymph nodes (mes lnn, 15/15). primary and/or secondary follicles were observed in both organs. t cell zones were mildly to moderately depleted in both the spleen (12/15) and mes lnn (13/15). in seven cases, the splenic red pulp was very cell-rich and predominantly composed of macrophage-like mononuclear cells. in 13 cases, mes lnn exhibited mild to severe histiocytosis, mainly of marginal sinuses, which was often associated with marked dilation of the sinuses. fcov-infected cats without fip generally exhibited secondary follicles both in the spleen and mes lnn. in the spleen, these were normal (2/13) or hyperplastic (9/13) and in only two cases were mildly depleted. in the mes lnn, secondary follicles were normal (3/13) or hyperplastic (10/13). t cell zones were mainly normal (11/13) in the spleen and often hyperplastic (7/13) in the mes lnn. myeloid/histiocyte antigen (m/h ag)-positive monocytes/macrophages represented less than 25% of the cells in the splenic red pulp of spf cats; they were more numerous in some fcov-infected cats without fip and often represented the dominant cell population in cats with fip ( figs. 1a and 2a and b) . in spf cats, up to 50% of cells in the red pulp were cd18-positive; in fcov-infected cats without fip, the number of cd18-positive cells was often higher and generally surpassed 75% in cats with fip (figs. 1b and 2c and d). in the mes lnn, the m/h ag was only expressed by scattered sinus histiocytes in all groups of cats. in spf cats and fcov-infected cats without fip, cd18 expression was also restricted to few, faintly positive sinus histiocytes. whereas in cats with fip, sinus histiocytes generally exhibited a strong cytoplasmic and peripheral cd18 reaction (fig. 3) . bone marrow activity was moderate to high in all groups of cats. in the majority of spf cats, m/h ag expression was seen in up to 50% of nucleated cells. the number of positive cells was often higher in fcov-infected cats without fip, whereas m/h agpositive cells generally represented the dominant cell population in cats with fip (fig. 1c) . in spf cats, the amount of cd18-positive cells was rarely above 25%, whereas it ranged between 25% and 75% in fcovinfected cats without fip. in cats with fip, more than 75% of nucleated cells were cd18-positive in the majority of cases (figs. 1d and 4a and b) . the demonstration of fcov antigen by ih yielded negative results in haemolymphatic tissues of all cats, except for the granulomatous lesions of cats with fip (see above). in general, cytokine transcription levels were very variable (fig. 5) . in spf cats, all cytokines were constitutively transcribed. gm-csf mrna was only detected in 43% (6/14), but all other cytokines were detected in more than 60% of the samples. il-1b, il-10, il-12 p40, tnf and particularly m-csf were transcribed at relatively high levels (fig. 5a) . cats with fip and fcov-infected cats without fip also exhibited constitutive transcription of all cytokines, with lowest rates of detection for gm-csf (4/ 13 (31%) fcov-infected cats without fip; 9/15 (60%) cats with fip). comparing the three groups of cats, average il-1b, il-10 and tnf transcription levels were highest in fcov-infected cats without fip (fig. 5a) . the increase in transcription was significant in the case of il-10 when comparing the fcov-infected cats without fip with the fip cats (table 2 ). in contrast, cats with fip exhibited the lowest il-12 p40 and m-csf transcription levels (fig. 5a) . the reduction in transcription of these cytokines was significant in cats with fip compared to spf cats (table 2) . in spf cats, all cytokines were constitutively transcribed, confirmed by demonstration of mrna in at least 60% of the samples. highest average transcription levels were observed for tnf, followed by m-csf (fig. 5b) . cats with fip and fcov-infected cats without fip also exhibited constitutive transcription of all cytokines, with at least 58% of samples positive for the respective mrna. comparing the three groups of cats, average il-6, g-csf and m-csf transcription levels were lowest in fcov-infected cats without fip (fig. 5b) . the reduction in transcription of these cytokines was significant in fcov-infected cats without fip compared to fip cats (table 2 ). in cats with fip, there was evidence for increased transcription of il-1b and m-csf and decreased transcription of il-12 p40 and tnf (fig. 5b) . the reduction in il-12 p40 transcription was significant in comparison to both other groups of cats. the increase in il-1b transcription and the decrease in tnf transcription were significant in comparison to spf cats (table 2) . in the red pulp, the vast majority of nucleated cells express the myeloid/histiocyte antigen. peroxidase anti-peroxidase method. papanicolaou's haematoxylin counterstain. bar = 100 mm. (c) spf cat. in the red pulp, a few cells express cd18. in the follicle, cd18 expression is restricted to a few cells in the center (follicular dendritic cells). avidinbiotin peroxidase complex method. papanicolaou's haematoxylin counterstain. bar = 100 mm. (d) cat with fip. in the red pulp, the vast majority of nucleated cells express cd18. in the follicle, cd18 expression is restricted to a few cells in the center (follicular dendritic cells). avidin-biotin peroxidase complex method. papanicolaou's haematoxylin counterstain. bar = 100 mm. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.) in spf cats, constitutive transcription was mainly seen for il-1b, il-10, tnf and m-csf, as mrna from all other cytokines was often not detected (fig. 5c) . in general, transcription levels for all cytokines were lower than in the spleen and mes lnn, and il-6, g-csf and gm-csf were transcribed in the lowest amounts. the overall cytokine transcription pattern was similar in both other groups of cats (fig. 5c) . in fcovinfected cats without fip, il-6 and gm-csf transcription was not detected at all. average il-10 and m-csf transcription levels were highest in cats with fip (fig. 5c ). there was no evidence of statistically significant differences in the transcription of cytokines in the bone marrow of the three groups of cats. there was no evidence to suggest a relationship between cytokine transcription patterns and type and distribution of fip lesions or the presence or absence of fip lesions in the mes lnn and splenic serosa, respectively. also, there was no distinct evidence of higher transcription levels of any cytokines examined either in the spleen or the mes lnn, of those cats where a cell-rich red pulp or a moderate to intense sinus histiocytosis in the mes lnn were observed. we have evaluated the activity and composition of haemolymphatic tissues in naturally fcov-infected cats with particular emphasis on the macrophage population and the transcription levels of cytokines that are produced by, and/or act on, monocytes/ macrophages (namely il-1b, il-6, il-10, il-12 p40, tnf, g-csf, m-csf and gm-csf). this is the first study to evaluate the constitutive transcription of cytokines in feline haemolymphatic tissues. the spleen, mes lnn and (although at a generally far lower level) bone marrow constitutively transcribe all cytokines examined, which indicates their role in the homeostatic balance of healthy cats. although the study was performed on a relatively homogeneous group of young adult spf cats, high variability in individual transcription levels was observed. a similarly high variability has previously been described in isolated feline monocytes (kipar et al., 2001b (kipar et al., , 2006 . such an obviously wide biological range of cytokine levels renders comparative studies, in particular on natural diseases, difficult. not unexpectedly, therefore, equally variable cytokine transcription levels were seen in both fcov-infected cats without fip (again a relatively homogeneous group of cats with regard to age and previous spf status) and cats with fip (a group heterogeneous both in age and background). the haemolymphatic tissues of both fcovinfected cats without fip and cats with fip exhibited evidence of proliferation and activation of monocytes/ macrophages and their precursors. bone marrow of these two groups contained increased numbers of m/h ag-positive, myelomonocytic cells (horny et al., 1990) . in particular in cats with fip, m/h ag-positive cells were also very numerous in the splenic red pulp, where they represent monocytes or recently bloodderived macrophages (rugtveit et al., 1996; kipar et al., 2005) . in both organs, the same cell types table 2 statistical analysis of differences in the relative quantity of cytokine transcription levels in the spleen, mesenteric lymph nodes and bone marrow exhibited b 2 -integrin upregulation, as shown by increased cd18 expression. in the bone marrow, where increased cd18 expression was most pronounced in cats with fip, it is a feature of myelomonocytic cell maturation (kim and feldman, 2002) and can be induced by cytokines, such as gm-csf and m-csf (aglietta et al., 1993; kamps et al., 1999) . in cats with fip, the mes lnn exhibited pronounced sinus histiocytosis, again with b 2 -integrin upregulation, represented by intense cd18 expression in particular on the cell surface. like in the splenic red pulp, this indicates activation of the macrophages (freyer et al., 1988; fernandez-segura et al., 1996) . in contrast to the splenic macrophages, the majority of sinus macrophages in the mes lnn were m/h ag-negative, which suggests they originated from local macrophages and thus proliferated locally, instead of being monocytes recruited into the lymph nodes (rugtveit et al., 1996) . interestingly, cytokines known to be produced by macrophages and to stimulate macrophages to proliferate and increase in activity, such as g-, m-and gm-csf, il-6 and/or tnf (aglietta et al., 1993; orikasa et al., 1993; springer, 1994; kamps et al., 1999) were not upregulated in either group of cats despite the obvious abundance of activated macrophages in the haemolymphatic tissues. accordingly, a systemic effect seems likely, possibly via cytokines released from circulating monocytes, as they (a) trigger fcov viraemia, (b) are activated in the course of fip (gunnmoore et al., 1998; kipar et al., 1999 kipar et al., , 2005 meli et al., 2004) and (c) produce the respective effector cytokines (clark and kamen, 1987; brandt et al., 1988; sallerfors and olofsson, 1992; aglietta et al., 1993; kerst et al., 1993; springer, 1994; kipar et al., 2005) . with regards to the lymphocyte populations and the overall cytokine transcription patterns, however, major differences were seen between the groups of cats. healthy fcov-infected cats without fip exhibited the generalised b and t cell hyperplasia already described in a previous study (kipar et al., 1999 (kipar et al., , 2001a and displayed overall significantly higher il-10 levels in the spleen as well as significantly lower il-6, g-csf and m-csf levels in the mes lnn when compared to cats with fip. reduced il-6 and csf transcription could be an effect of il-10 which is known to downregulate these cytokines in humans (de waal malefyt et al., 1991a; hannen et al., 1999) . il-10 also inhibits release of other cytokines, such as il-12, tnf and il-1b (de waal malefyt et al., 1991a; koch et al., 1996) . this might explain why in fcov-infected cats without fip, transcription levels of these cytokines were not significantly higher than in spf cats despite the abundance of activated macrophages in the tissues (de waal malefyt et al., 1991a; trinchieri, 1993) . in a fip dna vaccination study where il-12 was co-expressed with viral antigens, enhanced susceptibility of cats to fip was observed in the presence of il-12 (glansbeek et al., 2002) . these findings suggest that prevention of il-12 release is essential to avoid disease in fcov-infected cats. il-10 reduces the antigen-specific proliferation of t cells by downregulating mhc ii expression in monocytes and macrophages (de waal malefyt et al., 1991b ) which might in fcov infection limit the specific immune response despite the persistent antigen challenge in the course of prolonged viraemia (gunnmoore et al., 1998; kipar et al., 1999; meli et al., 2004) . il-10 also stimulates nk cells and upregulates both fcg receptor i expression and antibody-dependent cellular cytotoxicity in monocytes (te velde et al., 1992; moore et al., 2001) . thus, it might contribute to viral clearance and account for the generally lower viral load observed in healthy fcov-infected cats in comparison to cats with fip (kipar et al., 2006) . il-10 may also contribute to the b cell hyperplasia present in healthy fcov-infected animals (kipar et al., 2001a) , as it induces both the proliferation of antigen receptor-activated b cells and ifn-g secretion (rousset et al., 1992; lauw et al., 2000) . on the other hand, il-10 downregulates the b 2integrin expression in monocytes (petit-bertron et al., 2003) which may reduce their capacity to adhere to endothelial cells and thereby to initiate the fipfig. 5 . box and whisker plots, demonstrating levels of cytokine transcription in spf cats, fcov-infected cats without fip and cats with fip. the amount of target was calculated by 2 àdct , resulting in evaluation of the experimental samples as an n-fold difference relative to the calibrator fgapdh (kipar et al., 2001b) . the plots display the range of values, the minimum and maximum, low (first) and high (third) quartiles and the median. (a) cytokine transcription levels in the spleen; group: (1) spf cats, (2) fcov-infected cats without fip, (3) cats with fip; (b) cytokine transcription levels in mesenteric lymph nodes; group: (1) spf cats, (2) fcov-infected cats without fip, (3) cats with fip; (c) cytokine transcription levels in bone marrow; group: (1) spf cats, (2) fcov-infected cats without fip, (3) cats with fip. specific vasculitis (kipar et al., 2005) . taken together, our results indicate that il-10 is a key cytokine in fcov infection, ensuring an effective specific immune response, but avoiding the inflammatory processes associated with the development of fip (kipar et al., 2005) , by inhibiting the virus-induced macrophage activation. in lymphatic tissues, il-10 is produced by monocytes/macrophages and b and t cells (benjamin et al., 1992; moore et al., 1993; dean et al., 2003) . it remains to be clarified which cells in the spleen are responsible for the il-10 production in fcov-infected cats without fip, whether il-10 upregulation represents a direct viral effect on these cells and which factors determine whether il-10 is produced or not. in cats with fip, lymphatic tissues exhibit generalised t and b cell depletion, albeit with evidence of previous activation (kipar et al., 2001a) , and significantly lower il-12 p40 transcription levels than both other groups of cats. il-12 p40, the inducible monomer of il-12 is secreted predominantly by activated mononuclear phagocytes and dendritic cells (trinchieri, 1993) . the reduced production of il-12 observed in lymphatic tissues in this and a previous study (dean et al., 2003) , and in brains with fip lesions (foley et al., 2003) in the presence of abundant activated macrophages in haemolymphatic tissues and granulomatous infiltrates in cats with fip indicates impairment of the immune response, in particular of the th1 response (trinchieri, 1993; o'garra, 1998) . monocytes/macrophages produce il-12 when in contact with activated t cells and under the influence of ifn-g and gm-csf in particular (shu et al., 1995; kennedy et al., 1996) . interestingly, neither ifn-g (data not shown) nor gm-csf were transcribed at lower levels in haemolymphatic tissues of cats with fip despite the obvious t cell depletion in these animals. also, il-10 is obviously not responsible for the reduced il-12 transcription as overall il-10 levels were not elevated in cats with fip (koch et al., 1996) . results of this investigation are difficult to compare with those of a previous study which examined viruspositive and virus-negative lymphatic tissues of cats with fip (dean et al., 2003) ; there, the presence of virus was associated with increased il-10 and decreased il-12 transcription, but potential differences between fcov-infected cats with and without disease were not assessed. still, the general t cell depletion (haagmans et al., 1996; kipar et al., 1999 kipar et al., , 2001a de groot-mijnes et al., 2005) together with the obvious lack of significant ifn-g production in organs or lymphatic tissues with fip lesions (dean et al., 2003; foley et al., 2003) indicate impairment of the t cell-mediated macrophage stimulation in fip. as previous studies did not identify a direct effect of fcov on t cells (haagmans et al., 1996; dean et al., 2003; de groot-mijnes et al., 2005) , a lack of t cell activation by macrophages has to be considered, possibly due to fcov-induced impairment of antigen presentation and/or inhibition of il-12 production by monocytes/macrophages. the latter seems likely due to the fact that fcov induces high antibody titres (osterhaus et al., 1977; kipar et al., 1999; meli et al., 2004) and that fcg ligation can lead to suppression of il-12 transcription in macrophages (grazia cappiello et al., 2001) and indicates direct inhibition of il-12 production by fcov. we also demonstrated significantly reduced tnf transcription, but increased il-1b transcription in the mes lnn of cats with fip in comparison to spf cats. considering that both these cytokines are generally released under similar circumstances and have similar functions (abbas and lichtman, 2003) , a direct, selective effect of fcov on the function of sinus histiocytes appears likely. increased il-1b production may contribute to both the remittent fever observed in the course of fip and the systemic changes in cats with fip, such as the generalised activation of endothelial cells (dinarello, 1994; kipar et al., 2005) . the reduced tnf production, however, cannot readily be explained, in particular as a previous study provided evidence of increased tnf production in viruspositive, depleted lymphatic tissue of cats with fip (dean et al., 2003) . in summary, our findings suggest that, regardless of the development of fip, fcov induces proliferation and activation of monocytes/macrophages which is likely mediated by fcov-infected circulating monocytes. thereby, a constant supply of cells for viral replication and spread is ensured. in parallel, fcov provokes a specific systemic immune response. healthy, long-term fcov-infected cats mount an effective t and b cell response with general t and b cell hyperplasia; here, the immune response might be limited by upregulation of il-10. the presence of fip lesions, however, is associated with t and b cell depletion and reduced il-12 production in lymphatic tissues. a lack of il-12 might be responsible for the obviously impaired cellular immune response and a failure to reduce/limit the viral load in fip (kipar et al., 2006) . despite this fip is associated with general activation of monocytes/macrophages, as reflected by their upregulation of adhesion molecules and cytokine production (dean et al., 2003; kipar et al., 2005) . this indicates that the development of granulomatous inflammatory processes and fatal fip are the consequence of exaggerated activation of monocytes/macrophages. the cells that are directly affected by fcov and the mechanisms that elicit these effects need to be investigated further. it also needs to be clarified whether the virus or the immunophenotype of the individual cat are responsible for the variable effect of fcov infection on the host's lymphatic tissue. fcov virulence is associated with deletions in non-structural genes of yet unknown function and increased viral replication (vennema et al., 1998; kennedy et al., 2001; kipar et al., 2006) . further studies have to clarify whether viral gene products interact with effector molecules, such as cd40, cd40 ligand or mhc ii, or have a direct effect on gene transcription in infected cells. cellular and molecular immunology risk of feline infectious peritonitis in cats naturally infected with feline coronavirus granulocyte-macrophage colony stimulating factor and interleukin 3: target cells and kinetics of response in vivo human b-cell interleukin-10: b-cell lines derived from patients with acquired immunodeficiency syndrome and burkitt's lymphoma constitutively secrete large quantities of interleukin-10 cellular composition and interferon-gamma expression of the local inflammatory response in feline infectious peritonitis (fip) effect of recombinant human granulocyte-macrophage colony-stimulating factor on hematopoietic reconstitution after high-dose chemotherapy and autologous bone marrow transplantation the human hematopoietic colonystimulating factors in vivo cytokine response to experimental feline infectious peritonitis virus infection natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease interleukin 10 (il-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of il-10 produced by monocytes interleukin 10 (il-10) and viral il-10 strongly reduce antigen-specific human t cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class ii major histocompatibility complex expression the biological properties of interleukin-1 topographic distribution of cd18 integrin on human neutrophils as related to shape changes and movement induced by chemotactic peptide and phorbol esters inflammation and changes in cytokine levels in neurological feline infectious peritonitis modulation of surface cd11/cd18 glycoproteins (mo1, lfa-1, p150,95) by human mononuclear phagocytes adverse effects of feline il-12 during dna vaccination against feline infectious peritonitis virus suppression of il-12 transcription in macrophages following fc gamma receptor ligation detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis apoptosis and t-cell depletion during feline infectious peritonitis cytokine-mediated regulation of granulocyte colony-stimulating factor production interleukin 1 alpha mrnaexpressing cells on the local inflammatory response in feline infectious peritonitis systemic vascular lesions in feline infectious peritonitis acute myeloid leukemia: immunohistologic findings in paraffinembedded bone marrow biopsy specimens antibody, immune complexes, and complement activity fluctuations in kittens with experimentally induced feline infectious peritonitis role of macrophage colony-stimulating factor in the differentiation and expansion of monocytes and dendritic cells from cd34+ progenitor cells cd40/cd40 ligand interactions are required for t celldependent production of interleukin-12 by mouse macrophages deletions in the 7a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis interleukin-6 is a survival factor for committed myeloid progenitor cells activated fes protein tyrosine kinase induces terminal macrophage differentiation of myeloid progenitors (u937 cells) and activation of the transcription factor pu.1 cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis histopathological alterations of lymphatic tissues in cats without feline infectious peritonitis after long-term exposure to fip virus evaluation of lymphatic tissue activity in cats with spontaneous feline infectious peritonitis cytokine mrna levels in isolated feline monocytes morphological features and development of granulomatous vasculitis in feline infectious peritonitis natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats high level il-12 production by murine dendritic cells: upregulation via mhc class ii and cd40 molecules and downregulation by il-4 and il-10 proinflammatory effects of il-10 during human endotoxemia quantitative real-time pcr for the measurement of feline cytokine mrna high viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (fcov) type i and in naturally fcov-infected cats interleukin-10 interleukin-10 and the interleukin-10 receptor cytokines induce the development of functionally heterogeneous t helper cell subsets induction of macrophagic and granulocytic differentiation of murine bone marrow progenitor cells by 1,25-dihydroxyvitamin d3 seroepidemiology of feline infectious peritonitis using transmissible gastroenteritis virus antigen an overview of feline enteric coronavirus and infectious peritonitis virus infections adherence influences monocyte responsiveness to interleukin-10 interleukin 10 is a potent growth and differentiation factor for activated human b lymphocytes expression of the l1 antigen (calprotectin) by tissue macrophages reflects recent recruitment from peripheral blood rather than upregulation of local synthesis: implications for rejection diagnosis in formalin-fixed kidney specimens granulocyte-macrophage colonystimulating factor (gm-csf) and granulocyte colony-stimulating factor (g-csf) secretion by adherent monocytes measured by quantitative immunoassays activated t cells induce interleukin-12 production by monocytes via cd40-cd40 ligand interaction traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm il-10 stimulates monocyte fc gamma r surface expression and cytotoxic activity. distinct regulation of antibody-dependent cellular cytotoxicity by ifn-gamma, il-4, and il-10 interleukin-12 and its role in the generation of th1 cells feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence we wish to thank dr. m. linenberger, division of hematology, department of medicine, university of washington, usa, for kindly providing the sequence of the feline m-csf gene to design primers and probes. we are grateful to dr. c. leutenegger key: cord-021452-9rukc80y authors: bergman, robert l. title: miscellaneous spinal cord diseases date: 2009-05-15 journal: consultations in feline internal medicine doi: 10.1016/b0-72-160423-4/50054-8 sha: doc_id: 21452 cord_uid: 9rukc80y nan spinal cord diseases in cats vary in the severity and in progression of neurological dysfunction. diagnosis of spinal cord disease in cats can be a challenge. clinical signs often are vague and insidious. advanced imaging techniques have improved diagnostic capabilities and recognition of new disorders. infectious inflammatory disease is the most common categorical differential diagnosis in cats with spinal cord dysfunction. other common disease categories include neoplasms, trauma, and degenerative disorders. 1 veterinarians must think beyond the more common differential diagnoses to consider unusual diseases and different diagnostic approaches. this chapter emphasizes newly recognized spinal cord diseases and provides a review of the current literature. signs of neurological dysfunction dictate the neuroanatomical localization of a lesion within the spinal cord. spinal reflexes and paraspinal hyperesthesia assist with lesion localization. localization is specified to the spinal cord regions c1-c5, c6-t2, t3-l3, and l4-s2, based on upper motor neuron or lower motor neuron signs of limb weakness. cats with spinal cord compressive disease or meningomyelitis often exhibit paraspinal hyperesthesia. pathology of the spinal cord tissue itself usually does not have hyperesthesia as a clinical sign. orthopedic, polyneuropathic, myopathic, and neuromuscular junction disorders can mimic signs of spinal cord dysfunction. careful interpretation of the neurological examination differentiates among these disorders (see chapter 49) . signalment and history aid in formulation of a list of probable differential diagnoses. young cats are more likely to be diagnosed with feline infectious peritonitis (fip), lymphosarcoma, or a congenital anomaly. middle-age and older cats may be diagnosed with nonlymphoid neoplasia or intervertebral disc disease. history is important for determining temporal onset (acute, insidious, or episodic) and progression (rapid, gradual, or static). trauma, vascular insults, and some inflammatory and neoplastic diseases present acute in onset. cats with spinal cord dysfunction require thorough physical examination and routine laboratory diagnostic testing. other disorders that cause paresis can mimic spinal cord disease; for example, neuropathy, myopathy, junctionopathy, polyarthropathy, and cardiovascular disease. routine laboratory testing consists of a complete blood count (cbc), serum chemistry profile to include creatine phosphokinase (ck) enzyme activity, and urinalysis. evaluation of ck activity aids in identification of a myopathy, which can mimic spinal cord dysfunction. patient infection with feline immunodeficiency virus (fiv) or feline leukemia virus (felv) must be identified. additional serology for infectious disease is dependent on suspicion of other diseases. survey radiography of the spine is recommended for cats with spinal cord dysfunction. sedation or general anesthesia often is necessary to allow for proper patient positioning and relaxation of the spine. some findings are nonspecific, but discospondylitis, vertebral tumors, or spinal trauma usually have more obvious radiographic abnormalities. orthogonal or multiple views are recommended strongly, because a single view may not always provide complete information about the extent of the lesion (figure 51-1). myelography consists of injection of a nonionic contrast medium (0.3 to 0.45 ml/kg of iohexol [240 mg/ml] or iopamidol [200 mg/ml]) into the subarachnoid space of the low lumbar spine (l6-l7 or l5-l6) or the cerebellomedullary cistern. myelography is an imaging technique used commonly to identify the location and extent of spinal cord compression. 2 additional information may be used when combining the myelographic findings with computed tomography (ct). magnetic resonance imaging also is a more sensitive technique for evaluation of the spinal cord tissue. cerebrospinal fluid (csf) analysis is useful for detection of evidence of spinal cord disease, particularly when an inflammatory disorder is suspected. however, in most cases, a definitive diagnosis is not provided by csf analysis alone, even in cats with overt cns inflammatory disease. 3 exceptions include finding the inciting organism in the csf (e.g., cryptococcus neoformans) or identifying neoplastic cells (e.g., lymphosarcoma). 3 collection of fluid from the lumbar region may be preferable, because csf flows in a caudal direction. 4 additional diagnostic procedures include electrophysiology, csf protein electrophoresis, serology, and exploratory surgery. infectious inflammatory diseases account for 31 per cent of all feline spinal cord diseases. 1 common infectious inflammatory spinal cord diseases include fip, cryptococcosis, felv infection, and toxoplasmosis. approximately 15 per cent of cases of meningomyelitis in cats are bacterial or suspected to be bacterial in origin. 1 bacterial infections may occur secondary to hematogenous spread or, more likely, as a result of direct extension of a local wound (e.g., cat bite abscess). 5 pasteurella spp. and staphylococcus spp. are common pathogens. discospondylitis often is caused by a bacterial infection and involves the intervertebral disc and associated vertebral endplates. discospondylitis has been reported infrequently in cats and, if not treated appropriately, may progress to severe neurological dysfunction. 6, 7 polioencephalomyelitis, an inflammatory disease of unknown cause, is associated with 8 per cent of cases of feline spinal cord disease 1 and may present with clinical signs of paraparesis. 5 although not well described in the literature, eosinophilic/histiocytic meningomyelitis accounted for 6 per cent of inflammatory spinal cord diseases in cats. 1 feline immunodeficiency virus. fiv has been reported to cause a degenerative myelopathy that can be detected histologically with changes including myelin sheath splitting and intramyelinic vacuoles. clinical signs of spinal cord dysfunction are not evident in experimentally infected cats or in cats with naturally occurring fiv infection. 8 presenting signs and pathogenesis. fip accounts for more than half of the infectious inflammatory causes of myelitis in cats, and 16 per cent of all spinal cord diseases reported in cats. 1 clinical signs of fip result from the immune response of susceptible cats when infected by a mutant form of the feline enteric coronavirus (fecv), which reproduces within macrophages. 9, 10 the dry, or noneffusive, form of the disease is associated most commonly with cns signs as opposed to the "wet" or effusive form, which involves the visceral organs and causes abdominal effusion. pyogranulomatous inflammatory lesions involve the meninges, choroid plexus, and ventricular system. the immune response associated with fip causes a vasculitis and an ependymitis that subsequently may obstruct flow of csf. 11 signs of systemic illness occur in approximately 79 per cent of cats with fip. 1 typical signs include weight loss, anorexia, intermittent fever, and ocular changes (anterior uveitis or chorioretinitis). about one third of cats with fip have presenting clinical signs of neurological dysfunction. 12 young, purebred, sexually intact male cats are at a higher risk for developing fip. 13 a genetic susceptibility of about 50 per cent exists for development of fip. 10 all cats in one study of patients with neurological signs of fip were from multiple-cat households. 9 although younger cats are most susceptible to fip infection, cats of any age can develop the disease. in a case series of cats with spinal cord-related signs, more than 75 per cent were younger than 2 years of age. 1 most cats with fip have intracranial signs but often manifest signs of spinal cord dysfunction: pelvic limb ataxia, generalized ataxia, and paraspinal hyperesthesia. in a small case series of cats with confirmed fip, four of 10 had paresis or paralysis as the presenting clinical sign. 12 paresis was evident in two of seven of these cats with diffuse fip. 12 in another study, 28 of 29 cats with fip had histological lesions that predominated in the cervical spinal cord and brain. 1 diagnosis. antemortem diagnosis of fip is difficult. diagnosis is suspected based on assimilation of history, signalment, hematology, and other supportive diagnostic tests that include serology, csf analysis, findings on imaging, and tissue biopsies. a typical history includes acquisition of the cat from a cattery or shelter, and a fever that waxes and wanes and does not improve with antibiotic therapy. 10 common hematological and biochemistry abnormalities include neutrophilia or lymphopenia, low albumin with increased globulins, or a high serum fibrinogen. 10 serological testing and polymerase chain reaction studies to assess for viral load can be beneficial. 10 serology only confirms exposure to feline coronavirus. some cats with fip may have high antibody titers, but this is not absolute. a recent study of histopathologically confirmed cases of fip found that serological testing provided further support for tissue biopsy procedures. 14 high antibody titers (1:1600) provide a 94 per cent probability of active fip infection. 14 a titer that was positive but below 1:1600 suggested only a 44 per cent probability that cats had fip. 14 the titers in 10 per cent of cats with fip were negative, which suggests a compromised immune system. 14 definitive diagnosis of fip is made by histopathology of abdominal organs obtained by tissue biopsy. immunofluorescent assay/immunohistochemistry techniques can detect presence of coronavirus antigens within macrophages. 14 diagnostic evaluation of the cns aids in an indirect diagnosis of fip. results of csf analysis often reveal a marked increase in protein concentration and a neutrophilic pleocytosis. 11, 15 comparisons of antibodies in serum and csf may provide additional information, but false negatives and false positives are possible. presence of antibodies in csf must be interpreted in light of blood-brain barrier breakdown. adjunctive comparison of other infectious disease antibody titers in serum and csf can assist with determination of intrathecal production of antibodies. 10 common abnormalities on mri and ct include presence of hydrocephalus and periventricular contrast enhancement. overall the most consistent diagnostic findings in cats with the cns form of fip include a positive coronavirus igg titer in csf, a high serum total protein concentration, and abnormalities in brain imaging. 9 treatment. no treatment has been proven effective for fip, and the long-term prognosis is poor. 10 overall mortality rate is 95 per cent. 10 supportive therapies consist of antiinflammatory doses of prednisone (1 mg/kg/day po) and immunomodulation with cyclophosphamide or interferon. a recent report found use of recombinant feline interferon combined with corticosteroids more effective in cats with the effusive form than with the noneffusive form (n = 1) of fip. 16 other therapeutic recommendations include a diet with high nutritional value and stress reduction. 10 presenting signs and pathogenesis. cryptococcus neoformans is a saprophytic fungal organism that can cause systemic or focal disease. transmission occurs through inhalation of the organism that lives in the soil or bird excrements. cns signs are reflective of meningitis or focal granuloma formation within the brain parenchyma. fungal masses within the extradural space cause secondary compression ( figure 51 -2). 17 cryptococcal infection can cause focal spinal cord disease in some cats. the mean age for cats infected with cryptococcus is 6 years; however, the age range can vary. 18, 19 approximately 58 per cent of cats diagnosed with cryptococcus spp. were considered primarily outdoor cats. 18 systemic signs are variable and commonly include depression/lethargy, fever, poor body condition, or anorexia. 18 in one case series, approximately 50 per cent of the cats with cryptococcosis had cns signs, 42 per cent had ocular signs, and 32 per cent had respiratory signs. 18 cutaneous lesions also can occur. another case series reported that only 9 per cent of cats showed signs of neurological dysfunction. in this series, nasal signs were more common. 19 clinical signs of spinal cord dysfunction, including paraspinal hyperesthesia and paresis, have been reported in at least one case series. 18 c. neoformans accounted for 9 per cent of infectious causes of spinal cord disease in cats. 1 diagnosis. csf analysis is one of the most useful diagnostic tests in cats with cns cryptococcosis. neutrophilic and eosinophilic pleocytosis often are present. in some cases, the organism is identified. diagnosis also is based on detection of capsular antigen using a latex agglutination test in serum and csf. cats with focal granulomas in the cns may have negative antigen titers. 17 latex agglutination tests can have falsenegative results (or interference), which makes definitive diagnosis difficult. 18 in these cases, cultures, cytology, or histopathology of other tissues, such as skin, may be necessary. also important is documentation of the felv and fiv status of cats with cryptococcosis, because concurrent infection may be common. 18 additionally, cats with other concurrent viral infections tend to have a higher incidence of treatment failure. 20 treatment. treatment of cns cryptococcosis consists of long-term administration of systemic antifungal agents. itraconazole and fluconazole are considered the drugs of choice. fluconazole (5 to 15 mg/kg po q12h) is recommended, because it crosses the blood-brain barrier readily and has high lipid solubility. duration of treatment ranges from 6 to 10 months; however, a longer duration may be required to prevent relapse. 21, 22 antiinflammatory doses of corticosteroids help to decrease the inflammation and edema that can worsen neurological signs during treatment. therapeutic monitoring is based on clinical response and serial serum antigen titers. antigen titers often remain positive for a considerable period of time after clinical signs have resolved. 19 cats that have a reduction in antigen titer during the course of treatment have a better prognosis. 20 surgical removal of a fungal granuloma may be considered in conjunction with antifungal therapy. 17 presenting signs and pathogenesis. toxoplasma gondii is an infrequent cause of spinal cord disease in cats; it was reported as the cause for only 3 per cent of all infectious diseases resulting in spinal cord dysfunction. 1 t. gondii is a protozoal coccidian parasite for which cats are the definitive host. transmission occurs congenitally through the placenta from an infected queen or more commonly by ingesting the organism. healthy cats may be positive on serology but rarely develop clinical disease. predisposing factors for clinical disease include immunosuppression as a result of fiv and/or felv infection, administration of corticosteroids or chemotherapy, and diabetes mellitus. toxoplasmosis causes a nonsuppurative meningoencephalomyelitis. the organism also may infect muscle and peripheral nerves. 5 systemic signs include anorexia, weight loss, fever, and pneumonia. diagnosis. definitive diagnosis of toxoplasmosis is difficult. a suspected diagnosis is based on clinical signs, the exclusion of other cns diseases, serology, and response to treatment. 11 the amount of csf pleocytosis is variable, and the cellular differential count usually consists of mononuclear cells. albuminocytologic dissociation may be the only abnormality. t. gondii-specific igg and igm can be assayed in serum and csf. paired titer evaluations may detect an increase in serum igg; however, the disease course still may be static. 11 an igm titer greater than 1:256 may indicate an active or recent infection. antibodies in csf are compared with serum antibody titers for accurate interpretation of blood contamination and intrathecal antibody production. a definitive diagnosis is made by detecting the organism in a tissue biopsy. clindamycin (12.5 mg/kg po q12h for 4 weeks) is recommended for treatment of cns toxoplasmosis. 23 an alternative drug therapy is trimethoprim-sulfonamide (15 mg/kg po q12h). 23 one author reported a fair to good outcome in three cats treated for toxoplasma-induced myelitis. 5 clinical signs can be residual and response to therapy may be slow. 11 clinical presentation and pathogenesis. feline leukemia virus, an oncogenic retrovirus, can cause spinal cord dysfunction. felv can cause myelopathy by indirect and direct pathogenic mechanisms. felv can predispose to the spinal form of lymphoma indirectly or cause a degenerative myelopathy directly. felv-associated myelopathy reflects primary pathology within the spinal cord. 24 light microscopic examination revealed swollen axons and myelin sheaths in the brain stem and spinal cord of affected cats. immunohistochemical staining revealed felv antigens in neural tissue. a previously reported case of degenerative myelopathy in a felv-positive cat may have been felv-associated myelopathy. 25 this disease is associated with chronic infection with felv. cns signs develop on average 3 years after the first positive felv test. 24 mean age of affected cats is 9 years. 24 signs of felv-associated myelopathy include progressive ataxia and hyperesthesia, and paralysis develops within 1 year after onset of paraparesis. 24 urinary incontinence occurs in a small percentage of cats. diagnosis and treatment. a suspected antemortem diagnosis is based on ruling out other diseases. positive felv tests should heighten suspicion for this disease. csf analysis usually is not helpful. 24 advance imaging studies have not been evaluated in cats with felv-associated myelopathy. myelography is normal. no treatment options have been described. neoplasia is a common cause of spinal cord dysfunction in cats. with regard to relative incidence in one case series, neoplasia affected 28 per cent of cats diagnosed with spinal cord dys-function. 1 lymphosarcoma made up 38 per cent of neoplasiarelated spinal cord cases; however, this disease is becoming less common with the reduction in incidence of felv infection. 1, 26, 27 lymphosarcoma presenting clinical signs and pathogenesis. spinal lymphoma historically has been the most common cause of spinal cord neoplasms in cats. cns lymphoma accounted for 12.1 per cent of all cases of lymphoma and, of these cases, 88 per cent had spinal cord involvement. 28 the disease is especially common in young felv-infected cats, with a mean age reported between 3.6 and 4 years. 28, 29 cats younger than 3 years of age make up approximately 70 per cent of the cases. clinical signs associated with spinal lymphoma may be associated with a focal myelopathy that can occur in any region of the spinal cord. paresis has been reported in approximately 80 per cent of cats with spinal lymphoma. 28, 29 evidence of spinal hyperesthesia may be focal or multifocal with more extensive distribution. 29 the disease course can be rapidly progressive, with some cats showing signs for a week or less. 28, 29 neurological signs are related to the location of the lymphoma. although lymphosarcoma generally is a multicentric disease, more than 85 per cent of cats with cns involvement lack systemic signs or hematological changes. 29 renal lymphoma is likely to metastasize to the cns. diagnosis. evidence for systemic disease on physical examination includes enlargement of lymph nodes and abdominal organs. a cbc may show anemia, leukopenia, and thrombocytopenia. circulating lymphoblasts may be present on a differential white blood cell count. a positive correlation between serological testing and spinal lymphoma has been reported. 28, 29 the safest and most reliable method of obtaining a diagnosis of cns lymphoma is confirmation of the presence of lymphoma in other visceral organs. bone marrow aspiration may be diagnostic for neoplasia in up to 81 per cent of cases with this disease. 29 csf analysis is not always diagnostic for lymphosarcoma because of its extradural location. one case series reported that 6 of 17 cats had neoplastic lymphocytes in the csf. 28 myelography can determine lesion extent and detect presence of extradural, intradural-extramedullary, or intramedullary involvement. an extradural lesion is the most common myelographic finding. fluoroscopic aspiration and cytology may allow definitive diagnosis of the spinal lesion. 29 mri may detect intramedullary lesions. treatment. positive felv status in cats has been shown to be a negative prognostic indicator for spinal lymphoma. 30 the prognosis for cats with paresis or paraplegia is considered poor. treatment options for spinal lymphoma consist of chemotherapy, surgical resection, and focal irradiation. 31 no superior treatment strategy for chemotherapy has been documented. currently, multidrug protocols are advocated. 27, 29 a laminectomy procedure facilitates diagnosis and decompression until other therapies can take effect. clinical presentation and pathogenesis. nonlymphoid tumors involving the spinal cord are less common in cats. tumors may be categorized based on expected locations: intramedullary, extramedullary/intradural, and extradural. intramedullary tumors are considered uncommon and make up 10 per cent of all reported spinal cord neoplasms in cats. 1 documented tumors include astrocytoma and ependymoma. 32, 33 intradural/extramedullary tumors make up 12 per cent of spinal neoplasia cases and include meningiomas, meningeal sarcomas, and malignant nerve sheath tumors. 1 feline meningiomas usually occur rostral to the foramen magnum; only 4 per cent of meningiomas found in cats affected the spinal cord. 34 levy, mauldin, kapatkin, et al reported five cases of spinal meningiomas in cats: one in the cervical region, three in the thoracic spine, and one in the lumbar spine. 36 another case report described a meningioma affecting the spinal cord at the c6-c7 spinal cord segment. 35 extradural spinal cord compression can result from spinal canal masses or tumors of the surrounding bone and vertebrae. these make up about 40 per cent of spinal cord-associated neoplasms in cats, with vertebral and bone tumors accounting specifically for 29 per cent of all spinal tumors. 1 reported tumor types include chondrosarcoma, lipoma, osteosarcoma, and multiple myeloma. 36 nonlymphoid spinal neoplasia typically occurs in older cats, with a median age of 12 years in one case series. 36 these tumors are not associated with felv or fiv infection. clinical signs of myelopathy are dependent on tumor location. focal pain and paresis are most typical. intramedullary neoplasms usually do not cause spinal hyperesthesia until later in the disease course. the clinical course of spinal neoplasms also varies. chronic progressive spinal dysfunction may be expected; however, peracute signs (e.g., pathological vertebral fracture) may present (figure 51-3) . diagnosis. the process of diagnosis of nonlymphoid tumors begins with survey spinal radiographs. evidence of bony lesions can be evident in osteosarcoma and multiple myeloma. myelography determines extent and location of spinal involvement. advanced imaging (ct and mri) may assist further with determination of lesion extent. findings on csf analysis often are nonspecific. fluoroscopic aspiration or surgical biopsy may yield a definitive diagnosis. treatment. specific treatment regimens are based on histopathological diagnosis of the tumor. treatment often consists of palliative corticosteroids (i.e., prednisone 0.5 to 1 mg/kg/day po) to control edema, and pain management. surgical removal/debulking of various tumor types has been described and may improve survival times. 36 a reasonable survival time can be expected for cats with meningiomas after surgical resection. 36 osteosarcomas may be associated with long survival times and appear to be less aggressive than the canine form of this disease. 36 a treatment regimen reported for multiple myeloma in a maine coon cat 6 years of age consisted of a combination of chemotherapy and irradiation. 37 clinical presentation and pathophysiology. spinal trauma is an important cause of spinal cord dysfunction in cats. cats have been the subject of multiple laboratory studies of spinal cord injury. [38] [39] [40] [41] [42] [43] [44] information learned from this research must be interpreted from the standpoint that the mechanism of spinal cord trauma is controlled in the laboratory environment. naturally occurring spinal trauma in cats is not a well-described phenomenon because cats often do not survive the inciting incident. spinal injury occurs more frequently in younger cats. cats (n = 69) in a retrospective study of spinal injury were 9 years of age or younger; 39 per cent were younger than 1 year of age, and 29 per cent were between 1 and 3 years of age. 45 the mean age of another study of 30 cats was 3.6 years (range 2 months to 12 years). 46 common causes of spinal trauma are height-related and vehicular injuries. in a large report of high-rise syndrome in cats, 10 per cent sustained spinal fractures or luxations. 47 other sources of trauma include dog attacks and gunshot wounds. 45, 46 compression-type fractures occur in more than two thirds of the cases with spinal fractures. 46 twenty per cent of cats with spinal injuries also have acute disc extrusions secondary to the trauma. 46 spinal cord contusion injury without evidence of fractures also may occur after a fall. 46 other injuries that occur in conjunction with spinal trauma include pneumothorax, pulmonary contusions, abdominal organ trauma, and head trauma. although all segments of the spine are susceptible to trauma, the cervical and sacral/caudal regions are much less likely to sustain injury. the thoracic and lumbar regions make up 51 per cent and 32 per cent of spinal column injuries, respectively. 45 traditionally, the most likely location for spinal column injury is at a site characterized by a transition from rigid stability to less stability, such as t13-l1 or l7-s1. this was challenged recently in a larger study consisting of 69 cats, in which 51 per cent of the spinal trauma cases occurred between t8 and t12. 45 the segments between l2-l3 and l4-l5 also were significant sites in 43 per cent of the cases. the t11 through t12 vertebrae have been reported to be affected in 45 per cent of spinal injuries in cats. 46 a case series reported by voss showed that 50 per cent of spinal fractures were located between the l3 and l6 vertebrae. 48 diagnosis. in cases of suspected spinal injury, the neurological examination is performed with caution to minimize movement of the cat with suspected spinal instability. evaluation of deep pain perception is most important with regard to determining prognosis. spinal radiography using orthogonal views should localize the lesion. radiography documents spinal alignment during that time without knowledge of the amount of displacement at the time of the injury. the entire spine should be radiographed, because multiple spinal fractures are common. advanced imaging of the spine has been recommended because plain film radiography and myelography may underestimate the degree of fractures or luxations present. 48 myelography or mri defines the extent of spinal cord compression more accurately. 46 treatment. goals of therapy are to prevent further mechanical damage to the spinal cord and reduce secondary injury processes. treatment recommendations often are adapted from laboratory studies that involve species other than cats. drawing firm conclusions for optimal treatment of spinal trauma in cats is difficult. management of a cat with spinal trauma should focus first on systemic stabilization. management consists of following the abcs of trauma. airway is assessed for patency and adequate ventilation. appropriate fluid therapy helps to maintain cardiovascular function. aggressive fluid therapy is important to maintain spinal cord perfusion. 49 hypotension is one factor shown to worsen outcome in human beings with spinal cord injury. isotonic crystalloids (lactated ringer's solution or 0.9 per cent sodium chloride) at shock doses, initially (60 ml/kg/hr iv in cats) are given to effect until heart rate, capillary refill time, and pulse quality improve. hetastarch (6 per cent) is a large molecular weight colloid that consists of a branched polysaccharide, amylopectin. its molecular properties provide a long intravascular half-life. the dose is 10 to 20 ml/kg given to effect up to 40 ml/kg/hr. hetastarch is administered intravenously to cats to effect up to 10 to 15 ml/kg; the dose is increased in 5 ml/kg increments every 5 to 10 minutes to avoid nausea and vomiting. hypertonic saline (7 per cent) also may be used to expand blood volume quickly. the dose (4 to 5 ml/kg) is administered as an intravenous bolus over 3 to 5 minutes. the disadvantage associated with the use of hypertonic saline is that it remains in the vascular space for only 15 to 60 minutes. blood products also are used to expand volume and provide increased oxygen delivery. whole blood is administered intravenously at a dose of 4 to 10 ml/kg/hr, over 4 to 6 hours in stable patients and faster in unstable patients. the goal is to restore the hematocrit to 25 to 30 per cent and albumin to more than 2 g/dl. use of high-dose methylprednisolone sodium succinate (mpss) is becoming more controversial but is still considered standard of care in human medicine. experimental spinal cord injury studies in cats have shown that increased lactate levels in spinal cord immediately after injury most likely were attributed to decreased spinal cord perfusion. 41 high doses of mpss 30 minutes after induced trauma attenuated the secondary injury process dramatically. the intent of high-dose mpss is to provide adequate tissue concentrations of steroid at the site of injury. the original dose for this regimen was 30 mg/kg iv initially, then a dose of 15 mg/kg given 2 and 6 hours later, followed immediately by a 2.5 mg/kg/hr infusion, which is continued for 42 hours. the total dose of mpss administered is 165 mg/kg. 43 recent prospective clinical studies that have used modifications of this protocol have come under intense scrutiny for various reasons, including statistical manipulation, lack of proven efficacy, and increased rates of complications in human beings and dogs. [50] [51] [52] [53] [54] administration of mpss is time dependent and has shown efficacy if administered within 30 minutes of the injury. 55 use of high-dose mpss is not recommended for administration if the time has been longer than 8 hours after sustaining the injury. supportive medical management alone is useful if spinal instability is not detected or when there is financial constraint. 56, 57 cats may not tolerate body splints. 56 strict cage confinement is relied upon for 4 to 6 weeks after the injury to initiate the healing process. 58 surgical management. surgical management of spinal trauma is recommended in cases of instability or severe spinal cord compression. 58 the timing of surgery relative to the injury is somewhat controversial; however, adequate medical stabilization before surgery is essential. early decompression has been supported in the laboratory setting in cats, but the optimal time to perform surgery still is unknown. 59 immediate surgical decompression of the affected site is a controversial subject in human spinal trauma, and some studies have not shown a benefit to early surgery. 60, 61 the technique of surgical stabilization depends somewhat on the fracture type. decompression alone may be sufficient in some cases in which instability is not present. 45 decompression was needed in cats that sustained displacement of the intervertebral disc or endplate into the spinal canal. 45 a dorsal laminectomy, preserving the articular processes, suffices for adequate decompression. common techniques of internal spinal fixation/stabilization include the use of pins with polymethylmethacrylate and spinal stapling. spinal stapling involves the use of rigid intramedullary pins that are secured to the spine after reduction of the fracture/luxation. intramedullary pins are secured to the lamina at the base of the spinous process (figure 51-4) . 62 spinal stapling is considered technically easier to perform than other described forms of internal stabilization. limited information is available for the long-term outcome. problems associated with this type of surgery include fragility of the spinous processes and migration or breakdown of implants. 45 a recent case series involving 16 cats with thoracolumbar trauma described using a figure-8 tension band technique as a modification of spinal stapling. 48 complications from this technique were not observed. 48 successful use of pins and polymethylmethacrylate to stabilize a lumbar fracture in a cat has been described. 63 this form of treatment provides significant stability, particularly for rotational forces. 64 optimal placement of pins within the vertebral body may be difficult because of the small size of typical feline vertebral bodies. complications of this procedure include pin migration, pin breakage, pneumothorax, and additional trauma to soft tissue structures. outcome for cats with spinal trauma is guarded. survival rate in one study was only 60 per cent. 46 cats that did not survive were euthanized or died within 4 days of the injury. cats with spinal fractures and absence of deep pain perception almost always have a hopeless prognosis. the return of motor function does not equate necessarily with return of voluntary urination. 48 spinal walking: from laboratory to clinics. prognosis for return of voluntary motor function in cases of absent deep pain perception generally is considered grave. however, the cat has been the subject of extensive experimental work studying the return of ambulation after spinalization, or complete transection of the spinal cord. "spinal walking" is a clinical term used for return of ambulation in an animal with no deep pain perception in the pelvic limbs. in the laboratory setting, this phenomenon is known as spinal locomotion. pelvic limbs are under no voluntary control, and the thoracic limbs move asynchronously with the pelvic limbs when on a treadmill. spinal locomotion may be evident within a few days of the injury. 66 the spinal cord generates this pattern of limb movement, which allows for the placement each foot, weight-bearing, and alterations of speed with change in treadmill velocity. 65 the animal also is capable of stepping over objects placed in its way. 66 cats have been trained to develop spinal locomotion after complete experimental spinal cord transection at t13. the underlying mechanism may be the result of a spinal locomotor generator. 66 spinal locomotion is dependent on the development and preparation of a spinal locomotor pattern generator, stimulation of cutaneous receptors, alterations of intraspinal neurochemistry, and input from the midlumbar spinal cord. 66 plasticity occurs within the spinal cord as a result of training. lesion location within the spinal cord also can affect the ability to walk; for example, a lesion at the l3-l4 spinal segment is not conducive to development of spinal locomotion. 65 spinal walking in a cat with a complete spinal cord injury is much less likely to occur without training. 67 animals with complete spinal cord transections and no training can begin to take steps within weeks of the injury. 65 cats with naturally occurring spinal trauma had a low success rate in development of spinal locomotion after injury. reasons for the low success were attributed to less controlled spinal injury and inadequate physical therapy/training. training for 30 minutes daily 5 days a week provides an 87 per cent success rate of weight-bearing in the pelvic limbs. 68 without appropriate rehabilitation the rate drops to 33 per cent. 69 variability exists among cats as to when walking movements begin to occur. 66 repeated training of a cat by placing the thoracic limbs on a nonmoving platform and the pelvic limbs on a treadmill resulted in better walking and weight-bearing ability in the pelvic limbs. 65 this process involves the use of a treadmill, tail support, and various forms of stimulation. 70 cutaneous stimulation is important for afferent sensory input. younger animals tend to have a better recovery rate for walking. 66 training activities resulted in almost all cats regaining the ability to walk. early intensive training allowed for better walking. 66 spinal locomotion is maintained only for a finite period after discontinuation of training activities and begins to show decline after 12 weeks. 71 long-term management of a deep pain-negative cat. much has been learned in cats after experimental spinal cord injury regarding optimal medical management of deep pain-negative cats. 70 bladder expression is required at a minimum of twice daily. some cats urinate without expression, but the bladder is not emptied completely. stimulation of the perineum initiates a mass reflex and partial emptying of the bladder. 70 researchers report that treadmill training also stimulates urination and defecation in cats with complete spinal cord injuries. 70 inadequate emptying of the bladder predisposes to chronic urinary tract infections 70 (see chapter 48) . suggestions for care to prevent this problem include adequate bladder expression and water intake. 70 chronic bladder infections weaken the muscular wall, further complicating manual emptying of the bladder. 70 fecal elimination usually can occur without assistance and is aided by perineal stimulation. 70 diarrhea and constipation still can occur as complications. clinical presentation and pathogenesis. intervertebral disc disease (ivdd) is recognized commonly in cats with approximately 27 published cases. 72 several case series have been published in recent years. [73] [74] [75] the incidence of ivdd as a significant clinical problem compared to other diseases that affect cats has been reported to be 0.12 per cent. 74 earlier clinical reports of disc disease in cats were postmortem studies that described cervical and, to a lesser extent, lumbar disc disease in older cats. [76] [77] [78] [79] [80] these discs were mostly hansen type ii, with bulging of the annulus fibrosus into the spinal canal, and were described as incidental findings. characteristics of a degenerated intervertebral disc suggest some degree of chondroid degeneration of the discs. 72 more recent literature describes hansen type i, with extrusion of nucleus pulposus into the spinal canal, and recognizes this type to be the most common form of disc-related spinal cord compression in cats. 74 ivdd also can occur spontaneously in cats having no history of trauma. 74 ivdd occurs more frequently in middle to older aged cats. mean age for all reported cases is 7 years. 72 the age range varies somewhat in different reports, between 3 and 9 years, 73 and 4 and 17 years (mean age of 9.8). 74 no gender or breed predilections exist for ivdd in cats. clinical signs of disc disease vary on lesion location and in severity and can consist of back pain and paresis/plegia. lesion involvement in the thoracolumbar region of the spinal cord is common. 72 cervical disc disease is uncommon in cats, with two reported cases confirmed by necropsy, and one presumed case diagnosed with mri. [81] [82] [83] disc spaces between the t11 and l2 vertebrae are affected in 50 per cent of cats with clinical signs of ivdd. 72 the l4-l5 disc interspace also is a common site in 26 per cent of the reported cats with ivdd. 72 ivdd at l7-s1 disc was described in a cat with lower motor neuron signs, flaccid tail, and urinary and fecal incontinence. 84 diagnosis. survey spinal radiographs may reveal typical evidence of disc disease: narrowed disc spaces and evidence of mineralized material in the intervertebral foramen. 74 collection of csf is performed to eliminate other potential inflammatory diseases. findings on csf analysis in cats with ivdd are not specific and may show a mild neutrophilic pleocytosis and increased protein concentration. myelography is used to localize the site of the disc extrusion/herniation more precisely. computed tomography can detect hyperdense material within the spinal canal at the affected disc space. 74 findings on mri suggestive of ivdd include evidence of dehydration of the disc with loss of signal intensity on t2-weighted sequence. 83 treatment. conservative medical management has been used successfully in cats with ivdd; however, in severe spinal cord compression, this form of treatment should not replace surgery. based on a limited number of case reports, medical management alone may result in a poor outcome. 72 conservative management still may be a better option in cases with a small amount of extruded disc material in the canal. 83 medical management usually consists of pain control with use of a combination of narcotics and corticosteroids. corticosteroid therapy (prednisone 0.5 to 1 mg/kg/day po) is used short-term in combination with strict cage confinement for 4 to 6 weeks. physiotherapy also may aid the long-term outcome of neurological function. surgical decompression for removal of extruded disc material may be accomplished with use of either a hemilaminectomy or dorsal laminectomy procedure. 73, 74 surgery offers a higher rate of success and more rapid and complete neurological recovery when compared with conservative treatment. 75, 85 many cats still have residual neurological deficits that include paresis and urinary and/or fecal incontinence. 73, 74 clinical presentation and pathogenesis. fibrocartilaginous embolism (fce), or embolic myelopathy, has been described in many species including cats (table 51-1) . [86] [87] [88] this is a rare disease in cats, with about 7 per cent of spinal cord diseases attributed to vascular causes. 1 in this disease, a small portion of fibrocartilaginous tissue, which is presumed to be intervertebral disc material, occludes the vascular supply to the spinal cord, resulting in an acute onset of asymmetrical spinal cord dysfunction. typically, fce is nonprogressive and not painful. lesions in the cervical and lumbar spinal cord regions have been reported. the mean age from the limited case reports available is 10.2 years, with a range between 8 and 12 years of age. diagnosis. diagnosis of fce is based on elimination of other causes of myelopathy. csf analysis may reveal a neutrophilic pleocytosis and an increased protein concentration. 87, 88 similar abnormalities also have been reported with intervertebral disc disease and may simply indicate necrosis. 73, 83 case reports have lacked definitive imaging results, except in one case in which myelography showed evidence of intramedullary swelling. 88 mr images can be expected to show an increased signal intensity relative to surrounding tissues of the spinal cord parenchyma on a t2-weighted sequence. treatment. treatment strategies have been extrapolated from treatment options recommended in other species with fce. this consists of high doses of mpss (if the drug can be administered within 8 hours of the onset of clinical signs), adequate fluid therapy, bladder management, and supportive care. once the cat is stabilized, physical therapy may aid in recovery. prognosis in these cases is difficult to predict because the literature in this area shows some bias as definitive diagnosis requires necropsy. prognosis is presumed guarded to fair in cats that have deep pain perception intact. syringomyelia is an abnormal fluid-filled cavity within the parenchyma of the spinal cord. hydromyelia often occurs with syringomyelia and is defined as dilation of the central canal. pathophysiology of syringohydromyelia is associated with alterations in flow of csf often secondary to a congenital anomaly, infectious disease process, or trauma. syringohydromyelia has been reported in cats but is not well described. 89 clinical signs include paraspinal hyperesthesia and paresis. the syrinx can be detected using mri. treatment usually is directed toward the underlying cause. an antiinflammatory dose of prednisone (0.5 to 1 mg/kg/day) may reduce edema and inflammatory response. clinical presentation and pathogenesis. diverticulum within the subarachnoid space results in accumulation of csf and compression of the spinal cord, which causes neurological dysfunction. these diverticula are not true cysts but rather leptomeningeal cavitations that are filled with csf. 93 spinal arachnoidal cysts have been reported to cause paresis in cats. [90] [91] [92] [93] another case report documented an intradural epithelial-lined cyst found at the vertebral body of c7 in a 2 1 /2year-old female burmese cat. 93 location of these cyst formations is variable in cats and can occur in the cervical, thoracic, and lumbar spine. the cause of arachnoidal cysts is unknown, but may be related to factors that include previous trauma, inflammation, and developmental or congenital malformations. 92 an arachnoidal cyst in a 7-year-old spayed female domestic shorthair cat with paraparesis was associated with a lordotic malalignment of the caudal thoracic spine. 92 affected cats usually are young to middle-age with a range between 2 and 7 years of age. clinical signs usually are chronic and progressive and reflect the location of the cyst. duration of clinical signs is chronic and progressive in onset. a cat in one report showed signs for only a few weeks. 90 diagnosis. diagnosis of spinal arachnoidal cysts is made using myelography, ct-myelography, or mri. the diverticulum is identified with myelography as a teardrop shape within the subarachnoid space ( figure 51-5) . 92 magnetic resonance imaging can document a spinal arachnoidal cyst on a t2weighted sequence as an area of hyperintensity. 93 treatment. reports of treatment protocols for spinal arachnoidal cysts in cats have been limited. surgical fenestration has been reported. 90,91,93 a hemilaminectomy or dorsal laminectomy is used to expose the cystic structure for dural fenestration and possible excision of the cyst. outcome in these cases has been excellent with complete recovery. residual neurological deficits may occur. histopathology of the cyst wall is recommended to rule out other lesions. palliative medical management consists of antiinflammatory doses of corticosteroids. prevalence of diseases of the spinal cord of cats intervertebral disc disease and myelography inflammatory cerebrospinal fluid analysis in cats: clinical diagnosis and outcome. australian college of veterinary scientists science week analysis of cerebrospinal fluid from the cerebellomedullary and lumbar cisterns of dogs with focal neurologic disease: 145 cases (1985-1987) spinal cord diseases in cats bacterial discospondylitis in a cat lumbar diskospondylitis and meningomyelitis caused by escherichia coli in a cat neuropathology in cats experimentally infected with feline immunodeficiency virus: a morphological, immunocytochemical and morphometric study diagnostic features of clinical neurologic feline infectious peritonitis recommendations from workshops of the second international feline coronavirus/feline infectious peritonitis symposium spinal cord disorders an axial ct image after a myelogram of the lumbar spine from a 7-year-old male castrated domestic shorthair cat with a history of progressive upper motor neuron paraparesis. the cat also had a spinal malformation inflammation and changes in cytokine levels in neurological feline infectious peritonitis epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals comparison of different tests to diagnose feline infectious peritonitis back to the cat": feline spinal cord disease use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis a cryptococcal granuloma in the brain of a cat causing focal signs feline cryptococcosis: a retrospective evaluation clinical and serologic evaluation of cats with cryptococcosis cryptococcal infection in cats: factors influencing treatment outcome, and results of sequential serum antigen titers in 35 cats cryptococcosis in cats: clinical and mycological assessment of 29 cases and evaluation of treatment using orally administered fluconazole infectious diseases of the dog and cat toxoplasmosis and neosporosis feline leukemia virusassociated myelopathy in cats degenerative myelopathy in a cat feline lymphoma and leukemia therapeutic choices for the medical management of feline lymphoma. waltham feline medicine symposium feline spinal lymphosarcoma: a retrospective evaluation of 23 cats spinal lymphoma in cats: 21 cases feline lymphoma (145 cases): proliferation indices, cluster differentiation 3 immunoreactivity, and their association with prognosis in 90 cats principles of treatment for feline lymphoma spinal cord astrocytoma in a cat a glioma in the spinal cord of a cat meningiomas in animals use of magnetic resonance imaging for diagnosis of a spinal tumor in a cat nonlymphoid vertebral canal tumors in cats: 11 cases (1987-1995) multiple myeloma in cats: variable presentation with different immunoglobulin isotypes in two cats effects of a single large dose of methylprednisolone sodium succinate on experimental posttraumatic spinal cord ischemia. dose-response and time-action analysis the neuroprotective pharmacology of methylprednisolone correlation of methylprednisolone levels in cat spinal cord with its effects on (na + k + )-atpase, lipid peroxidation, and alpha motor neuron function lactate and pyruvate metabolism in injured cat spinal cord before and after a single large intravenous dose of methylprednisolone effects of multi-dose methylprednisolone sodium succinate administration on injured cat spinal cord neurofilament degradation and energy metabolism evaluation of an intensive methylprednisolone sodium succinate dosing regimen in experimental spinal cord injury pretreatment with alpha tocopherol enhances neurologic recovery after experimental spinal cord compression injury management of spinal trauma in 69 cats survival rates and outcomes in cats with thoracic and lumbar spinal cord injuries due to external trauma high-rise syndrome in cats: 207 cases tension band stabilization of fractures and luxations of the thoracolumbar vertebrae in dogs and cats: 38 cases combined medical and surgical treatment after acute spinal cord injury: results of a prospective pilot study to assess the merits of aggressive medical resuscitation and blood pressure management methylprednisolone for acute spinal cord injury: an inappropriate standard of care is the role of steroids in acute spinal cord injury now resolved? high dose methylprednisolone in the management of acute spinal cord injury -a systematic review from a clinical perspective gastric hemorrhage in dogs given high doses of methylprednisolone sodium succinate complications of methylprednisolone sodium succinate therapy in dachshunds with surgically treated intervertebral disc disease evaluation of time-dependent spread of tissue damage in experimental spinal cord injury by killedend evoked potential: effect of high-dose methylprednisolone nonsurgical management of thoracic and lumbar spinal fractures and fractures/luxations in the dog and cat: a review of 17 cases management of vertebral column fractures in dogs and cats: 211 cases (1977-1985) spinal fracture or luxation reversible spinal cord trauma in cats. additive effects of direct pressure and ischemia an evidence-based review of decompressive surgery in acute spinal cord injury: rationale, indications, and timing based on experimental and clinical studies current use and timing of spinal surgery for management of acute spinal cord injury in north america: results of a retrospective multicenter study principles of vertebral fracture management use of pins and methylmethacrylate in stabilization of spinal fractures and luxations the rotational stabilizing effect of spinal fixation techniques in an unstable vertebral model recovery of locomotion in the cat following spinal cord lesions determinants of locomotor recovery after spinal injury in the cat locomotor capacity attributable to step training versus spontaneous recovery after spinalization in adult cats effects of training on the recovery of full weight bearing stepping in the adult spinal cat return of weight supported locomotion in adult spinal cats chronic spinal cord-injured cats: surgical procedures and management retention of hindlimb stepping ability in adult spinal cats after the cessation of step training feline intervertebral disc disease: a review of the literature intervertebral disc extrusion in six cats intervertebral disk disease in 10 cats spontaneous lumbar intervertebral disc protrusion in cats: literature review and case presentations disc protrusions in the cat: distribution of dorsal protrusions along the vertebral column disc protrusions in the cat: age incidence of dorsal protrusions disc protrusions in the cat: ventral protrusions and radial splits degeneration of the intervertebral disc in the cat protrusion of the intervertebral disc in the cat intervertebral disc syndrome in the cat intervertebral disc protrusion in a cat acute intervertebral disc extrusion in a cat: clinical and mri findings lumbosacral disc disease in a cat radiographic diagnosis: intervertebral disc extrusion in a cat fibrocartilaginous embolic myelopathy in a cat tetraparesis in a cat with fibrocartilaginous emboli fibro-cartilaginous embolism in a cat syringomyelia and hydromyelia in dogs and cats subarachnoid cyst in a cat correlative imaging findings in seven dogs and one cat with spinal arachnoid cysts spinal subarachnoid cyst in a cat intradural epithelial cyst in a cat key: cord-022555-a7ie82fs authors: nan title: digestive system, liver, and abdominal cavity date: 2011-12-05 journal: the cat doi: 10.1016/b978-1-4377-0660-4.00023-5 sha: doc_id: 22555 cord_uid: a7ie82fs nan these complex pathways highlight the need to consider the whole cat and not just the cat's gastrointestinal vomiting can be defined as the ejection of part or all of the contents of the stomach and/or upper intestine through the mouth, usually in a series of involuntary spasmodic movements. the disturbances in gastrointestinal (gi) motility are coordinated with respiratory and abdominal muscle contractions and mediated by the central nervous system (cns). vomiting begins with retching, a series of brief negative intrathoracic pressure pulses that coincide with positive abdominal contractions. these pressure changes occur as a result of repeated herniations of the abdominal esophagus and cardiac portion of the stomach into the esophagus. during retching, food freely moves back and forth in the esophagus, which is now dilated because of the ingesta. ultimately, the diaphragm rapidly moves cranially, resulting in positive intrathoracic pressure that leads to expulsion of these contents. 12 vomiting is such an active process that it seems to involve the whole cat, and so it is little wonder that it concerns owners so much. since vomiting is mediated by the cns with input and influence from just about anywhere in the body, it is important to summarize this physiology so it can be appreciated when managing clinical cases. vomiting results from stimulation of the "vomiting center," which is located in the brainstem; there are four main pathways that stimulate the vomiting center, 12 and these are summarized below and in figure 23 (though not all older cats have grown out of this habit). some extragastrointestinal problems, such as hyperthyroidism and renal disease are more likely to occur in older cats. most texts and references instruct clinicians to distinguish between vomiting and regurgitation, with the latter noted as being quite passive. 3, 11, 12 in practice, it can be hard to make this distinction, because it is the author's experience that cats with esophageal disease can have quite forceful, spasmodic movements when ejecting ingesta by regurgitation-although it is also possible for regurgitation to be a passive process. given that the physiology of vomiting, as described above, results in ingesta being forced to and then evacuated from the esophagus, it is hardly surprising that it can resemble regurgitation. fortunately, regurgitation and esophageal disease do vary from vomiting in other ways! vomiting 3. blood and urine testing 4. imaging (radiography, ultrasonography) 5. biopsy samples 6. treat and manage underlying problem the decision to proceed to steps 4 and 5 is based on the assumption that the prior steps have narrowed down the underlying cause as gastrointestinal, pancreatic, or hepatic in origin. the important aspects of the clinical history are given in chronic kidney disease. the author has found that some cats with dental disease can gorge their food, resulting in vomiting; so, paying attention to the state of the teeth and gums is important. of course, some cats have multiple problems, and correction of dental disease may not resolve vomiting if there is another process. in the examination, it is also important to note consequences of both the underlying process and the vomiting itself; these include the demeanor of the cat, hydration status, and abdominal pain. the physical examination findings, together with the clinical history, help determine the next appropriate steps. well cats that are not continually vomiting and are appropriately hydrated, with no other specific signs, may be treated as outpatients by fasting them for 24 hours, then returning to food with a bland diet, such as plain cooked chicken or commercial, low-residue prescription diets designed for this purpose. follow-up is important to ensure signs do not progress. cats with nonspecific signs may require supportive care with subcutaneous or intravenous fluids and perhaps analgesia (with opioids). if clinical signs do not resolve, the pursuit of a specific diagnosis should be attempted. the practitioner must ask the following important questions: • are ancillary tests appropriate? • is supportive care necessary? • are any medications required? routine serum/plasma biochemistries, hematology, urinalysis, and total thyroxine (t 4 ) (for older cats) testing is not only important to distinguish primary from secondary gastrointestinal disease but to look for consequences of vomiting that may need to be addressed, such as hydration status and electrolyte abnormalities. careful interpretations should be made. severe azotemia, even with hyperphosphatemia, can occur as a result of primary gastrointestinal disease, and the distinction from renal disease usually requires an assessment of urine specific gravity. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity (fpli) tests are useful markers of intestinal and pancreatic disease, 7, 8, 9, 10 but it is important to note that they mostly do not give a precise diagnosis. more detail about the utility of these tests is noted below in the section approach to the cat with diarrhea. is usually preceded by the cat licking its lips, salivating, or making attempts to swallow. regurgitated ingesta is often in a tubelike structure and if undigested can be covered with frothy saliva. partially digested food suggests vomitus, and the presence of bile or digested blood confirms this. it is important to determine if the cat vomits regularly. many owners have seen their cats vomit on a regular basis with no evidence of the cat being unwell, and this is noted frequently in the veterinary literature. 3, 12 hairballs can cause gastric irritation, and it may be that eating quickly also stimulates the peripheral sensory receptors that contribute to vomiting. if a cat does vomit regularly, it is important to assess if the cat is presenting for a change in the vomiting pattern (e.g., frequency or timing in relation to eating) and if the cat is unwell in any way, such as anorexia or weight loss. the pattern of vomiting is important in all cases, because cats presenting with acute gastritis usually have a sudden onset of frequent vomiting compared with those with chronic disease processes that may vomit every few days. the timing in relation to eating can be helpful, because the stomach should empty by 6 to 8 hours after a meal; so, vomiting longer than 8 hours after a meal can suggest motility or retention disorders. the description of the vomitus can be helpful. if bile is present, the pylorus is not obstructed; the presence of blood (digested or fresh) indicates ulceration. hair in the vomitus can indicate hairball gastritis, and the possibility of trichobezoar obstruction should be considered. access to foreign bodies or toxins is an important aspect of the clinical history. has the cat been seen playing with an insect, mouse, or other prey? are there any medications unaccounted for (e.g., a dropped aspirin tablet)? are lilies present in the house? vomiting is the major sign of gastric disease, but given the number of potential organ systems that can be involved, a thorough physical examination should be undertaken. because linear foreign bodies are a common cause of vomiting, all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of string caught there. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see . a thorough examination may reveal specific signs, such as a palpable thyroid nodule and tachycardia in the case of hyperthyroidism or palpably small kidneys with and analgesia, many cats recover uneventfully. one survey assessed that 83% of cats undergoing exploratory laparotomy survived the hospitalization, and although complications occurred in 26% of cats, these were more likely to be associated with the underlying disease process and not surgery or anesthesia. 4 laparoscopy is not readily available in all veterinary clinics. this alternative is less invasive and allows exploration of the abdomen but not as thoroughly as with laparotomy. organs are usually exteriorized for biopsy. there is the possibility of anesthetic complications associated with insufflating the abdomen. endoscopy is the least invasive procedure and is the only alternative that allows examination of the intestinal lumen. this option limits the parts of the gastrointestinal tract that can be biopsied; it does not allow examination or sampling of any other part of the gastrointestinal tract and does not enable full-thickness biopsy samples. one study found that, of cats investigated for gastrointestinal disease, 9 of 33 cats (27%) had no pathology recognized proximal to the jejunum (i.e., the effective length of diagnostic endoscopes would have precluded diagnosis), and other organs were affected in 9 of 10 cats with inflammatory bowel diseases and 7 of 8 cats with intestinal small cell lymphoma. 1 careful case selection for endoscopy from survey ultrasonography can reduce the number of missed diagnoses from endoscopy, but the possibility still remains. the quality of endoscopically obtained biopsy samples varies greatly with the skill of the endoscopist. it has been stated that "it is exceedingly easy to take inadequate tissue samples with a flexible endoscope." 5 in an assessment of endoscopically obtained biopsy samples, two laboratories were compared, one that received samples from any practitioner and the other that received samples only from practitioners trained to take, mount, and submit endoscopy samples. all slides were reviewed by three pathologists who found that, of samples from the first laboratory, 15% of the slides were considered inadequate for diagnosis, 71% were considered questionable, and only 14% were adequate. by comparison, in the second laboratory (with samples from experienced practitioners) 0% of slides were inadequate, 21% were questionable, and 79% were considered adequate for diagnosis. 13 in the case of distinguishing between lymphocytic intestinal infiltrates (commonly known as inflammatory bowel disease) and lymphocytic neoplasia (small cell lymphoma), endoscopically obtained samples can give an incorrect diagnosis. 2 many of these problems can be minimized with experienced operators and careful case selection from prior ultrasonography. radiography is most useful for identifying foreign bodies or signs of intestinal obstruction from other causes. the major findings are noted below in the section intestinal obstruction. contrast radiography can aid the diagnosis for both discrete and linear foreign bodies but should be used with caution, because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium irritates the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. 6 ultrasonography is a useful diagnostic adjunct and helps to detect and characterize localized thickening of the stomach or intestinal wall, lymphadenopathy, radiolucent foreign bodies, and changes in the size and echogenicity of the pancreas, liver, kidneys, or spleen. abdominal effusions can be assessed and sampled. ultrasound-guided fine-needle aspiration can be used to sample masses, bile, or peritoneal fluid. it should be recognized that in most cases of gastrointestinal disease, imaging will not give a definitive diagnosis and biopsy will be required, usually using either endoscopy or laparotomy. ultrasonography can be a considered as a means to "survey the field," assessing • the nature of the underlying disease, such as • thickened intestines with or without discrete layers • lymph node involvement • other organ involvement • the location of disease, for example, • diffuse or focal • proximal duodenum (reachable by endoscope) versus distal ileum these factors may be used to assess the appropriateness of endoscopy versus laparotomy to obtain diagnostic samples. most commonly used antiemetics all control vomiting by different mechanisms and include mirtazapine, metoclopramide, dolasetron/ondansetron, maropitant, and the phenothiazines (tables 23-2 and 23-3) . metoclopramide functions both as an antiemetic and prokinetic in cats, while cisapride functions solely as a prokinetic. mirtazapine, a piperazinoazepine, antagonizes the presynaptic alpha 2 -adrenergic receptor, increasing noradrenergic and serotonergic neurotransmission; the primary mechanism targeted for its use is as an antidepressant in humans. mirtazapine is also a potent antagonist of the postsynaptic serotonergic receptors (5-ht 2 and 5-ht 3 ) and histamine h 1 receptors. because of its antiserotonergic and antihistaminic effects, mirtazapine is used as an entiemetic and appetite stimulant in cats. anorexia is a common clinical problem in ill cats, and in some anorexic or partially anorexic cats the use of an appetite stimulant as adjunctive therapy to nutritional support (i.e. feeding tubes) may be of clinical benefit. prior to the development of mirtazapine, cyproheptadine was used as an appetite stimulant in cats, with variable clinical results. recently, the pharmacokinetics and pharmacodynamics of mirtazapine have been reported in cats. in a group of healthy cats, mirtazapine was found to be an effective appetite stimulant, with a shorter half-life than that reported in humans. the recommended oral dose is 1.88 mg/cat every 48 hours. 55a in humans, age and kidney and liver dysfunction affect mirtazapine metabolism (hepatic cyp 450 enzymes) and clearance (excreted in urine and feces), suggesting that dose adjustment may be necessary. 69a side effects reported in cats treated with mirtazapine include behavior changes (vocalization and interaction), tremors, muscle twitching, and hyperactivity. 9a,55a metoclopramide is both an antiemetic and prokinetic drug that acts peripherally on the gastrointestinal tract and centrally within the central nervous system (cns). at low doses metoclopramide inhibits dopaminergic (d 2 ) transmission, and at higher doses it inhibits serotonergic 5-ht 3 receptors in the chemoreceptor trigger zone (crtz). 15, 23 metoclopramide also acts peripherally as a prokinetic at the level of the gastrointestinal smooth muscle of the stomach and duodenum, triggering gastric emptying and duodenal contractions. multiple mechanisms mediate metoclopramide's prokinetic activity, including augmentation of acetylcholine release and increased smooth muscle sensitivity to cholinergic neurotransmission, which may in part be because of antagonism of dopamine, but more recently, serotonergic 5ht 4 receptor activation has been suggested. 23, 56 metoclopramide has been reported to increase the lower esophageal sphincter tone in humans, 20 although in cats metoclopramide's affect on the lower esophageal sphincter is reported to be weak. 32 adverse central nervous system, extrapyramidal signs occur secondary to dopamine (d 2 ) antagonism, including excitement and behavior changes. extrapyramidal signs are most often seen at the higher doses needed to block 5-ht 3 receptors. because of metoclopramide's prokinetic properties, an intestinal obstruction should be ruled out prior to its use. dopamine is a less important neurotransmitter in the chemoreceptor trigger zone of cats than alpha 2adre nergic and 5-ht 3 -serotonergic receptors, suggesting that d 2 -dopaminergic antagonist may be a less effective antiemetic in cats. clinically metoclopramide commonly controls vomiting in cats, although this clinical response may be secondary to 5-ht 3 antagonism and/ or its prokinetic effects. 32, 44 extrapolated from the short elimination half-life of metoclopramide in dogs (90 minutes), frequent be clinically significant side effects. phenothiazines have the potential to lower the seizure threshold; their use is not recommended in patients with a known seizure history. other cns-associated side effects linked to d 2 antagonism occur at higher doses and produce extrapyramidal signs, including rigidity, tremors, weakness, and restlessness. antagonism of the histaminergic receptors carries the risk of sedation. because of the need for frequent dosing (0.2 to 0.4 mg/ kg subcutaneously every 8 hours) and the risk of hypotension and sedation, the clinical use of phenothiazine antiemetics is limited to hospitalized patients with refractory vomiting and should be avoided in patients who are dehydrated or hypotensive. cisapride is a serotonergic 5-ht 4 agonist that increases propulsive gastrointestinal motility from the lower esophageal sphincter to the colon. cisapride binds serotonergic 5-ht 4 receptors in the myenteric plexus, increasing the release of acetylcholine in gastrointestinal smooth muscle. in dogs cisapride has greater prokinetic activity in the stomach relative to metoclopramide. 29 cisapride has no direct antiemetic effect, although it is indicated in a vomiting cat with colonic dysmotility secondary to megacolon. colonic distention can trigger the vomiting reflex in cats. cisapride induces colonic smooth muscle contractions in cats with megacolon that is dependent on the influx of extracellular calcium and is only partially cholinergic dependent. 30 other potential indications include refractory generalized ileus or gastroesophageal reflux. dosage recommendations based on the pharmacokinetics in healthy cats is 1.5 mg/kg orally every 12 hours. 41 prior to the use of cisapride, an intestinal obstruction should be ruled out because of its strong prokinetic effects. side effects reported in humans are cramping and diarrhea. potentially life-threatening side effects include qt prolongation and ventricular arrhythmias, the primary concern in humans that led to cisapride's removal from the market in the united states. 47 in cats qt prolongation associated with cisapride administration requires 20 times the therapeutic dose. 37 because of the risk of prolongation of the qt interval and ventricular arrhythmias, the concurrent use of cisapride and dolasetron is not recommended. 14 other potential drug interactions associated with cisapride include concurrent therapy with azole antifungals (ketoconazole and itraconazole), because of their inhibition of hepatic cyp3a isoenzyme system and the inhibition of cisapride metabolism. 47 diet trials are commonly used in cats with idiopathic gastrointestinal signs or in cats with suspected or known intermittent dosing or delivery by a constant rate infusion (cri) is necessary. empirical dosing in cats is 0.2 to 0.4 mg/kg subcutaneously or orally every 8 hours or 1 to 2 mg/kg/day as a cri. approximately 25% of metoclopramide is excreted in the urine, thus dose reduction is recommended in cats with underlying renal azotemia. 42 dolasetron and ondansetron are selective serotonin antagonists that inhibit central and peripheral 5-ht 3 receptors. their main antiemetic effect is through antagonism of the peripheral 5-ht 3 receptors in the gastrointestinal tract. in cats 5-ht 3 antagonism of the crzt is also likely important in the antiemetic effect of dolasetron and ondansetron. dolasetron and ondansetron were originally used for vomiting secondary to chemotherapy because of their superior clinical efficacy. the clinical use of dolasetron and ondansetron in cats has not been associated with reported side effects, and experimental studies report minimal toxicity in animals at doses 30 times the antiemetic dose. 15 side effects reported in humans include headaches, elevated liver enzymes, rare hypersensitivity reactions, prolongation of the qt interval, and arrhythmias. 14, 24 dolasetron is commonly used for parenteral administration and ondansetron for oral administration, dictated primarily based on the tablet sizes available and cost. recommended dosing of dolasetron is 0.5 to 1 mg/kg intravenously every 24 hours and ondansetron 0.5 mg/ kg orally every 12 hours. maropitant is a neurokinin-1 (nk-1) receptor antagonist, blocking the binding of substance p to the nk-1 receptors located in the emetic center, crtz, and the enteric plexus. 55 in cats maropitant has been reported to be efficacious in treating xylazine-induced vomiting and motion sickness. 31 recommended dosing in cats is 1 mg/ kg intravenously, subcutaneously or orally every 24 hours for up to 5 days. 31 maropitant is reported to be well tolerated in cats. prochlorperazine and chlorpromazine are considered broad-spectrum antiemetics by antagonism of d 2dopaminergic, histaminergic (h 1 and h 2 ), and cholinergic (muscarinic) receptors within the crtz and, at high doses, the alpha-adrenergic receptors (alpha 1 and alpha 2 ) within the vomiting center. in cats alpha 2 -receptors play a key role in emesis (recall xylazine is the emetic of choice in cats), suggesting cats may be more sensitive to the antiemetic effects of the phenothiazines. prochlorperazine and chlorpromazine produce an antiemetic effect at relatively low doses, thus avoiding profound sedation; although, because of antagonism of the alpha-receptors, vasodilation and hypotension can hyperacidity alone is not considered a common cause for vomiting in cats, but famotidine is effective in treating vomiting in cats associated with gastric ulcers or gastritis. recommended dosage in cats is 0.5 mg/kg every 12 to 24 hours. ranitidine is also a competitive inhibitor of the h 2 receptor associated with gastric parietal cells. in addition, ranitidine increases lower esophageal sphincter tone and functions as a prokinetic agent (increasing gastric emptying and stimulating intestinal motility, including colonic motility), because of its anticholinesterase food hypersensitivities. dietary strategies used to control vomiting in cats focus on either a highly digestible diet or an elimination (novel protein/carbohydrate or hydrolyzed protein) diet. 72 the empirical use of elimination diets in cats is reported to be relatively successful, with approximately 50% of cats with idiopathic gastrointestinal signs responsive to a novel protein/carbohydrate diets within 2 to 3 days. 28 interestingly, traditional diet trials are recommended for a minimum of 8 to 12 weeks, but in this group of diet-responsive cats with chronic gastrointestinal disease, clinical improvement was reported within days. 28 thus if a cat is going to be diet responsive, clinical improvement to a diet trial should be noted relatively early. highly digestible diets enable more effective absorption and assimilation of nutrients in the face of a compromised digestive tract. these diets contain highly digestible proteins and carbohydrates, moderate to low fat, soluble fiber but low concentrations of insoluble fiber, and are supplemented with omega-3 fatty acids. these diets are recommended when food allergy or intolerance is suspected. these diets contain a single highly digestible novel carbohydrate source and novel protein source. alternatively, diets formulated with hydrolyzed proteins can be used as an alternative to novel protein/carbohydrate diets. see tables 23-4 and 23-5 for information on gastrointestinal ulcers. famotidine has no direct antiemetic effect but is a competitive inhibitor of the histamine (h 2 ) receptors associated with the gastric parietal cells. the h 2 -receptor is the dominant receptor involved in gastric acid secretion. h 2receptor antagonism is reported to result in a 70% to 90% reduction in acid production. 13 famotidine is more effective at suppressing gastric acid secretion relative to ranitidine. famotidine is well tolerated, although, with chronic therapy, there is the potential for hypoacidity and gastric bacterial overgrowth. in humans dose reduction is recommended in association with renal dysfunction. 21 famotidine is not an inhibitor of the hepatic microsomal cytochrome p-450 enzyme system, therefore significant drug interactions are not anticipated. activity. 40, 54 significant drug interactions associated with hepatic microsomal cytochrome p-450 enzyme system inhibition are not a clinical concern with ranitidine. 46 an adverse effect to be aware of in cats treated with ranitidine is transient hypotension associated with ranitidine administered as an iv bolus. 19 in humans dose reduction is recommended in patients with renal azotemia. 39 ranitidine is effective in decreasing gastric acid in cats. 22 ranitidine would be a logical choice in a cat with gastrointestinal ulceration and/or atony. the reported dosage recommendation for ranitidine in cats is 3.5 mg/ kg orally every 12 hours or 2.5 mg/kg intravenous every 12 hours. 19 omeprazole omeprazole is a proton pump inhibitor that targets the h + /k + atpase pump on the luminal surface of partial cells. omeprazole is effective at suppressing parietal cell acid secretion, and its effects persist for ≈24 hours after drug withdrawal because of drug accumulation in the parietal cell (by ion trapping). indications for omeprazole therapy are for the treatment and prevention of nonsteroidal antiinflammatory drug (nsaid)-induced ulcers. 9 omeprazole is enteric coated to prevent its degradation by gastric acid; therefore oral formulations should not be crushed. based on human studies, omeprazole is a hepatic microsomal cytochrome p-450 enzyme inhibitor with known drug interactions with diazepam. 2 the extent of clinically significant drug interactions in cats has yet to be studied. omeprazole is reported to be effective in reducing gastric acid secretion in cats. 22 the recommended empirical dosage in cats is 0.5 to 1 mg/kg orally once daily. long-term use in humans 33 and dogs 11 is associated with gastric polyps and parietal cell hyperplasia, respectively, but the effect of long-term use in cats is currently unknown. sucralfate is a disaccharide complexed with aluminum that dissociates to sucrose octasulfate and aluminum hydroxide upon exposure to gastric acid. the sucrose octasulfate spontaneously polymerizes, producing a viscous material capable of binding ulcerative lesions in the gastric mucosa. once bound to the exposed mucosa, it prevents back diffusion of h + , inactivates pepsin, absorbs bile acids, and increases mucosal prostaglandin synthesis, collectively supporting ulcer healing. sucralfate is not systemically absorbed but does prevent the absorption of drugs capable of chelating with aluminum, including fluoroquinolones, tetracyclines, and digoxin. if sucralfate is indicated in a cat being treated concurrently with fluoroquinolones, tetracyclines, or digoxin, the recommendation is to administer the other drug 2 hours prior to the administration of sucralfate to optimize drug absorption. clinical indications for the use of sucralfate in cats are for the treatment of gastric ulcers and esophagitis. 36 dosage recommendation in cats is 250 mg orally every 12 hours. sucralfate can be crushed, suspended in water, and administered as slurry. diet trials are used in some cats with diarrhea if the underlying cause is from known or suspected food hypersensitivities. dietary management includes either a highly digestible diet, an elimination (novel protein/ carbohydrate or hydrolyzed protein) diet (see above for both), or a diet high in fiber. 72 high-fiber diets contain a mixture of both soluble and insoluble fiber that can be beneficial in patients with signs of large bowel diarrhea. insoluble fiber, such as cellulose, functions to increase the bulk of the stool, bind fluid, and regulate intestinal motility. soluble fiber, including fruit and vegetable pectins and beet pulp, functions as a source of butyric acid that can be used by the colonic mucosa and decreases proinflammatory cytokines. 69, 72 cobalamin cobalamin (vitamin b 12 ) is an essential vitamin needed by a number of different enzymes, including key enzymes involved in methionine metabolism and the conversion of methylfolate to tetrahydrofolate needed for dna synthesis. cobalamin and folate are intimately linked, and hypocobalaminemia can lead to a functional deficiency of folate. 57 ingested cobalamin requires intrinsic factor binding for enterocyte absorption at the level of the ileum. hypocobalaminemia is commonly associated with distal small intestine diseases in cats, including inflammatory bowel disease. in addition, low cobalamin has a negative impact on enterocyte function; therefore in many cats with intestinal disease and hypocobalaminemia, cobalamin supplementation is necessary for resolution of clinical signs. 60, 64 quantification of serum cobalamin levels is recommended in cats with clinical signs of small bowel diarrhea, ones suspected to have an infiltrative disease of the small intestine (inflammatory bowel disease or gastrointestinal lymphoma), or ones with pancreatic dysfunction. when hypocobalaminemia is identified, supplementation is recommended mannanoligosaccharides, inulin, chicory, and lactosucrose. 72 reports on the use of prebiotics in cats are limited to their use in healthy cats; healthy cats fed fructooligosaccharides were reported to have a trend toward an increase in fecal concentrations of lactobacilli and a decrease in concentration of c. perfringens and e. coli relative to the controls. 65 to date no reports are available on the use of prebiotics in cats with gastrointestinal disease. probiotics and prebiotics potentially have a supportive role in the treatment of gastrointestinal disease in cats. the important clinical consideration in the use of probiotics as an adjunctive therapy is to ensure the use of live nonpathogenic microorganisms that have been documented to colonize the intestinal tract of cats. gastrointestinal flora co-evolve with their host. gastrointestinal microorganism colonization varies among species and within each individual animal. the distribution of fecal microflora for a given individual is considered unique but stable over time. 68 antimicrobial and antiparasitic therapies for the treatment of feline diarrhea are indicated based on the specific diagnosis of infectious diarrhea, bacterial enteritis, or as adjunctive therapy for inflammatory bowel disease. infectious pathogens more commonly associated with feline diarrhea include bacterial enteropathies (clostridium, campylobacter), protozoal enteropathies (tritrichomonas foetus, giardia spp.), and helminthic enteropathies associated with ascarids, hookworms, whipworms, and tapeworms. only the more common anthelminthic, antimicrobial, and antiprotozoal therapies are discussed below (tables 23-6 and 23-7) . more information about antimicrobials and antiparasitics is found under specific infections in the discussions of infectious enteritis and gastrointestinal parasites. fenbendazole is an anthelmintic used to treat common helminth infections, including ascarids, hookworms, whipworms, and a single species of tapeworm, taenia pisiformis. giardia spp. are also considered susceptible to fenbendazole. fenbendazole binds beta-tubulin subunits of microtubules, interfering with their polymerization. side effects include vomiting and diarrhea, although both are considered rare. fenbendazole is not approved for use in cats in north america but is commonly used clinically, and an empirical dosage of 50 mg/kg (250 µg/cat every 7 days) while the underlying cause of cat's malabsorption is being investigated and at initiation of targeted therapy. probiotics probiotics are ingested live microorganisms intended to benefit the host, specifically to support the microflora environment of the gastrointestinal tract as well as to provide an overall benefit to the body's immune function by immunomodulation. 8, 18, 51 probiotics chemically modify ingesta and intestinal mucus, as well as affect immune cells, enterocytes, and goblet cells within the intestinal mucosa through direct receptor interactions and indirectly through the action of cytokines. the microorganisms commonly used are nonpathogenic bacteria and yeast that have a vital role in gastrointestinal health, including lactobacillus spp., enterococcus faecium, bifidobacterium spp., and saccharomyces spp. for example, lactobacilli synthesize b vitamins, digestive enzymes, and folate coenzymes. 63 clinical indications for the use of probiotics are diverse, including primary gastrointestinal disease, chronic renal disease, and pancreatitis. 71 the rational use of probiotics in the treatment of gastrointestinal diseases include their ability to modulate gastrointestinal flora, minimize colonization by pathogenic bacteria, and decrease the likelihood of bacterial translocation. 17 in healthy cats, lactobacillus acidophilus is reported to reduce fecal clostridium counts. 45 when lactobacillus acidophilus was used adjunctively with antimicrobial therapy, fecal shedding of campylobacter was reduced in cats with campylobacter-induced diarrhea relative to cats treated with antimicrobials alone. 3 specifically, in cats with gastrointestinal disease, available research supports the probiotic enterococcus faecium as clinically beneficial in resolving diarrhea in kittens. 16 relative to the control group, the kittens treated with probiotics had increased fecal bifidobacteria and blood iga concentrations and decreased fecal counts of clostridium perfringens. prebiotics are dietary supplements used to select for the more beneficial enteric flora, support gastrointestinal function, and prevent the overgrowth of pathogenic bacteria, including salmonella, escherichia coli, clostridium, or campylobacter. for a food additive to be considered a prebiotic, it must be nondigestible by the gastrointestinal tract (resistant to gastric acidity, gastrointestinal hydrolysis and absorption), yet fermentable by gastrointestinal microflora to short-chain fatty acids to stimulate the growth of "good" intestinal bacterial. 72 prebiotics include nondigestible oligosaccharidescommonly, oligofructose, fructo-oligosaccharides, pyrantel pamoate is a nicotinic anthelmintic used primarily for the treatment of ascarids, but its spectrum of activity also includes hookworms and the stomach worm, physaloptera spp. pyrantel is toxic to susceptible parasites through its selective action on their nicotinic acetylcholine receptors, resulting in depolarization and spastic paralysis. pyrantel is not approved for use in cats but is considered safe in cats and is commonly used clinically. the dosage recommendation in cats is 5 mg/ kg orally once, repeat in 3 weeks, and finally repeated in 3 months. metronidazole is a nitroimidazole antibiotic with an anaerobic antibacterial spectrum with antiprotozoal activity against giardia spp. in an anaerobic environment, metronidazole is converted to unstable intermediates (nitroso free radicals) that disrupt bacterial dna synthesis. immunomodulatory properties capable of inhibiting cell-mediated immunity have been described for metronidazole, although its immunomodulatory properties are reported at dosages well beyond what is recommended for clinical use, 62 raising questions about the clinical use of metronidazole as an adjunctive therapy for treating inflammatory bowel disease. 34, 43 resistance to metronidazole is considered rare. 43 the most common adverse reaction is gastrointestinal upset, including inappetence, anorexia, nausea, and vomiting. profuse salivation can occur in cats after oral administration of metronidazole base (formulation used in standard tablets), which has lead to the use of metronidazole benzoate (a compounded formulation not approved by the food and drug administration) in some cats because of its better oral palatability. 61 at high doses (>200 mg/ kg/day) benzoic acid is reported to be neurotoxic in cats, but with appropriate clinical dosing of metronidazole benzoate benzoic acid toxicity is unlikely. 6 dose-related metronidazole toxicity in cats results in cerebellovestibular ataxia secondary to gamma-aminobutyric acid (gaba) inhibition at dosages greater than or equal to 58 mg/kg/day 12, 52 ; clinical signs include nystagmus, head tilt, ataxia, seizures, and obtundation. in cats with inflammatory bowel disease, the dosage recommendation for the metronidazole base is 10 to 15 mg/kg/day. metronidazole benzoate contains approximately 60% metronidazole base by weight, translating to an empirical dosage of 20 mg/kg/day of metronidazole benzoate (equivalent to 12.4 mg/kg/day of metronidazole base). 61 little is known about the safety of chronic metronidazole use in cats, but oral metronidazole has been reported to disrupt dna within feline peripheral mononuclear cells following 7 days of therapy. 61 this metronidazole-induced genotoxicity is reversible and is no longer detected 6 days after antibiotic therapy is discontinued. ronidazole is a nitroimidazole antibiotic (similar to metronidazole) and available as a powder-on-feed antibiotic. ronidazole is not approved for use in cats but has immunosuppressive therapies used in cats with inflammatory bowel disease include glucocorticoids, cyclosporine, and chlorambucil (tables 23-8 and 23-9 ). more information on the treatment of inflammatory bowel disease is found elsewhere in this chapter. glucocorticoids are considered first-line therapy in the treatment of cats with inflammatory bowel disease. glucocorticoids bind their intracellular glucocorticoid receptors, modifying the expression of genes with glucocorticoid response elements. immunomodulation is achieved through inhibition of cytokine release and response, including decreasing leukocyte phagocytosis, chemotaxis, and antigen expression. the more common side effects in cats include gastrointestinal ulceration, opportunistic infections (e.g., urinary tract infections), pancreatitis, and diabetes mellitus. cats are less susceptible to iatrogenic hyperadrenocorticism than dogs. initial therapy is usually with oral prednisone or prednisolone. prednisone is a prodrug that is metabolized to its active form prednisolone. cats are reported to be less efficient in the conversion of prednisone to prednisolone 27 ; therefore prednisolone may be preferred in cats, especially in cats refractory to prednisone therapy. been used off-label to effectively treat tritrichomoniasis in naturally and experimentally infected cats (30 mg/kg orally every 12 hours for 14 days). 25 t. foetus reduces nitroimidazoles to their nitroso free radicals. ronidazole has been reported to have better in vitro and 10-fold higher in vivo activity against t. foetus relative to metronidazole. 25, 35, 49 ronidazole resistance is beginning to be reported in t. foetus isolates from cats with diarrhea. 26 side effects include hepatoxicity and neurotoxicity. neurotoxicity is associated with high doses and has been reported in cats. 59 the use of ronidazole is recommended only for confirmed cases of t. foetus, and dosing should not exceed 30 mg/kg once daily in cats, especially in cats at risk for neurotoxicity. ronidazole is not registered for human or veterinary use in the united states; therefore its use in cats requires owner informed consent and client education of the potential human hazards. immunosuppressive therapies are considered the standard of care for cats with gastrointestinal biopsies consistent with inflammatory bowel disease (lymphoplasmacytic or eosinophilic inflammation). the common alternative forms of glucocorticoids can be considered in specific patient populations. in patients with severe malabsorption, injectable dexamethasone may provide improved bioavailability and clinical response. also dexamethasone maybe preferred in patients with a history of heart failure, fluid retention, or hypertension because of its lack of mineralocorticoid activity relative to prednisone/prednisolone. dexamethasone's potency is 4 to 10 times that of prednisolone; therefore a dose reduction is necessary when prescribing dexamethasone (the dexamethasone dose is one seventh that of prednisolone). 4, 10 budesonide is an oral, locally active, highpotency glucocorticoid that is formulated to be released in the distal gastrointestinal tract (based on the ph differential between the proximal and distal small intestine), where it is absorbed and is locally immunomodulating at the level of the enterocyte. the amount of systemically absorbed budesonide is minimized, because 80% to 90% of the budesonide absorbed from the gastrointestinal tract undergoes first-pass metabolism in the liver. some systemic absorption does occur, as evidenced by a blunted adrenocorticotropic hormone (acth) stimulation test in dogs treated with budesonide at 3 mg/m 2 for 30 days. 66, 70 the use of budesonide in cats remains anecdotal, with a suggestive empirical dose of 0.5 to 1 mg/cat/day. initial glucocorticoid therapy for cats with inflammatory bowel disease consists of antiinflammatory (0.5 to 1 mg/kg/day) to immunosuppressive (2 to 4 mg/kg/ day) dosages, with dosages based on the potency of prednisone/prednisolone. the goal of therapy is to achieve clinical remission and slowly taper the dose of glucocorticoids to the lowest dose that will control the cat's clinical signs. 67 some cats may be completely weaned off therapy, while others require long-term lowdose therapy. the tapering of therapy should be slow, with a 25% to 50% dose reduction every 3 to 4 weeks. cyclosporine is considered a second-tier immunosuppressive drug used to treat inflammatory bowel disease in cats. use of cyclosporine in the treatment of diarrhea associated with inflammatory bowel disease in cats is extrapolated from its use in dogs to treat glucocorticoid refractory inflammatory bowel diarrhea. 1 cyclosporine suppresses t-lymphocyte-mediated inflammation in the gastrointestinal tract secondary to suppression of inflammatory cytokines. specifically, cyclosporine attenuates t-lymphocyte activation and proliferation through the inhibition of interleukin-2 (il-2) production. side effects of cyclosporine in cats include dose-dependent inappetence and vomiting, which may occur at the onset of therapy and are generally responsive to dose reduction. other less common side effects reported in cats are opportunistic infections, including toxoplasmosis 5 and hepatoxicity. the microemulsion formulation of cyclosporine has higher oral bioavailability and less variable pharmacokinetics. 58 a suggested initial dosage of cyclosporine is 4 mg/kg every 12 or 24 hours. serum cyclosporine levels can be used to monitor for excessive trough plasma concentration (>400 ng/ml) as determined using a highperformance liquid chromotography (hplc) analytical method. 53 chlorambucil is a slow-acting nitrogen mustard that alkylates and effectively cross links dna, leading to altered protein production. the immunosuppressive effects of chlorambucil are the result of its cytotoxic effect on lymphocytes, similar to other nitrogen mustards. bone marrow suppression is considered mild to moderate and is rapidly reversible. neurotoxicity and myoclonus has been reported in a cat accidently overdosed with chlorambucil. 7 chlorambucil is used as a second-tier drug in cats to treat immune-mediated disorders, in part because of ease of administration and its low risk of myelosuppression. for the treatment of inflammatory bowel disease, the recommended dosing in cats is 2 mg/cat every 48 hours in cats greater than 4 kg and 2 mg/cat every 72 hours in cats less than 4 kg. 50 chlorambucil is commonly used in combination with glucocorticoids in the treatment of immune-mediated diseases, including inflammatory bowel disease, 48 overlooked. awareness about feline esophageal diseases is low, the clinical signs are often not specific, and imaging beyond survey radiographs may be required for diagnosis. the esophagus is composed of four layers (from inner to outer): mucosa, submucosa, muscularis, and adventitia (there is no serosal layer). in the dog, the muscle layer is entirely composed of skeletal muscle, but in cats, the distal third of the esophagus is composed of smooth muscle. the upper esophageal sphincter prevents reflux of esophageal contents into the pharynx and minimizes aerophagia. the lower esophageal sphincter prevents gastroesophageal reflux and relaxes during swallowing to allow food and fluid to enter the stomach. clinical signs of esophageal disease include drooling, dysphagia, pain on swallowing (odynophagia), and, most classically, regurgitation. weight loss may occur secondary to inadequate food intake when disease is severe or chronic. other clinical signs, such as anorexia, cough, dyspnea, and fever, may occur if complications such as aspiration pneumonia or esophageal perforation occur. regurgitation is passive expulsion of food or fluid from the esophagus. the food is undigested and often accompanied by mucus and saliva. mucosal erosions may produce frank blood in the regurgitated material. regurgitation must be differentiated from vomiting (table 23 esophageal disease is uncommon in the cat when compared with dogs, but it is also likely that problems such as esophagitis and esophageal strictures are often salivation, retching, and abdominal contractions. the vomitus consists of partially digested food from the stomach and/or intestines and may be mixed with bilestained fluid. some cats will have both vomiting and regurgitation. expectoration may also be confused with vomiting or regurgitation. expectoration is associated with coughing, but cats that cough excessively may also stimulate vomition so that a careful history is needed to characterize the clinical signs correctly. coughing may also occur in cats that have aspirated as a result of regurgitation. drooling, dysphagia, and odynophagia are most commonly seen with conditions of the oropharynx and/or proximal esophagus. odynophagia is most commonly associated with esophagitis and foreign bodies. dysphagia and regurgitation together most commonly indicate oral or pharyngeal dysfunction; if regurgitation is not accompanied by dysphagia, esophageal dysfunction is likely. 55 regurgitation in cats with esophageal disease is caused by obstruction or muscular dysfunction. causes of obstruction include vascular ring anomaly, foreign object, stricture, and neoplasia. causes of muscular dysfunction include congenital disease, esophagitis, myopathies, neuropathies, and dysautonomia. regurgitation may occur immediately after eating if the lesion is in the proximal esophagus. however, a dilated esophagus provides a reservoir for food and fluid so that regurgitation may not be associated in time with eating. young cats with signs of esophageal disease should be suspected of congenital defects, such as vascular ring anomaly, or a foreign body. adult cats with esophageal disease may have a recent history of general anesthesia, administration of certain oral medications, or ingestion of irritant chemicals. acute onset of clinical signs may suggest a foreign body, while chronic, slowly worsening signs may indicate a stricture or tumor. all cats suspected of esophageal disease should have a minimum database as part of the diagnostic plan (complete blood cell count, serum chemistries, urinalysis, and other tests as indicated by age or concurrent diseases, such as serum total t 4 and blood pressure measurement). an important part of diagnosis is observation of the cat while eating food, to localize the location of the dysfunction. if the cat is unwilling to eat while in the veterinary clinic, the owner can make a video of the cat eating at home for the clinician to view. the general diagnostic approach to regurgitation in cats is found in figure 23 -3. plain and contrast radiography and endoscopy are important diagnostic tools for esophageal disease. fluoroscopy is valuable for the diagnosis of motility disorders, but availability is limited to universities and referral centers because of the cost of equipment. ultrasonography is limited to evaluation of * references 1, 8, 11, 15, 38, 43. the cervical esophagus and a small segment of abdominal esophagus between the cardia of the stomach and the diaphragm. the entire esophagus should be evaluated with cervical and thoracic radiographs. thoracic radiographs may also show evidence of complications such as aspiration pneumonia or esophageal perforation. the normal esophagus is not visualized on plain radiographs, but may be seen if food or fluid are retained or a foreign body or mass is present. radiographic contrast agents useful for esophagrams in cats include liquid or paste barium. a water-soluble iodinated contrast agent (e.g., iohexol, gastrografin) is preferred if there is any risk the esophagus is perforated, because these agents are less irritating and more rapidly reabsorbed. esophagrams are most useful for diagnosis of luminal obstructions, extraluminal compression, mucosal irregularities, and possibly alterations in motility. dilute liquid barium can be administered with a syringe or it may be mixed with canned food, especially if a motility disorder or stricture is suspected. multiple lateral radiographs are taken rapidly, starting within 10 seconds of swallowing the contrast agent. contrast is rapidly cleared from the normal esophagus by peristalsis. if the contrast in the esophagus terminates abruptly, an obstruction is likely. if the contrast is retained throughout the esophagus, muscular dysfunction is suspected. some conditions, such as esophagitis, are difficult to diagnose radiographically, because contrast agents may or may not adhere to ulcerated mucosa. flexible endoscopy is a noninvasive diagnostic tool for esophageal disorders and is often used if plain and contrast radiographs have failed to establish a diagnosis. it is most sensitive for diagnosis of masses, ulcers, perforations, and obstructions. in addition, it is often possible to retrieve foreign bodies using endoscopy as well as to assist with dilatation of strictures or placement of gastrostomy feeding tubes if required. biopsy of the esophageal mucosa is more difficult than biopsy of gastric or intestinal mucosa and is not commonly performed with the exception of mass lesions. esophagitis may result from various causes of inflammation, such as contact irritation from foreign bodies (including trichobezoars lodged in the esophagus), chemical irritants or caustic medications, gastroesophageal reflux, persistent vomiting, hiatal hernia, or general anesthesia. inflammation disrupts the esophageal mucosa and exposes the submucosa. an important part of the treatment plan is identification and treatment of the underlying cause. clinical signs include dysphagia, regurgitation, salivation, and repeated swallowing, although signs may be absent in cats with mild esophagitis. cats with odynophagia may repeatedly extend the head and neck while swallowing. if the esophagitis or underlying disease is severe, weight loss and dehydration may occur secondary to anorexia. if the submucosa and muscularis are damaged, strictures may form as a result of the production of fibrous connective tissue and compromise the esophageal lumen. 54 neoplasia is an important cause of esophageal stricture in humans, but not in cats. most cases have single strictures, but multiple strictures are possible. in two studies, the mean stricture diameter was reported as 5 mm. 26, 32 most strictures are less than 1 cm in length. clinical signs associated with strictures appear 5 to 14 days after the esophageal injury and may be present for weeks before definitive treatment is pursued. regurgitation typically occurs immediately after eating, although if the stricture is long standing, a pouch may form cranial to the lesion where food accumulates. survey radiographs may be normal in cats with esophagitis and strictures, but are useful to rule out other causes for the clinical signs, such as a foreign body, or to detect related problems, such as aspiration pneumonia. in some patients, dilation of the esophagus with fluid or air may be seen. 45 a contrast esophagram may disclose irregularities of the mucosa in cats with severe esophagitis. segmental dilation may occur with severe inflammation. strictures may be diagnosed with an esophagram (figure 23-4) ; however, in some cases, it may be difficult to differentiate a stricture from intramural thickening (e.g., because of neoplasia). endoscopy is useful for diagnosis of esophagitis; findings include mucosal erythema, hemorrhage, and erosions or ulcerations. if gastroesophageal reflux is present, the lesions will be most severe in the distal esophagus, and the lower esophageal sphincter may be dilated. endoscopy is often used for definitive diagnosis of esophageal stricture as well as to visualize the lesion during treatment by bougienage or balloon catheter dilation. strictures appear as a ring of white fibrous tissue that narrows the esophageal lumen. if endoscopy is performed after a barium esophagram, 24 hours should be allowed to elapse between the procedures or the barium will obscure visualization with the endoscope. 19 general anesthesia is an important cause of esophagitis (sometimes leading to stricture formation) in cats, probably because gastroesophageal reflux appears to occur commonly in anesthetized cats.* for example, in a series of seven cats with benign esophageal stricture, recent anesthesia for ovariohysterectomy was the suspected cause in five cases. 1 clinical signs appeared up to 21 days after anesthesia. abnormal esophageal tissue was performed in two cases. the authors noted that the esophageal mucosa may appear grossly normal, but submucosal inflammation may be found on histopathologic examination of biopsies. a consequence of chronic severe gerd in humans is the development of metaplastic columnar epithelium (barrett esophagus) that replaces the normal squamous epithelium. one case series reported on barrett-like esophagus in three cats. 23 two cases were associated with hiatal hernia and one with cardial incompetence. drug-induced esophageal damage and stricture formation is well known in humans and cats (see . in humans over 70 drugs have been implicated, and most are antibacterials or nsaids. 30 implicated drugs in the cat include tetracycline, doxycycline, and clindamycin in tablet or capsule form administered without a food or water bolus. 4, 17, 32, 36, 37 clinical signs (dysphagia, regurgitation, salivation, anorexia) appear 3 to 16 days after drug treatment is started. strictures commonly form in the midcervical esophagus or over the heart base in the thoracic esophagus. doxycycline hyclate is most commonly associated with esophageal strictures in cats, and the principle reason for its irritating properties is an acidic ph. the monohydrate salt of doxycycline is less irritating and is marketed as tablets and a palatable paste licensed for use in dogs and cats in some countries. 48 in humans esophageal ulceration after doxycycline therapy is more common than stricture formation. although the development of strictures in cats would appear to be uncommon, it seems possible the incidence of esophagitis is underestimated, because the clinical signs (e.g., odynophagia, chest pain) may go unrecognized. esophageal transit studies of normal cats have shown that the passage time of dry-swallowed tablets and capsules is often prolonged (longer than 30 seconds). 20, 53 complete entrapment (retention for more than 4 minutes) in the midcervical region occurs commonly. however, a small bolus of food or water is sufficient to ensure immediate passage of the medication into the stomach. 20, 53 the risk of esophageal retention can also be lessened by coating a tablet or capsule with butter or a gel dietary supplement (nutri-cal; vétoquinol, fort worth, tex.). 21 one study determined that tablets or capsules administered using a one-step pill gun with flavored liquid (flavorx pill glide; flavorx, columbia, md.) or a pill delivery treat (greenies pill pockets; nutro products, franklin, tenn.) ensured an average transit time of 60 seconds or less. 6 delayed esophageal transit of medications allows tablets and capsules to disintegrate within the esophagus, exposing the mucosa to irritating chemicals. cats may be at risk of delayed esophageal transit, because they do not typically drink water with medication, and they do not have an upright posture. in addition, medications are often given to sick or dehydrated patients many preanesthetic drugs and induction agents reduce lower esophageal sphincter pressure. 27, 28 other predisposing factors may be intraabdominal surgery and a head-down position on the surgery table. reflux fluid with a ph less than 4 is likely to cause esophageal mucosal damage, as is prolonged contact time. esophageal defense mechanisms include clearance of the reflux fluid by peristalsis and neutralization of the acidic ph by the bicarbonate present in saliva. in a study of 40 kittens less than 15 weeks of age, risk of gastroesophageal reflux during anesthesia was evaluated with use of a laryngeal airway mask versus endotracheal intubation. 47 gastroesophageal reflux was observed in 50% of kittens with use of the laryngeal airway mask but more importantly in 22% of kittens with endotracheal intubation. the reflux episodes occurred shortly after anesthesia induction. in a study of 50 cats anesthetized with thiopentone or propofol, gastroesophageal reflux occurred in 14%. 16 reflux also occurred shortly after anesthesia was induced and lasted for a mean of 23 minutes. it is unknown why esophageal strictures form only in a small number of cats that experience gastroesophageal reflux during anesthesia. gastroesophageal reflux disease (gerd) is a commonly reported cause of esophagitis in humans, but it is rarely reported in cats when not associated with general anesthesia. 24, 33 the true incidence is unknown, and diagnosis may be hampered by scant knowledge about the clinical presentation and diagnosis. clinical signs and diagnostic procedures are as for other causes of esophagitis. in one case series of three cats, diagnosis of gerd was based on clinical signs, contrast radiography, and endoscopic findings. 24 biopsy and histopathology of obtained from a compounding pharmacy in most countries and can only be given orally. h 2 -receptor antagonists are competitive inhibitors that block parietal h 2 receptors and decrease the amount of gastric acid produced. proton pump inhibitors are noncompetitive inhibitors that act on the h + /k + atpase enzyme system at the secretory surface of gastric parietal cells. they are considered superior for decreasing gastric acid secretion and are therefore the first choice, despite their greater cost. 45 a drawback of proton pump inhibitors is that they must be administered orally. sucralfate may be beneficial for reflux esophagitis, because it binds to mucosal erosions in an acid environment and provides a protective barrier. it is given as oral slurry, ideally separate from meals or other medications. antibiotics are not commonly recommended unless aspiration pneumonia is present or the eroded mucosa is at risk of bacterial infection in a patient with severe disease or a compromised immune system. corticosteroids are often recommended for cats with esophagitis to reduce esophageal inflammation and impair the formation of fibrous connective tissue. however, the benefit of corticosteroids in cats with esophagitis has not been investigated and administration must be weighed against potential adverse effects, especially in patients with aspiration pneumonia. treatment of esophageal stricture typically requires dilation with either bougienage or a balloon catheter; both are used with endoscopic visualization under general anesthesia. appropriate analgesia should be provided, because dilating the stricture is painful. it does not appear that placement of a gastrostomy feeding tube is specifically required to recover from dilation procedures, although a tube may be placed in some anorexic cats to ensure nutritional intake and administer oral medications. a bougie is a long, narrow, oblong, mechanical dilator available in various sizes (typically 9-to 12-mm sizes are used in cats) that is gently passed through the stricture, usually over a guide wire. established criteria for selection of bougie diameter and dilation end points are not available. in one study, the initial bougie chosen was approximately the same size as the estimated diameter of the stricture, or no more than 2 mm larger. 8 once the first bougie is passed, subsequent bougies of increasing diameter are employed. two to four bougies of increasing size may be passed in a single session, with the goal of dilating the stricture without causing esophageal tear or perforation. determining when dilation should be stopped is a matter of clinical judgment. the procedure may be repeated as needed to maintain improvement; the total number of procedures required is variable. in one retrospective case series of eight cats treated with bougienage, the median number of procedures was 4.5, and a good outcome was achieved in 75% of the cases. 8 in some cases, the endoscope tip itself has been used for that may be at greater risk of esophageal retention of medication. all oral medication given to cats in tablet or capsule form should be followed with food or a liquid. mild esophagitis will resolve on its own, especially if an underlying cause can be removed or treated. frequent meals of canned food should be provided. cats with moderate to severe esophagitis will require medical therapy, and those with difficulty eating or weight loss may also require gastrostomy tube feeding. esophagostomy or pharyngostomy feeding tubes should be avoided in these patients. treatment is provided to control inflammation and promote healing while reducing gastric acid secretion and increasing lower esophageal sphincter tone. the length of medical treatment will vary from about one week to several weeks, depending on the underlying cause and severity of disease. medications indicated for esophagitis include prokinetics, h 2 -receptor antagonists, proton pump inhibitors, and sucralfate (table 23-11) . prokinetic drugs enhance gastric emptying and increase lower esophageal sphincter tone. metoclopramide also has antiemetic effects, which may be beneficial in patients with chronic vomiting. it can be administered by the subcutaneous (sc) route, an advantage in a vomiting or regurgitating patient. cisapride may be more effective at enhancing both gastric emptying and lower esophageal sphincter tone, but it must be stent placement has recently been described in cats with esophageal strictures with variable results. a 1-year-old cat presented with a 4-week history of dysphagia and regurgitation caused by a single cervical esophageal stricture after treatment with oral clindamycin. 18 guided balloon dilation was performed 6 times over a period of 3 weeks, but stricture formation always recurred. a self-expanding metal stent was placed using endoscopy and fluoroscopy after another dilation procedure. the cat did well eating a canned diet from an elevated position for 10 months, but by 12 months, the cat was no longer able to eat even liquid food and was euthanized. on necropsy, the stent had migrated and was obstructed by swallowed hair. in another case, a biodegradable self-expanding stent was used to successfully treat an 11-year-old cat that presented with a stricture in the cervical esophagus after anesthesia for dentistry. 3 balloon dilation was performed twice, but regurgitation recurred 5 days after the last procedure. the stricture was dilated a third time with a balloon catheter, and a tubular selfexpanding polydioxanone stent was placed with fluoroscopic guidance. the life span of the stent was estimated to be 10 to 12 weeks, sufficient time to allow healing of the esophagus. foreign bodies are less commonly found in the esophagus of the cat than in other gastrointestinal locations. reported foreign bodies include string, needles, fish hooks, and bones. trichobezoars may cause obstruction when they become lodged in the esophagus during vomiting ( figure 23 -5). recurrent esophageal trichobezoars have been infrequently reported in the literature. 12, 51 it is not known if an esophageal motility disorder is the underlying cause for recurrent obstructions. in one case, an esophageal diverticulum developed in association with recurrent trichobezoars. 12 treatment for recurrent trichobezoars includes prokinetic drug therapy (e.g., cisapride), moderate to high-fiber diets, and shaving of long-haired cats. common areas for foreign bodies to lodge include the thoracic inlet, the heart base, and the esophageal hiatus in the diaphragm. 5 obstruction of the esophageal lumen may be complete or partial. clinical signs include acute onset of gagging, salivation, repeated swallowing, dysphagia, and regurgitation. however, chronic esophageal foreign bodies have been reported in cats with dysphagia, intermittent regurgitation, and weight loss over a period of weeks or months. 2 bougienage when bougies or balloon catheters were not available. balloon catheter dilation has become a popular method in recent years. 26, 32, 38 although some clinicians feel this is a safer procedure than bougienage, there is no data in the literature to support this assumption. the catheter can be placed through the endoscope biopsy channel, alongside the endoscope, or with the aid of a preplaced guide wire. as for bougienage, established criteria for selection of balloon diameter and dilation end points are not available, and the clinician's best judgment must be used. various balloon sizes are available; in one study, the size was selected so that the inflated diameter was 4 mm larger than the stricture diameter. 32 the balloon is passed into the stricture with endoscopic guidance. it is then inflated to a predetermined pressure for 1 to 2 minutes to stretch the stricture, usually with saline, but contrast agents may also be used if fluoroscopy is used. as for bougienage, some cases may require more than one dilation procedure (typically two to four). cuffed endotracheal tubes are not appropriate substitutes for balloon catheters. regardless of the method used, after the dilation procedure, the endoscope should be used to look for other strictures and should be passed into the stomach to look for potential causes, such as causes of chronic vomiting. after treatment, medical management to decrease ongoing gastroesophageal reflux, resolve inflammation, and prevent further stricture formation should be instituted (as described previously). most cats are able to eat the day following the dilation procedure. corticosteroid treatment after dilation is controversial, and no controlled studies in animals are available. antibiotics are not routinely recommended. the prognosis for cats undergoing esophageal dilation is generally good based on the ability to eat canned food with minimal episodes of regurgitation. however, published studies show 10% to 30% of cats died or were euthanized despite multiple episodes of dilation, and up to 30% could only be fed liquid diets. 1, 8, 32, 38 even among cats with good outcomes, a return to a dry kibble diet may not be possible. the dilation technique employed may be dictated by the clinician's experience, the equipment available, and the cost. potential complications of both methods include esophageal tear or perforation, hemorrhage, infection, and aspiration. esophageal tears or perforations may lead to pneumothorax or pneumomediastinum. repeated stricture formation is also possible, leaving only less desirable treatment options, such as long-term percutaneous gastrostomy tube feeding or surgery. esophageal surgery is generally avoided whenever possible, because it is difficult and invasive (requiring a thoracotomy), with risk of serious complications, such as failure of anastomosis, necrosis, and stricture formation. closure of incisions in the esophagus is following uncomplicated foreign body removal, the esophagus should be carefully inspected for lesions and bleeding before the endoscope is withdrawn. food and water should be withheld for 24 to 48 hours. supportive care includes fluid therapy and analgesia; a gastrostomy feeding tube may be required in selected cases for nutritional support. broad-spectrum antibiotics are administered to control bacterial infection and therapy for esophagitis should be instituted as described previously. careful follow-up should include evaluation for stricture formation. if an esophageal perforation has occurred, conservative management may be sufficient if the defect is small. a broad-spectrum antibiotic should be administered along with other supportive care, such as fluid therapy and analgesia. feeding through a gastrostomy tube for several days is recommended as well as close monitoring for complications such as pleuritis. large perforations require thoracotomy for surgical repair. megaesophagus is a diffuse hypomotility disorder that may be classified as congenital versus acquired or idiopathic versus secondary to other diseases. it is uncommon in cats compared with dogs. at least two dog breeds have been identified with heritable congenital megaesophagus. a heritable form of megaesophagus has been suggested for cats, particularly for siamese cats, although no detailed studies have been performed. 13, 29 it is often frustrating to determine the underlying cause of acquired megaesophagus. megaesophagus may be a manifestation of neuromuscular diseases, such as dysautonomia or myasthenia gravis (see chapter 27) . megaesophagus may also develop secondary to esophagitis from chronic vomiting or gerd. 24, 43 other uncommon causes of megaesophagus are found in the literature. one case report describes a young cat with megaesophagus secondary to a large nasopharyngeal polyp that extended into the cervical esophagus. 10 megaesophagus resolved once the polyp was removed. in another report, a young cat with diaphragmatic hernia was diagnosed with megaesophagus and gastric dilation. 31 megaesophagus resolved with medical treatment and surgical correction of the diaphragmatic defect. clinical signs are typically those of esophageal dysfunction; regurgitation is the most consistently found sign. regurgitation may not be closely related in time to eating if the esophagus is markedly distended and holds food. cats with long-standing disease may suffer from weight loss or secondary rhinitis. the appetite is typically normal or increased. additional signs may occur if systemic neuromuscular disease is present. aspiration pneumonia may cause fever, dyspnea, and cough. two case reports describe cats with idiopathic cough, mucopurulent nasal discharge, and fever may be found if aspiration has occurred. trauma to the esophagus may cause esophagitis and even esophageal stricture. perforation of the esophagus by the foreign body may lead to pneumothorax, pneumomediastinum, or pyothorax with signs of depression, anorexia, fever, and dyspnea. if the perforation occurs in the cervical esophagus, swelling, cellulitis, and drainage of serous or purulent material may be noted. many foreign bodies are readily diagnosed with survey radiographs, especially if they are radiopaque. other radiographic findings include an esophagus dilated with fluid or air. radiolucent objects may be detected with an esophagram. care must be taken when performing esophagrams on cats that may have an obstruction, because aspiration is a concern. if abnormalities that could be consistent with an esophageal perforation (e.g., periesophageal gas or fluid, pleural effusion) are detected on survey radiographs, an aqueous iodine contrast solution should be used. removal of esophageal foreign bodies should be performed as soon as possible to minimize esophageal trauma and pressure necrosis. endoscopy can be used to confirm the diagnosis and often to remove the object. both rigid and flexible endoscopes may be used along with accessories such as various forceps and foley catheters. care should be taken to remove the object as atraumatically as possible, especially if the object is sharp or pointed. if the object is in the caudal esophagus and it cannot be grasped and removed, an attempt should be made to gently push it into the stomach, where it can be retrieved using laparotomy and gastrotomy. if esophageal perforation has occurred, esophagotomy is recommended and is described elsewhere. 5, 14 removal of fish hooks may require a combination of surgery and endoscopy. 5, 39 a surgical approach to the esophagus is made, but the esophagus is not incised; rather, the portion of the hook protruding through the esophagus is cut and removed, and the endoscope is used to retrieve the remainder. clinical sign is regurgitation, and most patients are underweight. a distended cervical esophagus may be palpated, and secondary aspiration pneumonia may occur. a history of regurgitation since weaning is very suggestive of a vascular ring anomaly, but other causes of regurgitation must be ruled out. survey radiographs show a dilated esophagus cranial to the heart, while the caudal esophagus is usually normal. the bulge of the aortic arch normally seen on a ventrodorsal radiographic view is absent. an esophagram is used to confirm the location of the obstruction and the severity of disease. definitive treatment is surgical repair of the vascular defect (i.e., ligation and transection of the ligamentosum arteriosum). some patients will require nutritional support through gastrostomy tube feeding and treatment for aspiration pneumonia before surgery. early diagnosis and surgical intervention brings the best prognosis for return of normal esophageal function. some affected cats are left with residual esophageal hypomotility, which is managed as for idiopathic megaesophagus. esophageal neoplasia is rare in the cat as in the dog. although parasitic granulomas caused by spirocerca lupi are associated with esophageal neoplasia in dogs, this parasite does not infect cats. both primary and metastatic esophageal tumors can occur in the cat. squamous cell carcinoma is the most common primary esophageal tumor in cats and is often found in the caudal two thirds of the esophagus. 7, 22, 25, 46 affected cats are middle aged or older. clinical signs are typically those associated with esophageal obstruction, such as regurgitation, dysphagia, odynophagia, and salivation. patients with advanced disease may suffer anorexia, depression, and weight loss. on physical examination, an esophageal mass may or may not be palpable. survey and contrast radiographs reveal esophageal dilation, a soft tissue mass, or periesophageal lesions that displace the esophagus. computed tomography is useful to identify periesophageal or intraluminal masses. definitive diagnosis is made with endoscopy and biopsy. mucosal biopsies are difficult to obtain, because the esophageal mucosa is tough; exfoliative cytology may also be helpful. treatment is rarely undertaken, because disease is often advanced at the time of diagnosis, and many patients have complications such as aspiration pneumonia. palliation may be attempted with chemotherapy or radiation, although data on efficacy is unavailable. in general, squamous cell carcinomas in other anatomic locations respond poorly to treatment. surgical resection may be attempted if anastomosis can be accomplished without excessive tension. megaesophagus and chronic vomiting associated with intermittent gastroesophageal intussusception. 35, 50 survey and contrast radiographs may identify a dilated esophagus (figure 23-6), but contrast fluoroscopy is the diagnostic tool of choice when available, because it allows for assessment of peristalsis. care must be taken with contrast studies because of the risk of aspiration. treatment of megaesophagus is largely symptomatic and supportive unless an underlying disorder can be identified and treated. frequent small meals are offered with the cat feeding in an upright position. the upright position should be maintained for at least 10 minutes after eating to allow for gravity-assisted passage of food into the stomach. this is best accomplished by having the owner hold the cat over their shoulder so that the esophagus is in a vertical position. 44 different types of diets should be offered to determine which is best for the individual patient; calorically dense diets may be beneficial for patients with weight loss. prokinetic drugs, such as cisapride, stimulate smooth muscle, but since most of the esophagus is skeletal muscle, the efficacy of such drugs is questionable for treatment of megaesophagus. prokinetic drugs also increase lower esophageal sphincter tone and may increase esophageal transit time, neither of which is desirable in patients with megaesophagus. vascular ring anomalies are congenital malformations of the great vessels that entrap the thoracic esophagus and cause obstruction. the most commonly reported anomaly is persistent right aortic arch. the esophagus is entrapped by the aorta on the right, the ligamentum arteriosum and the pulmonary trunk on the left, and the heart base ventrally. other vascular anomalies are rarely described in cats, such as a double aortic arch described in a siamese cat. 56 onset of clinical signs occurs around the time of weaning to solid food so that most affected cats are presented at less than 6 months of age. the most common surgery. 34 surgery is the treatment of choice for large defects, especially in young cats with congenital disease or cats that have failed medical management. various reconstructive surgical techniques have been described. 14 disorders of the hiatus are rare in cats. hiatal hernia is protrusion of the distal esophagus and stomach through the esophageal hiatus of the diaphragm into the thoracic cavity; the protrusion may be intermittent ("sliding") or persistent. other organs are occasionally involved, such as the omentum. 40 this is distinct from a gastroesophageal intussusception where the stomach is prolapsed into the lumen of the distal esophagus. 35, 49 both congenital and traumatic hiatal hernias have been described in cats. 9, 23, 41, 42, 52 congenital hernias appear to be more common than acquired hernias, and affected cats typically present with clinical signs before 1 year of age. it is suspected that increased inspiratory effort associated with upper airway obstruction, such as a nasopharyngeal polyp, may also lead to development of hiatal hernia. 23 hiatal herniation reduces lower esophageal sphincter pressure. clinical signs associated with hiatal hernia, such as intermittent vomiting and regurgitation, may be because of reflux esophagitis, hypomotility, or obstruction. large hernias and secondary aspiration pneumonia may be associated with respiratory distress. survey radiographs may reveal a gas-filled soft tissue density in the caudal dorsal mediastinum. an esophagram will show the gastroesophageal junction and gastric rugae cranial to the diaphragm (figure 23-7) . both fluoroscopy and endoscopy may be useful for diagnosis but are not typically necessary. the prognosis for cats with hiatal hernia is considered to be good. a trial of medical management (as for reflux esophagitis) for 1 month has been recommended before the stomach is a frequent site for gastrointestinal problems in cats, and the most common gastric problems are described in this chapter. some conditions such as gastric dilatation-volvulus are often reported in dogs but rarely reported in cats. in one report of three feline cases, all were associated with diaphragmatic hernia. 15 gastric parasites, the diagnostic approach to the vomiting cat, the gastric emptying time of normal cats is shorter than that of other mammals. in one study, the gastric emptying half-time for solid food in normal cats was 1.4 to 3.6 hours. 53 this implies prolonged fasting (longer than 8 hours) in preparation for anesthesia and surgery is unnecessary. the main clinical sign of gastric disease is vomiting, but it is important to note that vomiting is also associated with many nongastric problems, including concurrent intestinal disease, such as enteritis or colitis. vomiting patients therefore require a thorough physical examination and diagnostic plan to determine the cause. vomiting must be distinguished from regurgitation, which is primarily associated with esophageal disease (see table 23 -10). vomitus often contains food, hair, refluxed bile, and therapeutics for vomiting are covered elsewhere in this chapter. the anatomy of the feline stomach is similar to that of other mammals having a simple glandular stomach. most of the stomach is situated on the left side of the abdominal cavity. it has five regions, starting from the lower esophageal sphincter: cardia, fundus, body, antrum, and pylorus ( figure 23-8) . the pylorus of the cat is unique compared with other species in that it is narrow and has high resistance in order to maintain a tight seal ( figure 23 involve a wide variety of objects, including linear objects (e.g., dental floss, thread with or without a needle, tinsel, string). the owner may or may not be aware of the ingestion. ingestion of multiple foreign bodies may be seen in cats with pica ( figure 23-12 ). in one case report, a young domestic shorthair cat required gastrotomy for removal of 32 copper pennies. 43 some patients require multiple surgeries, because of repeated foreign body ingestion. 22 in such cases, a behavioral diagnosis should be sought and treatment instituted (see chapter 13) . trichobezoars (large masses of hair) also represent a type of foreign object. both long-and shorthaired cats may be affected. hair is normally ingested during grooming and is eliminated in vomitus and feces. cats lack the strong peristaltic contractions ("housekeeper" contractions) that clear the stomach of undigested contents normally found in other species. this may explain why cats seem to be susceptible to gastric trichobezoars. gastric motility dysfunction is suspected to cause repeated gastric trichobezoars in some cats. intestinal 3, 22 and esophageal 14, 59 obstruction with trichobezoars has also been documented. traditional treatments for cats with recurrent trichobezoars include regular grooming, shaving the hair coat of long-haired cats, flea control, or blood. fresh blood may appear as large or small clots. older blood clots have a brown "coffee ground" appearance. gastric bleeding may also cause melena. other clinical signs may be associated with gastric disease, such as anorexia, weight loss, pain, lethargy, bloating, and nausea. gastritis may be acute or chronic in nature, and this distinction may be useful in assessing the potential cause. for example, cats with acute gastritis may be suspected of foreign body or plant ingestion, drug or toxin exposure (see chapter 31), or dietary indiscretion. cats with chronic gastritis may be suspected of parasitism, helicobacter spp. infection, or dietary intolerance or hypersensitivity (see chapter 17) . chronic lymphocytic plasmacytic gastritis of unknown etiology is also a common cause of chronic vomiting. whenever possible, a specific underlying cause should be sought and treated. patients with sudden onset of vomiting may have an obvious cause in the history (e.g., dietary indiscretion), but in many cases, the cause is not apparent. abdominal radiographs should be taken if foreign body ingestion is possible, especially in a young cat. if the patient is systemically well, further diagnostic testing may be postponed pending response to therapy. treatment for uncomplicated acute gastritis is symptomatic and supportive. clinical signs are expected to resolve in 24 to 48 hours; if signs persist, re-evaluation and further investigation is warranted. subcutaneous fluid therapy using an isotonic balanced electrolyte solution may be used to correct mild fluid deficits (<5%). oral intake of fluids and food should be discontinued for up to 24 hours. a highly digestible diet, either commercial or homemade, is introduced with a gradual transition back to the normal diet over the next several days. antiemetic therapy may be indicated for acute uncomplicated gastritis if the vomiting is frequent or the cat has signs of nausea (see table 23 -3). protectants, such as kaolin and pectin, are difficult to administer to cats and are without proven efficacy. bismuth subsalicylate is controversial; it is considered contraindicated by some experts, because of the cat's sensitivity to salicylates, 39 yet is commonly used in clinical practice. cats ingest foreign bodies less commonly than dogs. in one study of 208 cases of gastrointestinal foreign body ingestion, only 12% were in cats. 22 foreign body ingestion is most likely to be seen in young cats and may a b taken just before surgery to ensure the object has not moved further down the gastrointestinal tract. postoperative management after gastrotomy includes maintenance of hydration and electrolyte balance. hypokalemia is common with anorexia and vomiting and should be treated by supplementation of iv fluids with 20 to 40 meq/l potassium chloride (not to exceed 0.5 meq/kg/hour). refractory vomiting should be treated with an antiemetic. a highly digestible diet can be introduced the day after surgery. in general, the prognosis for recovery is good. in one study, 88% of cats with gastrointestinal foreign bodies survived to discharge. 22 those cats that did not survive had linear foreign bodies of long-standing duration with subsequent peritonitis. helicobacter are spiral or curved gram-negative bacteria that inhabit the glands, parietal cells, and mucus of the gastric antrum and fundus. helicobacter contain large amounts of urease, which alters the ph in the vicinity of the bacteria and allows for colonization of the acidic environment of the stomach. in the early 1980s, the discovery of the association of helicobacter pylori with gastric disease (gastritis, peptic ulcers, and neoplasia) in humans revolutionized treatment of those diseases. since then, helicobacter spp. have been associated with gastric disease in various veterinary species, including cats and dogs. several helicobacter spp. (e.g., h. heilmannii, h. bizzozeronii, h. felis) have been identified in cats, some of which have the potential to infect humans, although transmission is thought to be rare. 19, 48 the prevalence of helicobacter infection in cats varies geographically and may be very high (>40%) in some locations. 1, 27, 37, 47, 58 the importance of helicobacter as a cause of gastric disease is cats is unclear; the bacteria may be found in the stomach of both clinically normal cats and cats with gastritis. the prevalence of helicobacter infection is not higher in cats with gastritis compared with normal cats. 61 determination of the role of helicobacter is also hampered by the paucity of controlled clinical trials that evaluate eradication of gastritis and clinical signs in infected cats. an immune response to infection characterized by gastric lymphoid hyperplasia is common, although the local immune response in cats is generally less severe than the response in humans infected with h. pylori. to date gastrointestinal ulcers have not been associated with helicobacter infection in cats. recent studies have suggested a possible association between helicobacter infection and gastric lymphoma in cats, although more research is needed to confirm the association and understand the pathogenesis. 7, 32 helicobacter spp. may be commensal in most cats, and perhaps loss of tolerance explains the development of gastritis in some individuals. 49 another possibility is that the inflammatory response is normally well managed and disease may treatment of underlying dermatologic disorders, and administration of semisolid petroleum laxatives. more recently, commercial diets have been formulated for control of trichobezoars. cats with recurrent trichobezoars causing illness and suspected motility disorders may benefit from treatment with prokinetic drugs such as cisapride. clinical signs of gastric foreign bodies are variable but typically involve intermittent or persistent vomiting because of gastric outflow obstruction, distention, and mucosal irritation. gastric obstruction may be partial or total. patients with complete obstruction will present with more dramatic signs, including anorexia and depression. the base of the tongue should always be examined, because linear foreign bodies are sometimes anchored either in this location, or they may be lodged in the pylorus, causing intestinal plication. gastric foreign bodies may also be asymptomatic and found incidentally. 5 physical examination may be unremarkable or may reveal dehydration or abdominal pain. if the stomach is markedly distended, the foreign body may be palpable in some patients. survey radiographs are always indicated when foreign body ingestion is suspected. radiopaque foreign bodies may be readily diagnosed, although some, along with radiolucent objects, will require a contrast study for diagnosis ( figure 23-13) . barium is commonly used as a contrast agent, although if gastric perforation is suspected, an aqueous iodinated agent is preferred. ultrasonography is also useful for detection of gastrointestinal foreign bodies. 56 removal of some foreign bodies can be attempted endoscopically, particularly if the object does not have sharp edges and is not too large. successful removal of fish hooks, particularly single-barb hooks, using endoscopy has been described. 35 otherwise, foreign objects are best removed using gastrotomy through a ventral midline laparotomy. a radiograph should always be to know when treatment should be attempted. one expert recommends treating only patients with clinical signs of gastritis that have biopsy-confirmed helicobacter infection with a treatment regimen of amoxicillin (20 mg/kg, every 12 hours, po), clarithromycin (7.5 mg/ kg, every 12 hours, po) and metronidazole (10 mg/kg, every 12 hours, po) for 14 days. 49 a common dilemma would be determining the treatment of choice for patients with lymphoplasmacytic inflammation of the stomach and small intestine and confirmed helicobacter infection. are such patients best treated for inflammatory bowel disease, helicobacter infection, or both? currently, guidelines for determining the best treatment approach are lacking. also, few studies on the efficacy of combination therapy have been conducted in cats. long-term eradication of infection may be difficult, and histopathologic resolution of gastritis may not be possible, which raises the question of whether helicobacter is the true underlying cause. 38, 41 in one study, two cats with clinical gastritis and helicobacter infection were treated with oral metronidazole, amoxicillin, and bismuth subsalicylate for 3 weeks and were also fed a commercial elimination diet. 25 posttreatment gastric biopsies were obtained a mean of 7 weeks after the cessation of treatment. resolution of clinical signs occurred rapidly, and clearance of helicobacter spp. was achieved at that time point, but gastric inflammation persisted in post-treatment biopsies. in another study, 13 cats with asymptomatic helicobacter infection were treated with oral omeprazole, amoxicillin, metronidazole, and clarithromycin for 14 days. 26 treatment failed to eradicate infection in 4 of the cats based on molecular analysis of post-treatment gastric biopsies. it is unclear if treatment failure is because of recrudescence or reinfection. the reader is referred to excellent reviews of helicobacter in cats for more information. 27, 38, 48 chronic gastritis chronic gastritis is common in cats with chronic intermittent vomiting. ollulanus tricuspis is a worm that infects the stomach of cats, causing chronic gastritis, and it is difficult to diagnose (see below, gastrointestinal parasites). the worm is occasionally found on histologic examination of gastric biopsy samples. 9 it is reasonable to treat empirically (fenbendazole 10 mg/kg, once daily, po × 2 days) for this parasite when the cause of gastritis is not apparent. 49 the frequency of vomiting in cats with chronic gastritis is highly variable, ranging from once or twice per week (and not necessarily every week) to more than once daily. most patients are otherwise well, although other clinical signs (inappetence, anorexia, depression, or weight loss) are possible depending on disease severity. results of routine laboratory testing are typically normal but may show neutrophilic leukocytosis, result when there is an abnormality of the immunoregulatory system. 21 the most commonly used methods for diagnosis of helicobacter infection in cats are based on gastric specimens obtained during endoscopy (or laparotomy): exfoliative cytology, histopathologic examination of biopsy specimens, and rapid urease testing of biopsy specimens. 28 however, it is important to note that even when helicobacter organisms are identified, the infection may not be the cause of the patient's clinical signs, and other causes of vomiting should always be evaluated. exfoliative cytology is the least expensive and most easily performed diagnostic test. in one study, it was also the most sensitive diagnostic method when compared with urease testing and histologic examination. 20 brush cytology samples gathered during endoscopy are airdried on microscope slides and stained with wright's stain. the slide is examined at 100× magnification under oil immersion. spiral bacteria are readily seen if present. at least 10 oil-immersion fields on two slides should be examined before determining a specimen is negative for helicobacter-like organisms. 28 since helicobacter produce abundant urease, a rapid urease test (e.g., clotest, ballard medical products, draper, utah) may be used for diagnosis. 38 the kit consists of an agar gel impregnated with urea and a ph indicator. a gastric biopsy sample is applied to the gel, and if urease is present, ammonia will form and change the ph (and thus the color) of the gel. the gel may change color rapidly (within 30 minutes), but 24 hours must elapse before the test can be considered negative. 28 the more rapidly the color changes, the higher the bacterial load. both false-positive and false-negative results are possible with rapid urease testing for various reasons, giving the test a sensitivity of 70% to 90%. 28, 37 histopathologic examination of gastric biopsy samples using hemotoxylin and eosin (h&e) or silver stains is highly sensitive and specific in human studies for detection of helicobacter-like organisms. the organisms are not equally distributed; so, examination of biopsy specimens from multiple sites will increase sensitivity. the bacteria may be seen in mucus on the surface epithelium as well as in the gastric pits, glandular lumen, and parietal cells. organisms may also be seen submucosally within gastric lymphoid follicles. 46 histopathologic examination of biopsy samples also allows for assessment of other abnormalities. mild to severe lymphocytic-plasmacytic or lymphocytic gastritis may be present. in humans combination therapy with antibiotics and antisecretory drugs is recommended to reduce the risk of gastric ulcers and cancer from h. pylori infection. treatment is highly successful at eradicating both clinical signs and histologic changes in the gastric mucosa. since helicobacter infection is common in cats, yet no clear pathogenic role has been established, it is difficult cases. 29 depending on the underlying cause and severity of disease, abdominal pain, anorexia, lethargy, pale mucous membranes, and drooling may also be seen. cats with neoplastic disease may have prolonged clinical signs and are more likely to present with anorexia and weight loss. 29 cats with perforated ulcers may or may not present with signs of shock. diagnosis may be problematic because the clinical signs and physical examination findings are often not specific, even in cats with perforated ulcerations. 8 the causes of gastric ulceration in cats are not well characterized. in dogs the most common cause is the administration of ulcerogenic drugs, particularly nsaids, either alone or in combination with corticosteroids. several cases of nsaid-induced gastroduodenal ulceration or perforation have been reported in cats. 8, 34, 45 additional cases may be reported in the future, because long-term administration of these drugs is gaining in popularity for treatment of chronic diseases such as osteoarthritis. nsaids cause direct mucosal damage and interfere with prostaglandin synthesis. although inhibition of the cox-1 enzyme is thought to be the cause of adverse effects, such as gastric ulceration, even cox-2-selective drugs have been associated with adverse effects, and safety in sick cats is not well evaluated. recently, guidelines for the long-term use of nsaids in cats were published by the international society of feline medicine and the american association of feline practitioners. 51 the recommendations include administering nsaids either with or shortly after food, withholding therapy if inappetence or anorexia develops, determining dose based on lean body weight, and titrating to the lowest effective dose. neoplastic causes of gastric ulceration include systemic mastocytosis, mast cell tumor, lymphosarcoma, adenocarcinoma, and gastrinoma (zollinger-ellison syndrome). cats with chronic renal disease may suffer mucosal damage from uremic toxins and increased gastric acid production secondary to hypergastrinemia (because of decreased renal metabolism of gastrin). 18 hepatic disease is a cause of gastric ulceration in dogs but is uncommonly reported in cats. 23 recent anesthesia and surgery have been implicated as a cause of gastric ulceration and perforation, perhaps through hypovolemia, hypoperfusion, or stress. 8, 29 other non-neoplastic causes reported for gastric or gastroduodenal ulceration in cats include parasites (e.g., ollulanus tricuspis, toxocara cati, aonchotheca putorii, gnathostoma spp.), bacterial infections, toxins, inflammatory bowel disease, and foreign bodies. one case report describes a cat with severe gastric ulceration caused by intoxication with dieffenbachia leaves. 36 in some case reports, the cause for the gastric ulcerations could not be determined. a minimum database should be collected for cats suspected of gastric ulceration, to identify underlying diseases. anemia, usually regenerative, may be present. eosinophilia, or hypoproteinemia. survey and contrast radiographs are often normal. the most common finding on histopathologic examination of biopsy samples is lymphocytic plasmacytic (lp) gastritis ( figure 23-14) . some patients will also have concurrent evidence of lp inflammation in the small intestine, pancreas, and/or liver. such patients will be treated for their concurrent problem; treatment of inflammatory bowel disease, pancreatitis, and cholangiohepatitis is covered elsewhere in this chapter. some cats with chronic lp gastritis respond to treatment for dietary intolerance or hypersensitivity with a limited antigen diet (see chapter 17) . patients with moderate to severe lp gastritis may be best treated with a limited antigen diet and immunosuppressive therapy (prednisolone 1 to 2 mg/kg/day, po tapering to every other day at the lowest dose that controls clinical signs). patients that fail this initial treatment approach may require additional immunosuppressive therapy, such as chlorambucil (see table 23-9) . occasionally, cats with chronic gastritis are diagnosed with eosinophilic inflammation on histopathologic examination of biopsy specimens. treatment is similar to that for lp gastritis, although such patients should be evaluated for evidence of hypereosinophilic syndrome and eosinophilic enteritis. eosinophilic fibrosing gastritis was suspected to be caused by toxoplasmosis in one case report. 33 gastric or gastroduodenal ulcerations are uncommon in the cat compared with the dog and may be caused by a variety of disorders, both gastric and nongastric. 29 classical clinical signs include vomiting, hematemesis, and melena. however, in one review of eight cats, hematemesis and melena were present in less than one third of suturing of the ulcer site as well as collection of biopsy samples for histopathologic examination. the prognosis for recovery was excellent in two studies, particularly for cats with non-neoplastic causes of gastric or gastroduodenal ulceration. 8, 29 in one study of seven cats with perforated gastric or duodenal ulcers, the survival rate was low (14%). 23 disorders of gastric motility are better characterized in dogs than in cats. the most common clinical sign is vomiting of undigested food 8 hours or more after a meal. if outflow obstruction is present, vomiting may be projectile. there may also be a history of recurrent trichobezoars. various disorders are associated with impaired gastric motility, such as chronic gastritis, drug therapy (e.g., anticholinergic and narcotic drugs), dysautonomia, gastric neoplasia, metabolic disorders (e.g., hypokalemia), and temporary postsurgical gastroparesis. in some cases of chronic motility dysfunction, no cause can be identified. outflow obstruction may be caused by neoplasia, foreign bodies, and extragastric masses. pyloric stenosis is infrequently documented in young cats, often siamese cats. 4, 40, 55 since the range of underlying disorders is diverse, the diagnostic approach should allow for detection of both gastric and nongastric disorders. a minimum database (cbc, serum chemistries, urinalysis, feline leukemia virus [felv] and feline immunodeficiency virus [fiv] serology) is used to establish overall health status. radiographs are used to confirm presence of food in the stomach for longer than 8 hours. ultrasonography may detect gastric lesions, such as masses. endoscopy is used to identify outflow obstruction as well as other lesions, such as ulcers, and evidence of gastritis. assessment of gastric emptying using nuclear scintigraphy is the most accurate method but is limited to referral centers. gastric emptying times for liquids, canned food, and dry diets have been established using nuclear scintigraphy. 11, 16, 17 however, emptying times are variable, depending on the amount and type of diet fed as well as the amount of water ingested. even the shape of kibble affects emptying time. 2 radiographic contrast series are widely used, but gastric emptying times are variable for barium in either liquid form or mixed with canned food. contrast radiography using liquid barium (8 to 10 ml/kg) is performed in a fasted patient. radiographs are taken immediately after administration of the barium and again at 15 and 30 minutes, in some cases, also at 1 and 3 hours. liquid barium is expected to enter the duodenum no more than 30 minutes after administration, and the stomach should be completely empty of barium within 3 hours. the clinician should be aware that some cats with gastric motility disorders will have other findings will be dependent on the presence of underlying diseases; for example, azotemia and isosthenuria may indicate renal disease. electrolyte and acidbase abnormalities may be because of chronic vomiting and anorexia. survey and contrast radiographs and ultrasonography are primarily useful to rule out other causes for the clinical signs, such as foreign bodies. cats with perforated ulcers may have evidence of pneumoperitoneum (sometimes severe) on plain radiographs or ultrasonographs, and this is an indication for surgical exploration. 6, 8, 24, 31, 34 evidence of peritonitis on imaging studies should be followed with peritoneal fluid analysis. a definitive diagnosis may be made using endoscopy, which allows direct visualization of lesions and collection of biopsy samples. however, some cats with gastric ulceration present in poor condition, which may preclude the use of endoscopy because of anesthetic risk and risk of ulcer perforation. 29 the location of ulcers is typically pyloroantral or fundic in cats with nonneoplastic disease. 8, 29 areas of erosion may appear pale or hemorrhagic; the mucosa is often friable and bleeds easily. fresh or clotted blood may be seen in the stomach lumen. in some cases, mucosal ulceration must be distinguished from ulcerated tumors. nsaid-induced ulcers are typically found in the antrum and do not have marked mucosal thickening; ulcerated tumors frequently have thickened edges and surrounding mucosa. 49 biopsy samples should be taken at the periphery of the ulcer to avoid perforation. treatment should be directed at any underlying disorder. treatment for nsaid toxicity is described in chapter 31. general supportive measures include fluid therapy and electrolyte replacement; blood transfusion may also be required (see chapter 25) . gastric acid production can be decreased with the use of h 2 -receptor blockers or proton pump inhibitors, and sucralfate is used as a mucosal protectant (see table 23 -5). sucralfate may inhibit absorption of other oral medications and should be given 2 hours apart from other drugs. if vomiting is severe or persistent, antiemetic therapy is warranted (see table 23 -3). analgesia should be provided for painful patients; a good choice is the opioid buprenorphine (see table 6 -1). broad-spectrum antibiotic therapy is indicated for patients with significant mucosal barrier dysfunction, perforation, leukopenia and/or neutrophilia, fever, and melena. surgical intervention is warranted for patients with life-threatening hemorrhage, failure to respond to medical management, or evidence of perforation. 29 the entire abdominal cavity and gastrointestinal tract should be thoroughly explored to locate extragastrointestinal lesions, non-perforated ulcers, and multiple ulcers. in one case series, nonperforated ulcers were detected at laparotomy by association with adhesions or a gastric mass. 29 surgical management includes débridement and months. physical examination findings are nonspecific, although occasionally a gastric mass or gastric thickening may be palpated if the stomach is markedly enlarged. results of routine diagnostic testing are generally nonspecific; anemia may be associated with ulceration. survey or contrast radiography may reveal a mass ( figure 23 -15, a); other findings include delayed gastric emptying, impaired motility, and mucosal ulceration. ultrasonography is also useful for diagnosis and can be used to guide needle aspirates of masses ( figure 23-15 , b). endoscopy allows for visualization of lesions as well as the ability to obtain partial thickness biopsy samples. problems with interpretation of endoscopic biopsy samples include detection of necrosis, inflammation, and ulceration rather than the primary lesion. in dogs some neoplastic lesions are submucosal, making it very difficult to obtain diagnostic samples by endoscopy. therefore several biopsies should be taken and masses should be biopsied multiple times in the same place to sample deeper tissues. the center of ulcerated lesions should not be biopsied. surgical biopsies are more reliable for diagnosis. a normal gastric emptying time with liquid barium. barium can also be mixed with canned food and fed as a meal; retention of barium-containing food in the stomach for more than 8 to 12 hours is abnormal. gastric emptying time may also be established with the use of barium impregnated polyspheres (bips; med i.d. systems, grand rapids, mich.) and radiography. gastric emptying times for bips have been established in healthy fasted and fed cats as well as in sedated cats, 10,52 but the values do not correlate well with scintigraphic studies. 17 a mixture of small (1.5 mm) and large (5 mm) spheres are administered with food, and two to four radiographs are taken over the next 24 hours. the small spheres are intended to mimic liquid transit time and the large spheres solid transit time. however, studies assessing the clinical relevance of this method are lacking. one review concluded that bips are probably sufficiently sensitive to detect grossly delayed gastric emptying. 60 treatment of gastric emptying disorders is directed at identifiable causes. treatment for gastric ulcers, chronic gastritis, and foreign bodies is described elsewhere in this chapter. pyloric stenosis is managed surgically. if no outflow obstruction exists, treatment with prokinetic agents, such as metoclopramide or cisapride, may be beneficial (see table 23 -3). gastric tumors account for less than 1% of malignancies in dogs and cats. 30 benign gastric tumors are even less common than gastric malignancies. gastric smooth muscle hamartoma has been reported in one 11-year-old cat. 50 although adenocarcinoma is the most common gastric cancer of the dog, lymphoma is the most common gastric cancer in the cat. feline gastrointestinal lymphoma occurs as two major types: small cell (lymphocytic) and the more aggressive large cell (lymphoblastic) form. small cell lymphomas are more frequently enteric. 57 in one study of 12 cats with gastric lymphoma, diffuse large b-lymphocyte tumors of immunoblastic nuclear type predominated. 42 gastric lymphoma is not associated with felv, and the role of helicobacter in the development of gastric lymphoma in cats requires investigation. 7 adenocarcinoma, 12,13,54 plasmacytoma, 62 and gastric carcinoid 44 have also been described. the siamese cat may be predisposed to adenocarcinoma. 12, 54 as would be expected, most cats with gastric neoplasia are older cats. as for most gastric diseases, vomiting is the most common clinical sign of neoplasia. the vomitus may contain blood and melena may be present. other clinical signs include anorexia, weight loss, bloating, and depression. perforation of the tumor may occur, leading to pneumoperitoneum or septic peritonitis. clinical signs present gradually and are often present for weeks to surgical resection is the most common treatment for gastric neoplasia other than lymphoma ( figure 23 -16). the prognosis for most patients is poor, typically because of debilitation, concurrent diseases, and recurrent or metastatic disease. 30 the success of chemotherapy for lymphoma depends on cell type, with small cell tumors carrying a better prognosis than large cell tumors. in diarrhea can be defined as increased volume and/or increased frequency of defecation of stools with increased water content. approaches to diarrhea, as for any clinical sign, need to take into account the individual animal. for example, neoplasia is much less likely to occur in a kitten than in a geriatric cat. in many cases, the precise diagnosis of gastrointestinal disease cannot be reached without biopsy samples. the decision to obtain biopsy samples should follow a logical pathway that is appropriate to the cat's condition. these are summarized in figure 23 -17. for example, many cases of acute diarrhea in a well cat can resolve with limited or no intervention, and so do not require a precise diagnosis. the diagnostic steps are 1. signalment and clinical history 2. physical examination 3. fecal assessment 4. blood and urine testing 5. imaging (radiography, ultrasonography) 6. biopsy samples these steps do not include treatment/diet trials or other empiric therapies that are appropriate in many cases. steps 3 and 4 are often undertaken at the same time, and there is no definite order for these steps. they are divided here for reasons of clarity. in a younger cat, where infectious causes are more likely, thorough fecal testing is more important; in an older cat, extragastrointestinal diseases, such as hyperthyroidism, are more likely; so, blood and urine testing is more important, but fecal assessment should not be neglected. the decision to proceed to step 4 (and each subsequent step) should take into account several considerations. the main considerations in assessing and managing a cat with diarrhea are • is there an acute onset or a chronic time course? • are there any dietary changes or indiscretion? 36 consider treatment trials: antibiotics (e.g., amoxicillin/ clavulanate ؉ metronidazole). food trials with novel proteins (e.g., rabbit, kangaroo, venison). • is the cat well or unwell? • is there primary or secondary gastrointestinal disease? • is there small or large bowel diarrhea? the components of the clinical history for cats with diarrhea are detailed in table 23 -12. after establishing the cat's age, breed, vaccination, and deworming history, it is important to establish the duration and nature of the diarrhea. chronic diarrhea is usually defined as greater than 3 weeks in duration and mostly warrants at least some degree of a diagnostic workup, whereas acute diarrhea is often self-limiting in a well cat. a description of the feces helps determine whether the diarrhea is small or large bowel in origin (table 23 -13); this will affect how any investigations might proceed. important questions to ask concern frequency of defecation (and how this compares with the normal state), tenesmus (straining usually indicates large bowel diarrhea, since an irritated colon leads to urgency), volume of feces (smaller volumes are typical of large bowel diarrhea; larger volumes are more typical of small onset and duration of diarrhea acute versus chronic? acute diarrheas are abrupt in onset and of short duration, and generally they are self-limiting. chronic diarrheas persist usually longer than 3 weeks and fail to respond to symptomatic therapy. appearance of diarrhea quantity and quality of the stool (color, consistency, character, presence of blood or mucus)? loose to watery feces that contain fat droplets, undigested food, melena, and variable colors suggests small intestinal disease. the volume is always increased with small intestinal disease. loose to semisolid feces containing excess mucus and fresh blood (hematochezia) indicates large intestinal disease. the volume may be normal to slightly decreased with large intestinal disease. description of defecation process tenesmus (straining) and dyschezia (painful defecation)? these are hallmarks of large intestinal disease (e.g., inflammatory or obstructive lesions of the colon, rectum, or anus). frequency is normal to slightly increased with small bowel disease, but greatly increased with large bowel disease. associated physical signs vomiting, anorexia, weight loss, and dyschezia may help localize the disorder to a specific part of the gastrointestinal tract. clinical signs relating to problems in other organs or body systems should be noted and may suggest a more generalized disease. vomiting may occur as a consequence of small intestinal inflammation in some cats with diarrhea. weight loss may result from decreased caloric intake (anorexia), decreased nutrient assimilation (maldigestion/malabsorption), or excessive caloric loss (protein-losing enteropathy or nephropathy). weight loss is observed uncommonly with large bowel disease. in many cases, the answers to these questions are obvious. for example, a cat may seem well but has had access to lilies (the author has seen diarrhea as a primary presenting sign for this!) or has a palpable abdominal mass. substantial weight loss is an indicator that further investigations are warranted sooner rather than later. if the decision is made for empiric management and outpatient care, it is vital to follow up either by scheduling a recheck visit or calling the client, because simple acute problems can turn into complicated chronic problems. if the diarrhea has been present for less than a week and the cat has no weight loss, dehydration, fever, or palpable abdominal abnormalities, it is appropriate to manage the cat as an outpatient. even in the absence of fecal testing, it is appropriate to deworm the cat (see the section gastrointestinal parasites). the cat should be fasted for 24 hours (12 hours, if less than 4 months old) and then fed a bland diet (such as plain, cooked, skinless chicken, or low-residue prescription diets designed for cats with gastrointestinal problems). it is appropriate to maintain the cat on the low-residue diet for at least 7 to 10 days and then slowly reintroduce the regular diet. fecal assessment is mostly used to assess infectious agents, such as parasite-associated diarrhea, but the importance of assessing feces, even when parasitic or bacterial infections are not suspected, should not be underestimated. gross examination of feces can determine if melena or fresh blood or mucus are present to help distinguish large from small bowel disease when the owner's observations may be misleading. occult fecal blood can be an indicator of gastrointestinal inflammation in cases of subtle disease, and undigested starches and fats can indicate maldigestion or malabsorption. 8 for assessment of feces for parasites, the fecal sample should ideally be fresh (<1 hour old). refrigeration (for no longer than one week) can preserve ova, oocysts, and cysts but not protozoal trophozoites. feces should be assessed by a. to assess for trophozoites bowel), how formed the stool is (from soft stool to cow-pat consistency to liquid tea; usually more watery stool relates to small intestinal disease), color (darker indicates digested blood), and presence of any mucus or blood (presence relates to large bowel). most household toxins, such as plants, cause signs additional to diarrhea such vomiting or neurological signs, 11 but it is important to ascertain if the cat has had access to anything unusual. likewise, it is important to find out if the cat has had any possible exposure to dietary indiscretions; this can include if the cat has been seen with or is known to hunt prey including insects. cockroaches carry pathogenic bacteria 12, 18 and other prey such as birds and rats can carry salmonella; salmonellosis in cats has been dubbed songbird fever. 6 simple causes of self-limiting diarrhea include dietary change (either a new flavor or a new style of food, such as dry food for the first time); so, the owner must also be quizzed if anything new has been offered, either new cat food or treats (such as greasy fish or chicken). although the physical examination will usually determine how unwell a cat is, the owner's impressions are also important, because cats can hide signs from strangers, particularly in a practice setting. lethargy and inappetence are important signs, as ill cats typically do not eat well. the cat's general demeanor can be an indicator of how unwell a cat is and therefore dictate the extent of diagnostic testing required. this can be noted by assessing how interested the cat is in its surroundings or any behavior changes from previous visits, such as if a normally difficult-to-handle cat is placid. body weight should be assessed and, if possible, compared with that of previous visits (even those noted on a clinical record from another veterinarian). the body condition score (bcs) should also be assessed and can be very important when there is no prior weight information. dehydration is usually a sign that a cat needs more involved management. abdominal palpation should be performed to assess pain (where?), any masses (foreign bodies, lymph nodes, or even focally thickened intestines, such as with neoplasia), or turgid intestines. fever often indicates infection but can also reflect neoplasia or other inflammatory changes. a thorough examination of all body systems should always be performed, no matter what a cat presents for. in the case of diarrhea, extragastrointestinal signs can be of vital importance, such as a palpable thyroid and tachycardia suggesting hyperthyroidism. after the clinical history has been taken and the physical examination performed, the veterinarian must make the important decisions of whether any interventions are required and whether the patient should be factors affecting interpretation include whether the growth is a heavy and pure growth of a known pathogen, such as salmonella, campylobacter, yersinia, or clostridium difficile. further information about the relevance of culture and pcr results is contained below in the section infectious enteritis. investigations begin by assessing if the diarrhea is the result of primary gastrointestinal disease or secondary to another process, by performing routine serum/plasma biochemistries, hematology, urinalysis, and total t 4 (for older cats). in most cases of secondary gastrointestinal disease, diarrhea is not usually the primary presenting complaint, but since the approach to investigations and management diverge so much, this is an important step to take. biochemistry and urine tests may also show the consequences of diarrhea, such as dehydration and electrolyte abnormalities. a. can aid in the visualization of internal structures of some protozoa 3. fecal flotation (preferably with centrifugation) a. to find cysts, oocysts, and ova fecal culture should be undertaken with the understanding that bacteria will be cultured; so, interpretation is based on the relevance of the positive culture result. used to evaluate the smear for the presence of trophozoites, such as giardia spp. and tritrichomonas foetus. 1. place peppercorn size amount of feces on a warm slide and mix with a drop of 0.9% saline (smear must not be too thick, because trophozoites will be easily missed). 2. apply coverslip. 3. evaluate systematically for motile organisms using the 10× magnification. 4. confirmation at 40× magnification. adding iodine to a wet mount through the edge of the coverslip can aid in the visualization of internal structures of some protozoa. the direct wet preparation must be examined without any stain for motility first, because staining the preparation kills the organism. methylene blue is useful for identifying trophozoites, particularly those of entamoeba histolytica. this method has little to no diagnostic value for the diagnosis of bacterial-associated diarrhea. used to find cysts, oocysts, and ova in feces. standing (gravitational) flotation methods are easier and quicker but have much poorer sensitivity than centrifugation methods. 2 solutions used in centrifugation flotation methods include zinc sulfate and sheather sugar. 1. weigh out 2 to 5 g of feces. 2. mix feces with approximately 10 ml of flotation solution. 3. pour mixture through a tea strainer into a beaker or fecal cup. 4. pour strained solution into a 15-ml centrifuge tube. 5. fill tube with flotation solution so that a slight positive meniscus forms, being sure not to overfill the tube. 6. place a coverslip on the tube, and put the tube in the centrifuge. 7. make sure the centrifuge is balanced. 8. centrifuge at 1200 rpm (280× g) for 5 minutes. 9. remove the tube and let stand 10 minutes. 10. remove the coverslip, and place it on a glass slide. systematically examine the entire area under the coverslip at 100× magnification (i.e., 10× objective). you may wish to use the 40× objective lens to confirm your diagnosis and make measurements; however, with practice, most parasites can be identified using the 10× objective (100× magnification). (fpli) are useful markers of intestinal and pancreatic disease, [14] [15] [16] [17] but it is important to note that they typically do not give a precise diagnosis. cobalamin and folate are water-soluble vitamins and are readily found in commercial cat foods so that dietary insufficiency is rare, and decreased levels are almost always because of gi disease. these vitamins are taken up by specific receptors in different areas of the small intestine. chronic inflammatory gastrointestinal disease may damage the receptors and lead to decreased serum concentrations of one or both vitamins, provided the disease process is severe and long standing enough to deplete body stores. serum cobalamin and folate concentrations may also be decreased in cats with exocrine pancreatic insufficiency (epi). trypsin-like immunoreactivity is a pancreasspecific marker, and assessment of serum tli is used for diagnosis of epi and pancreatitis in the cat, although the sensitivity of the assay for pancreatitis is low. pli is a marker for pancreatic inflammation and is more sensitive than tli for the diagnosis of pancreatitis. since inflammation of the small intestine may be seen concurrently with pancreatitis, serum tli and pli are useful adjunctive tests in the diagnosis of diarrhea. tli, pli, and cobalamin are stable in serum at room temperature for several days, but folate is unstable so that samples for cobalamin/folate analysis should be frozen (table 23-14) . samples submitted for folate concentration should not be hemolyzed, because red blood cells contain high levels of folate. in addition, folate is light-sensitive, and samples should be wrapped to exclude light. severe lipemia may interfere with common assays for tli and pli. the main utility of these tests are to indicate that further investigation of gastrointestinal disease is warranted. when a cat presents for weight loss with no overt signs of gi disease, decreased cobalamin or folate can indicate that further investigations with imaging and, ultimately, biopsy sampling are warranted. many clients are more willing to proceed with hematology can be normal in some cats, with changes expected, and so should not be used to rule out any condition. it can be useful, for example, if there is a left shift neutrophilia, indicating acute infection, or eosinophilia, reflecting parasitism. monocytosis can suggest chronic disease that was not suggested by the clinical history. in the case of acute onset diarrhea, the cat may be unwell as a consequence of the diarrhea (e.g., from dehydration) and not because of the cause of the diarrhea. if rehydration is required (with intravenous or subcutaneous fluids, depending on severity of illness), then it is important that biochemistry tests are performed before fluid administration so that any diagnostic clues are not lost by alteration of the profile from the fluid therapy. fever and neutrophilia may indicate the need for antibiotic therapy. if infection is suspected, fecal sampling (see step 4) should occur before starting antibiotics. if a cat is unwell from dehydration, then further testing may not be warranted. the clinician should be alert that linear foreign bodies can result in diarrhea (see the section intestinal obstruction). diarrhea of chronic duration (greater than 3 weeks) does require a more thorough investigation at the outset. however, if clinically well, the cat can be managed as an outpatient in the first instance, at least while waiting for results of diagnostic testing. a diet trial with a novel protein is appropriate for a well cat with stable weight. as with any patient managed as an outpatient, follow-up is vital and, in this scenario, includes scheduling revisits. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity invasive diagnostics when a specific marker of the disease in the organ involved has been recognized. caution should be exercised, because either cobalamin or folate may not be reduced with gi disease. in one study of small cell lymphoma, only 78% of cats were hypocobalaminemic, 7 meaning that if this was the only instigating factor to investigate, nearly one fourth of cats would not have been investigated further. also, cobalamin may be reduced in nonalimentary illness. 1 2. to detect hypocobalaminemia that may indicate the need for supplementation for clinical improvement. 13 to recognize pancreatic pathology when fpli is increased. 17 it is important to note that an elevated value gives no indication of the nature of the pancreatic pathology. 4. to make a diagnosis of epi when the ftli is low. 15 it should be noted that epi can result from other pathology that may require further investigations. veterinarians if the patient is new to the practice) are usually helpful. body condition scoring (using a 5-point or 9-point scale) for every cat seen is helpful in recognizing those that are underweight. weight loss often occurs with loss of muscle mass in cats, and muscle mass can be assessed over the ribs and pelvis as well as scapulae and nuchal crest. thickened intestines are also a subjective finding; it is the author's opinion that thickened intestines are actually intestines with increased turgidity, since differences between normal intestines and those with inflammatory infiltrates can be as little as 0.5 mm. perhaps more important during the history taking and physical examination are those signs that can point to extragastrointestinal disease. when confronted with a cat showing weight loss or vomiting or diarrhea (or a combination of signs), the clinician should start with trying to distinguish the signs as being either primary gastrointestinal or secondary signs. examples of clues pointing to extragastrointestinal diseases include tachycardia and palpable thyroid nodule, indicating hyperthyroidism, or polydipsia/polyuria, which has a variety of causes but is not typical of primary intestinal disease. inflammatory bowel disease has traditionally been considered an immune-mediated disease. the local immune system of the intestinal mucosa no doubt plays an important role, but recent work has also shown the importance of the normal bacterial population in perpetuating and, perhaps, even initiating pathology. it is known for certain that ibds are an expression of an overanxious immune response, with a recent study 104 indicating increases in inflammatory (il-6), type-1 immunity (il-12 p40), and immunomodulatory (transforming growth factor [tgf]-beta, il-10) cytokines. other researchers have found an association with bacterial counts (enterobacteriaceae, e. coli, and clostridium spp.) and abnormalities in mucosal architecture, indicating that mucosal bacteria are involved in the etiopathogenesis. 65 we can summarize these theories by saying that ibds are likely to be a consequence of hypersensitivity reactions to antigens from the intestinal lumen (e.g., bacterial, parasitic, or dietary antigens). this hypersensitivity may occur because of failed immunoregulation (suppressive function) of the gut-associated lymphoid tissue (galt). it is known that granulomatous colitis in boxer dogs is associated with infection, 143 and pathogens may well be found in at least some cases of ibd in cats that cause the immune response and subsequent inflammatory infiltrate of the lamina propria typically seen. although not described specifically in cats, chronic intestinal inflammatory change can impair motility. inflammatory bowel disease (ibd) refers to intestinal inflammatory infiltrates of the small or large intestine (or both) of unknown etiology. the term ibd should strictly be applied to mean idiopathic ibd, thus excluding inflammatory enteritis because of food sensitivities, although common usage has led to ibd referring to intestinal inflammatory infiltrates of both known and unknown causes. ibd is not a diagnostic end point but a description of a series of intestinal diseases that have similar histopathology. recent efforts by the world small animal veterinary association (wsava) gastrointestinal standardization group have led to both diagnostic and classification guidelines 35, 168 ,169 that encompass chronicity, nonresponse to symptomatic treatment, no specific cause found, as well as histologic confirmation of non-neoplastic intestinal inflammatory changes. there are no obvious breed or gender predispositions, and although cats of any age can be affected, inflammatory intestinal diseases are more likely to occur in middle-aged to older cats (5 to 10 years of age or older) than in younger cats. presenting clinical signs include vomiting, diarrhea, and weight loss with increased or decreased appetite. these signs can occur in isolation or together. weight loss without vomiting or diarrhea deserves special mention because not only have several studies 38, 58 shown this to be the most common presenting sign for ibd, but many veterinarians do not consider primary intestinal disease without the presence of vomiting or diarrhea. weight loss despite normal to increased caloric intake can represent poor absorption of food because of small intestinal disease, although it can also represent maldigestion associated with exocrine pancreatic insufficiency or increased metabolism associated with hyperthyroidism, or even lack of energy utilization associated with diabetes mellitus. conversely, appetite may be reduced, most likely because of nausea. if the large bowel is affected, signs are typically discomfort when defecating, resulting in frequent small volumes of diarrhea, often with mucus and blood; if the large bowel alone is affected, there may be no weight loss. physical examination findings are often nonspecific, but the most consistent findings for small intestinal disease are weight loss (or being underweight in a cat not seen previously) and palpably "thickened" intestines. noting a cat as underweight can be subjective, and prior recorded weights (even from previous paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma than those with ibd, but this change was also seen in 12% of cats with normal small intestine. 177 inflammatory bowel diseases require histologic findings obtained from biopsy samples for diagnosis, but diagnosis should not be made solely on these findings. the wsava international gastrointestinal standardization group has proposed "an all encompassing definition of inflammatory bowel disease" that comprises clinical criteria, imaging criteria, as well as pathophysiologic criteria. 169 the clinical criteria for the diagnosis of ibd include there are no typical laboratory findings in ibd, and many cats may have entirely normal results from routine biochemical and hematologic investigations. moderate liver enzyme elevations may be seen 5,38,58,66 even in the absence of recognizable hepatic pathology, and this may reflect subclinical secondary hepatic disease, secondary cholestasis, or showering of the liver with inflammatory cells from the small intestine through the portal circulation. 66 other changes can reflect consequences of the intestinal disease, such as azotemia or hemoconcentration reflecting dehydration, 58 or hypokalemia reflecting inappetance. 38 the chronic inflammation may be reflected by neutrophilia, monocytosis, 5, 38, 58, 66 or hyperglobulinaemia. 38 hypocobalaminemia can reflect ileal inflammation, and low serum folate can reflect proximal small intestinal inflammation. 149 typical ultrasonographic findings consistent with ibd are focal or diffuse intestinal wall thickening ( figure 23-19) ; normal wall thickness is less than or equal to 2.8 mm for the duodenum and less than or equal to 3.2 mm for the ileum, 50 and large mesenteric lymph nodes with hypoechoic changes may be seen. one study found that ultrasonographic findings correlated with histologic grade of ibd. 5 there is no clear distinction between ultrasonographic changes from ibd and those from small cell lymphoma. one recent therapeutic trial, follow-up visits are vitally important. many cats with small intestinal disease may show initial improvement simply because of the diet having lower residue, since there is decreased substrate for intestinal bacteria to digest and lower osmotic potential. the corollary of this is that failure of one novel protein diet does not mean that all novel protein diets will fail. when food sensitivities are responsible for gastrointestinal clinical signs in cats, the responsible food ingredient is usually a dietary staple. commonly incriminated ingredients are beef, fish, wheat, and corn gluten. 55 a careful dietary history is therefore important. large bowel inflammation typically improves with higherfiber diets, 38, 58 and attempting a trial with such a diet is certainly appropriate. immune suppressive therapy is the mainstay of ibd treatment, and glucocorticoids, such as prednisolone, are most commonly used. sulfasalazine use for large bowel signs has not been critically evaluated but seems safe and effective. in cats with substantial weight loss or severe clinical signs, such as chronic diarrhea, the author prefers to start with corticosteroid therapy, even if dietary causes have not yet been ruled out. the diet should also be changed to one containing a novel protein, and, if and when clinical signs resolve, an attempt is made to wean the cat from corticosteroid therapy, hopefully to the point of being discontinued. a diet challenge can then be used to confirm the diagnosis of food sensitivity. there are no universal guidelines for doses of corticosteroids. the author prefers the use of orally administered prednisolone to reduce the chance of side effects and will choose the starting dose based on the severity of disease. the starting dose is usually 2 mg/kg, once daily, po (10 mg/cat/day for most cats) starting 10 days after biopsies have been obtained to allow time for the mucosa to heal. if there is an improvement noted after a recheck at 2 weeks, the higher dose is maintained for a further 2 to 4 weeks, at which point, many cats are back to their normal weight and are not exhibiting clinical signs. if this is the case, the corticosteroid dose can be weaned down to 1 mg/kg, po (often 5 mg/cat/day) for several months, with continued rechecks scheduled to assess weight, clinical signs, and diet. the goal is to wean down to the lowest effective dose. if hypocobalaminemia is present, cobalamin supplementation may be required. 144 cobalamin is administered parenterally at 250 µg/cat subcutaneously weekly for 6 weeks, then every second week for 6 weeks, then monthly. owners can be shown how to inject their cats (as practitioners routinely do with diabetics). clinicians often consider the assessment of histologic samples to be out of their hands; however, it is important to work with the pathologist by providing good quality samples and a good clinical history, as well as having an open dialogue if the findings are not within expectations. for example, with lymphocytic/plasmacytic infiltrations, the pathologist has the difficult task of distinguishing diseased from normal tissue in a site that is laden with lymphocytes in the healthy state. once deciding the tissue has pathology, the pathologist's next task is distinguishing inflammatory infiltrate from neoplastic infiltrate with normal, mature lymphocytes (as seen in small cell lymphoma). inflammatory change also results in changes to normal tissue architecture, with thickened villi, edema, or erosion of the epithelium being typical changes. clinicians should expect morphologic descriptions as well as assessments of degree and type of inflammation. these difficulties are further compounded with the recognition that histologic grading of mild, moderate, or severe does not necessarily correlate with severity of clinical signs. this means that a cat with severe clinical signs of weight loss and vomiting or diarrhea may have only mild histologic changes (and vice versa). concurrent inflammation of the pancreas and liver with intestinal inflammation was first described in the mid-1990s, 171 and despite constant reference to this phenomenon at conferences and veterinary websites, there has been little description since then, though one study found 70% of ibd cases had liver inflammation and 30% had pancreatic inflammation. 6 the term "triaditis" has frequently been used, but the author prefers to spell this "tri-iditis" to distinguish it from inflammation of the hepatic portal triads. there has been no assessment of prognosis when the pancreas and/or liver are involved, but the author has found no difference in prognosis. many cases diagnosed with intestinal inflammatory infiltrates have these changes because of dietary sensitivity. in one study, 29% of cats with histologic gastrointestinal changes improved with dietary elimination therapy alone. interestingly, improvement was noted within 4 days compared with the longer duration of 8 weeks often recommended for improvement of dermatologic manifestations of food sensitivities. this careful study made note of the cat's prior diets and likely dietary causes of sensitivities. 55 another study found dietary therapy to be unsuccessful in 52 of 60 cats but no specifics of diets tried are noted. 58 as with any national cancer institute working formulation (nci wf) system. 116 for most veterinarians in practice, the most important distinction is the histologic grade, because low-grade (lymphocytic or small cell) lymphoma has a much better prognosis (and requires different treatment) compared with high-grade (often lymphoblastic) or intermediategrade lymphoma. for the purposes of simplicity and practicality, only small cell lymphoma and high-grade lymphoma will be addressed here. the prognosis and treatment for intermediate-grade intestinal lymphoma should be considered as for high-grade lymphoma. small cell lymphoma was first described in human pathology in 1966. 122 earlier, small lymphocytes were considered end-stage cells without the ability to divide. in cats small cell lymphoma is most commonly associated with the gastrointestinal tract or skin. 163 small cell neoplasia can be a confusing concept, since our traditional ideas of malignant neoplasia focus on rapidly dividing cells. the confusion is compounded by various terms used in the literature, such as lymphocytic lymphoma, low-grade lymphoma, well-differentiated lymphoma, or diffuse lymphoma; another term, epitheliotropic malignant lymphoma predominantly applies to small cell lymphoma, and other papers fail to distinguish these lymphomas from lymphoblastic lymphosarcoma (the traditional, aggressive form). "small cell lymphoma" seems to be most widely used term, though the author prefers "lymphocytic lymphosarcoma," since it is more descriptive. intestinal small cell lymphoma can be considered as a severe lymphocytic intestinal infiltrate, the most common form of which is commonly called ibd. not only is lymphocytic ibd hard to distinguish histologically from lymphocytic lymphosarcoma, but the approaches and treatments are similar. several reports have suggested a relationship between the two conditions in that inflammatory infiltrates may become neoplastic over time. 27, 43, 90 prevalence the true prevalence of intestinal small cell lymphoma is unknown, but several recent studies have indicated similar rates to inflammatory bowel diseases, with kleinschmidt et al noting 10 small cell lymphoma cats compared with 14 with intestinal lymphocytic infiltrates, 74 evans et al reporting 10 cases compared with 12 with ibds 40 , and baral et al diagnosing 8 cases compared with 10 with ibds. 6 traditionally, 90% of feline lymphosarcoma is regarded as intermediate or high grade, 163 but this may not be the case within the gastrointestinal tract. fondacaro et al found 75% of gastrointestinal lymphoma to be lymphocytic 43 ; a more recent paper found some cats seem resistant to conventional therapy. if this is the case, the diagnostic findings should be re-assessed to ensure no steps were missed or findings disregarded; the cat should be reexamined to look for emergence of other signs; and the pathologist who reads the histology should be contacted to recheck the findings. some cases of apparently resistant ibd are actually food sensitive, but it can be difficult to find the incriminating diet source, and commercial diets are not always effective. if underlying infectious causes have been entirely ruled out and the practitioner is certain of the diagnosis of idiopathic disease, immune suppressive therapy can be increased by either increasing the dose of prednisolone or using other agents, such as chlorambucil, typically at 2 mg/cat, po, every second day. it has been suggested that cats with eosinophilic inflammation may be more likely to be refractory to standard therapy. side effects of immunosuppressive therapy are rare but include inducing diabetes mellitus, immune suppression, delayed healing, and gastrointestinal ulceration. reported doses of sulfasalazine to manage large bowel ibd are 10 to 20 mg/kg, po, once daily for 7 to 10 days. 174 because this drug is usually only available as 500 mg tablets, one eighth of a tablet, providing a dose of 62.5 mg, is usually appropriate for most cats. in some countries, it is possible to have a compounding pharmacist formulate the drug into more convenient tablet sizes or as an oral suspension. cats are generally regarded as susceptible to salicylates, and possible side effects include vomiting or diarrhea, or anemia. the exact pharmacodynamics of this drug are not known; so, caution for extended use should be exercised and the drug withdrawn if any possible adverse signs are noted, but there are anecdotal reports of extended use of this drug without adverse consequences. a survey of the online veterinary cancer registry (http://www.vetcancerregistry.com) identified 6% of all submitted feline tumors to be intestinal tumors. approximately 74% of reported feline small intestinal tumors were lymphomas. adenocarcinomas accounted for 17%, and other tumor types reported included mast cell tumors and leiomyosarcomas. 135 "lymphoma in veterinary medicine: no longer a oneword diagnosis" was the title of an editorial in a recent issue of the veterinary clinical pathology journal, 95 and this is nowhere truer than in the feline gastrointestinal tract! a recent study classified 50 cases of feline gastrointestinal lymphoma both histologically and immunophenotypically, and it found eight different categories according to the revised european and american lymphoma/world health organization (real/who) classification system and six categories according to the approximately equal numbers of high-grade and lowgrade gastrointestinal lymphoma. 84 older cats are more at risk of small cell lymphoma, with mean or median ages reported from 9 to 13 years. younger cats with the disease have, however, been recognized. 40, 43, 73, 84 no breed or gender predispositions have been definitively recognized. two larger studies have suggested a skew to males with 28 males compared with 22 females in one report, 43 and 24 males compared with 17 females in the other 73 ; most other studies looking at gender and breed did not clearly distinguish between lymphoblastic and lymphocytic neoplasia. clinically, it is impossible to distinguish cats with ibds from cats with small cell lymphoma. this is hardly surprising when even histologic distinction can be difficult! therefore cats will present with weight loss or vomiting or diarrhea at a similar frequency to those with ibd. weight loss has been recognized as a presenting sign in 82% to 100% of cases, diarrhea in 25% to 60% of cases, and vomiting in 25% to 73% of cases, with various combinations of these signs also possible. other variable signs are lethargy and inappetence or, conversely, polyphagia. 40, 43, 73, 84 these findings can be summarized by stating that cats with gastrointestinal small cell lymphoma can present with any combination of signs relating to the gastrointestinal tract. intestinal small cell lymphoma is typically a diffuse disease, and therefore multiple areas of the alimentary tract are usually affected. in studies where different locations of the small intestine were assessed, the jejunum was most commonly affected (100%), with the ileum frequently affected (93% to 100%), and duodenal pathology slightly less prevalent (83% to 90%). 40, 84 although the numbers of cats assessed in these studies are small, the important fact that the duodenum is not always affected needs to be recognized, which has important implications for how biopsy samples are obtained, because lesions beyond the duodenum are likely to be beyond the reach of an endoscope. further difficulties in precise diagnosis may arise, since non-neoplastic lymphocytic infiltrates (e.g., ibd) are often found in other locations along the intestinal tract. 27, 40, 84 the stomach is also affected in 14% to 40% of small cell lymphoma cases. 40, 43, 84 although not fully assessed, involvement of the colon appears rare. 84 local lymph node involvement is common, being noted in up to 59% of cases. 84 this percentage may be even higher, because many studies assessed lymph node cytology from ultrasound-guided fine-needle aspirates, which may miss spread to the lymph node, because the population of neoplastic lymphocytic cells is indistinguishable from the normal population of lymph node cells. histology is required to assess changes in lymph node architecture. liver involvement is not uncommon but not thoroughly assessed. one study noted liver lymphocytic neoplasia in 8 of 38 cats with small intestinal lymphocytic neoplasia, 73 another found 5 of 15 affected cats in which the liver was biopsied, 84 another noted 2 of 4 cats had liver involvement, 27 and a further study detected neoplasia "in the lymph nodes, liver, or both" in all 10 cats with intestinal small cell lymphoma. 40 the pancreas may also be involved. 73, 84 this may be akin to the noted association of lymphocytic inflammation of intestine, pancreas, and liver 171 that has been dubbed tri-iditis. ultrasound findings may not suggest extragastrointestinal involvement. in the case of liver pathology, ultrasonography may show no changes in as many as 75% to 80% of cases. 40, 84 focal nodular changes and he patomegaly have been recognized as ultrasonographic signs of hepatic small cell lymphoma. 7 both lymphocytic ibd and lymphocytic neoplasia are often recognized simultaneously in the same cat, 40, 84 and numerous authors have suggested that lymphocytic ibd may be a precursor to intestinal lymphoid neoplasia. 90, 125 if this is the case, then antigenic factors, such as bacterial population changes or food sensitivities, could be considered primary initiating factors for small cell lymphoma since they are potential underlying etiologies of ibds. 79 however, neoplasia also requires genetic mutations to occur (often affecting regulation of cell death and cell survival), and these may be initiated by the inciting antigenic factors or the ongoing inflammatory changes. 154 as opposed to other feline lymphoid neoplasia, no association has been made with felv infection. 27, 43, 73, 125 intestinal lymphocytic lymphosarcoma begins in the superficial mucosa and progresses to involve the entire mucosa and submucosa; then advancing in a perivascular pattern into the tunica muscularis, eventually infiltrating all four intestinal tunics. 43 lymph node and other organ (such as liver or pancreas) involvement likely represent metastasis through lymphatics and perhaps hematogenously. more distant metastasis is not reported. serum or plasma biochemistry and hematologic findings are typically nonspecific. however, this testing is important as part of the diagnostic workup to rule out extra-gi disease, such as hyperthyroidism or diabetes mellitus. common biochemistry findings are mild to moderate increase of liver enzymes, such as alanine aminotransferase (alt), aspartate aminotransferase (ast), and/or alkaline phosphatase (alp). 27, 40, 43, 84 as with ibds, these liver enzyme changes may or may not represent overt hepatic disease. 67 albumin may be reduced 43 but is normal in most cases 27, 43, 84 ; azotemia may be present and may be of prerenal origin or represent concurrent renal disease. in one study, 25 of 32 cats were hypocobalaminemic; 1 of 27 cats had low folate, but 10 of 27 had elevated folate; and 12 of 16 cats had increased ftli. 73 hematologically, a mature neutrophilia with or without monocytosis is sometimes present, representing the inflammatory response; lymphopenia may be present as a stress response. anemia may be present and may occur as a result of chronic slow gi blood loss, and in some cases, ulceration, or it may be because of chronic disease; hemoconcentration is also possible, reflecting dehydration. 27, 40, 43, 84 palpable or ultrasonographically visible thickened intestines (30% to 41% of cases) 27, 40, 43, 84 or mesenteric lymph nodes (20% to 50% of cases) 27, 40, 43, 84 are no more or less likely to be present in comparison with ibds. there are no defined ultrasound guidelines for cats with intestinal small cell lymphoma, because most prior papers do not distinguish between small cell and lymphoblastic neoplasia. 54 ,113 a more recent paper found 9 of 15 cats undergoing ultrasound examination had diffuse small intestinal wall thickening, with a mean of 4.3 mm (range, 3.4 to 5.0 mm; median, 4.5 mm), and focal mural thickening of 20 mm was noted in one cat. 84 in many cases, against expectations, intestinal wall layering was preserved. these findings also mean that 5 of 15 cats had ultrasonographically normal intestinal wall thickness (≤2.8 mm for the duodenum and ≤3.2 mm for the ileum). 50 if affected, jejunal lymph nodes may appear as hypoechoic and enlarged; in the same study, 12 of 15 cats had lymph node changes with a mean diameter of 15.9 mm (range, 6.5 to 30 mm; median, 10 mm) 84 compared with the normal diameter of less than or equal to 5.0 mm. 132 none of these findings can definitively distinguish small cell lymphoma from ibds; although one recent paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma (figure 23 -20) than those with ibd, this change was also seen in 12% of cats with a normal small intestine. however, thickening of the muscularis layer together with lymphadenopathy was recognized in 26% of those cats with small cell lymphoma compared with 4% of those with ibd and 2% of cats with no small intestinal pathology. 177 biopsy samples and histopathology are required for definitive diagnosis. an example of jejunal and mesenteric lymph node appearance at laparotomy is shown in figure 23 it is difficult to distinguish between lymphocytic inflammation and small cell lymphocytic neoplasia in any location; some histopathologic features that might help in differentiating the ends of the spectrum may include therefore become known as the fondacaro protocol. this consists of a combination of prednisolone and chlorambucil given orally by the client at home (table 23-15 ). the rationale is that a slow alkylating agent, such as chlorambucil, is more appropriate to use for the slowly dividing, well-differentiated lymphocytes that cause disease. this can be contrasted to the aggressive chemotherapeutic agents required for the rapidly proliferating cells in lymphoblastic neoplasia that is typically associated with lymphosarcoma. reported response rates to this protocol are excellent, with 59% to 76% of cats achieving complete clinical remission, reported median survival times ranging from 20 to 30 months for those cats responding to therapy, and reports of individual cats surviving as long as 76 months. 43, 73, 84 the original reported protocol comprised prednisolone (10 mg/cat, po or 2 mg/kg, po) given daily with chlorambucil pulsed by administration of 15 mg/m 2 for 4 days every 3 weeks. a more recent study 73 dosed prednisolone similarly, but chlorambucil was given as continuous therapy of 2 mg/cat, po every second or third day. no mucosal congestion, edema, or fibrosis in lymphocytic neoplasia, 43 compared with ibd 4. epitheliotropism, or homing of neoplastic t lymphocytes to the mucosal epithelium in lymphocytic neoplasia 27 these features can be seen in figure 23 -22. each of these criteria may be useful but are unlikely to be definitive. further studies that may not be routinely available but which may be helpful are immunophenotyping; most reports have found purely t lymphocytes in most cases of intestinal small cell lymphoma 27, 73, 84, 116 (figure 23-23 ). 2. clonality; the detection of a clonal population of cells, as recently described for intestinal lymphocytic lymphosarcoma, 101 would be closest to providing the basis for definitive diagnosis. effective treatment of feline intestinal small cell lymphoma was brought to light by fondacaro et al 43 and has cat to be weaned off corticosteroids, with chlorambucil continued as monotherapy (as is often the case with humans). iatrogenic diabetes mellitus usually needs to be managed with insulin therapy, at least initially (see chapter 24) . high-grade lymphoma or lymphosarcoma is the traditional style of aggressive, rapidly dividing lymphoid neoplasia that carries a much poorer prognosis than small cell lymphoma. most early studies do not distinguish grade of neoplasia; so, the prevalence of low-grade and high-grade alimentary lymphoma are difficult to assess. several recent studies found a similar prevalence of each, 84 ,116 but the seminal paper describing small cell lymphoma found only 17 cases of lymphoblastic lymphoma compared with 50 cases of small cell lymphoma. 43 this ratio of approximately one high-grade gi lymphoma case for every three low-grade cases more closely approximates the rate found in the author's practice. the reported median ages of affected cats range from 10 to 12 years, but cats as young as 1 year old have been diagnosed. most papers note that males are overrepresented, and siamese cats may also be overrepresented although most affected cats are domestic shorthairs. 43, 49, 90, 116, 176 precise signalment is difficult to determine from the literature, because many papers assess all anatomic locations of lymphoma without necessarily breaking down epidemiologic data for each anatomic site. also, there are few comparisons to a reference population. the association of lymphoma with felv infection is well established and documented 139 and is covered in chapter 28; fiv has also been shown to be lymphomagenic. 12, 139 since the control of felv through vaccination began in the 1980s, nonretroviral-associated lymphoid neoplasia has become more common, and the rates of intestinal lymphoma have, in fact, increased since felv infection rates have decreased. 86 the underlying causes for this increase are not known. the association with inflammation from ibds was noted for small cell lymphomas, and perhaps there is a spectrum from lymphocytic ibd to small cell lymphoma to high-grade lymphoma. that some cats are more likely to have inflammatory changes become neoplastic is suggested by a paper noting higher lymphoma rates in cats with vaccine-associated sarcomas (a neoplastic condition where the role of chronic inflammation is well noted). 89 similar protocols are used in humans with both lowgrade (i.e., lymphocytic) lymphosarcoma and chronic lymphocytic leukemia. 117,151 some studies with humans have indicated that continuous therapy with chlorambucil results in prolonged survival, 64 although metaanalyses have not been able to determine optimum dosing and scheduling of administration of chlorambucil or other alkylating agents in these conditions in humans. 20, 72 although we do not have enough data to critically compare pulsed therapy to continuous dosing, the study assessing continuous dosing appeared to have a lower number of cats completely responding, although those cats that did respond had a longer median survival 73 than those in the studies assessing pulsed chlorambucil dosing. 43, 84 the differences may also relate to the definitions used for complete response. the chlorambucil dose of 2 mg/cat, po every second day (or third day) is often chosen because of the ready availability of 2 mg coated tablets, the breaking of which can expose the owner to these cytotoxic medications. chlorambucil can be compounded into smaller doses, thus allowing daily dosing of 1 mg capsules. the author has used this dose to apparent good effect, but there has been no critical assessment. it is unknown whether involvement of lymph nodes or other organs, such as the liver, affects prognosis. the only study of substantial size to include extra-gi locations found anatomic location was not prognostic for response or survival time. 73 in another study, of the five cats with liver involvement, two cats did not survive more than 5 months, yet the other three lived longer than 2 1 2 years, with two surviving longer than 4 1 2 years. 84 a study of hepatic small cell lymphoma suggests the density of neoplastic lymphocytes may influence survival, and density may relate to the stage of the disease when diagnosis occurs. 7 adverse effects of chlorambucil are rare, but gastrointestinal signs, myelosuppression, and myoclonus have all been reported. gastrointestinal signs, such as vomiting, diarrhea, or inappetence, can be difficult to distinguish from continuation of the gastrointestinal disease diagnosed. these signs are usually self limiting. myelosuppression is also possible with thrombocytopenia reported. 57, 84 monoclonus has been reported on one occasion. 17 it is ideal to check hematologic parameters every 2 months for cats receiving chlorambucil. continuous therapy using lower doses of chlorambucil may be less likely to lead to these adverse effects. high doses of corticosteroids can induce diabetes mellitus, and thus blood glucose should be checked regularly. if diabetes occurs, the author has found that budesonide (1 mg budesonide is generally considered to be equivalent of 5 mg prednisolone) can be substituted for prednisolone, since it has reputed lower systemic effects (though no assessments of this drug's effectiveness in cats have been made). an alternative is for the of distinction of intestinal layering as shown in figure 23 -24. the area of lymphomatous infiltration is hypoechoic, because it contains a uniform cell population without much reactionary fibrous tissue. mesenteric lymphadenomegaly is common (figure 23-25) , as are changes in other organs, such as kidney, liver, or pancreas. ascites may also be seen. 54, 113 although ultrasonographic distinctions predominate, there is considerable overlap between ultrasonographic findings with small cell lymphoma and high-grade lymphoma. the clinician must not lose sight of the fact that microscopic distinctions are required to diagnose either condition. cytologic diagnosis of high-grade lymphoma from fine-needle aspirates (fna) is much more likely than with small cell lymphoma. this is because there is usually a focal lesion, and the neoplastic cells are a monomorphic population of large, immature cells (i.e., that are not normally seen in tissue). sometimes, mixed lymphoid whether the underlying cause is retroviral or chronic inflammation or anything else, the pathogenesis of highgrade intestinal lymphoma, as with small cell lymphoma and other neoplasia, depends on chromosomal changes that affect regulation of cell growth and death, resulting in malignant transformation and clonal expansion of immature lymphocytes. 152 metastasis can occur in one third to two thirds of cases, 90,92 with involvement of mesenteric lymph nodes most commonly noted, but spread to liver, spleen, kidneys, and thorax is also possible. 90 a recent survey of gastrointestinal lymphoma found that most cases (37 of 50) involved the small intestine (including 3 that also involved the stomach and 4 that also involved the large intestine), and 4 of 50 cases involved the large intestine only. 116 cats with high-grade alimentary lymphoma often present similarly to those with other gastrointestinal diseases. typical clinical signs are weight loss, anorexia, lethargy, vomiting, diarrhea, or a combination of these signs. repeated studies have found cats with no vomiting or diarrhea; in one study, 13 of 28 cats had only anorexia or weight loss on presentation. 90 cats with large bowel pathology usually present, as with other causes of colitis, with increased urgency, and small, frequent amounts of diarrhea, often with blood or mucus. cats with large bowel neoplasia of any form can present for constipation caused by intestinal obstruction. palpation of an abdominal mass has been recognized in 59% to 85% of cases, 43, 90 but the corollary of this is that 15% to 41% of cases did not have a palpable mass. it is also important to note that up to 50% of cats with intestinal small cell lymphoma, and a number with ibds, have palpable mesenteric lymph nodes; so, a palpable abdominal mass is not a specific indication of high-grade neoplasia. many cats have palpably thickened bowel loops. 124 hematology and plasma or serum biochemistry findings are also nonspecific. increased liver enzymes may or may not indicate liver involvement. anemia may be recognized and can be non-regenerative, reflecting chronic disease or slow blood loss, or regenerative if there is more substantial blood loss associated with mucosal ulceration. hypoalbuminemia can be because of blood loss or intestinal protein loss. hypercalcemia of malignancy is a possibility but not commonly reported. despite nonspecific signs, laboratory testing is important to rule out extra-gi diseases and help manage consequences of enteric disease, as with small cell lymphoma and ibds. ultrasonography commonly shows a focal intestinal thickening (of 5 to 25 mm) with partial or complete loss although noted as the next most common intestinal neoplasia, after the various forms of lymphoma, adenocarcinoma is seen relatively infrequently in practice. most cats are more than 10 years old, 32,76,126 males may be overrepresented, and several studies have recognized a distinct overrepresentation of siamese cats. 32, 76 three distinct forms have been described 140 : cats typically present with nonspecific signs of gastrointestinal disease but can present with obstructive signs. cats with large bowel neoplasia can present for tenesmus or hematochezia and even constipation, if the lesion is obstructive (or partially obstructive). on physical examination, an abdominal mass is palpable in approximately 50% of cases, but other findings are usually nonspecific. anemia can be found if mucosal ulceration has occurred, but there are no distinctive laboratory findings. lesions can occur anywhere along the intestinal tract. one study of 100 cases found 40% of feline intestinal adenocarcinoma lesions were present at the ileum or ileocolic junction. 32 twenty-five to 50 percent of cases have metastasis at the time of the diagnosis, and this is a poor prognostic indicator. 32, 76, 145 radiology may show a mass lesion or intestinal obstruction, and ultrasonography can localize lesions to an intestinal origin. the ultrasonographic appearance of the proliferative, circumferential, outwardly expansile populations (of immature lymphoblasts and mature lymphocytes) are seen if a germinal lymphoid follicle is aspirated, and precise diagnosis may be difficult if there are a large number of lymphoblasts. 160 fna samples are best obtained with ultrasound guidance. the cytologic sample quality is greatly improved by not aspirating when the needle is visualized in the mass but merely "pecked" into the mass so that the needle is merely acting to finely "core" the mass. on removing the syringe and needle, the hub of the needle is removed before drawing air into the syringe, the hub is replaced, and the sample within the needle is expressed onto a slide. usually, the decision to diagnose by cytology from fna is based on the ultrasonographic appearance of a mass. since there is substantial crossover of ultrasonographic appearance of intestinal masses, laparotomy for excision is often performed with the affected bowel submitted for histology. except when intestinal obstruction has resulted, there is no therapeutic benefit of excising a gastrointestinal lymphoma (which requires excision and anastomosis), but there is minimal room for doubt when a histologic diagnosis is achieved. the response to therapy for high-grade intestinal lymphoma is significantly worse than that for small cell lymphoma. 43, 124 further, response to therapy for highgrade intestinal lymphoma appears to be worse than for lymphoma in other anatomic locations. 142 precise remission rates and survival times are difficult to quantify, because many studies assess lymphoma from multiple locations and do not necessarily differentiate response of gastrointestinal lymphoma or report the grade of lymphoma. with remission rates reported from 18% to 80% 43,92,176 and a median survival time of up to 41 weeks (range, 4 to 120 weeks), 176 it can be said with some certainty that some cats respond to therapy for reasonable durations. multiple authors have noted that the best prognostic indicator is response to an initial treatment cycle, 92,100,176 which should prompt clinicians to encourage owners to start therapy and decide whether to continue based on the cat's response. there are several published chemotherapeutic protocols, 43 ,92,100,142,176 but all follow the same principles of using medications to target specific phases of the cell division cycle (such as l-asparaginase and vincristine) with other medications that interrupt multiple phases of the cell cycle (cyclophosphamide and doxorubicin). targeting the cancer cell in different ways enables more cells to be killed, reduces the toxicity of the individual drug used, and reduces the likelihood of resistance to a specific drug. several authors have noted increased success with the addition of l-asparaginase and doxorubicin to protocols. 92, 162, 176 chemotherapy for lymphoma is covered in more detail in chapter 28, oncology. look promising, but only two cats assessed had gastrointestinal mast cell neoplasia. lomustine was used unsuccessfully in one cat with sclerosing mct; another cat with sclerosing mct received eight treatments of vinblastine and had a survival time of greater than 4 years. 56 adenomatous polyps have been reported in the duodenum 87 and ileum 106 and can result in intussusception. 133 cats of asian ancestry, predominantly siamese, are greatly overrepresented, and most reported cases have been males. 87 cats usually present for vomiting or hematemesis that, surprisingly, can be very acute in onset; complete intestinal obstruction may result. 87, 106, 133 resection is curative, with survival times of more than 4 years reported. 87 eosinophilic sclerosing fibroplasia has recently been described in a series of 25 cases and is not strictly neoplasia. 29 the ulcerating mass lesions that can occur anywhere from the stomach to the colon are often grossly and histologically mistaken for neoplasia. there appears to be no breed predisposition or age predisposition (with ages ranging from 14 weeks to 16 years), but 18 of 25 cases (72%) were castrated males cats compared with 7 of 25 (28%) female spayed cats. eighty-four percent of cats presented for vomiting, 68% presented for weight loss, and 7 of 12 (58%) cats had peripheral eosinophilia. all cases had a palpable abdominal mass. the pyloric sphincter was the most common site, and lesions in this location were mostly considered unresectable. fourteen of 25 cats (56%) had bacterial colonies within microabscesses and necrotic foci within the lesion. the bacteria recognized were predominantly gram-negative rods, but antibiotics did not seem to be clinically effective. the bacteria are suspected to initiate the lesions, having been embedded after foreign body penetration. there are no specific treatment recommendations, but excision, where possible, would be prudent; corticosteroids appear to be helpful adjunctive therapy. survival times are difficult to estimate since many cats were euthanized because neoplasia was suspected and follow-up times were short (up to 6 months) for the remaining cats. 29 there are very few reports of intestinal leiomyosarcomas in cats, 8, 159 which have been reclassified as gastrointestinal stromal tumors. 99 these tumors may be more likely to arise from the ileocecocolic junction. resection, if possible, is usually recommended, with survival times of 3 to 4 months reported before recurrence. the author owns a cat with this tumor where resection was not form is better described than the annular, constrictingband form with minimal outward enlargement. in these cases, sonographically, a solitary segmental intestinal mural mass is present and characterized by circumferential bowel wall thickening with transmural loss of normal sonographic wall layers. the thickening can vary in echogenicity but may be hypoechoic and may be symmetric or asymmetric. there is no definitive distinction, however, from lymphosarcoma, mast cell tumor, smooth muscle origin tumors, or even segmental benign inflammatory bowel disease. 126 surgical resection is the treatment of choice. there seem to be two distinct groups in terms of survival time postresection: 1. short-duration survival (euthanasia or death within 2 weeks of surgery) 2. long-duration survival (mean survival time of 15 months, 76 with a number of cats surviving greater than 2 years) 76, 109 because clean margins improve prognosis, for large bowel adenocarcinoma, subtotal colectomy may be required for complete excision. 145 because of the potential for success after resection, it is recommended to excise unidentified masses at the time of surgery. 145 other forms of intestinal neoplasia are recognized infrequently, and include intestinal mast cell tumors, adenomatous polyps, eosinophilic sclerosing fibroplasia, gastrointestinal stromal tumors (leiomyosarcoma), and hemangiosarcoma. mast cell tumors (mct) are often cited as the third most common form of feline gastrointestinal tumor, 85 but the intestines are a far less common site than cutaneous, splenic, or hepatic mast cell neoplasia. 85, 124 masses are usually segmental nodular thickenings that occur in older cats. 140 the masses are indistinguishable ultrasonographically from other tumors, such as lymphoblastic lymphosarcoma. 126 a recent series of 50 cases described a variant of feline intestinal mast cell tumor, dubbed sclerosing mast cell tumor, for which neoplastic cells form a trabecular pattern with dense stromal collagen. additionally, eosinophilic infiltrates were moderate to marked in most cases. these cases can be confused histologically with eosinophilic enteritis, gastrointestinal stromal tumor, or fibrosarcoma. 56 surgical resection is recommended, but lesions are commonly infiltrative or metastasize widely, and there are few reports of successful treatment. lomustine (dosed 50 to 60 mg/m 2 , po every 4 to 6 weeks) has recently been assessed as adjunctive chemotherapy for mast cell neoplasia in various locations, 123 and results recognized in that many intestinal bacteria can be found in healthy animals. 70 further antibiotic administration can result in increase of other bacteria. 69 fungal causes of diarrhea are usually recognized from histology of biopsy samples. it remains to be seen whether the recent ready availability of pcr panels looking at a number of infectious causes of diarrhea will be beneficial for recognizing pathogens that had previously been misdiagnosed or a hindrance for readily recognizing commensal organisms not necessarily causative of the clinical signs being investigated. the most common viral, bacterial, and mycotic causes of diarrhea in cats are described below. parasitic gastrointestinal diseases are covered later in this chapter. viral causes of diarrhea are not usually specifically diagnosed, since, with the exception of the canine fecal elisa possible, and the cat appears healthy 24 months past diagnosis (figure 23-27 ). cats with intestinal hemangiosarcoma often present with anemia, and the disease appears to be highly metastatic. 33 the intestines appear grossly thickened by dark red tissue. 138 the small and large intestines seem to be affected with similar frequency. removal of macroscopic disease is recommended, but often the full extent of the severity is only recognized at surgery. 33 the prognosis is poor. suspicions of infectious causes of diarrhea should be aroused in younger cats, cats from shelters, or cats with immune suppression. when considering infectious causes of diarrhea, clinicians should assess whether the diarrhea is large bowel or small bowel in origin and correlate this with specific pathogens that are likely to cause clinical signs as shown in table 23 -16. to increase the diagnostic yield of fecal examination for parasitic causes of diarrhea, wet smears and appropriate fecal flotations should be performed on fresh fecal samples (<1 hour old). it is appropriate to administer broad-spectrum anthelminthics, even if fecal tests are negative. bacterial and viral causes of diarrhea should be considered when the cat is systemically unwell with fever. fecal culture should be performed in these circumstances, but the limitations of this testing need to be yersinia enterocolitica current theory of fip pathogenesis involves initial infection with fecv and then mutation to fipcv in small numbers of susceptible individuals. 112,165 routine serologic testing for fecv in cats with diarrhea would neither prove correlation with the clinical signs nor affect how the disease is managed and so is not recommended. cats with fecv diarrhea should be managed with symptomatic therapy of fasting, then reintroducing a bland diet and supportive care with fluid therapy if necessary. other viruses, such as astrovirus, reovirus, rotavirus, and torovirus-like agent, have been recognized to cause diarrhea in cats, but their roles as pathogens are unclear. they are not routinely recognized in practice, since electron microscopy of fecal samples is necessary for diagnosis and is not routinely performed. management is supportive care with appropriate fasting, then reintroduction of bland diets and fluid and electrolyte replacement if necessary. 51 successful identification of a known bacterial pathogen from a fecal sample does not necessarily mean that the agent found is the cause of disease in the cat. although a number of bacterial pathogens have been demonstrated to cause diseases when specific pathogen-free (spf) cats are experimentally infected, 42 these same organisms can be found in healthy cats. 93 the differences between healthy and diarrheic cats that have bacteria found in their feces may relate to virulence factors of the organism, or host factors (local or systemic immunity) of the cat. there is no definitive answer for this quandary. the author's opinion is that • if a diarrheic cat is systemically unwell and has a fever, then feces should be cultured. • if an organism is isolated that is known to cause signs consistent with those the cat is showing, the cat should be treated appropriately. campylobacter diarrhea is usually caused by c. jejuni. clinical signs of infection are poorly documented, but most cats are asymptomatic. younger cats are more likely to have clinical signs and hemorrhagic, mucoid diarrhea has been reported. diagnosis can be from culture of feces or swabs, and the organism is quite hardy; so, it usually survives transport to the laboratory. 28 in individual cases, the organism has not been cultured after antibiotic treatment, 45,46 but it is not definitively proven that antibiotic therapy affects the natural course of the disease. antibiotics that can be used are amoxicillin-clavulanate (15 mg/kg, every 12 hours, po) for parvovirus, routine definitive tests are not available. clinical signs of panleukopenia (feline parvovirus infection) are more likely to occur in kittens, with the highest morbidity and mortality occurring between 3 and 5 months of age. subclinical cases in older (susceptible) cats probably go unrecognized. the organism is very stable in most environments, and infections mostly occur from environmental contact. peracute cases can result in death within 12 hours, with little or no warning signs. acute cases often have fever, depression, and anorexia, with signs beginning approximately 3 to 4 days before presentation. vomiting is usually bile tinged and unrelated to eating. diarrhea does not always occur, and when it does, it is usually later in the course of the illness. leukopenia is not pathognomonic, because this can also occur with acute bacterial infection (e.g., salmonellosis can present identically). 53 commercially available elisa tests for canine parvovirus antigen in feces can detect feline parvovirus; however, shedding may have ceased by the time clinical signs occur, and vaccination can result in positive test results for up to 2 weeks. 111 aggressive fluid therapy, usually at twice maintenance rates, is usually required. broad-spectrum antibiotic coverage is used to prevent or treat secondary bacterial infection from viral injury of intestinal mucosa. parenteral antibiotics are preferred to prevent the possibility of further gastrointestinal irritation. the author recommends calculating iv doses and introducing appropriate amounts of antibiotics to the fluids bag to create a constant rate infusion (cri); cefazolin can be used in this way at 100 mg/kg/24 hours, and betalactam cris are commonly used in human medicine. 127 aminoglycosides or fluoroquinolones can be used concurrently at routine doses if fever persists after 24 hours or the cat is moribund on presentation, but care must be used with these agents. aminoglycosides are potentially nephrotoxic, and fluoroquinolones have been reported to result in cartilage damage in growing animals, although this has not been demonstrated clinically in cats. 134 fluoroquinolone retinal toxicity has been seen in all animals. cats that survive the first week usually recover, and prior infection imparts lifelong immunity. 28 vaccinations are highly effective for disease prevention. feline enteric coronavirus (fecv) mostly causes mild, self-limiting diarrhea and must be distinguished from feline infectious peritonitis coronavirus (fipcv), which is essentially always fatal and for which diarrhea is not a typical sign (but is possible). the most widely accepted or fluoroquinolones, such as enrofloxacin (5 mg/kg, once daily, po) for durations of 14 to 21 days. 44 macrolides, such as erythromycin (10 to 15 mg/kg, every 8 hours, po), are regarded as the drug of choice for humans but can cause gastrointestinal side effects. 93 clostridium difficile has been recognized in up to 5% of diarrheic cats. 93 clinical signs are typically acute onset watery diarrhea and anorexia. diagnosis has been made with detection of toxin a or toxin b in fecal samples using elisa. although these tests have not yet been validated for cats, they may prove to be a useful aid to diagnosis 94 and are available for testing of equine feces at some commercial laboratories. nontoxigenic strains exist; so, positive culture alone does not ensure diagnosis. metronidazole (10 mg/kg, every 12 hours, po) for approximately 7 days is the treatment of choice. 93 clostridium perfringens typically results in large bowel diarrhea with tenesmus, mucus, and hematochezia, but small bowel signs can also be seen. 42 pcr testing for enterotoxin a is commercially available and may prove to be a useful adjunct in diagnosis. antibiotics that can be used include metronidazole (10 mg/kg, every 12 hours, po), tylosin (10 to 20 mg/kg, twice daily, po), or amoxicillin-clavulanate (22 mg/kg, every 12 hours, po) for 7 days. 94 escherichia coli is a ubiquitous organism within the feline intestinal tract, and it would be unusual not to successfully culture e. coli from the feces of both healthy and unwell cats. when e. coli is associated with clinical signs of gastrointestinal disease, it is mostly as an opportunistic pathogen, with overgrowth resulting from changed environmental conditions, such as inflammation from other pathology or another pathogen. there are also specific strains of e. coli that are true pathogens because of virulence factors not present in commensal e. coli; these include enteropathogenic e. coli and enterotoxigenic e. coli, which both induce a watery diarrhea, and enterohemorrhagic e. coli, which produces a diarrheal syndrome with copious bloody discharge and no fever. 77 pcr testing is commercially available to identify pathogenic strains of e. coli 16,148 ; although not offered at routine veterinary laboratories, this testing is available to veterinarians, and laboratories offering these services can readily be found online. diagnosis should also document histologic lesions corresponding to the strain of e. coli identified. 77 there is emerging resistance to e. coli worldwide in all species of animals, including humans. this includes the typical therapies for gram-negative bacteria of beta-lactamenhanced penicillins and fluoroquinolones. 167 a major risk factor is prior antibiotic usage, because commensal organisms are exposed to antibiotics. pcr testing does not enable antibiotic sensitivity testing, and fecal culture may not be able to distinguish pathogenic from nonpathogenic strains; so, sensitivities may not be an accurate reflection of the pathogenic organism. pcr testing for genes that impart resistance to e. coli have recently been described but are not yet commercially available. 34 in some circumstances, supportive care with fluid and electrolyte replacement may be all that is required while the cat's immune system combats the infection. empiric therapy could include beta-lactam-enhanced penicillins (such as amoxicillin-clavulanate at 20 mg/kg, every 12 hours, po), fluoroquinolones (such as enrofloxacin at 5 mg/kg, once daily, po), or cefovecin (8 mg/kg, every 2 weeks, sc), but the clinician must be aware of possible drug resistance. salmonella typhimurium infection is possible from ingestion of infected prey, infected food sources, or from a contaminated environment, including the veterinary hospital. the resulting clinical signs depend on the number of infecting organisms, the immune status of the cat, and the presence of concurrent diseases. infection rates in cats (and humans) have been correlated with seasonal bird migrations, 153 and the illness has been dubbed songbird fever, 52 but there is no distinction between this and other salmonella infections. clinical signs usually begin 3 to 5 days after exposure, starting with fever (often >40° c [104° f]), malaise and anorexia, and progressing to diarrhea, vomiting, and abdominal pain. hematology can show leukopenia with a left shift and nonregenerative anemia, and biochemistry results are usually nonspecific. diagnosis is based on isolation of the organism by culture or identification with pcr, but care should be taken to correlate pathogen identification with clinical signs since, as with most gi pathogens, the organism can be isolated from healthy animals. 147 as with e. coli, antibiotic resistance is widespread, 120 with one united kingdom survey finding the multiple drugresistant strain dt104 to be the most frequent bacteriophage type identified. 115 treatment should be reserved only for those cats showing systemic signs, because routine antibiotic use in treating salmonellosis induces drug-resistant strains and prolongs the convalescent excretion period. 52 antibiotic choice should be based solely on sensitivity findings, since resistances are so widespread and unpredictable. 98 this means that if the organism has been identified by pcr, then culture of feces must also be undertaken. the duration of treatment must be long enough to eliminate fecal excretion of the organism, prevent the chance of relapse, and reduce the chance of resistance developing; up to 28 days has been advocated. 4, 164 these cautions are particularly important because of the zoonotic potential of salmonellosis. linear foreign bodies have traditionally been considered more common than discrete foreign bodies in cats, 10,14,41 but a study from a primary care facility indicated only 33% of foreign body cases were because of linear foreign bodies. 60 the larger case load of linear foreign bodies at referral institutions noted in earlier studies may indicate the abilities of primary care practitioners to recognize and effectively deal with discrete foreign body obstructions. most studies have found that cats with intestinal foreign bodies are generally younger (mean, 1.0 to 2.7 years), with a notable exception being obstruction from trichobezoars where three of five cats in one study were 10 years or older; the greatest risk factor appears to be length of hair coat. 9 no specific breed predispositions have been described but siamese and siamese-related cats have been noted to have oral fixations 13 and so may be expected to be overrepresented with intestinal foreign bodies. clinical signs will vary depending on the type of foreign body (linear or discrete), the position of obstruction, and the time since obstruction. most cats present for anorexia or vomiting. partial obstruction can result in diarrhea (which can be bloody). foreign body obstruction is typically considered an acute condition, with duration of obstruction because of a linear foreign body, measured from the onset of clinical signs to diagnosis, reported to range from 1 to 10 days. 10,41,60 however, one paper demonstrated chronic, intermittent, gastrointestinal disease from a linear foreign body of a 1-month duration 175 demonstrating that partial obstruction can result in a chronic course. physical examination may or may not reveal abdominal pain, palpable abdominal mass (or plication), dehydration, or fever. all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of a linear foreign body. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see figure 3-8) . life-threatening consequences can result from the interactions of local and systemic factors that arise from intestinal obstruction. locally, damage to the mucosa from traction and pressure of the foreign object can cause hemorrhage, ischemia, and necrosis. systemically, hypovolemia, toxemia, and acid-base and electrolyte imbalances can ensue. complete intestinal obstruction by discrete masses results in gas and fluid distention of the lumen proximal other bacterial causes of diarrhea have been reported in cats, such as yersinia enterocolitica, 48 yersinia pseudotuberculosis, 63 clostridium piliforme (tyzzer's disease), 71 and anaerobiospirillum sp. 36 specific diagnosis of these (and other bacterial infections) may be found in the course of investigation. management follows the principles of supportive care and appropriate antibiosis based on sensitivity testing. small intestinal bacterial overgrowth (sibo) has not been specifically described in cats. the criteria defined for dogs is a fasting bacterial count in duodenal juice of greater than 10 5 organisms/ml 11 and is often recognized with other chronic gastrointestinal diseases. 130 healthy cats appear to have at least this number of upper intestinal bacterial with a range of 10 5 to 10 8 /ml recognized. 68 bacterial overgrowth could potentially occur with ileus or intestinal inflammation of any underlying cause. foul-smelling small bowel diarrhea with no specific pathogen recognized may be an indicator of this condition, as could an increase in bacterial metabolites, such as folate. if suspected, it is appropriate to manage with broad-spectrum antibiotics, such as metronidazole (10 to 15 mg/kg, every 12 hours, po) or amoxicillin (10 mg/ kg, every 12 hours, po) for an extended duration such as 21 to 28 days. alterations in intestinal flora have been recognized after such treatment 69 ; however, any advice for this "condition" is entirely empirical. all efforts should be directed at identifying a precise underlying cause. mycotic and other infectious agents are only rarely recognized as intestinal pathogens in cats. diagnosis is made by histologic and microbial analysis of samples obtained at biopsy. possible agents include histoplasma capsulatum, 24 aspergillus spp., candida albicans, 140 and pythium insidiosum. 119 intestinal obstructions arise most commonly as a result of neoplasia in older cats and foreign body ingestion predominantly in younger cats. 10, 41, 60 less common causes include intussusception 83 and granulomatous inflammation (e.g., from fip) 59 ; tapeworm infection, with greater than 30 worms acting as a linear foreign body, has also been reported. 173 other listed causes are volvulus, intestinal torsion, incarceration of bowel in a hernia, adhesions or stricture, intramural abscess or hematoma, and congenital malformations. 140 may not occur if the obstruction is partial or intermittent, or if vomiting results in less fluid present. since most foreign body obstructions in cats are proximal, identifiable dilatation may not be recognized for this reason. 78 linear foreign bodies present further challenges for radiographic recognition; the following typical radiographic signs 129,137 may or may not be present: to the obstruction. most gas accumulation is a result of swallowed air, which is predominantly nitrogen that cannot be absorbed by the intestinal mucosa. gas also arises from bacterial fermentation. fluid accumulates as a result of increased secretions (saliva, bile, and secretions of gastric, pancreatic, and small intestinal origin) and retention of fluid already ingested, and it can be augmented by local hemorrhage. 39 since most intestinal obstructions in cats do not reach the midjejunum, 60 reabsorption of fluids that normally occurs at the jejunum and ileum is impaired. 39 linear foreign bodies, such as string, dental floss, or elastic toys, require proximal anchoring, usually under the tongue or in the pylorus (for example, by part of a toy attached to elastic). peristalsis moves the free end of the "string" through the intestinal tract, resulting in pleats of intestines around the foreign body. as the foreign body is forced against the intestinal mucosa, the mucosa becomes edematous, and even partial penetration affects mucosal integrity, allowing systemic entry of bacteria. intraluminal bacterial populations increase for both discrete and linear foreign bodies as a result of stasis. mucosal permeability can be affected by prolonged luminal distention, allowing entry of bacteria and toxins systemically or into the peritoneal cavity. direct entry of bacteria to the peritoneal cavity, causing septic peritonitis, can result from perforation of the intestinal wall from linear foreign bodies or sharp discrete foreign bodies, such as toothpicks or plastic toys. definitive diagnosis requires identification of the foreign body retrieved at surgery or in some cases, by endoscopy. this may be aided greatly prior to surgery by diagnostic imaging. however, imaging findings, particularly in the case of partial obstructions, may be subtle enough that obstruction of no identifiable cause is recognized or no overt signs are apparent. laboratory findings are not helpful in the precise diagnosis but are important to assess fluid and electrolyte balances that must be corrected. cats rarely help practitioners by ingesting radiopaque objects, but on the rare occasions that they do, these can be observed easily on plain radiographs. nonopaque foreign bodies depend on dilatation of the intestine from gas and fluid accumulation proximal to the obstruction for radiographic recognition (figure 23-28) . one study has suggested that if the jejunal diameter is greater than 2.5 times the length of the cranial end plate of the second lumbar vertebra, then intestinal obstruction is the most likely abnormality. care must be taken that the jejunal and not duodenal diameter is measured and that the radiographs must be positioned strictly lateral, because an oblique view can alter the measurement of the lumbar vertebra. 1 however, dilatation of an obstructed intestine successful treatment of foreign body obstruction requires evacuation or removal of the foreign body as well as correction of any bacteremia or endotoxemia, acid-base or fluid imbalances. discrete foreign body obstruction requires surgery or endoscopy to remove the object. in some specific circumstances, linear foreign body obstruction may be managed conservatively by cutting the anchor point below the tongue and allowing the cat to pass the foreign body by peristalsis. however, the decision to manage a cat conservatively must be done with the cat hospitalized, with fluid therapy and antibiotic coverage and a clear recognition on behalf of the practitioner and the owner that surgery may subsequently be required. cutting a sublingual linear foreign body may be achieved in a conscious cat by applying pressure with the thumb of one hand in the intermandibular space to elevate the tongue and gently grasping it using gauze with the other hand while a second person cuts the line with a suture cutter. there is a chance of a small nick on the sublingual surface. if the cat will not tolerate the procedure, sedation is appropriate. when cutting the line, the nature of the linear foreign body should be assessed (i.e., is it more or less likely to cut mucosa). in one study, 10 19 cats with linear foreign bodies were managed conservatively with 10 cats subsequently requiring surgery. the authors of that paper created guidelines that will be adapted here. conservative management should be attempted if the cat • is presented acutely (within 2 days) after known ingestion of a linear foreign body • has a sublingually fixed linear foreign body that can be cut • has no overt signs of peritonitis • altered gas pattern with luminal gas collecting in small bubbles instead of normal curved tubular columns. this can be subtle when there is only minimal involvement of the intestine but overt when involving the entire small intestine. commashaped gas patterns are more likely to occur with linear foreign bodies. 1 contrast radiography can aid diagnosis for both discrete and linear foreign bodies but should be used with caution because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium is irritating to the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. 137 ultrasonography is a very useful diagnostic tool, particularly for discrete foreign bodies, where, in most cases, there is overt distention of the small intestines with intraluminal fluid apparent (figure 23-31) . this modality has not been extensively assessed as an adjunct to diagnosis of foreign body intestinal obstruction in cats specifically, although there are several papers assessing dogs and small numbers of cats that agree with its utility. 114,156,161 linear foreign bodies are more difficult to assess ultrasonographically, but plicated bowel can be recognized, sometimes with the foreign body seen as a hyperechoic line centrally. 156 si a technique has been described for removal of linear foreign bodies by making a single enterotomy incision proximally and passing a red rubber catheter over the linear foreign body aborally, milking the foreign body within the catheter through the colon for retrieval from the cat's anus by an assistant. 2 this technique is not always effective, because it can be hampered if the foreign body is knotted or does not run smoothly through the red rubber catheter. 102 if the affected bowel segment demonstrates evidence of necrosis or perforation on the mesenteric border of the intestine, resection and anastomosis should be performed. necrosis is indicated by dark discoloration, thin intestinal wall, poor arterial pulsation, poor capillary bleeding, or lack of peristalsis. end-to-end anastomosis can be accomplished using a simple interrupted appositional pattern or a modified simple continuous appositional pattern with the same type of suture material used for enterotomy closure. 14,88 intraabdominal masses causing intestinal obstruction are often presumed to be neoplastic but can also be of infectious origin. resection, where possible, is always recommended, because resection of neoplasia (if no metastasis) can offer a good prognosis, 76, 87, 109, 145 and infectious causes may be managed with adjunctive therapy after definitive diagnosis. intestinal obstruction in older cats is more likely to be secondary to neoplasia. any neoplasia can cause obstruction, but adenocarcinoma 32,76 and adenomatous polyps 88, 106 are reported to cause obstruction more often surgical intervention is mandatory if • clinical signs (e.g., vomiting or anorexia) persist or deterioration occurs with conservative management • the cat has overt signs of peritonitis • the linear foreign body is fixed at the pylorus some authors disagree with attempting conservative management, since a perforated intestine from a linear foreign body reportedly carries a 50% mortality rate, 41, 88 and early surgical intervention is never an incorrect decision. this should be balanced with the observation that cats can carry a linear intestinal foreign body, such as an elastic cord for a 1-month duration without intestinal perforation. 175 however, fishing line, for example, would not be so forgiving! surgery to remove an intestinal foreign body (figures 23-32 and 23-33) should be considered an exploratory laparotomy. that is, the aim of the surgery is not only to remove the foreign body but to assess the entire intestinal tract and abdomen for other foreign bodies or pathology. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction, thus delaying healing and creating the potential for surgical dehiscence. linear foreign bodies require multiple enterotomy incisions, since pulling the object out through a single incision could create iatrogenic intestinal perforation. the anchor point (either sublingual or pylorus by gastrotomy) must be released in the first instance. enterotomy incisions are closed with 5/0 synthetic, monofilament, absorbable suture material, such as polydioxyanone (pds) or equivalent, in either a simple interrupted or simple continuous pattern. 14,88 removal of a discrete foreign body (a piece of leather) at laparotomy. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction. this is the same cat as in the radiology image in figure 23 -28. affected bowel is required, with anastomosis of the healthy tissue. there appears to be no benefit to enteroplication, which can result in significant ileus. there is no benefit to performing resection-anastomosis if the intussusception does reduce manually. 15, 25 the prognosis depends on the underlying disease process and the chronicity of the intussusception, and therefore how debilitated the cat is at presentation, however, prognosis is mostly good, with survival reported in up to 80% of cases, though recurrence can occur in some cats with idiopathic disease, often at different locations of the intestinal tract. 15 constipation is defined as infrequent or difficult defecation associated with retention of feces within the colon and rectum. prolonged constipation results in harder than other types of neoplasia. please refer to the sections on intestinal neoplasia earlier in this chapter for more details. granulomatous inflammation causing a single focal intestinal lesion can lead to obstruction in the same way that neoplastic change can. feline infectious peritonitis (fip) can present as focal lesions, 59 often in the colon or ileocecocolic junction. in the case of fip, the focal lesion is usually an indicator of multisystemic disease; so, resection does not help prognosis. the fungus-like organism, pythium insidiosum has also been reported to cause granulomatous lesions, resulting in intestinal obstruction 119 from large extraluminal masses that are approximately fist sized. resection with adjunctive itraconazole (10 mg/kg) for 2 months after surgery was a successful treatment. intussusception refers to invagination or prolapse of one portion of the intestine into the part of the tract that either precedes or follows it. there is a bimodal age distribution with intussusceptions in older cats, most likely associated with neoplasia (or ibd in some cases) 25 ; underlying causes for younger cats are ill defined and may be idiopathic in many cases, 15,25 but associations with parasitism and, in one case, a linear foreign body, have been made. siamese and burmese cats seem to be overrepresented. the most common locations are the ileocolic region and the jejunum. 15, 25, 83, 110 affected cats present with nonspecific signs of gastrointestinal disease, such as anorexia and lethargy. vomiting is not necessarily a presenting sign; diarrhea may occur. abdominal palpation reveals a mass in most cases. plain and contrast radiography only show evidence of obstruction and usually do not help define that the bowel has intussuscepted. 15, 25, 83, 110 ultrasonography is very useful for diagnosis, because a distinctive pattern of alternating hypoechoic and hyperechoic concentric rings (figure 23-34) is present in transverse sections. 25, 110 sometimes, the target lesion seen can be hard to distinguish from the pathology of other intraabdominal masses, such as lymph nodes, and in these cases, the size of the lesions can help, because the width will always be greater than 11 mm with an intussusception (because the sum of at least four intestinal wall widths cannot be less) and is often greater than16 mm. 110 surgical correction is always required, and manual reduction is typically not possible because there is usually significant venous infarction, edema, and congestion (figure 23 -35) as well as adhesions from fibrin and effusions from the affected bowel. 15, 25 if the intussusception does not reduce manually, resection of the and drier feces that become impacted, and this is known as obstipation. 140 chronic, recurrent constipation and obstipation can result in megacolon, which refers to persistent increased bowel diameter that is not responsive to therapy. megacolon is not a specific disease entity; it may be considered the most advanced stage in the spectrum of chronic constipation. 18 in most cases, constipation can be managed quite simply if the underlying cause is determined and dealt with. a comprehensive list of causes of constipation is noted in table 23 of course, multiple factors can interact. for example, an older cat may have renal disease and so will be dehydrated to some degree and have arthritic hips and so be reticent to squat. the presenting signs of constipation are usually evident to owners and include straining in the litter box and producing hard dry feces, if at all. sometimes, however, owners can misinterpret signs. cats can strain because of lower urinary tract problems, and, if no urine is produced, some owners assume the problem is because of constipation. some constipated cats can intermittently have diarrhea because of direct colonic irritation from hard dry feces and so may present for diarrhea and not constipation. cats can also present for less specific signs, such as anorexia, lethargy, weight loss, and even vomiting. 140,170 vomiting can occur because of colonic receptors stimulating vagal afferent endings, which, in turn, can stimulate the chemoreceptor trigger zone. 61 sometimes owners are concerned that their cat is defecating less, but the cat has just changed its diet to a much lower-residue diet and so is producing less feces. a full dietary history is an important aspect of the initial assessment. physical examination should confirm presence of feces in the colon and assess the degree of impaction. the presence of feces can usually be confirmed by abdominal palpation. in constipated cats, the colon is often palpated as a long firm tube extending cranially; sometimes, feces can be palpated to and around the colic flexure. alternatively, the feces may be palpated as large, discrete fecal concretions (that can sometimes be hard to distinguish from intraabdominal masses such as lymph nodes). if there is any doubt of the presence or degree of fecal impaction, survey abdominal radiographs should be taken. a lateral view taken in a conscious cat should be adequate to confirm the diagnosis. the physical examination should also assess for contributing causes, including musculoskeletal conditions. any recent trauma should be taken into account. the hips and lumbosacral region should be assessed for pain. the degree of flexion and extension of the hips should be gently assessed. the lumbosacral spine can be assessed by running two fingers on either side of the spinous processes. the cat will flinch in painful areas. any arthritic change is magnified in an underweight cat, since there may be less muscle mass and the joints may bear a heavier load. any suspicions of underlying musculoskeletal abnormalities can be confirmed with radiographs. neurologic assessment should also be performed. subtle changes just affecting colonic innervation will not be apparent on physical examination alone. however, an assessment of proprioception, placing reflexes, and gait should at least be performed to assess for lumbosacral spinal cord disease. anorectal abnormalities or lesions should be evaluated. impacted or infected anal sacs can lead to reticence to defecate; and therefore anal sacs should be assessed and expressed. because this is painful for most cats, the cat should be held by an experienced assistant. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb positioned externally. a rectal exam can be performed with a well-lubricated (gloved!) middle finger, feeling over the pelvic rim for masses as well as assessing if the colon closes over (squeezes) the finger. if the colon feels open around the finger, this can be an indicator of impaired colonic innervation but does not imply that this is a permanent change. if there are impacted feces continuing to the anus, rectal examination is not possible until this has been cleared. if a cat finds anal gland expression or rectal examination too painful to tolerate (based on the clinician's judgment), these procedures should be done under sedation. hydration and electrolyte status are also important factors in the constipated cat. chronic renal disease is defined by azotemia (in conjunction with inadequately concentrated urine), which means the cat must be dehydrated to some degree. plasma or serum biochemistry and urinalysis can be used to diagnose renal disease, assess degree of renal disease, or recognize prerenal dehydration. electrolyte changes including hypokalemia and hypocalcemia may also contribute to reduced colonic smooth muscle function. 140 in young to middleaged cats of apparent good health and hydration, blood difficulty defecating and pass hard, dry stools but do not have fecal impaction at the time of examination. after the obstructing feces have been removed, steps must be taken to ensure colonic motility and smooth passing of feces. medical management of constipation traditionally involves laxatives and prokinetic agents. these may not be required in straightforward cases. as long as there is no obstructive lesion, cisapride at 2.5 mg/ cat, every 12 hours, po 82 is very safe and can be instituted with a view to reducing the dose to once daily after 10 to 14 days and discontinuing if signs remain abated. doses of up to 7.5 mg/cat, every 8 hours, po have been reported. cisapride is only available from compounding agencies in most countries. an osmotic or lubricant laxative (table 23 -18) may be used concurrently at reduced doses as necessary. reducing the fecal bulk produced is an important part of long-term management. traditional dietary recommendations are to increase the amount of fiber. 18, 26, 140, 170 increased dietary fiber results in production of shortchain fatty acids, which have been demonstrated to stimulate feline colonic smooth muscle contractions. 128 however, dietary fiber is also classified as a bulk laxative and so, by definition, will increase fecal bulk. in humans dietary fiber has been considered a mainstay of therapy for constipation, but a recent review concluded that many patients with more severe constipation have worsening symptoms when increasing dietary fiber intake. 103 because megacolon is believed to be the end result of chronic dilatation, 140,170 it is the author's firm belief that initial dietary efforts should be directed to reducing fecal bulk and thus introducing a low-residue diet. reduced dry matter intake reduces stool volume, 31 and the author has found that recurrence rates of constipation reduce greatly when cats are transitioned to entirely wet food diets. wet food diets also help ensure adequate water and urine assessments are usually not required at an initial presentation for constipation. in all cases, the same principles of management apply: 1. ensure removal of obstructing feces 2. ensure colonic motility and smooth passage of feces 3. reduce fecal bulk 4. ensure adequate hydration 5. manage underlying problems the first step is to ensure obstructing feces are removed. in simple cases, the cat will evacuate feces after use of a glycerin or sorbitol pediatric rectal suppository. another option is administration of a microenema, such as microlax (mcneil consumer healthcare, fort washington, pa.), which contains 5 ml of sodium lauryl sulfoacetate. these products act to lubricate the colon wall and therefore facilitate the passage of feces. the author prefers to use one or two of these within the consult room to observe the cat defecating (the cat must be provided with a litter tray!). the outside tube should be lubricated with the suppository contents before carefully inserting and then expressing the rest of the contents. there are also stimulant laxatives (containing bisacodyl) and emollient laxatives (containing sodium docusate) that have reportedly been used. 140 if a rectal suppository vial cannot easily be inserted because hardened fecal content obstructs its entry, a more substantive enema will be required (sometimes requiring sedation or anesthesia), and this is covered in the next section on management. some cats present for the abdominal wall with the other hand, but great care must be taken with this maneuver, because the devitalized colon can be perforated more easily. 140,170 enemas as described are painful for the cat, and opioid analgesia is recommended at the time of anesthesia. opioids can reduce peristalsis in humans, 96 but having evacuated the bowel, the pain relief is more important than this transient effect. an alternative to enemas is administration of an oral polyethylene glycol (peg 3350) solution (e.g., colyte, golytely). a nasoesophageal tube is placed and the solution is given as a slow trickle (6 to 10 ml/kg/hour) over 4 to 18 hours. defecation usually results in 6 to 12 hours. in a retrospective study of 9 cats, median time to defecation was 8 hours and the median total dose of peg 3350 was 80 ml/kg. 26a no adverse effects were noted. a cat that has been obstipated needs supportive therapy when discharged. there are no controlled comparisons of the various therapies noted in table 23-21; the author prefers cisapride 2.5 mg, every 12 hours to every 8 hours, po 82 (first thing in the morning, when the owner returns from work, when the owner goes to bed), and lactulose syrup 2 ml/cat, every 12 hours, po. a cat that has been so severely obstipated that an enema under anesthesia is required can be expected to continue these medications lifelong. to reduce fecal bulk and decrease the opportunity for recurrence, low-residue canned foods (or sachets) are preferred for cats that have become obstipated. some cats may benefit from high-fiber diets. as with simple initial episodes, canned food helps maintain adequate hydration, and at home subcutaneous fluids may be used additionally in cats with chronic kidney disease. with repeat episodes or severe obstipation, investigations for an underlying cause should be thorough and include evaluation for colonic mass obstructions. a review of published cases indicated that 96% of cases of megacolon are accounted for by idiopathic megacolon (62%), pelvic canal stenosis (23%), nerve injury (6%), or manx sacral spinal cord deformity (5%). 170 although most cases are idiopathic, an attempt should be made to identify and treat any specific underlying causes. megacolon is not specifically defined in cats. it has been described as "generalized colonic dysfunction manifesting as severe colonic dilation and fecal impaction," or a "severely and irreversibly dilated and hypomotile" colon 140 and "a subjective evaluation of the diameter of the colon, usually based on radiographic assessment." 18 there are specific radiographic guidelines for humans with megacolon, in that a colonic diameter of more than 6.5 cm at the level of the pelvic brim is considered diagnostic. 118 intake and therefore help maintain hydration. however, increased dietary fiber is beneficial for some cats, and trial and error may be required to determine whether a high-fiber or low-residue diet will be of benefit to each individual cat. in one report, 15 cats with recurrent constipation refractory to traditional medical and dietary management were successfully treated with a psylliumenriched dry extruded diet. 48a after 1 month on the diet, 14 cats had no clinical signs of constipation. the remaining cat was clinically normal after 2 months on the diet. improvement was noted in 10 of 15 cats after only 7 days of dietary therapy. measures should be taken to ensure adequate hydration. maintaining adequate hydration is particularly relevant for cats with chronic kidney disease that have impaired ability to conserve water. changing to wet food diets helps increase water intake. some cats with chronic kidney disease may need additional fluid support, such as subcutaneous fluids administered by the owner at home on a regular basis. underlying problems may be minor and simple to manage, such as an anal gland abscess, or more involved, such as reduced pelvic outflow, as a result of prior trauma. arthritis is a common underlying factor in many older cats and may be managed with prudent use of nonsteroidal agents (see chapter 26) . in cases of obstipation, the cat is more likely to be debilitated to some degree; so, laboratory investigations to assess plasma or serum biochemistry parameters as well as hematology and urinalysis are ideal. any hydration deficit or electrolyte abnormalities should be corrected before the anesthesia that is often required to remove the obstructing feces. rectal suppositories and microenemas are usually ineffective in obstipated cats. enemas are often required to remove impacted feces in such circumstances. the enema solution must be warmed and introduced slowly to avoid vomiting. the typical volume required is 5 to 10 ml/kg (so, up to approximately 50 ml/cat). the enema solution can be an isotonic electrolyte solution or tap water, and mild soap can be added (but any soap used must not contain hexachlorophene, which is neurotoxic if absorbed); mineral oil can be used (5 to 10 ml/cat) as a lubricant or docusate as an emollient (5 to 10 ml/cat), but the two agents must not be used together since docusate promotes mucosal absorption. sodium phosphate-containing enemas must not be used, because they can induce severe hypernatremia, hyperphosphatemia, and hypocalcemia in cats. often, the enema solution alone is insufficient to reduce the fecal mass, and manual manipulation of the feces by abdominal palpation is required. sometimes the feces must be broken down by a gloved finger perrectum while the colon is massaged manually through radiographically, in the lateral view, the normal colon should be approximately the same diameter as the length of the body of the second lumbar vertebra. 81 in cats, however, "there are no published guidelines for determining megacolon, so, diagnosis of abnormal colonic dilatation is subjective." 75 however, one author has suggested that "as a rule of thumb, the diameter of the colon should be less than the length of the body of the seventh lumbar vertebra (l7)." 105 this author continues, "enlargement of the diameter of the colon beyond 1 1 2 times the length of the body of l7 is indicative of chronic large bowel dysfunction and an explanation must be sought." 105 a recent paper found that 15 of 20 cats with no gastrointestinal disease had a colon diameter greater than the length of l7; however, no assessment of constipated cats was made. 1 in practice, many cats with megacolon have a colonic diameter far exceeding this guideline ( figure 23 -36). one study of 11 cats with megacolon found the mean diameter of the colon was 2.7 times greater than the length of the seventh lumbar vertebra (median, 2.4; range, 1.8 to 3.3), 107 but in general, objective descriptions of this condition are lacking in the veterinary literature. the definition of megacolon in cats should include functional as well as radiographic guidelines. in the absence of broadly recognized radiographic recommendations, the author proposes that the o'brien rule-ofthumb guidelines 105 (as noted above) be introduced until a more comprehensive study can establish other radiographic diagnostic criteria (or confirm these). the author therefore proposes to define megacolon as dilatation of the colon, to more than 1.5 times the length of the seventh lumbar vertebra, which is refractory to medical and dietary management. practitioners can expect the radiographic assessment of colonic dilatation to exceed this guideline in cats with megacolon and, conversely, there are likely to be cats having colonic distention greater than this amount that will respond to medical and dietary management and can therefore not be defined as having megacolon. by the definition used above, megacolon is refractory to medical and dietary therapy; so, to be defined as having megacolon, a cat may have had several episodes of obstipation managed by enema as well as dietary trials (with both low-residue and high-fiber diets) and medical therapy with cisapride and an osmotic or emollient laxative; yet the cat will still obstruct with feces. in these circumstances, the only possible therapy is subtotal colectomy. subtotal colectomy refers to surgical excision of 95% to 98% of the colon, whether it is grossly diseased or not with preservation of the ileocolic junction (icj). this approach has resulted in a more favorable clinical response than when the icj is also excised. 22, 170 when preserving the icj, it has been noted that, in some rare cases, it can be difficult to join the proximal segment of colon to the distal piece of descending colon because of the tethering effect of the ileocecocolic blood vessels. in these cases, sacrificing these vessels and removing the icj (i.e., total colectomy) is recommended to facilitate approximation of the ileum to the distal colonic segment. 21 a recently described technique using a biofragmentable anastomosis ring, compared with sutured anastomoses, showed no discernible effect on prognosis. 131 prognosis following subtotal colectomy is generally good. a review of multiple papers, totaling over 100 cats that had undergone subtotal colectomy, found the most commonly reported perioperative complication was diarrhea or loose stools immediately after surgery. in the majority of individuals, stool consistency improves without further treatment so that within 1 to 6 weeks of the surgery soft, formed stools are developed. diarrhea can persist in a small number of cases. in the longer term, in some cats, constipation can eventually return, but this can usually be managed by dietary and medical therapies. 172 pathology of the rectum or anus is relatively rare in cats and therefore poorly described in the veterinary literature. consequently, published information is often not referenced, suggesting it expresses the authors' opinions. readers are directed to surgical texts for details and approaches about surgical corrections. the anal sacs are paired cutaneous evaginations situated between the internal and external sphincter muscles. these sacs store secretions from alveolar and sebaceous glands that reside within the sacs. each anal gland has an associated duct that opens to the skin surface just lateral to the anus. 136,155 normal anal gland secretions have only very recently been described 47 and vary markedly; the color can be white, brown, orange, yellow, tan or gray, and consistency can range from watery to thick and creamy, with two thirds of cats having solid portions within the secretion. on microscopic examination, epithelial cells are commonly seen, with most cats having some neutrophils present. bacteria are commonly recognized as are, on some occasions, yeasts. bacteria seen in this study were mainly gram-positive cocci (63%) or gram-negative cocci (30%). gram-negative or grampositive rods were also seen but were rarely the dominant bacterial population. 47 with such a wide range of normal secretions, it is difficult to diagnose any pathology from the nature of the secretion alone. however, blood is infrequently recognized, and neutrophils are typically present in only small numbers in normal secretions. anal sac diseases described in cats include impaction, inflammation (sacculitis), infection, abscessation, and neoplasia (essentially the same as in dogs). 140,155 it has been contended in dogs that sacculitis and abscessation are an extension of impaction. it is not known in dogs or cats what the predisposing causes are, but suggested underlying reasons are loose stools (that are less effective at expressing the sac during defecation), local swelling or edema occluding the duct, and obesity. 155 the author's observations have also indicated that constipation can result in anal sac impaction because of less frequent expulsion of the sac contents; the resultant pain of the anal sac impaction can lead to further constipation, thus establishing a cycle. the retention of secretions may predispose to sacculitis, but impacted anal sacs do not always result in inflammation. abscessation is a likely sequel to sacculitis. 155 cats usually present for licking, scratching, or biting at the perineal area and can present for scooting (or dragging their anus) as dogs do. other presenting signs can be inability to sit or settle, a lump seen by the owner, or a generally unwell state. expression is the only management required for impacted (and not infected) anal sacs. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb externally. this is painful for most cats; so, the cat should be held by an experienced assistant, and it is sometimes not possible without some degree of sedation. with frequent episodes, underlying causes should be investigated. sometimes, trial-and-error diet change to manipulate the nature of the feces to either more (highfiber diets) or less (low-residue diets) bulk help reduce the frequency of episodes. obesity should be managed by reduced caloric intake, but dietary management for this should also take into account the nature of the feces. overt infection may be recognized by pus secretion from the anal sacs, which will have a high numbers of neutrophils. this can be managed by broad-spectrum antibiotics, such as amoxicillin/clavulanate or cephalosporins. a single treatment with a nonsteroidal antiinflammatory drug, such as meloxicam, can be given in animals with appropriate hydration and without other illness. anal sac abscesses often present already open and draining. many heal well by secondary intention with antibiotic treatment until they are closed over; so rechecks are required before the completion of an antibiotic course. large abscesses may require surgical drainage with the insertion of a penrose drain and management as for a cat fight abscess. it must be remembered that wounds in this area are easily re-infected by fecal contamination. recurrent impaction, sacculitis, or infection may require anal sacculectomy (as in dogs). this procedure should be delayed until infection is cleared. the procedure is similar to that performed in dogs. reports of anal sac/gland neoplasia were confined to sporadic case reports, 97,108 until a large case series was recently published. 141 in this study, 64 cases of anal gland carcinoma were recognized at a private diagnostic laboratory during a 12-year period, with submissions from 62 practices. this indicates that, for most practices, this condition will be seen, at most, once every 12 years. affected cats ranged in age from 6 to 17 years (median and mean, 12 years); female (mostly spayed) cats were overrepresented (61% of cases), and siamese cats may have been over-represented (7.8% of cases). the number of siamese cats with anal sac neoplasia was 3 times greater than the number of siamese cats in the laboratory reference population. affected cats presented for dyschezia, recurrent constipation, change in the nature or volume of feces, b). a low-residue wet diet is recommended to reduce fecal bulk during the healing period. rectal prolapse occurs as a result of a disease process that causes chronic straining, such as intestinal conditions that result in diarrhea and tenesmus 2. conditions that result in constipation or other intestinal obstruction 3. lower urinary tract diseases 4. dystocia and/or perineal swelling or ulceration, sometimes with purulent or hemorrhagic discharge. most tumors were originally interpreted as and initially managed as anal sac abscess. presumptive metastasis in liver, lung, or abdominal lymph nodes was recognized by physical examination or radiography in six cats; one cat was hypercalcemic. excision appeared to be curative (with a 3-to 4-year follow-up period) in 3 of 29 cats undergoing surgery for resection or debulking (others had only incisional biopsy performed). for the remaining 36 cats with known postsurgical outcome, median survival was only 3 months, with a 19% 1-year survival rate (with none of these cats surviving to 2 years). 141 atresia ani is a developmental defect of the anal opening or terminal rectum (see figure 41 -4). kittens usually present within days or weeks of birth with abdominal distention, discomfort, tenesmus, restlessness, vomiting, and/or loss of appetite. there are several anatomic variations 22 : • type i: a membrane over the anal opening remains, with the rectum ending as a blind pouch just cranial to the closed anus. • type ii: the anus is closed as in type i, but the rectal pouch is located somewhat cranial to the membrane overlying the anus. • type iii: the rectum ends as a blind pouch cranially within the pelvic canal (rectal atresia), whereas the terminal rectum and anus are normal. • type iv: occurs in females and atresia ani exists with a persistent communication between the rectum and the vagina (rectovaginal fistula). this fistula can occur with a normal anal opening as well. most reported cases have been type iv, 23, 146, 150, 158, 166 and this has also been recognized with concurrent sacrococcygeal agenesis. 3 surgical correction has been described for type ii 157 and type iv 19,91 atresia ani in cats. the reader should consult these references for surgical advice; possible complications include megacolon after prolonged obstruction, postsurgical anal stricture, and fecal incontinence because of sphincter dysfunction. foreign bodies in cats rarely obstruct the gastrointestinal tract distal to the jejunum 60 ; however, large fecal balls resulting from constipation can, additional to constipation or obstipation, cause distention of the anus. this distention can result in inflammation of the anal sphincter with loss of tone ( figure 23-37, a) , which, in the author's experience, is temporary with correction of the underlying cause of constipation. it can take some weeks for the dilated anus to return to normal (figure 23 -37, with antibiotics, such as cephalosporins, and regular cleansing. prolapses are usually classified in three ways. 62 first degree: prolapse of only mucous membrane 2. second degree: prolapse of full rectal wall thickness 3. third degree: prolapse is sufficient to bring mesorectum outside the anus the prolapsed rectum is obvious but must be differentiated from ileocolic intussusceptions, which have been described with neoplasia. 37 this distinction can be made by inserting a thermometer through the anus alongside the prolapsed mass. insertion will not be possible for an intussusception but will be for an anorectal prolapse. the prolapsed tissue must be assessed for viability, and management must include determining and managing the underlying cause as well as management of the prolapse. in simple cases where the mucosa is viable, the prolapse can be reduced with lubrication and gentle pressure. a temporary purse-string suture may be required to prevent recurrence. perineal dermatitis is often confused with gastrointestinal or urogenital disease, because there are often copious sebaceous secretions that can mimic fecal or urinary secretions. perineal dermatitis can result from flea or other allergies but also fecal or urine scalding associated with diarrhea or urinary incontinence, respectively. skin fold dermatitis can also occur in obese cats ( figure 23-38 ). episioplasty has been described to correct this, 121 but the author has found that stringent dieting can result in improvement while managing the skin fold dermatitis these populations. 48 the reported prevalence for each parasite varies greatly with the population studied, the geographic location of the population, and the sensitivity of the diagnostic test used to study that population. 48 the presence or absence of diarrhea is not a reliable predictor of whether a particular cat is infected with or shedding a parasite. 42 in fact, most cats with diarrhea do not harbor enteric protozoa. 48 on the other hand, most cats with diarrhea because of enteric pathogens will shed those organisms, often intermittently. it is important to remember that infection with most gastrointestinal parasites may not cause clinical signs. therefore detection of a pathogenic parasite in a cat with diarrhea does not necessarily prove causation. 48 a search should always be undertaken to identify other causes of diarrhea prior to convicting a cat of having diarrhea because of a particular parasite. in addition, co-infections or the presence of other noninfectious causes of diarrhea can result in more severe diarrhea that is often refractory to treatment for the parasite. treatment will be more rewarding if all potential causes of diarrhea are identified in the patient. enteric parasites with zoonotic potential occur commonly enough that cats, particularly those with diarrhea and who are owned by immunocompromised persons, should be evaluated for those pathogens. 26, 27 the following is a discussion of the most common enteric parasites found in cats. for more on parasite prevention and control, see chapter 8, and for more on zoonotic enteric parasites, see chapter 34. ollulanus tricuspis is an almost microscopic nematode worm infecting the stomach of domestic and wild cats. 5 the worm measures less than 1 mm long. 3 the larvae of o. tricuspis develop and hatch within the uterus of the female worm. they develop to maturity in the stomach of the cat where it is capable of re-infecting the host. 3 the worm is transmitted to other cats that ingest the vomitus of an infected cat. 41 clinical signs shown by infected cats include vomiting, anorexia, and weight loss. 2, 5 histologic findings in infected cats include lymphocytic-plasmacytic gastritis, lymphoid hyperplasia, and mucosal fibrosis. gross lesions may be absent, or the cat may develop nodular gastritis. 41 one report suggested the parasite may have been a contributing factor in the carcinogenesis of a gastric adenocarcinoma in an infected cat. 10 160. a common theme when discussing the prevalence of most gastrointestinal parasites in cats is that they occur more commonly in younger cats and in cats housed in crowded conditions, such as catteries and shelters. it is likely an increased chance for transmission exists in lungs. after further development in the lungs, the parasite migrates up the trachea and is swallowed. adult s. felis and s. planiceps burrow into the wall of the small intestine, while adult s. tumefaciens lives in the colonic mucosa. ova may be shed in the feces or hatch in the intestinal tract. autoinfection occurs if larvae become infective and penetrate the intestinal wall before being shed. ova and larvae that are shed develop into freeliving adult worms. 11 the prepatent period is between 7 and 10 days. 1, 11 clinical signs and diagnosis signs of a strongyloides spp. infection are usually absent. 1, 40 lung migration may cause cough or respiratory distress. the presence of the parasite in the intestinal tract may result in diarrhea and weight loss. 11 strongyloides tumefaciens is associated with the formation of small, worm-filled nodules in the colon. 40 identification of strongyloides spp. larvae using the baermann fecal concentration technique is required to diagnose most infections. unless the infection is heavy, examination of a fresh fecal smear is insensitive for identification of these larvae. 1 the nodules formed by s. tumefaciens infection can be visualized during colonoscopy. histopathology of the biopsied nodules should reveal many adult worms. 40 infection with strongyloides spp. can be treated with fenbendazole, 11 pyrantel pamoate, 40 thiabendazole, 1,11 or ivermectin. 3 to evaluate efficacy, repeat a fecal examination 2 to 3 days after the treatment ends. because of the presence of free-living adult worms in the environment and the ability of larvae to cause infection by penetrating intact skin, prevention is difficult. keeping cats indoors in warm, humid climates may be an owner's only means of preventing infection with strongyloides spp. parasites. infections with trichuris vulpis rarely occur in cats and are considered to be clinically unimportant. 3, 14 the two species of roundworms commonly infecting cats are toxocara cati (figure 23-39) and toxascaris leonina (figure 23-40) . the latter also has the ability to infect dogs. 14 cats are infected with t. cati in several ways. most commonly, infection is by ingestion of contaminated food, water, or infected paratenic hosts such as rodents. transuterine transmission has not been reported. 14 the diagnosis of infection with o. tricuspis is difficult, because ova are not shed in the feces; rather, the vomitus must be examined for worms or larvae. the worms may also appear in gastric mucosal biopsy samples. 41 a report of 131 cats undergoing endoscopic examinations found the parasite in gastric biopsy samples from 4 cats. 5 fenbendazole may be effective in treating infections with o. tricuspis. 41 preparations with febantel may also be expected to successfully treat these infections. transmission can be prevented by appropriately treating infected cats. other cats should not be allowed to ingest infected vomit. this parasite is of no zoonotic concern. physaloptera another parasite rarely inhabiting the stomach in cats is in the genus physaloptera. larger than ollulanus tricuspis, this blood-sucking worm infects cats that have ingested intermediate hosts, such as cockroaches, crickets, or flour beetles. 11 preying on transport hosts, such as mice that have eaten an intermediate host, is another way cats become infected with this parasite. clinical signs of infection with physaloptera spp. include vomiting, anorexia, and melena. a diagnosis of physaloptera infection can be made after identifying the ova in the patient's feces or adult worms in the vomitus. occasionally, the worms may be seen during gastroscopy. the adult worms must be differentiated from ascarids. 11 infection can be treated with ivermectin, pyrantel pamoate, or fenbendazole. 3 because there is no migratory phase of the life cycle, the treatment does not need to be repeated. 11 three species of strongyloides infect cats. strongyloides felis infects cats in india and tropical australia, 1,43 s. tumefaciens is a rare parasite of cats in the southeastern united states, 3 and s. planiceps is found in cats in malaya and japan. 1 strongyloides stercoralis, found in dogs and humans, produces experimental infections in cats, but natural infection with this species has not been observed. 1 feline infection with strongyloides spp. is considered by most to be rare. however, one report from australia identified s. felis in 169 of 504 necropsied cats. 43 infection with strongyloides spp. occurs after ingestion of infective larvae. infection can also take place after the larvae penetrate the skin of the cat. 11 ingested larvae penetrate the intestinal wall and migrate through the diaphragm into the lungs. after cutaneous penetration, the larvae enter the venous circulation and enter the clinical illness because of roundworm infection is uncommon. illness, when it does happen, most often occurs in kittens 26 signs may be mild and can include vomiting, 14 diarrhea, weight loss, poor growth, and a "pot belly." 26 a heavy infection with t. cati can result in catarrhal enteritis. severe infections can lead to intestinal obstruction and, possibly, perforation. 26 much less dramatic changes arise after infection with t. leonina, although enteritis may occur. 14 roundworms are frequently diagnosed with a fecal floatation. the centrifugal floatation technique is more sensitive than the simple fecal floatation technique many hospitals use. 14 occasionally, adult worms will be passed with the feces. the goals of treating roundworms include disease prevention in an individual cat or kitten, prevention of environmental contamination by cats defecating outside, and the prevention of zoonotic infections. many effective and safe anthelmintics are available (table 23-19) . benzimidazoles, such as fenbendazole, act on the parasite's microtubular structure, leading to disintegration of the worm's intestines, muscular layer, and hypodermis. 14 pyrantel in the pamoate formulation is poorly absorbed and causes paralytic parasite death. macrocyclic lactones, such as milbemycin, also lead to paralytic parasite death. these compounds act on the parasite's gamma-aminobutyric acid (gaba)-and glutamate-controlled ion channels. these channels are lacking in tapeworms, accounting for the lack of efficacy against these parasites. 14 lastly, emodepside (a cyclic octadepsipeptide) has been combined with praziquantel in the product profender (bayer animal health). this topical parasiticide has been shown to be both safe and effective. 14 these drugs appear to be so safe that overdosing is almost impossible. 14 kittens can be dewormed starting at two weeks of age and again at 4, 6, 8, 12, and 16 weeks. 26 older kittens and adults can be dewormed every month to 4 months. 14 because of the safety of these drugs, the possibility of false-negative tests and, more importantly, the zoonotic potential of these infections, perhaps all kittens should be dewormed, not just those testing positive. roundworm ova are very hardy and can remain infective for years. 14 they survive sewage treatment and composting, and there is no practical means of decreasing the ova population once the environment is contaminated. thus it is best to attempt to prevent contamination in the first place. when practical, keeping cats indoors allows appropriate control of potentially transmammary infection occurs, but only if the queen is acutely infected late in pregnancy. chronically infected queens do not pass t. cati ova in their milk. 14 after ingestion, t. cati larvae migrate through the small intestinal wall, into the liver, and then to the lungs where they are coughed up and swallowed. these larvae then infect the small intestine. some of the migrating larvae become encysted in the cat's muscle tissue. larvae from ova ingested through the milk tend not to undergo migration and mature directly in the small intestine. 14 the prepatent period is approximately 8 weeks. infection with t. leonina occurs after ingestion of infective ova or an infected paratenic host. unlike t. cati, very few t. leonina larvae migrate through the cat's tissues. most develop in the wall of the small intestine. the prepatent period is 7 to 10 weeks. toxascaris leonina ova can become infective within 8 days of being passed in the feces when the ambient temperature is 27° c but normally require 3 to 4 weeks. 14 lungs, and kidneys. ocular larval migrans results in granulomatous retinitis that is often misdiagnosed as retinoblastoma in older children. 3 this can lead to unnecessary enucleation. toxocara cati appears, however, to be less important than t. canis as an infection in humans. 3 the species of hookworms that infect cats are ancylostoma tubaeforme and ancylostoma braziliense (see figure 23 -39). they are reported to be an uncommon infection in cats. 26, 34 ancylostoma braziliense can also infect dogs. hookworm infections occur after ingesting food or water contaminated with hookworm larvae or eating contaminated fecal material. if the pet cat is allowed outdoors, attempts at preventing hunting may reduce the possibility of infection. keep children's play areas, such as sand boxes, inaccessible to cats when children are not at play. feeding only well-cooked food can prevent infection by contaminated food. finally, empirical, preventative deworming for cats that go outdoors should be performed 3 to 4 times yearly. any less frequently does not lead to an appreciable decrease in the prevalence of the parasite. 7 roundworms easily infect humans who ingest the ova, particularly children. visceral larval migrans occurs after infection with toxocara canis in humans. infection can lead to the formation of nodules in the brain, liver, tapeworm infections are well tolerated by the cat. usually there are no signs of infection other than finding segments on the feces or attached to perianal hair. because both d. latum and spirometra tapeworms absorb vitamin b 12 across the cuticle, megaloblastic anemia is possible, but unlikely. 11 tapeworm infections are diagnosed by identifying the typical appearance of the segments 8 or the egg packets within the segments. 26 the segments of t. taeniaeformis are flat, while those of d. caninum have been described as appearing like a grain of rice. the segments should be handled carefully, because they are friable and rupture may result in exposure of the handler. 8 the operculated ova of d. latum and spirometra spp. must be differentiated from trematode ova. even though tapeworm infections are well tolerated, cats should be treated for reasons of owner discomfort and public health concerns (see table 23 -19) . these infections are easily treated, because drug treatment is highly effective. re-infection must be controlled using preventative measures, especially flea control to prevent re-infection with d. caninum. praziquantel and infected paratenic hosts. the larvae can survive for months in the tissues of paratenic hosts. 14 infection also occurs after larval migration through the skin. in either case, the worm matures in the small intestine. 14 unlike dogs, transmammary infection has not been reported in cats. 3, 14 the prepatent period is between 19 and 28 days, depending on the route of infection. the time to patency after transcutaneous infection is longer than for direct colonization. infective l3 larva develop 2 to 7 days after the ova are passed. 3 developing larvae attach to the mucosa of the small intestine where they ingest copious amounts of blood. because the worms can remove a significant volume of blood from kittens, weakness from iron-deficiency anemia 34 or blood-loss anemia may be noted. 14 melena and diarrhea may also be recognized. signs are uncommon in adult cats. identification and treatment of hookworm infections are similar to that for roundworm infections (see table 23 -19) . hookworm larvae are not as hardy as roundworm eggs. soil contamination may be a temporary problem in areas that experience a hard frost. 3 hookworm larvae will not develop in temperatures less then 15° c or greater than 37° c. frequent, appropriate disposal of feces, cleaning surfaces with a 1% bleach solution, and deterring hunting may prevent infections. migration through the skin of persons coming into contact with the larvae of a. braziliense is the most common cause of cutaneous larval migrans, particularly in the southeastern united states. 3 this is an erythematous, pruritic skin eruption often found on the soles of the feet of infected children. the tapeworms most commonly found in cats are dipylidium caninum and taenia taeniaeformis. diphyllobothrium latum, spirometra spp., and echinococcus multilocularis occasionally infect cats. the latter is important, because it can lead to alveolar echinococcosis in humans. 8 spirometra tapeworms are found in north america (s. mansonoides) and far-east asia (s. mansoni and erinacei), while d. latum prefers temperate climates. 11 unnoticed, but the cat may cough or experience hemoptysis. 3 diagnosis involves demonstration of fluke ova in the feces. although therapy may be unnecessary, 11 praziquantel or epsiprantel are effective in eliminating the intestinal population of the fluke. platynosomum spp. are flukes living in the gall bladder, bile ducts, 46 and pancreatic ducts. 3 these flukes are most prevalent in the southeast united states and caribbean islands 3 and require two intermediate hosts. the first host is a snail, while the second intermediate host is a lizard, toad, gecko, or skink. 46 cats become infected with this fluke after ingesting an infected second intermediate host. the prepatent period for the fluke is 8 weeks. 46 most infections are subclinical. if clinical signs do occur, they may include weight loss, vomiting, diarrhea, icterus, hepatomegaly, or abdominal distention. diagnosis involves identification of ova shed in the feces using a fecal sedimentation method 46 or by finding adult flukes in the gall bladder or bile ducts during abdominal surgery. treatment involves administering praziquantel (20 mg/kg, q24h, po for 3 to 5 days) and/or surgical removal of the flukes. 3 two species of coccidians are the most common to infect cats, isospora felis and isospora rivolta (figure 23-41) . the genus isospora may be renamed cystoisospora. these are epsiprantel are safe and effective. fenbendazole is effective against t. taeniaeformis, but not d. caninum. without controlling exposure to intermediate hosts, tapeworm infections are difficult to eliminate. flea control is imperative in eradicating infections with d. caninum. controlling predation helps prevent ingestion of t. taeniaeformis-infected rodents. infection with d. caninum occurs in young children who are most likely to eat fleas. infection results in only minimal signs of illness. 8 the larval stage of t. taeniaeformis is of little zoonotic importance. 3 although cats are uncommonly infected with echinococcus multilocularis, potentially life-threatening alveolar damage occurs in north american humans infected with this tapeworm. 8 plerocercoids of spirometra spp. can penetrate the mucous membranes or open skin wounds of humans and migrate around the subcutaneous connective tissue, forming nodules, a condition called sparganosis. 3 megaloblastic anemia, as a result of vitamin b 12 deficiency, may occur in humans infected with d. latum or spirometra spp. tapeworms. 11 alaria marcianae flukes reside in the intestinal tract of cats and the mammary glands of lactating queens. miracidia hatch underwater from ova shed in the feces and penetrate the skin of a snail. after further development, cercariae penetrate the skin of leopard frog tadpoles and are able to survive the metamorphosis to the adult frog. 3 if the tadpole is eaten by a snake, bird, or mammal, the parasite enters the host's tissues but does not undergo further development. after a male or nonlactating female cat ingests the infected intermediate host, the parasite penetrates the wall of the small intestine, passes through the diaphragm, and enters the lungs for further development. finally, the parasite is coughed up and swallowed to complete maturation and reproduce in the small intestine. if, however, an infected host is ingested by a lactating queen, the parasite migrates through the tissues to the mammary glands, rather than the lungs. 3 once shed in the milk, the parasites develop into mature adults in the kittens. some of the mesocercariae remain in the mammary glands to infect future litters. clinical signs associated with worms in the small intestine are uncommon. 11 migration through the lungs often goes species-specific obligate intracellular parasites. 4, 13 they are able to survive in the environment for months. 13 a detailed description of the coccidial life cycle can be found elsewhere. 4, 13 simply put, direct transmission is by ingesting oocyst-contaminated food or water or by grooming contaminated body parts. indirect transmission occurs after ingesting a mechanical vector or the infected tissues of paratenic hosts. 39 after ingestion by a cat, the oocyst excysts in the small intestine and enters the enterocyte where further development occurs. 26 the parasite may also migrate through the intestinal wall to form cysts in mesenteric lymph nodes. these cysts may serve as a source for reinfection. 4, 13 the prepatent period is 4 to 11 days 26 and the shed oocyst becomes infective after several days of exposure to warmth and moisture. 13 infection with isospora spp. is usually subclinical. 26 signs, if they occur, range from mild, transient watery diarrhea to severe mucohemorrhagic diarrhea with vomiting and resultant dehydration and weight loss. 4, 13 signs are most commonly recognized in severely infected neonatal kittens, 26 particularly those with concurrent illness, and arise because of small intestinal congestion, mucosal erosion, or villus atrophy. 39 signs may also be noted in immunosuppressed adult cats. 26 isospora species are readily found in fecal floatation or wet-mount examinations. shedding can be intermittent, but most cats with diarrhea caused by coccidial infection shed large numbers of oocsyts. 39 fortunately, in most cats, the diarrhea from isospora spp. infection is self-limiting. 26 in fact, if a kitten is persistently shedding oocysts despite appropriate treatment 13 or the parasite is identified in an adult cat with chronic diarrhea, attempts should be made to identify co-infections or other diseases that may cause diarrhea. 39 anticoccidial drugs are either coccidiostatic or coccidiocidal (table 2320) . coccidiostatic drugs are the most commonly used drugs for individual pet cats. trimethoprim-augmented sulfadiazine (tribrissen; intervet/schering-plough animal health, summit, nj) or another sulfa-containing antibiotic, sulfadimethoxine (albon; pfizer animal health, madison, nj), can be used. supportive care for severely affected kittens, such as parenteral rehydration, should be used as needed. coccidiocidal drugs are often reserved for use in densely populated situations such as catteries or shelters. 26 however, many veterinarians are now using them as a first-line defense against isospora spp. infection. 13 ponazuril (marquis oral paste; bayer animal health, shawnee mission, kan.), formulated for horses, is effective and can be safely administered to cats. for more on the use of ponazuril in cats, see chapter 46. a related drug, diclazuril, is also available and may be administered once at 25 mg/kg po. 13 while not available in north america, toltrazuril (baycox, bayer animal health) may be administered once at 30 mg/kg po or 15 mg/kg po once daily for 3 days. 32a a second course of therapy 10 days later may be required to completely eliminate the oocysts. sanitation is very important, because the oocyst requires several days to become infective. frequent removal of feces, preferably daily, is recommended to prevent re-infection and transmission to other cats. 39 controlling a cat's ability to hunt reduces the chance of ingesting an isospora-infected rodent. control of mechanical vectors, such as cockroaches and flies, is also useful. 13 since a cat can become infected after grooming an infected cat's perineum, consideration should be given to treating all cats in contact with the patient. 39 in addition, catteries and shelters should ensure all food is well cooked, litter boxes are cleaned daily, and surfaces are well cleaned with steam 13 or 10% ammonia. 39 where recurrent isospora spp. infections are a problem, prophylactic treatment of all 2-to 3-week-old kittens with ponazuril should be considered. 13 despite all wellintentioned efforts at hygiene and treatment, isospora spp. infection can still be transmitted to other cats. 13 because these are species-specific parasites, transmission of i. felis and i. rivolta from cats to humans does not occur. the flagellated protozoal parasite, giardia duodenalis, has seven microscopically indistinguishable genotypes or assemblages. 26 assemblages a and b infect humans, while assemblage f is harbored by cats. cats will occasionally harbor assemblages a and b. 39 infection with g. duodenalis occurs after ingesting cystcontaminated feces, by grooming an infected cat or from contaminated fomites. 37 re-infection may occur by selfgrooming. only a small number of cysts need be ingested to establish an infection. in humans as few as 10 cysts are required to cause infection. 37 after ingestion of infective cysts, trophozoites begin to excyst in the stomach. 37 this process is completed in the proximal duodenum. 26 the trophozoites adhere to enterocytes along the length of the small intestine using the ventral suction disk. intermittent shedding of immediately infective cysts begins 5 to 16 days after infection. 28 proteins released during encystment of the trophozoites are detected by the fecal antigen tests. 37 cysts may adhere to the perianal region, facilitating re-infection by self-grooming. 28 occasionally, trophozoites are found in examinations of fresh, watery feces. these do not survive for long and are not infective. 4 the mechanisms of disease induced by g. duodenalis are still unclear. after the trophozoite attaches to the brush border of the enterocyte, the tight junction between cells is disrupted, increasing intestinal permeability. 37 the brush border becomes attenuated, further exacerbating malabsorption of water, electrolytes, and other nutrients. 39 the alteration in intercellular adhesion results in t-lymphocyte activation and mucosal cell injury. 37 infection also promotes mucosal cell apoptosis (preprogrammed cell death). 28 in addition, small intestinal bacterial overgrowth may accompany g. duodenalis infections, resulting in more severe clinical signs. 39 fortunately, most cats infected with g. duodenalis show no clinical signs. 28, 39 the most common sign is acute, transient, small bowel diarrhea 28 without systemic illness, such as fever or vomiting. 39 less commonly, a cat might have profuse, watery malodorous diarrhea 37 with mucus. 39 also possible, but uncommon, is weight loss 26, 28 or abdominal pain. 37 the severity of clinical signs exhibited in an individual cat depends on the age and general health of the cat. 37 cats co-infected with cryptosporidium felis or tritrichomonas foetus may have more severe diarrhea that is more difficult to control, 39 as will the presence of bacterial overgrowth. the diagnosis of g. duodenalis requires demonstration of trophozoites or cysts in a fecal examination, or detection of encystment proteins or giardial dna in a fecal sample. a reliable diagnosis may be difficult to obtain for several reasons. cysts are small, easily missed, and must be differentiated from plant debris or yeast. 37 trophozoites are short lived outside the body and can only be found in very fresh, watery feces or, better yet, in diarrheic feces collected directly from the cat's rectum. 37 shedding of cysts is usually intermittent, and the intensity of shedding varies greatly. 28, 37 because of these difficulties, the absence of the organism in a fecal sample does not eliminate it as the cause of diarrhea. it is often necessary to test multiple fecal samples, using at least two different techniques in order to find the organism. 37, 39 the easiest test to perform is a fecal smear or wet mount examination to identify trophozoites or cysts (figures 23-42 and 23-43 ). the sample examined should be very fresh, warm, diarrheic feces. 39 one drop of feces is placed on a slide along with a drop of 0.9% saline or lugol iodine. 28 trophozoites are identified by their characteristic structure (table 2321) . the motile trophozoites have a motion described as appearing like the back and forth rolling motion of a falling leaf. since lugol iodine stain kills the trophozoite, there will be no motion to detect. 28 this test is not very sensitive; however, with trained examiners, the test has a high specificity. increased sensitivity can be gained by performing a centrifugal flotation using zinc sulfate. the sample should be warm, fresh feces or feces refrigerated for no more than 2 days. 28 the processed sample is examined for the same structures as the wet mount. the sensitivity of examining one sample is 70% 28 and increases as more samples are examined. the sensitivity of looking at three samples is 95% 26, 28 ; therefore the test is not considered negative until three specimens have been found free of the organism. 39 a fecal antigen test that identifies the encystment protein is available. the snap giardia antigen test (idexx laboratories) uses fresh or frozen feces, or feces refrigerated for less than 7 days. 34 since the antigen is continuously shed, this test avoids the problem of intermittent shedding of the whole organism. 28 the sensitivity of the test is 85%, with a specificity of 100%. 35 by combining the antigen test with a zinc sulfate fecal centrifugal flotation, the sensitivity improves to 97.8%. 11 it is unknown how long the antigen remains in the feces after treatment. thus a zinc sulfate centrifugal flotation examination should be used to evaluate therapeutic efficacy. 28, 39 the use of this test in cats without diarrhea is controversial, because these cats are unlikely to shed cysts. the zoonotic significance of a positive antigen test in a cat not shedding cysts is unknown and may cause confusion. 39 polymerase chain reaction detection of giardia dna is available, but the test has not been standardized across all diagnostic laboratories. one needs to ensure the laboratory performing the test has validated it for assemblage f. the test may also be used to identify cats harboring the zoonotic assemblages a and b. the sensitivity of this test is unknown. 39 two commonly available drugs are used most frequently to treat infections with g. duodenalis (see table 23 -20) . fenbendazole may be effective and can be used in pregnant queens 28 and in cats co-infected with roundworms, hookworms, and taenia spp. tapeworms. 39 however, in one small study, only four of eight cats infected with both g. duodenalis and cryptosporidium felis stopped shedding giardia permanently after receiving fenbendazole. 29 febantel, in the combination product drontal plus (bayer animal health), is converted to fenbendazole. when six experimentally infected cats received 56.5 mg/ kg of febantel q24h po for 5 days, four of them stopped shedding g. duodenalis cysts. 39 metronidazole has been the traditional drug used to treat g. duodenalis in pets. 28 the drug is also useful for treating concurrent small intestinal bacterial overgrowth and clostridial infections. 39 the administration of metronidazole may eliminate shedding in 67% of cats. 26 neurologic side effects may occur at the dose recommended for treatment of giardia (see above, therapeutics for vomiting and diarrhea). the use of a giardia vaccine was ineffective in clearing infection by itself. 44 the combination of fenbendazole and metronidazole has been suggested as the initial treatment of choice for g. duodenalis infections. 39 although controlled studies are lacking, they may work synergistically by acting on two different targets within the parasite. 28 febantel would be expected to have the same synergism with metronidazole. drug therapy may not be necessary in cats without diarrhea that are infected with g. duodenalis, 37 because it is uncommon for a cat to carry the assemblages required to infect humans. the veterinarian may be obligated to treat a healthy cat if the owner wants to treat, the owner is immunocompromised, or the goal is eradication of an infection from a multicat home or prevention of parasite transmission to giardia-naïve cats is attempted. 28 what may appear to be treatment failure is more likely to be re-infection. in addition to drug therapy, steps should be taken to prevent re-infection. all cats with diarrhea positive for g. duodenalis should be treated along with their housemates. 37 sanitation is imperative in the fight against re-infection and transmission of g. duodenalis. dispose of old litter pans and scoops and use disposable litter boxes during treatment. when the infection is eliminated, not just controlled, new litter boxes and scoops may be purchased. bathe all cats during treatment to remove cysts from the hair coat. since giardia spp. cysts are susceptible to desiccation, 28 blowdry all cats using a warm air blower, paying particular attention to the perineal area. disinfect bowls, housing, and other utensils with bleach. 28 in addition to antiprotozoal drugs and sanitation, supportive care may become necessary. probiotics and a highly digestible, bland diet may be offered to cats with small bowel diarrhea, while a high-fiber diet may be useful for those few cats with large bowel diarrhea. 39 where required, hydration and electrolyte imbalances must be corrected and antiemetics used to control vomiting. therapy can be evaluated by retesting feces with a zinc sulfate centrifugal flotation examination 1 to 3 days after the end of treatment and again 3 weeks later. a positive test immediately posttreatment is most likely because of therapeutic failure. if the cat is negative immediately after treatment ends, but is positive 3 weeks later, re-infection is likely. 28 since the fecal antigen test may remain positive long after the infection is eradicated, this test is inappropriate for evaluating therapy. 28, 39 re-treatment of fecal flotation-positive, recovered cats may be handled in a manner similar to the positive healthy cat mentioned above. 28 cats with diarrhea that continue to shed cysts may be re-treated for g. duodenalis infection along with dietary modification and empirical treatment for other common intestinal parasites. however, serious consideration should be given to investigation into other potential causes of diarrhea. 28 the giardia vaccine has been found to be ineffective in preventing infection 37 and production has been discontinued. 39 this means prevention of giardia infection involves avoiding exposure, stress and re-infections. providing a clean environment, feeding only processed foods, and controlling potential transport hosts will help reduce the chances of exposure. isolation of cats with diarrhea may be important, too. 39 municipal sanitation control is difficult as the cyst survives for weeks in cool, moist environments. 28 cysts are also able to survive water treatment and can pass through attempts at water filtration. 37 giardiasis is associated with debilitating diarrhea in some humans, particularly those who are immunocompromised. 35 however, cats do not commonly carry the assemblages needed to infect humans. transmission of g. duodenalis from cats to humans is rare and unproven. 28 still, it seems prudent to consider the owner's health when contemplating management of giardial infections in cats. to avoid human health risks, cats with diarrhea that test positive for g. duodenalis should be treated with the goal of controlling the diarrhea. 39 since no treatment for g. duodenalis is completely effective or 100% safe, treatment of positive cats without diarrhea should only begin after a discussion of the benefits and risks of the treatment with the owner. 39 tritrichomonas foetus is best known for causing bovine reproductive infections. it is an obligate anaerobic parasite 26 that also colonizes the lower intestinal tract of cats. there are enough differences between the two isolates that the feline isolate does not cause disease in heifers and vice versa. 39 the parasite depends on the host's normal intestinal flora and secretions for obtaining nutrition. 7 a report from the united states of purebred cats tested at an international cat show found t. foetus in 36 of the 117 cats tested, a prevalence of 31%. 23 this parasite seems to have a higher prevalence in purebred cats than nonpurebred cats. a study of pet cats visiting veterinary hospitals across the united states reported 12 of 32 purebred cats were positive for t. foetus, while only 5 of 141 nonpurebred cats were positive. in this same study, 12 of the 17 positive tests were from purebred cats. 45 a study from the united kingdom of diarrheic fecal samples sent to a veterinary diagnostic laboratory reported similar results. purebred cats represented 14 of the 16 cats testing positive for t. foetus. the u.k. study also found the siamese and bengal breeds each represented 6 of 14 positive cats; only two other breeds tested positive. 25 transmission like most other protozoal parasites, t. foetus is transmitted by ingestion of the parasite, in this case, the trophozoite. unlike most of the other parasites, t. foetus does not form cysts and only survives up to 3 days outside the body in moist feces. 47 a cat becomes infected through the use of a shared litter box with an infected cat. after walking into the box, the parasite is transferred from the infected feces of one cat to the paws of the other. infection then occurs through ingestion of the trophozoites during grooming. 47 after infection, t. foetus colonizes the distal ileum and colon, 15 followed by shedding of infective trophozoites 2 to 7 days later. 19 there are several mechanisms by which t. foetus causes diarrhea. these include alteration of the cat's normal bacterial flora population, increases in local inflammatory cytokine concentrations, production of enzymes, and direct mucosal injury. the resulting injury leads to plasmacytic-lymphocytic 49 and neutrophilic colitis. 37 although most infections involve only the mucosa of the colon, one study reported two of seven cats with diarrhea and t. foetus infections as having trophozoites in deeper layers of the colonic wall. 49 co-infection with cryptosporidium felis 17 or giardia duodenalis 39 can be associated with increased numbers of t. foetus trophozoites and increased severity of diarrhea. signs of infection are most frequent in kittens and young cats, although infections without clinical signs can occur. 39 adult cats, however, may also show signs of t. foetus infection. the most common sign is a foulsmelling large bowel diarrhea with increased frequency of defecation, 39 mucus, blood, 15 and flatulence. 37 the consistency of the diarrhea may wax and wane, but the presence of diarrhea does not. 47 cats with diarrhea are otherwise in good health and maintain their body condition. 15, 39 severe diarrhea can result in anal swelling and fecal incontinence. 39 diarrhea may respond to the use of antibiotics because of changes in the cat's intestinal microbial flora. however, it always returns at the cessation of therapy. 39, 47 many cats experience a spontaneous resolution of the diarrhea within 2 years of diagnosis. 15, 38 since t. foetus causes reproductive infections in heifers and bulls, there is speculation the parasite also infects the reproductive tract in cats. tritrichomonas foetus was found in the uterus of a queen with pyometra. 9 however, in a study of 60 breeding male and female cats from 33 catteries, no cytologic or molecular evidence of t. foetus was found in the reproductive tract. the authors reported colonic infection with t. foetus in 15 of the 60 cats representing 22 of the 33 catteries. 24 detection of the trophozoites in a sample of feces is the most expedient means of diagnosing an infection with t. foetus (figure 23-44 ). an index of suspicion is required, because the clinical presentation of t. foetus infection is often mistaken for infection with giardia duodenalis. if a cat is not responding to treatment for that parasite, consider t. foetus as a cause of the diarrhea. the sample required for the diagnosis of t. foetus is a fresh, nonrefrigerated sample of watery feces. refrigeration kills the trophozoites, and they are not found in normal feces. 34 the sample may be freshly passed diarrhea, feces collected using a wire loop passed into the colon, or collected by a colonic flush using a red rubber catheter and 10 ml of saline. 47 a wet mount or smear examination of the feces should be performed on all cats with diarrhea. examination of multiple samples may be required to find the t. foetus trophozoites with this technique because it is insensitive. 39 the trophozoites must be differentiated from giardia duodenalis based on structural differences and motility patterns (see table 23 -21). the trophozoites of t. foetus can be cultured using the inpouch tf system (biomed diagnostics). this test is more sensitive than the fecal wet mount examination and detects 1000 trophozoites per sample. 15 the number of parasites shed by a cat with diarrhea is high enough to be routinely detected with this method. 18 the test should be performed in-house, because the parasite is unlikely to survive the trip to the laboratory. 47 the test pouch is inoculated with 50 µg of freshly collected feces, about the size of a peppercorn. 18 any more than this increases the chances of bacterial overgrowth. 15 the pouch is incubated at 25° c and examined under the microscope for motile trophozoites every other day for 12 days. the pouch should be tapped gently to dislodge the parasites, which tend to collect along the seams. 15 the test is considered negative if parasites are not found after 12 days. one benefit of this system is that it does not support growth of giardia duodenalis or pentatrichomonas hominis. 18 if a fecal wet mount examination and culture are both negative and infection with t. foetus is still under consideration, a pcr test can be performed. this test detects dna from live or dead trophozoites, but is more expensive than other diagnostic methods. 47 this test is more sensitive than the other two methods and can detect 10 parasites per sample. 16 the sample size is 200 mg of feces not contaminated by litter preserved in 3 to 5 ml of rubbing alcohol shipped at room temperature. 15 trophozoites of t. foetus are sometimes found in colonic biopsy samples adhered to the surface or in the lumen of crypts. 15 the most effective drug for the treatment of t. foetus in cats is ronidazole. 17 the drug has a bitter taste and should be compounded into capsules. veterinary staff and owners should use gloves when handling ronidazole. 15 if a confirmed relapse occurs, another course of treatment may eliminate the parasite. 39 diarrhea may take several weeks to resolve after elimination of the parasite, because significant colitis is often present. 47 effectiveness of treatment can be evaluated by performing fecal pcr tests 2 and 20 weeks after the end of treatment. 15 apparent treatment failures may occur because of re-infection, co-infection with giardia duodenalis or cryptosporidium felis, or the presence of another concurrent diarrhea-causing disorder. a more worrisome cause for treatment failure is a recent report of parasite resistance to ronidazole in two cats. 22 fortunately, diarrhea ultimately resolved in both cats despite the continued presence of the parasite. if the cat retests negative and the diarrhea is not improving after 2 weeks, consider the possibility that another disease may exist. nonspecific treatment for diarrhea is unhelpful 37 and may prolong the duration of diarrhea. 15 diarrhea may respond to antibiotics as they alter the intestinal flora population; however, once treatment is stopped, the diarrhea will return. 47 an important and potentially serious adverse effect of ronidazole administration in cats is a reversible neurotoxicity. onset of signs often begins within 1 week of the onset of therapy and may last between 1 and 4 weeks after cessation of therapy. 38 these signs can include depression, ataxia, seizures, 47 behavioral changes, weakness, hyperesthesia, and trembling. 38 neurotoxicosis usually requires only supportive care along with discontinuation of the drug. the neurologically affected cat should be retested for the parasite, because it may have been eliminated. 38 because of the potential for neurotoxicity, the use of ronidazole should be restricted to cats with confirmed infections with t. foetus. 47 crowded conditions should be avoided, because transmission of t. foetus trophozoites is more efficient in these settings. 39 cats testing positive should be isolated from other cats during treatment. 37 providing a clean environment will help prevent transmission of trophozoites. although there is a report of an infection in one immunocompromised person, transmission of t. foetus trophozoites from cats to healthy humans has not been reported. 39 still, prudence dictates handling feces infected with t. foetus trophozoites carefully. recent genetic evaluations have shown that most feline infections with cryptosporidium spp. are with c. felis; not, as previously thought, with c. parvum. 36 cryptosporidium parvum seems to be limited to farm animals. 4 cryptosporidium felis is an obligate intracellular parasite infecting the small intestine. 4 infective oocysts are ingested from contaminated feces during self-grooming of contaminated body parts and from contaminated food and water. 32, 39 after infection, the parasite attaches to the brush border of the enterocyte. the prepatent period is 3 to 6 days, 39 and the oocysts are infective as soon as they are shed, making this a very contagious disease. 20 like most intestinal parasites, shedding is often intermittent. the pathogenic effects of c. felis infections are not well understood. direct cytotoxicity and inflammation causes villus atrophy and decreased surface area for absorption of water, electrolytes, and other nutrients. 20, 32 apoptosis (preprogrammed cell death) of the mucosal cells may be accelerated, adding to the malabsorption. 20 most infections with c. felis are subclinical. 39 signs, if present, range from a mild, self-limiting small bowel diarrhea 33 to chronic intermittent small bowel diarrhea. 32 severe diarrhea with weight loss and anorexia may also occur. 32, 33 clinically apparent infections are most common in kittens, adult cats with concurrent gastrointestinal diseases, and cats co-infected with giardia duodenalis or tritrichomonas foetus. 39 cats with co-infections may experience more severe clinical signs. 32 a fecal flotation, which should be performed on all cats with diarrhea, may reveal c. felis if there are large numbers of oocysts (figure 23-45) . the fecal floatation test, however, is often negative 39 because of intermittent shedding. the parasite is small and floats in a higher plane than helminth ova; the high-power lens and appropriate adjustment of the microscope stage is required to find the parasite. 32 the small size of the oocyst makes identification difficult, particularly if the examiner is not specifically looking for them. 34 a modified ziehl-neelsen stain of a thin fecal smear may help in the identification of the oocysts. 39 this technique works well in humans with large numbers of oocysts. 33 once signs resolve or the oocyst numbers decline, a single examination of a stained smear becomes insensitive. when only one sample is available, testing for c. felis antigen is a good choice. 34 the prospect microplate assay (alexon biomedical, sunnyvale, calif.) is more sensitive and specific for the diagnosis of c. felis than is the examination of a stained smear. 6 immunofluorescent antibody testing is available from some laboratories. fecal c. felis dna can be detected using pcr testing. this test is available at many veterinary diagnostic laboratories; however, at present, there is no test standardization among laboratories. 39 the clinical and zoonotic significance of a positive pcr test combined with an oocyst negative test is unknown. 39 therefore a positive pcr test in a cat without diarrhea presents a confusing situation for the attending veterinarian with regard to recommendations for the owner. unfortunately, there are no completely effective and safe treatment protocols available for c. felis. 32, 39 a concerted attempt to find other causes of diarrhea should take place prior to convicting a cat of having diarrhea solely from c. felis infection. most reports on therapy for c. felis are uncontrolled and anecdotal. a number of drugs have been discussed. azithromycin for at least 10 days appears safe but produces variable results. 39 paromomycin, an oral aminoglycoside, may be effective. however, one study reported acute renal failure in 4 of 32 cats receiving the drug. deafness also occurred in three of those four cats. 21 nitazoxanide is a drug approved for treating humans with diarrhea caused by cryptosporidium spp. infections. the administration of nitazoxanide to cats at 25 mg/kg q12h po for at least 5 days 39 up to 28 days 32 may be effective. however, nitazoxanide is a gastrointestinal irritant and commonly results in vomiting and foul-smelling diarrhea. co-infections with giardia duodenalis and/or tritrichomonas foetus are more difficult to control. if diarrhea from c. felis infection improves but does not resolve at the end of therapy, the duration of treatment may be prolonged. 39 additional diagnostic testing should also be performed to ensure the only cause of the diarrhea is infection with c. felis. environmental control of c. felis is difficult, because it is extremely hardy. it is resistant to chlorination and most disinfectants. 32 oocysts remain viable at temperatures above freezing up to 65° c. 4 the parasite is difficult to filter and survives treatment at municipal water treatment facilities. 20 steam-cleaned housing and utensils may be beneficial in controlling parasite numbers, and they are susceptible to 5% ammonia solutions; however, the required contact time is 18 hours. 39 cryptosporidium spp. are relatively species specific, and there are no reports of waterborne outbreaks of human cryptosporidiosis associated with c. felis. 32 cryptosporidiosis can cause life-threatening diarrhea in hivpositive persons. 20 fortunately, humans are rarely infected with c. felis. 39 in fact, the zoonotic species most commonly found in humans (often veterinary students), is c. parvum found in young heifers. 4 regardless of a person's health, feces from a cat with diarrhea should be handled carefully. if a cat infected with cryptosporidium spp. is owned by an immunocompromised person, a pcr test may be useful in determining the species of the parasite and its zoonotic risk. like other coccidians, toxoplasma gondii is an obligate intracellular parasite. 12 domestic cats and other felids are the only animals that shed oocysts. any warmblooded animal, including humans, can be infected with this parasite. toxoplasma gondii can be transmitted by ingestion of infective oocysts in fecally contaminated food or water after ingestion of tissue cysts through carnivorism, or by transplacental or trans-mammary transmission of the parasite. the parasite enters into one of two cycles, depending on the host species. the enteroepithelial cycle only occurs in cats and results in shedding of oocysts after sexual reproduction of the parasite. after a cat ingests an infective oocyst or a tissue cyst, the parasite enters the mucosal cells of the small intestine, where it may undergo development and sexual reproduction, after which oocysts are shed. 12 the prepatent period after ingesting an infective oocyst is 19 to 48 days, while shedding after ingesting tissue cysts starts in 3 to 10 days. 4 fecal shedding, which occurs only after initial infection, lasts for 2 to 3 weeks 4,31 and the oocysts become infective 1 to 5 days after they are shed. 12 the extraintestinal cycle occurs in any animal, including cats. after ingestion, the parasite penetrates the cells of the small intestine and rapidly replicates in the enterocytes and associated lymph nodes into tachyzoites. after hematogenous and lymphatic spread, tachyzoites infect cells in all tissues of the body. 4 tissues most commonly infected include the brain, liver, pancreas, and lungs. 30 if a pregnant queen becomes infected, tachyzoites cause placentitis, after which they infect the fetus. 13 in 3 weeks, the host's immune response slows parasite replication, and the resultant bradyzoites form tissue cysts 30 in the brain, striated muscle, and liver, and they remain viable for the life of the animal. 4 immunosuppressive drugs or disease may dull the suppression of parasite division by the host immune system and allow the slowly dividing bradyzoites in tissue cysts to begin rapid division, thereby reactivating the infection with tachyzoites. 30 none of the forms of t. gondii produces a toxin. rapid replication of tachyzoites within a cell leads to rupture of the cell and necrosis of the tissue in which they are located. 12 the most commonly injured tissues are the brain, lungs, liver, and pancreas. prenatal infection leads to more severe illness, because the immature immune system is unable to slow down replication by tachyzoites, allowing continued damage to tissues. prenatal infection is more likely to result in ocular infections, and neonatal death is usually caused by pulmonary or hepatic infection. 30 type ii and iv hypersensitivities may be involved in the pathogenesis of chronic disease from bradyzoites in tissue cysts. 30 kittens infected perinatally can be stillborn or die shortly after birth. they may also suffer from hepatomegaly and ascites, central nervous system signs resulting from encephalitis, respiratory distress, or uveitis. 12, 13 clinical signs of infection in healthy adult cats are uncommon (box 23-2). 31 diarrhea from enteroepithelial development of the parasite is rare. 39 cats that develop clinical disease often have an episodic course with vague signs 30 that depend on the body system affected. onset of illness may be acute or chronic, and the most commonly affected organs include the brain, lungs, liver, heart, pancreas, and the eyes. 13 signs are the result of spread of tachyzoites after initial infection or after reactivation of tissue cysts. cats suffering from uveitis may develop lens luxation and glaucoma. the best way to identify a cat shedding t. gondii oocysts is to demonstrate them with a centrifugal fecal flotation technique using sheather sugar solution. the oocysts are about a quarter of the size of isospora felis oocysts ( figure 23-46 ). 12 oocysts of t. gondii are morphologically indistinguishable from hammondia or besnoitia spp. oocysts. 13 detection of fecal t. gondii dna using a pcr test can be used to definitively differentiate t. gondii oocysts from similar coccidians. 31 it is probably best, however, to assume suspicious oocysts are those of t. gondii until proven otherwise. proving infection with t. gondii is responsible for a cat's systemic illness is also difficult. finding tachyzoites in cytology samples is uncommon. they are most likely to be identified from body cavity effusions. 13 the most common method of identifying an infected cat is by detecting t. gondii-associated immunoglobulins using immunofluorescent antibody or elisa techniques. since cats are infected for life, a seropositive cat has been infected at some point in its life. however, use of serology alone is insufficient to diagnose an active t. gondii infection. serum immunoglobulin m (igm) is produced within 1 to 2 weeks after infection, but increased igm titers may persist for months to years. serum immunoglobulin g (igg) begins to rise later; in some cats, igg may not be detectable for 4 to 6 weeks. 12 by the time igg is detectable, shedding will have ceased. maternally acquired igg persists in kittens for 8 to 12 weeks. 13 a rising igg titer is associated with an active infection, but the degree of increase is not associated with the severity of the clinical signs. if a cat becomes seronegative, it is more likely the titer has fallen below the sensitivity of the test rather than the parasite has been eliminated from the body. 31 because of the vague nature of the clinical signs, many cats are presented later in the course of the disease. by this time, they may have switched from igm to igg production or passed the time of maximal igg production. thus a negative igm titer or a lack of rising igg titer does not rule out t. gondii infection. 30 also, reactivation of tissue cysts is rarely associated with rising igg titers. 31 ultimately, the diagnosis of an active systemic t. gondii infection requires demonstration of an igm titer greater than 1 : 64 or a fourfold increase in igg titers over a 2-to 3-week period along with signs consistent with toxoplasmosis, the exclusion of other disorders that may cause the clinical signs, and response to appropriate anti-t. gondii treatment. 31 although serum igm titers may be increased in otherwise healthy cats, increased igm titers in cerebrospinal fluid or aqueous humor only occurs in cats with active cns or ocular infections. 30 the goals of treating a cat infected with t. gondii are to reduce shedding of oocysts and to control the clinical signs in sick cats. shedding can be reduced by using ponazuril, 13 toltrazuril, or high doses of clindamycin. the drug options for treating a sick cat include clindamycin, trimethoprim-augmented sulfadiazine, or azithromycin for at least 4 weeks (see table 23 -20) . recurrences are more common if the cat is treated for less than 4 weeks. 13, 30 the antifolate drug pyrimethamine may be more effective than trimethoprim, but megaloblastic anemia develops in many cats. supplementation with folinic acid (5 mg/cat, once daily, po) or brewer's yeast (100 mg/kg, once daily, po) may prevent or reverse the anemia. 12 no drug clears all of the tissue cysts; so, cats remain infected for life. if uveitis is also present, use appropriate topical, oral, or parenteral corticosteroids. for a cat with proven t. gondii-associated uveitis alone, a topical ocular glucocorticosteroid is the only required treatment; no antibiotics are necessary unless the uveitis is persistent or recurrent. 31 • wash hands after handling cats, especially if you are pregnant or immunocompromised. • remove fecal material from the home environment daily, since shed oocysts require a minimum of 24 hours to become infective. • do not have immunocompromised persons clean the litter box. if they must clean the litter box, they should wear gloves and wash hands thoroughly when finished. • use litter box liners, and periodically wash the litter box with scalding water and detergent. • wear gloves when gardening, and wash hands thoroughly when finished. • cover children's sandboxes when not in use to avoid fecal contamination by outdoor cats. • only feed cats cooked or commercially processed food. • control potential transport hosts, such as flies and cockroaches, that may bring the organism into the home. • filter or boil water from sources in the environment. • cook meat for human consumption to 80° c for 15 minutes minimum (because of uneven heating, microwave cooking does not kill all t. gondii 12 ). • freeze meat at −12° c for 24 hours. 12 • wear gloves when handling meat, and wash hands thoroughly with soap and water when finished. clinical signs such as malaise, fever, and muscle pain should begin to resolve in 2 to 3 days. 30 if there is no response within 7 days, switch to or add another drug. 31 if there is still no response, search for another condition that may cause the observed clinical signs. however, ocular and cns signs resolve more slowly and thoracic radiographic changes may take weeks to resolve. 30 some cns changes may never completely resolve. cats co-infected with feline immunodeficiency virus (fiv) do not respond to anti-t. gondii treatment as well as fivnegative cats respond. 12 feeding cats commercially processed cat food and avoiding undercooked or raw meat can prevent exposure to t. gondii. controlling hunting reduces access to paratenic hosts with infective tissue cysts. access to mechanical carriers of t. gondii, such as earthworms or cockroaches, should be minimized. human infection with t. gondii is common, more so in warm, humid climates where the prevalence of t. gondii seropositive persons approaches 100%. the number of persons seropositive for t. gondii is estimated to be around 500,000,000 worldwide. 12 infective oocysts are hardy and may remain viable in the environment for up to 18 months. 12 human infection most often occurs after eating raw or undercooked meat infected with tissue cysts or by transplacental infection. 31 seropositive cats are finished shedding and are unlikely to resume shedding even if the infection becomes reactivated. 31 cats found to be shedding oocysts should be quarantined at a veterinary hospital until shedding ends. oocysts of t. gondii have not been found on the hair coat 13 ; so, transmission of toxoplasmosis does not occur after touching a cat. 31 pregnant women infected with t. gondii for the first time, or chronically infected women who are also hiv positive, can transmit the parasite to their unborn child. transplacental infection can result in stillbirths, cns, or ocular disease. 30 more severe fetal disease may occur if the infection happens in the first half of the woman's pregnancy. 12 toxoplasma gondii infection of immunocompetent humans usually results in a self-limiting fever and malaise. 30 steps useful in preventing transmission of t. gondii to humans can be found in box 23-3. pancreatitis refers to inflammation of the pancreas only, with no implication of the underlying cause or pathology. for example, acute necrotizing pancreatitis (anp) with pancreatic auto-digestion, requiring predominantly supportive care by maintaining fluid and electrolyte balances and pain relief, must not be confused with chronic pancreatitis (cp) caused by lymphocytic infiltration, and commonly associated with lymphocytic inflammatory bowel disease (ibd), and often requires corticosteroids to manage. these two conditions (and others) can only be definitively distinguished histologically. in many cases, the clinical signs of cats with acute pancreatitis will resolve with supportive care before a precise diagnosis is reached and will thus remain undiagnosed. there are no formal classifications for feline pancreatitis, but most authors 78, 89, 90 use the terms • acute pancreatitis • acute necrotizing pancreatitis, characterized by severe peri-pancreatic fat necrosis • acute suppurative pancreatitis, characterized by neutrophilic infiltration • chronic pancreatitis, characterized by lymphocytic infiltration the exact prevalence of feline pancreatitis is unknown. necropsy studies from the 1970s to 1990s reported prevalence of feline pancreatitis ranging from 0.45% to 2.4%. 21,67 a more recent study 17 found 67% of 115 cats had evidence of pancreatitis. however, this included pancreatic pathology in 45% of apparently healthy cats, which suggests that mild pathology is unlikely to cause clinical signs. these studies all show lymphocytic pancreatitis to be significantly more prevalent than acute pancreatitis. this may underestimate the true prevalence of acute pancreatitis, since it is understood that no permanent histopathologic changes are present after resolution of acute pancreatitis. 89 it is also possible that studies assessing pathology in necropsy cases do not reflect clinical practice. there are no specific age, breed, or sex predispositions. although one study reported siamese cats to be at increased risk of acute pancreatitis, 33 subsequent studies have recognized the majority of cases are domestic shorthair cats, suggesting no specific breed predispositions. 22, 29, 60, 71 most studies have indicated older cats (8 to 10 years of age) are more likely to be affected, 22, 29, 60, 71 but these studies most likely underrepresent cats with less severe clinical disease for which definitive diagnosis may not be reached and which may be younger. no association has been made with a high-fat diet or obesity. in most cases of both acute and chronic pancreatitis, no specific cause is found, and the disease is primarily considered to be idiopathic. 22, 90 there are, however, some specific underlying causes that are sporadically recognized. these include infections with herpesvirus, 75 calicivirus, 37,49 feline infectious peritonitis (fip), 44 liver fluke 58 and pancreatic fluke, 26, 77 and toxoplasmosis. 20 however, a recent paper found no association between serum feline pancreatic lipase immunoreactivity (fpli) concentrations and toxoplasma gondii serology. 8 pancreatitis has also been recognized subsequent to trauma 81 and organophosphate poisoning. 33 the association of pancreatitis with inflammatory bowel disease and cholangitis is frequently mentioned (triaditis) but poorly described in the literature. 80 one study found 30% of ibd cases to have histologic evidence of pancreatic involvement, 6 and another found fpli concentrations were elevated in 70% of cases with histologically confirmed ibd. 3 it is the author's experience that many cases of pancreatitis recognized with ibd have no specific clinical signs attributable to pancreatitis and should therefore be diagnosed and treated as intestinal disease. diabetes mellitus is a recognized co-morbidity of pancreatitis in cats. a recent study found fpli concentrations were significantly higher in 29 diabetic cats compared with 23 non-diabetics. no association could be made between fpli concentrations and the degree of diabetic control. 23 one study found 5 of 13 cats (38%) histologically diagnosed with hepatic lipidosis were also histologically diagnosed with acute pancreatitis. it is not known if pancreatitis is a cause, consequence, or coincident disease of hepatic lipidosis. for example, anorexia associated with acute pancreatitis could predispose to fatty infiltration of the liver. however, the high rate of concurrent disease has important implications for ensuring cats with pancreatitis receive adequate caloric intake. 1 ongoing or recurrent pancreatitis may lead to pancreatic cysts 10 or exocrine pancreatic insufficiency, 74 which are both covered later in this chapter. although pancreatitis has been experimentally induced in cats, 18, 41, 56 the pathophysiology of spontaneous pancreatitis remains unknown. acute pancreatitis is initiated by an increase in secretion of pancreatic enzymes that leads to inappropriate cellular activation of trypsin and subsequently other digestive zymogens. these activated digestive enzymes lead to local effects including inflammation, hemorrhage, acinar cell necrosis, and peripancreatic fat necrosis. 43, 78, 86 chronic pancreatitis may result from any of several underlying processes: ongoing, low-grade acute pancreatitis episodes may instigate chronicity; chronic pancreatitis, with a predominance of lymphocytic inflammation has been induced experimentally within 5 weeks by narrowing the main pancreatic duct to approximately 25% of its normal diameter 18 ; and the association with ibd 80 may suggest an immune-mediated cause. the clinical signs of pancreatitis in cats are nonspecific. a review of eight prior series totaling 159 cases of acute pancreatitis in cats found anorexia (87% of cases) and lethargy (81%) to be the most common historical findings. 78 vomiting was recognized in 46% of cases, diarrhea in 12%, and weight loss in 47%. physical examination findings were similarly nonspecific with dehydration (54%) being the major finding; fever was recognized in only 25% of cases and abdominal pain in 19%. it is important to note that vomiting and abdominal pain, key features of pancreatitis in dogs, are not consistently recognized in cats. similar, nonspecific findings indistinguishable from ibd are recognized in cats with chronic pancreatitis. 3, 6 diagnosis because the presenting signs and physical examination findings are nonspecific, the diagnosis of pancreatitis can be challenging, requiring not only clinical suspicion but a combination of diagnostic modalities. for the most part, hematology and plasma biochemistry findings are unremarkable, although a combination of findings may increase clinical suspicion. for example, moderate elevations in liver enzymes, bilirubin, and glucose are present in approximately 50% of cases and hypocalcemia in approximately two of three of cases; hypocalcemia infers a poorer prognosis. hypoalbuminemia is seen in approximately one of three of cases and has important implications for fluid therapy. 78 amylase and lipase elevations are not reflective of pancreatitis in cats. 47 feline trypsinlike immunoreactivity (ftli) is the diagnostic test of choice of exocrine pancreatic insufficiency, but elevations in pancreatitis are not seen consistently enough to warrant use of this test for this purpose. 29, 47, 71 the biggest recent advance in feline pancreatic diagnostics has been the characterization of feline pancreatic lipase, 69 leading to the development of a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity (fpli). 70 it must be remembered, however, that an increase in fpli only tells the clinician that pancreatic pathology is present, but not the cause of pathology, which may be, for example, neutrophilic or lymphocytic pancreatitis or neoplasia, and it may or may not involve the intestines or liver. fpli should therefore be used as a screening test, with elevated results not suggesting a diagnostic end point. further, the high interassay variability of this test 70 would suggest that mild cases may be missed as shown in one study 24 and that the test may not be appropriate for serial monitoring. fpli is currently available as "spec fpl" from commercial laboratories and has a sensitivity of 79% and a specificity of 82% when 5.4 µg/l is used as the diagnostic cut off 25 compared with 3.5 µg/l, which is the listed reference range high point. in an acutely unwell cat (less than 2 days) with only mild to moderate signs of disease, further diagnostics may not be warranted, and many cats will improve with supportive therapy of balancing fluid and electrolytes, pain relief, and antinausea/vomiting therapy. cats with chronic duration of signs and acutely unwell cats that do not improve with supportive therapy warrant further diagnostics. the underlying disease process cannot be assumed from an elevated fpli; in one study of 63 cases, acute necrotizing pancreatitis could not be distinguished from chronic nonsuppurative pancreatitis by signalment, duration of signs, or clinical findings. 22 the major utility of diagnostic imaging is to rule out other differential diagnoses, such as an intestinal foreign body, and perhaps confirm that the pancreas is affected. radiography is non-specific for diagnosis of pancreatitis, but findings may include decreased abdominal detail (sometimes associated with ascites), soft tissue density in the right cranial quadrant of the abdomen, hepatomegaly, or gas-filled intestines 22,60,64 (see . additionally, thoracic radiographs may show pleural effusion. one study found 5 of 20 cats with pancreatic necrosis had such a change 60 ; the mechanisms resulting in pleural effusion are not precisely defined. ultrasonography has high specificity (>85%) but low sensitivity (<35%) for recognizing pancreatitis in cats, 22, 29, 60, 71 with findings dependent on operator skills, quality of equipment, and severity of lesions. typical findings are hypoechogenicity of the pancreas, which may be enlarged or irregular; hyperechogenicity of the peripancreatic fat; the possible presence of abdominal effusion; and abnormal findings with other organs, such as liver or intestine, may add to the clinical picture 22, 60, 64, . one study indicated that contrast-enhanced doppler ultrasonography can provide further diagnostic insights. 54 a recent study suggested that endosonography may be useful in cases where transabdominal ultrasonography is difficult, for example, because of obesity, hyperechoic mesentery, or excessive intestinal gas. 61 for more than 20 years, computed tomography (ct) has been a commonly used modality to confirm pancreatitis in humans, 55 but this reliability has not been demonstrated in cats, where sensitivity may be as low as 20%. 24, 29 definitive diagnosis of pancreatitis, including differentiation of the inflammatory process, can only be made by cytologic assessment of pancreatic tissue. in most cases, ultrasound-guided fine-needle aspiration (fna) of the pancreas is technically difficult because of the small dimension of the feline pancreas; there appears to be no assessment of feline pancreatic fna findings in the literature. gross inspection of the pancreas and samples for histologic assessment can be obtained during laparotomy 22, 64 (see or laparoscopy. 16, 79 because pancreatitis often occurs concurrently with pathology of other organs, 22 thorough evaluation of the abdomen by ultrasonography or gross inspection is recommended, as are multiple biopsies of, for example, intestines, liver, and mesenteric lymph nodes, where appropriate. clinicians may be reluctant to biopsy the pancreas because of perceived risks of deleterious effects. studies of pancreatic biopsy in healthy cats dispel the concern that the pancreas is unforgiving to mild manipulation and biopsy 16,42a and the author's clinical experience is consistent with these findings. supportive care comprising correction of fluid/ electrolyte imbalances, pain management, and nutritional support are the mainstay of therapy for cats with figure 23-49 gross appearance of pancreas at laparotomy; this was histologically diagnosed as chronic pancreatitis (i.e., lymphocytic infiltration was recognized). gross appearance of pancreas at laparotomy; this pancreas was found to be histologically normal. it does look smaller than is typically seen; pancreatic atrophy can look similar to this, grossly. pancreatitis. 78, 86, 89 specific underlying causes, when diagnosed, should be managed, as should concurrent diseases. follow-up evaluation is determined on a case-by-case basis; reduction or resolution of clinical signs is the main criterion for success of therapy. serial fpli values may be monitored when initial results are extremely high but are of limited value for mild increases because of assay variability. dehydration, acid-base and electrolyte abnormalities should be corrected during the first 12 to 24 hours. hypocalcemia, if present, should be treated with a calcium gluconate infusion of 50 to 150 mg/kg during 12 to 24 hours, with continued assessment of plasma calcium concentrations. plasma transfusions can be considered in cats with hypoalbuminemia. 78, 86, 90 although abdominal pain is not commonly described in cats with pancreatitis, it is likely to be present in most cases and may contribute to anorexia. historical concern about exacerbation of pancreatitis with opioids is no longer accepted, and this class of drugs is considered appropriate. meperidine (1 to 2 mg/kg sc or im) every 1 to 2 hours, butorphanol (0.2 to 0.4 mg/kg sc) every 6 hours, or sustained-release buprenorphine (120 µg/kg sc) every 72 hours are alternatives. 67, 78, 86 the author uses one dose of methadone (0.1 to 0.2 mg/kg sc, im, or iv) initially and places a fentanyl patch for longer-term pain management. the traditional recommendation for management of pancreatitis across all species has been nil per os for several days. this recommendation is appropriate for cats with severe vomiting, but there is no evidence to support this approach in cats that are not vomiting and that are eating normally. further, nutritional support is vital for those cats with concurrent hepatic lipidosis. if the cat is not eating voluntarily, nutritional support by tube feeding is often warranted. 67,78,86 a recent paper found nasogastric tube feeding of cats with pancreatitis was tolerated well and resulted in few clinically significant complications. 42 other reported nutritional strategies for cats with pancreatitis incorporate partial parenteral nutrition (ppn; 8.5% amino acids, 20% lipids), or total parenteral nutrition (tpn; 6% amino acids, 20% lipids, 50% dextrose), or both instead of enteral feeding. 14,39,53 cats do not seem to benefit from feeding of specially formulated low-fat diets; commercially available, veterinary liquefied diets appear to be well tolerated despite their high-fat contents. 86 other therapy may be appropriate in individual cases. all cats with pancreatitis that are vomiting should be treated with antiemetics. examples of drugs that can be used are 5-ht 3 antagonists, such as dolasetron (0.5 to 1.0 mg/kg iv or po, once to twice daily); ondansetron (0.1 to 0.2 mg/kg iv every 6 to 12 hours); and maropitant, an nk 1 -inhibitor (0.5 to 1.0 mg/kg sc once daily). these drugs are covered in detail earlier in this chapter under therapeutics for vomiting and diarrhea. dopaminergic antagonists, such as metoclopramide, are less effective antiemetic agents in cats than the other choices mentioned. 78, 89 in most cases, pancreatitis begins as a sterile process, and antibiotic therapy is controversial. pancreatic necrosis and inflammation may predispose to bacterial colonization of the pancreas as demonstrated in experimental models. 82, 84 this has not been demonstrated in spontaneous disease, and no comparison of outcomes has been made of cats with pancreatitis treated with or without antibiotics. cefotaxime (20 to 80 mg/kg iv, im) has been used to prevent bacterial colonization in experimental models. 83 other broad-spectrum cephalosporins or ampicillin may act similarly. antibiotic considerations are possibly more important for acute pancreatitis than for treatment of chronic disease. cats with demonstrated lymphocytic pancreatitis, with or without concurrent ibd or lymphocytic cholangitis, should be treated with corticosteroids (e.g., prednisolone, 1 to 2 mg/kg once to twice daily) with tapering to the lowest effective dose. there is no justification for use of corticosteroids in cats with acute necrotizing or acute suppurative pancreatitis, or cats for which the cause of pancreatitis has not been diagnosed histologically. use of corticosteroids in cats with pancreatic disease creates a risk of iatrogenic diabetes mellitus. surgical intervention is warranted to relieve any bile duct obstruction that may result or for the débridement of pancreatic abscesses or necrotic tissue; in many cases, cats will survive multiple years after such corrective surgery. 65 pancreatic cysts, pseudocysts, and bladders have been described sporadically in cats.* pancreatic cysts are lined by a single layer of cuboidal epithelium and do not communicate with the pancreatic duct; pseudocysts are enclosed by a wall of fibrous tissue, lacking the epithelial lining characteristic of true cysts and can form secondary to pancreatic inflammation; cystic dilations of the pancreatic duct are referred to as pancreatic bladder. true pancreatic cysts have been described in three cats 9,10,15 ; a congenital pancreatic cyst with associated inflammation was described as an incidental finding in an adult cat 15 ; multiple pancreatic cysts were described in a cat with concurrent polycystic disease in the kidney and liver 9 ; and a another cat had multiple recurrent pancreatic cysts with concurrent mild pancreatic inflammation and atrophy associated a with rapid clinical course resulting in diabetes mellitus. 10 cysts, pseudocysts, and bladders may be identified ultrasonographically or by ct. they may be benign, but the associated pancreatic inflammation and other sequelae, such as diabetes mellitus, may need to be managed. pancreatic bladders may result in biliary obstruction, and surgical correction may be required. pancreatic nodular hyperplasia is recognized quite frequently as an incidental finding in older cats or at necropsy. 45 neoplasia of the exocrine pancreas is rare in cats. its frequency was assessed in the 1970s when one study estimated 12.6 cases per 100,000 patients per year at risk, 52 and another found pancreatic tumors in 5 of 800 feline necropsies. 45 a more recent study recognized, from 15,764 feline admissions over a 20-year study period, only two cats with pancreatic adenomas (0.013% of admissions) and eight with pancreatic adenocarcinomas (0.05% of admissions). 62 adenomas appear as small, solitary or multifocal nodules and are not typically associated with adjacent pancreatic inflammation. they do not cause clinical signs, unless large, when any clinical signs result from the physical size and are usually an incidental finding. 45, 86 few generalities can be made about the presentation for pancreatic adenocarcinoma. the age range is large (4 to 20 years), there is no sex predisposition, and no clear breed predispositions are present. 62, 86 only cytology or histopathology can distinguish pancreatitis from pancreatic carcinoma in cats antemortem, yet it is important to differentiate the two conditions, because, in contrast to adenomas, pancreatic adenocarcinoma is associated with a grave prognosis. the presence of lesions consistent with metastases on radiography or ultrasonography may suggest malignancy, but one study could not distinguish neoplasia from pancreatic nodular hyperplasia ultrasonographically based on the appearance of the pancreas alone 32 (figures 23-53 and 23-54) . pancreatic adenocarcinomas in cats can result in a paraneoplastic dermatologic condition consisting of nonpruritic, symmetric alopecia affecting the face, ventral body, and medial aspect of the limbs of cats. the skin is usually glistening but not fragile, and there can be crusty lesions on the footpads.* the pathogenesis of this dermatologic disease is unknown. in one case, surgical excision of the pancreatic carcinoma resulted in resolution of dermatologic disease, indicating that the process is reversible (although signs recurred as the tumor re-emerged). 73 diabetes mellitus is a recognized complication of pancreatic adenocarcinoma. the mechanism is unknown and may simply be secondary to compression or invasion of islet cells by the tumor. in some cats, diabetes is recognized ahead of pancreatic neoplasia. 31, 40, 62 obstructive jaundice has also been described with pancreatic adenocarcinoma. 13 most cases of pancreatic adenocarcinoma in cats have metastasized by the time of diagnosis, and most reported cases die or are euthanized within 7 days of diagnosis. 62 surgical excision is a potential option if neoplasia is confined to one limb of the pancreas, but recurrence is possible even if there is no evidence of metastasis and excision seems complete at the time of surgery. 73 exocrine pancreatic insufficiency (epi) is a condition caused by insufficient synthesis and secretion of pancreatic digestive enzymes from the exocrine portion of the pancreas. 66 in humans it has been reported that 90% of pancreatic acinar cells must be lost before clinical signs of epi are seen. 19 epi is considered rare in cats but is perhaps being recognized more frequently because of increased awareness. there are less than fifty cases described in the veterinary literature* with one of these papers describing only 16 cases from five institutions, with prevalence described as 0.01% to 0.1% of cats seen over a 15-year period. 74 in contrast to this, the gastrointestinal laboratory at texas a&m university recognized 1342 samples with serum ftli concentrations at or less than 8.0 µg/l, which is diagnostic for epi, out of 84,523 submissions, 66 which equates to 1.6% of cats with known or suspected gastrointestinal disease. all studies indicate a wide age range of cats can be affected, from kittens less than 6 months of age to cats more than 15 years old, with a median age of approximately 7 years. there is no apparent breed predisposition. 66, 68, 74 one paper recognized 10 of 16 (62.5%) cats to be male, 74 and another recognized 15 of 20 (75%) male cats, 68 suggesting a possible sex predisposition. chronic pancreatitis is believed to be the most common cause of epi in cats, 66 acinar atrophy (paa) is recognized as the most common cause of epi in dogs, and has been definitively described in two feline cases 74 and mentioned as a cause for three other cases. 85 other potential causes of epi include disruption of pancreatic enzyme flow at the duodenal papilla following duodenal resection 72 and pancreatic fluke infection (eurytrema procyonis), 2,26 and amyloid deposition and neoplasia are other possible causes of pancreatic cell damage that have not definitively been described in cats. 66 congenital pancreatic hypoplasia or aplasia has not definitively been reported in cats, but reports of epi in cats as young as 3 months of age 63, 74 suggest this possibility. since chronic pancreatitis is a common cause of epi and chronic pancreatitis has a strong association with ibd, many cats may have concurrent lymphocytic pancreatitis and enteritis. 66, 74, 85 therefore cats failing to respond to therapy for epi may require further diagnostics and management of an underlying condition. further, destruction of functional exocrine pancreatic tissue can also affect pancreatic endocrine tissue, resulting in concurrent diabetes mellitus. 35 several studies have indicated that all cats with epi will have weight loss when diagnosed, unless a kitten, in which case ill-thrift is recognized. 68, 74 diarrhea is not necessarily present, being described in 50% to 75% of cats; the nature of feces can vary from voluminous, malodorous stools that can be discolored (yellow or pale), sometimes with steatorrhea, to normal feces in other cats. increased frequency of defecation and the presence of mucus in the feces of some cats can lead to the diarrhea being characterized as large bowel. only about 20% to 30% of cats are polyphagic, some described as having a ravenous appetite; conversely, some cats present with anorexia. vomiting has also been described. since cats with epi often have concurrent disorders, such as ibd, the clinical signs recognized may reflect the concurrent disease and not necessarily epi alone. physical examination findings are similarly nonspecific, with thin/ emaciated body condition being the most common finding. hematologic findings are non-specific, but a mild nonregenerative, normocytic, normochromic anemia may be recognized as well as lymphopenia or neutrophilia. plasma biochemistry results may show a mild to moderate increase in alanine aminotransferase (alt) and a mild increase in alkaline phosphatase in some cats. mild to moderate hyperglycemia may be seen, as may mild hypoglycemia or normoglycemia. 66, 68, 74 hypocobalaminemia is recognized in nearly all cats with epi. 66, 68, 74, 85, 87 this may be because of insufficient production of intrinsic factor, a cobalamin-binding protein only produced by the pancreas in cats and necessary for ileal absorption of cobalamin 27 ; it may also be because of failure of pancreatic enzymes to liberate cobalamin from binding by r protein in the duodenum or small intestinal bacterial overgrowth (sibo), not yet specifically described in cats. 74 folate concentrations may be reduced (because of concurrent intestinal malab sorption), 68 normal, 68, 74 or increased, 74 which may relate to reduced pancreatic bicarbonate secretion, secondary to severe hypocobalaminemia, 59 or associated with sibo. 7 none of these presenting complaints, physical examination findings, or routine testing results are specific to epi. therefore epi requires a degree of clinical suspicion and/or thorough diagnostics to ensure the diagnosis is not missed. a low level of serum ftli is diagnostic for epi. 66, 68, 74 samples can be sent to the gastrointestinal laboratory at texas a&m university from anywhere worldwide (with instructions about sample handling requirements on their website: http://vetmed.tamu.edu/gilab/). the reference range for serum ftli is 12 to 82 µg/l, with concentrations at or less than 8.0 µg/l diagnostic for epi. since the clinical signs and routine laboratory findings are nonspecific for epi, it is ideal to test serum for ftli in any cat with weight loss or ill-thrift. the texas a & m gastrointestinal panel also includes testing for levels of cobalamin, folate, and fpli, ensuring concurrent hypocobalaminemia will not be missed and potentially providing indications of other gastrointestinal disease. conversely, although a low level of serum ftli confirms a diagnosis of epi, it is not necessarily a diagnostic end point, since epi is so often recognized concurrently with other gastrointestinal disease. failure to respond to therapy should prompt the clinician to consider and investigate further for concurrent processes. most cats with epi can be successfully managed with dietary supplementation of pancreatic enzymes. commercial products (e.g., viokase [axcan pharma, birmingham, ala.], pancrezyme [virbac, fort worth, tex.], and creon [abbott laboratories, abbott park, ill.]) are available, and powder is considered more effective than tablets or capsules (some capsules can be opened and the contents sprinkled onto food, like powder). the required dose can vary quite substantially from cat to cat. it is appropriate to start with one teaspoon of powder with food twice daily, and adjustments can be made depending on the response; most cats accept the powder readily if it is mixed thoroughly through canned food, but other flavors (e.g., fish oil or brine from canned tuna) can be used to disguise the taste if necessary. raw pancreas (e.g., from beef or pork) may also be used, with 30 to 60 g twice daily an appropriate starting dose. 66 since most cats with epi are hypocobalaminemic, supplementation by subcutaneous injection is required (oral supplementation is not effective since cobalamin deficiency leads to cobalamin malabsorption). an appropriate dose for most cats is 250 µg, and it is usually given weekly for 6 weeks, then every second week for a further six doses; it is appropriate to continue dosing every month beyond that. owners can be taught to inject their cats at home (as owners of diabetic animals are taught to do with insulin). 66 because some cats may have sibo, antibiotics such as metronidazole (15 to 25 mg/kg po every 12 hours for 14 days) may be warranted. an elevation of folate may arouse suspicion of sibo, but it is appropriate to try antibiotics in a cat failing to respond to enzyme and cobalamin supplementation. concurrent diseases, such as lymphocytic, chronic pancreatitis, or ibd may need to be managed with corticosteroids, or diabetes mellitus with insulin. no studies have assessed specific dietary requirements in cats with epi. most cats respond to appropriate treatment, with a return to normal weight and normal feces. with ongoing therapy, cats can lead normal lives for a full life span. 83 the feline liver is a large, complex organ involved in a variety of essential metabolic, functional, and detoxification processes that can be affected, individually or collectively, by disease or dysfunction. cats have a unique set of liver diseases that occur more commonly in this species compared with the typical diseases that occur in dogs, and these include hepatic lipidosis, feline cholangitis syndrome, and infectious hepatopathies (e.g., fip, flukes, histoplasmosis, toxoplasmosis). 2, 15, 34, 58, 61 nevertheless, these conditions often present with characteristic clinical, laboratory, and histopathologic changes that are necessary for proper diagnosis and management. the goal of this section is to review the interpretation of clinical and laboratory changes that occur in these feline liver diseases, provide an approach for separating the more common diseases by their clinical footprint, and then discuss therapy of each liver disease based on our current level of understanding of hepatoprotectants, antioxidants, and drugs used for specific therapeutic purposes. the clinical signs of liver disease in cats are often vague and nonspecific; however, recognition of certain clinical and laboratory abnormalities and their association with liver disease can greatly aid the diagnostic process. the most common early clinical signs observed in cats with liver disease are anorexia, lethargy, and weight loss, which are signs present in many (if not most!) feline diseases. 2, 15 because these early indicators of disease do not point specifically toward liver disease, a delay in diagnosis will occur unless the clinician carefully considers all possibilities and performs other tests to further evaluate the situation. for example, feline hepatic lipidosis is the most common form of liver disease in cats in the united states, united kingdom, japan, and western europe, occurring with a prevalence of nearly 16% in one study. 2 however, the most common, and often only, clinical sign associated with onset of this condition is anorexia; the signs of serious hepatic disease (especially jaundice and vomiting) do not occur until later (days or weeks) in the course of the disease. 2, 21 recognition that anorexia in a cat, even for a few days, is a risk factor for development of hepatic lipidosis is essential, and this risk is increased in obese cats. 11, 21 further, the clinical signs of liver failure develop much more slowly; many cats with hepatic lipidosis present alert and responsive until much later in the course of the disease, thus delaying onset of appropriate therapy. a similar clinical situation exists for the second most common form of liver disease in cats, feline cholangitis syndrome. 15, 28, 58 this complex of diseases in the cat can be associated with signs ranging from anorexia and lethargy to vomiting and jaundice, and these signs can vary in severity and prevalence. the key point is that except for development of jaundice, there is no constellation of clinical signs that are classic clinical indicators of liver disease in cats. 15, 28 as with many feline diseases, the subtle clinical signs of anorexia, lethargy, or inactivity are often the only signs of illness and should be further investigated. there are few changes that occur in the complete blood count that are specific indicators of primary liver disease in cats. the most common finding is the presence of poikilocytes, which are red blood cells with an irregular shape, speculated to be caused by changes in membrane lipids as a result of liver dysfunction. 14 other abnormalities may occur, such as anemia of chronic disease or neutrophilia, but these findings are nonspecific and occur with variable frequency. perhaps the most important reason for obtaining a hemogram is in icteric cats, because this test is essential to help rule out hemolysis as the cause of the hyperbilirubinemia. the serum chemistry profile can be very helpful, but there are several critical points in interpretation of these values that are important to review. the hepatic transaminases (alanine aminotransferase [alt] and aspartate aminotransferase [ast]) are leakage enzymes but do not discriminate among hepatobiliary disorders, nor do they provide an indicator of severity or disease origin. thus although increases in alt may be noted in cats with liver disease, they are also present in a variety of other systemic infectious, inflammatory, neoplastic, and and protein-losing nephropathies can also cause loss of albumin and affect cholesterol, it is essential to evaluate the cat for these problems when interpreting these results. finally, bilirubin metabolism is a critical function of the liver, but interpretation of hyperbilirubinemia requires a careful consideration of bilirubin disposition. hyperbilirubinemia develps because of one of three possible causes: (1) excessive hemolysis of red blood cells (rbc) (also known as prehepatic icterus)-high bilirubin in the blood stream occurs because of an overload of the mononuclear/phagocyte system with heme pigments from rbc destruction, (2) hepatic parenchymal disease or insufficiency (also known as hepatic icterus)-resulting in lack of normal bilirubin metabolism in hepatocytes and regurgitation of the pigments into the blood stream when they are not taken up into cells and excreted in bile, and (3) disease of gall bladder, biliary tract, or pancreatic duct (also known as posthepatic icterus)resulting in obstruction of the bile ducts or loss of bile into the abdomen (duct or gall bladder rupture and bile peritonitis). 41 the bottom line is that in any cat with hyperbilirubinemia, an assessment of the packed cell volume and rbc morphology should be completed to determine whether icterus is caused by hemolysis. once hemolysis is ruled out, then assessment of primary endocrine diseases, including hyperthyroidism, feline heartworm disease, fip, and neoplasia.* alternatively, the cholestatic membrane-associated enzymes alkaline phosphatase (alp) and gamma glutamyltransferase (ggt) are especially useful for recognizing disorders involving biliary or pancreatic ductal components. unlike the dog, these enzymes will only increase modestly in cats, even in severe disease, and there is no glucocorticosteroid or drug induction of the enzymes to influence interpretation. 14, 37 thus increases in alp in the adult cat represent a release of enzyme from the hepatobiliary tree and should be considered clinically important. both alp and ggt are produced in other tissues than the liver, with the highest ggt activity present in the kidney and pancreas; however, sources other than the liver do not contribute to the activity of these enzymes in health. recent studies of the effects on these enzymes in cats with pancreatitis, cholangitis, extrahepatic bile duct obstruction (ehbdo), and hepatic lipidosis reveal some important characteristics in interpreting increases in these enzymes. 14 first, both alp and ggt are increased in cats with pancreatitis, cholangitis, or ehbdo, because inflammation in the biliary tree also affects the pancreatic ducts (and vice versa, figure 23-55) , and if the fold increases in these enzymes are similar, the diagnosis is likely one of the three. 15 conversely, in cats with hepatic lipidosis (without concurrent inflammatory disease of the biliary or pancreatic duct system), large increases in alp are observed, but ggt will remain normal or only slightly increased. thus if the increase in alp is 5 to 10 times, while ggt is not increased or is only increased 1 to 2 times, then the likely diagnosis is hepatic lipidosis. [14] [15] [16] other than enzymes on the biochemistry panel, which are of limited value for assessing liver function, there are several key tests that can be used to help assess liver function cats with elevated liver enzymes. these five tests found on most routine biochemistry panels are helpful functional indicators: cholesterol, bilirubin, glucose, albumin, and urea nitrogen (bun). however, none are immune to outside influences on their interpretation, including bilirubin and cholesterol, which are the most liver specific. in cats with severe liver disease or failure, bilirubin levels tend to be quite elevated, while bun, albumin, cholesterol, and glucose concentrations tend to be significantly decreased, reflecting inability to metabolize urea (lack of arginine), inability to produce albumin or cholesterol, and abnormal metabolization of glucose. however, these changes represent severe loss of liver function and thus are not sensitive indicators of liver function because the changes occur quite late in the course of the disease. nevertheless, in cats with elevated liver enzymes and clinical signs of liver disease, these values should be carefully assessed. because gi disease of hepatic failure. in nonicteric cats with severe liver disease or in young cats suspected of having a portosystemic shunt, serum bile acids are the more reliable indicator of hepatic insufficiency. 18 the measurement of serum bile acid concentrations, preprandially and postprandially, is the most reliable, readily available, and sensitive test of hepatic function in nonicteric cats. 5, 14 that being said, although increases in bile acids are accurate indicators of hepatic insufficiency, the levels cannot be used to assess severity of disease or the type of dysfunction. further, bile acid assays are most effective when paired samples (preprandial-and postprandial) are compared, because single, fasting, or random bile acid samples can result in a falsenegative (normal) result. however, cats will often not eat in the hospital or when they are sick, and this prevents collection of a postprandial sample. however, this does not invalidate the results, because if the result of the single bile acid sample is abnormal, it does reliably indicate liver dysfunction. an alternative to using serum for testing bile acids in cats is urine bile acid analysis. healthy cats excrete a small percentage of conjugated bile acids in the urine 14 ; however, in cats with liver disorders that cause increased serum bile acids (and especially cholestatic liver diseases) a significant increase in urine bile acid excretion occurs. when urine bile acids (uba) were collected 4 to 8 hours after a meal and measured (normalizing the value with urine creatinine: uba/ucr) and compared with serum bile acids in a study of 54 cats with hepatic disease, 17 cats with nonhepatic disease, and 8 normal cats, the results were highly correlated. 47 the utility of the urine bile acid test is that it does not require a paired sample (postprandial test), and it is not as affected as the serum test is by hemolysis or lipemia of the blood sample. normal cats will have an uba/ucr of less than 4.4 µmol/mg, while values greater than 4.4 are considered evidence of significant hepatic dysfunction. 47 it is well known that the liver plays a central role in coagulation homeostasis and is the single site of synthesis of many coagulation proteins, anticoagulant proteins, and fibrinolytic factors. vitamin k is one of the most common factors found to be inactive or deficient in cats with liver dysfunction, and it is essential for normal functioning of factors ii, vii, ix, and x; protein c and s; and thrombin. insufficient or inactive vitamin k can occur for a variety of reasons, including dietary restriction (e.g., anorexia or diet deficiency), disruption of the enteric microflora that synthesize vitamin k (e.g., chronic antibiotic therapy), diseases causing fat malabsorption (e.g., ibd, exocrine pancreatic insufficiency), ingestion of vitamin k antagonists, or liver dysfunction. 20 for example, in cats with hepatic lipidosis, approximately 25% will have an increased prothrombin time (pt), 35% will have an increased partial thromboplastin time (ptt), but 60% of cats will have increased pivka parenchymal disease versus disease of the biliary tree is completed by evaluating the clinical presentation, laboratory values, and imaging of the biliary tree and abdomen for possible evidence of biliary or pancreatic disease. a urinalysis is also an important part of the minimum database, and it is no different in a sick cat with suspected liver disease. in cats the presence of hyperbilirubinuria is abnormal at any urine concentration, because they do not conjugate bilirubin in their renal tubules. 14 however, like bilirubinemia, presence of bilirubin in the urine can occur because of any of the three possible causes of hyperbilirubinemia: prehepatic, hepatic, and posthepatic; thus further evaluation is necessary once bilirubin is detected. ammonium biurate crystalluria suggests the presence of hyperammonemia, which in the cat is either because of a congenital portosystemic shunt (less common in cats than in dogs) or because of severe, end-stage liver disease resulting in portal hypertension, which is typically caused by cirrhosis or advanced polycystic liver disease. 6, 41 the most common feline liver diseases are hepatic lipidosis and feline cholangitis syndrome, which are two diseases that often result in development of clinical or biochemical icterus. thus because hyperbilirubinemia is a more sensitive indicator of liver function than bile acids or other liver function tests, the need for further testing is moot. however, there will be circumstances when further assessment of liver function is indicated, and for this, serum bile acids, blood ammonia levels, and urine bile acids may be needed. there are several situations where liver function testing may be indicated, but the most common indications for additional testing would be a cat with persistently elevated liver enzymes of unknown origin, a cat that develops urethral obstruction because of urate stones (suggestive of portosystemic shunting) or a cat with possible polycystic liver disease. 5 one of the oldest tests of liver function, because of its association with development of hepatoencephalopathy, is measurement of blood ammonia levels. 38 however, although this test is the only practical way to diagnose hepatoencephalopathy in dogs, the test has a number of limitations, including differences in ammonia levels between arterial and venous (lower) samples and significant sample handling issues (ammonia is labile and results are affected by improper sample handling or lack of immediate measurement) that make its use difficult in practice. 43 in cats hyperammonemia is even less common than in dogs likely because of their highfunctioning urea cycle pathways 14 ; the assays have not been validated for feline blood in most laboratories, and as such, the test is not recommended as the sole indicator uncommon in cats, the most common causes are neoplasia (primarily of the pancreas, but cholangiocarcinomas can occur) or chronic pancreatitis, which can occur concurrently with cholangitis in cats, resulting in both intrahepatic and extrahepatic cholestasis in some cats. 23, 41 the bile ducts are affected in cats with chronic pancreatitis, because the feline biliary system and pancreatic duct system merge at the level of the pancreas to form a single duct that empties into the duodenum. thus in cats with either pancreatitis or biliary disease, recent evidence has shown that the inflammation affects both organs. 54, 59 further, in chronic pancreatitis, either persistent inflammation or development of fibrosis can result in dilation or obstruction of the common bile duct. 33 in cats with chronic ehbdo, the common bile duct will become widely dilated and tortuous, a finding easily seen on abdominal ultrasonography but a problem not easily managed (figures 23-56 and 23-57) . interestingly, the gallbladder is often not enlarged, and may in fact be small in cats with this condition, because the remaining fluid in the gallbladder is white bile (highly concentrated mucinous bile from which the pigment has been resorbed). 41 in addition, variable filling of the gall bladder is a normal phenomenon; thus gallbladder size is not an indicator of ehbdo. (proteins induced by vitamin k antagonists or absence). 20 nevertheless, although pivka is a very sensitive test for abnormalities of vitamin k function, most cats with liver disease that have a normal pt/ptt, but abnormal pivka do not represent clinical evidence of bleeding. in any case, abnormalities in the clotting cascade related to vitamin k deficiency in cats with liver disease are common, whether or not they show evidence of active bleeding. and because the balance of the coagulation system in a cat with liver disease can be disrupted by a procedure that initiates small amounts of bleeding (e.g., a biopsy), all cats with liver disease should be given vitamin k as a precautionary measure before and after invasive procedures, even if the clotting times (pt and ptt) are normal. this may be especially important in cats with hepatic lipidosis, because their vitamin k clotting status is likely to be even more affected by the concurrent anorexia and disruption of enteric microflora. 14 the dose of vitamin k 1 (phytonadione, aquame-phyton [merck, west point, pa.]) used prophylactically is 2.5 mg sc, im, or po q12h for 3 to 5 days, then weekly until recovered. see box 23-4 for a summary of the causes of icterus. cholestasis is the reduction of bile flow, which can occur at any point along the biliary tree; bile production occurs in hepatocytes, and flow is connected to the distal concentrating components (gallbladder and common bile duct) by the bile ductules. thus cholestasis can occur inside the liver's biliary tree (intrahepatic cholestasis) or outside the liver in the gallbladder and common bile duct (extrahepatic cholestasis). intrahepatic cholestasis most often occurs in diseases involving hepatocellular damage, leakage, or swelling, such as infections (e.g., bacterial cholangiohepatitis, toxoplasmosis, fip, or other diseases causing inflammation), infiltrative diseases (e.g., lymphoma), metabolic diseases (e.g., hepatic lipidosis), or diseases causing disruption of architecture (e.g., cirrhosis or severe polycystic disease). 41 intrahepatic cholestasis occurs in zone 1 of the liver lobules (periportal zone); at the level of hepatocytes, canaliculi or bile ductules; and is damaging to cells because of the emulsifying properties of lipid on membrane lipids. however, because the liver has a large reserve capacity, clinical icterus (e.g., jaundice) only occurs in the most severe cases when the liver is affected diffusely. thus severe or persistent intrahepatic cholestasis can serve to perpetuate the inflammation and cell damage if it is not corrected. extrahepatic cholestasis or extrahepatic bile duct obstruction (ehbdo) is less common than intrahepatic cholestasis and is most commonly associated with obstruction of the common bile duct. since gallstones are icterus is the result of cholestasis, and the underlying cause can be either hemolysis or hepatobiliary disease, for which further clinical examination will be needed to determine if rbc destruction or liver disease is occurring. in most hepatobiliary diseases of cats, cholestasis is occurring, but there may be no clinically apparent icterus because the degree of hyperbilirubinemia must be at least 2 to 3 times greater than the normal values to exceed the capacity of the liver to process the excess bilirubin. in cats with hyperbilirubinemia not caused by hemolysis, whether it is clinical or subclinical, there is no need for further evaluation of liver function (e.g., bile acid assays), because bilirubin is a more sensitive indicator of liver function than bile acids. the degree of hyperbilirubinemia does not suggest differentiation of intrahepatic versus extrahepatic cholestasis; however, the presence of acholic feces (white feces) is diagnostic for extrahepatic bile duct obstruction (ehbdo), because lack of stercobilinogen (the brown/black pigment in feces) is only found in cats with complete obstruction of the bile duct. finally, the presence of intrahepatic cholestasis and clinical icterus in a cat indicates a diffuse hepatobiliary disease, such as cholangitis or hepatic lipidosis, as focal liver disease, even if severe, will not cause clinical hyperbilirubinemia because of the tremendous reserve capacity of the liver for bilirubin uptake. portal hypertension is an abnormally high venous pressure in the portal system and is typically caused by increased resistance to portal blood flow. there are potentially three regional causes of portal hypertension: prehepatic (disease in the portal vein itself), hepatic (intrahepatic diseases causing compression or decreased flow), and posthepatic (diseases of the caudal vena cava, right heart or pulmonary vasculature). the most common cause of portal hypertension in the cat is cirrhosis or portal venous thrombosis, because portal vein hypoplasia (formerly known as microvascular dysplasia) is known to occur only in the dog, and the other causes of portal hypertension (budd-chiari syndrome, heartworm caval syndrome, pulmonary hypertension) are rare and more likely to occur in the dog. 41, 44 in any case, the clinically recognizable effects of portal hypertension are development of ascites (unusual in the cat), acquired portosystemic shunting (reported in cats), and development of hepatic encephalopathy (less common in cats than in dogs, because of their profound ability to handle protein wastes). 5, 36, 41 most cats and dogs that develop hepatic encephalopathy (he) secondarily to portal hypertension do so because of reduced liver function (because of portosystemic vascular shunting [pss] or cirrhosis and the acquired shunting that develops). cats can develop another form of chronic he because of hepatic lipidosis, but this is believed to be because of the combination of liver failure and prolonged fasting, resulting in arginine deficiency and impaired ammonia detoxification. 2 portosystemic vascular anomalies, also called portosystemic shunts or portovenous shunts (pss), although less common than in dogs, also occur in cats. these vascular anomalies can be either congenital or acquired, single or multiple in number, and occur as extrahepatic vascular shunts or within the liver itself (intrahepatic shunts). 5 the shunting of blood around the liver is the cause of hepatic atrophy and reduced hepatic function that results in an accumulation of toxins, particularly ammonia that leads to the development of hepatoencephalopathy. the two most common veins that serve as the connection point for the shunting portal venous blood are the caudal vena cava and the azygous. 5 in cats a single, extrahepatic, portocaval shunt is the most commonly reported form, and occurs in 75% of cats with pss. 5 as in dogs, specific breeds of cats may have pss more commonly, and these include domestic shorthair cats, burmese, siamese, persian, and himalayan breeds. 5 in contrast to dogs, males may be more predisposed to pss than females, but the clinical signs relate to the three body systems most affected: the central nervous system, gi tract, and urinary tract. the most common presenting complaints in cats are weight loss or poor/stunted growth, and dull, bizarre or lethargic behavior, especially after eating. signs of gi disease common in dogs, such as vomiting, diarrhea, or inappetence, are less common in cats, but in one report, 75% of cats with pss drooled. 5 finally, cats with pss often present with signs of lower urinary tract disease (e.g., hematuria, stranguria, or even obstruction) because of the development of urate uroliths (which are radiolucent, thus difficult to detect). 5 because the most common signs of he are apathy, listlessness, and decreased mental alertness, they are often not recognized specifically as indicative of brain dysfunction but as part of the constellation of signs of the liver disease. however, with progression of the has not been reported. the clinical presentation is typically nonspecific (the most common signs are vomiting, lethargy, and anorexia), and there are no laboratory changes that are suggestive of hepatic neoplasia. thus the diagnosis must be made by identification of disease, other signs will develop, including ataxia, salivation, stupor, or coma. the best and only practical diagnostic test for he is plasma measurement of ammonia levels. 43 however, as previously noted, the test has many technical issues that make its clinical utility in the practice setting difficult at best, and there are few laboratories that have validated ammonia measurement in the cat. cancer of the liver can occur as a primary disease (table 2322) or as a result of metastasis of neoplastic disease occurring elsewhere and, most typically, the abdominal cavity. the most common neoplastic infiltration of the liver that is not a primary liver tumor is lymphoma (figures 23-58 and 23-59), followed by visceral mastocytosis. 4 as with many other types of cancer, hepatobiliary neoplasia is most common in middle-aged to older cats, and it is relatively rare, with a reported incidence of 1.5% to 2.3%. 4 benign tumors, such as biliary cystadenoma ( figure 23-60) , carry a good prognosis if they are amenable to surgical resection. the incidence of metastatic neoplasia (including lymphoma and mast cell tumors) the most common clinical signs are related to spontaneous rupture of the enlarged and friable liver. affected cats may present with lethargy, anorexia, pale mucous membranes, and a heart murmur secondary to anemia. clinical signs of liver disease are usually absent. hepatomegaly and hypotension may also be found. results of routine laboratory testing (mild to marked increases in alt and globulins while alp and ggt are typically normal) and ultrasonographic examination (hepatomegaly, generalized increase in hepatic parenchymal echogenicity) 4a of the liver may be supportive, but definitive diagnosis relies on histopathologic examination of a liver biopsy. fna of the liver is not helpful because amyloid is rarely detected with this method. hemostasis should be evaluated carefully before any biopsy procedure is planned. the most important differential diagnoses are fip, hepatic lipidosis, and hepatic lymphoma. scintigraphic imaging using i-123 serum amyloid p component has potential as a noninvasive test. 39a there is no specific treatment for amyloidosis in cats, so therapy is primarily supportive care (antioxidants, vitamin k, blood transfusion). attention should be paid to identification and control of any underlying chronic inflammatory disease. unfortunately, the long-term prognosis is poor as most affected cats die of intra-abdominal bleeding. survey abdominal radiography is the simplest and most readily available imaging modality to assess structures in the abdominal cavity. radiographs are most useful to assess liver size, will reveal large hepatic masses, and provide evidence of radiopaque masses or other abnormalities in the abdomen. however, the preferred imaging modality used to assess hepatic structures in cats with suspected liver disease is abdominal ultrasonography (aus). the reasons why ultrasonography is a more useful tool for assessment of the liver in cats are numerous, but because feline liver diseases are primarily diffuse, infiltrative, or metabolic diseases that also affect the biliary tree, ultrasonography is the only imaging tool that will give reliable diagnostic information. this widely available diagnostic tool can be helpful in determining liver size and parenchymal echogenicity, in identifying mass lesions, evaluating the biliary tree and gallbladder, quantifying flow (doppler techniques), and identifying vascular anomalies. 29 as with all diagnostic modalities, the skill and experience of the operator is vital to accurate procurement and interpretation of the images. further, it is important to remember that although ultrasonographic images are extremely useful in the clinical evaluation of a cat with possible liver disease, the images themselves do not represent a histologic diagnosis. structural abnormalities by hepatobiliary imaging and subsequent examination of the tissue either by fna or biopsy techniques. historically, amyloidosis has been recognized as primarily a renal disease, especially in abyssinian cats. more recently, cases of hepatic amyloidosis without renal involvement have been diagnosed in siamese and related breeds, as well as in nonpedigreed cats. 4a,10a,30a the majority of cases have been described in australia, the united kingdom, and europe. amyloid a is deposited in the liver, probably in response to chronic inflammation in another organ. in the siamese breed, a genetic component may contribute. 48a the amyloid a protein occurring in the siamese breed differs from that known in the abyssinian breed. 48a contraindicated in cats, because they may cause a lethal shock reaction. 40 a similar reaction may be seen with penetration of the larger bile ducts or gallbladder with a large-bore biopsy needle, because these tissues have a significant autonomic innervation in the cat that may result in bradycardia and shock following the procedure. 40, 48, 54 it is particularly important to recognize this as a risk in cats with ehdbo or dilated bile ducts, and this risk factor reiterates the need for ultrasound examination of the liver prior to making biopsy decisions. nonetheless, owners should be informed of these potential risks, in addition to the risk of bleeding from biopsy sites in any cat undergoing liver sampling. 7, 48 biopsy techniques liver biopsies, whether they are obtained by needle, laparoscopy, or surgical means, should be taken from a location that represents the primary liver pathology, handled appropriately to ensure accurate interpretation of the sample, and the histopathologic description should be interpreted according to the guidelines set by the wsava standards for clinical and histologic diagnosis of canine and feline liver disease. 42, 61 guidelines for obtaining and handling surgical biopsies of the liver are reviewed elsewhere 27 and will not be further discussed. because needle aspirates/biopsies, tru-cuttype biopsies, and laparoscopic biopsies are commonly used to obtain liver tissue in cats, the benefits and limitations of each of these techniques will be discussed. as a general rule, the more tissue that can be obtained, the better the pathologist's interpretation of the tissue abnormalities will be. for example, most pathologists believe that at least six portal areas are necessary to make a diagnosis of inflammation liver disease in cats. 42 this will require either a 16-or 18-gauge needle size or larger piece of tissue than is obtained with smaller needles or an aspirate. the amount of tissues required to view at least six portal areas is approximately 15 mg, and 5 mg will be required for culture of the tissue. 42 if other analyses of the tissues are considered (e.g., metal analysis), approximately 20 to 40 mg of liver is needed. 42 a typical laparoscopic cup biopsy forceps will provide 45 mg of liver tissue, a 14-g tru-cut-type biopsy needle provides 15 to 20 mg, and an 18-g needle biopsy provides only 3 to 5 mg of liver tissue. 42 thus, depending on the clinical circumstances and considered differentials, the best approach for obtaining the needed tissue must be considered prior to planning the procedure. fine-needle aspiration to obtain liver tissue for cytologic examination is commonly performed in cats with liver disease for good reason. the procedure is inexpensive, easy to do, is relatively low risk, and often requires only sedation to complete. 57 further, samples obtained by this method can be diagnostic for hepatic lipidosis, hepatic lymphoma or other round cell tumors, and in for the most common liver diseases of cats (hepatic lipidosis, feline cholangitis syndrome, and neoplasia/ lymphoma), aus examination provides a useful means of obtaining clinical clues and tissue to support or refute the differentials. for example, in cats with hepatic lipidosis, the liver is quite enlarged and typically diffusely hyperechoic, while in cholangitis or other inflammatory diseases, the liver is more often diffusely hypochoic. 29 however, these sonographic findings are very nonspecific and can easily lead to errors in diagnosis if the tissue is not subsequently sampled for confirmation. 24, 35 thus one of the most important utilities of the aus is the ability to obtain liver tissue (either by aspiration or guided-needle biopsy) and for aspiration of the gallbladder to obtain bile for culture. 24, 49 these techniques alone have made the aus an extremely important diagnostic tool in the evaluation of liver disease in cats. the diagnosis of most liver diseases requires a histopathologic sample of liver tissue, and this is particularly true in the most common feline liver diseases, which tend to be diffuse diseases affecting the entire liver. cats with one of these diffuse diseases can be sampled randomly using any one of these commonly employed techniques: ultrasound-guided fine-needle aspirates (fna), ultrasound-guided needle biopsy, laparoscopic biopsies, or biopsies obtained surgically. some types of neoplasia (particularly round cell tumors) and vacuolar hepatopathies (hepatic lipidosis) can often be diagnosed by cytology using fna techniques. however, differentiation of liver cell tumors (adenomas and carcinomas) and inflammatory diseases of the liver cannot be diagnosed without a larger sample of tissue and histopathologic examination. 30, 50 further, even in cats with classic hepatic lipidosis changes, concurrent diseases such as cholangitis or lymphoma can be missed if only fna techniques are employed. 60 thus it is essential to consider that in many liver diseases the lesions, although typically diffuse, may also have focal components; for example, inflammation may be throughout the liver, but fibrosis will be present only in focal areas. thus the results of fna or tru-cut needle biopsies should always be considered in the light of the clinical, laboratory, and ultrasonographic evidence. prior to scheduling a cat for a biopsy, the risk-tobenefit ratio of performing a liver biopsy should always be considered. this is primarily because heavy sedation or anesthesia will be essential in most cats undergoing a liver fna, and for all cats undergoing a liver biopsy (needle or otherwise). in addition to anesthesia risks, the use of automatic spring-loaded biopsy guns to obtain ultrasound-guided biopsies of liver tissue is equipment, the interested reader is referred to several recent reviews on the subject. 48, 54 to maximize the histopathologic accuracy, biopsies taken at laparoscopy or surgically should be taken from both normal-appearing and abnormal areas in the liver. further, if there is a need to obtain samples from the deeper tissues, the laparoscope can be used to direct a tru-cut needle biopsy to the best location for sampling. one of the major advantages of the laparoscopic technique is that it allows the operator to observe the biopsy sites for excessive bleeding, which is unusual, but if observed can be staunched by using pressure on the site, gelatin coagulation material placement, or electrocautery. with experienced operators, the complication rate for laparoscopy is very low (less than 2%), and most complications were because of anesthesia, bleeding, or air embolism. 48 finally, although not necessary to have direct visualization to obtain an aspirate of gallbladder bile, laparoscopy allows easy sampling of bile for culture, which is important in all cats with suspected inflammatory liver disease or hepatobiliary disease. once a diagnosis of liver disease is made in the cat, specific therapy for the cause (if available) should be instituted; however, for many feline liver diseases, no specific therapy is available, and thus hepatoprotective therapy is used concurrently to aid in the recovery of the liver from the insult. in this section, therapy of two of the most common diseases of the feline liver will be considered, with a special emphasis on nutritional aspects of treatment, nutraceutical therapy, and the unique needs of cats. the most common liver disease of cats is idiopathic hepatic lipidosis (figures 23-61 and 23-62) , a disease that results in liver failure because of a combination of factors including hepatic lipid accumulation, insulin resistance, fasting, and protein (especially arginine) deficiency. 2, 8, 9, 11 thus, unlike many diseases of the liver, the primary focus of therapy and the essential component for recovery is nutritional support. as in any patient with serious liver disease, initial therapy is always aimed at correction of any fluid or electrolyte abnormalities that may exist, because these may be profound if the cat has been vomiting. in addition, normalization of electrolytes is particularly important in cats that have been anorexic for an especially long time (1 to 2 weeks), because refeeding syndrome may be triggered with the initiation of feeding, resulting in sudden drops in potassium, phosphate, and magnesium. 1 although this phenomenon is less common and usually less profound in cats fed enterally versus areas where appropriate, definitive diagnosis of certain infectious diseases (e.g., histoplasmosis). 57 however, even with these relatively straightforward diseases, fna of liver tissue has significant limitations, the most important of which is the failure to accurately identify the primary disease. for example, although it is easy to make a diagnosis of hepatic lipidosis using this technique, a paper recently showed four cats that were incorrectly diagnosed with hepatic lipidosis instead of lymphoma because the fna samples were obtained from areas that did not have lymphoma infiltration. 60 in another study, reviewing the agreement between fna cytologic samples of liver and the histopathologic diagnosis, only 51% of the cases had overall agreement. 50 thus although cytology of fna samples of liver tissue in cats with diffuse hepatic disease remains a useful first step, it is important for the clinician to carefully interpret the results and discuss the potential limitations of this technique with owners. there are several needle biopsy techniques available for sampling liver tissue, but not all are suitable or safe for use in cats. the menghini technique is one such approach that is not suitable for use in cats, because it is a blind procedure using a large-bore needle that cannot be used with ultrasound guidance. 42 the second option among the needle biopsy techniques that is not recommended for cats is the biopsy gun device. tru-cut biopsy guns are operated by a triggering device that can result in the induction of a lethal vagotonic shock reaction in the cat immediately following the procedure. 40 for most ultrasound-guided liver biopsy procedures, either the manual or, preferably, the semiautomatic tru-cut device is recommended for use in obtaining needle biopsies from cats. as a general rule, the tru-cut device will advance into the liver to a depth of 2 cm; so, it is essential to carefully note the amount of liver tissue available during the ultrasound assessment before advancing the needle for tissue collection. properly obtained tru-cut needle biopsies are a valuable technique for obtaining a representative sample of liver tissue 61 ; however, because of the risk for bleeding or liver fracture with any movement, it is essential that cats be anesthetized for this procedure. laparoscopy is an intermediate step between needle biopsy and surgical laparotomy for obtaining liver tissue for histopathology in cats. 48, 54 this technique is becoming more widely used as more specialists are trained for this procedure that allows visualization of tissues to be biopsied without opening the entire abdomen. although this technique does require general anesthesia, the limited degree of invasiveness, the large biopsy sample size, and rapid patient recovery make laparoscopy a valuable tool for obtaining liver tissue, 48 and it can be used to obtain biopsies from the spleen, pancreas, kidneys, lymph nodes, or to aspirate the gallbladder. for a detailed discussion of laparoscopic techniques and lipidosis. the echogenicity of the parenchyma is uniformly increased, which is more apparent when compared with other ultrasonographic images presented in this chapter. additionally, the gall bladder is distended. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease. (courtesy dr. randolph baral.) liver gb figure 23-62 gross appearance of liver from a cat with hepatic lipidosis. note the pale tan and exaggerated reticular pattern. in most cases, the edges appear more rounded than is evident here. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease (same cat as in figure 23-61) . (courtesy dr. randolph baral.) those started on intravenous nutrition, it can be a significant source of morbidity if electrolyte replacement and monitoring are not carefully attended. once the cat is hemodynamically stable, the next step in treatment planning in cats with hepatic lipidosis is re-introduction of nutrition, which must include placement of a feeding tube (box 23-5). however, because many of these cats are extremely ill and are not good candidates for anesthesia, placement of a nasoesophageal (ne) tube to allow initiation of enteral feeding is often the most appropriate step for the first few days. when administering food through a feeding tube, there are several important points: 1. the food should be room temperature (not too hot or cold). 2. the tube should be flushed with water following feeding, to remove any particles of food or medication that may cause the tube to clog. 3. if the cat is volume sensitive, it is important to carefully calculate how much water is used for flushing the tube, because a significant volume of fluid can be infused, creating a potential fluid overload. if the cat is fluid sensitive, the total amount of fluids (amount in the food, amount added to food if blenderized, and amount of flush) must be determined, and the amount of fluid used in flushes or food preparation may have to be reduced. force feeding is to be strongly discouraged in these sick cats for several reasons: • it is highly stressful and will further increase the stress response and insulin resistance phenomena that are perpetuating the hepatic lipidosis. • it can be dangerous to the cat (aspiration) or operator (scratches/biting). • it is rarely able to meet the necessary nutritional goals set for the patient. • it may induce food aversion, a phenomenon unique to cats, but creating a profound aversion to the chosen food that can be lifelong. 12 although ne tubes are excellent choices for short-term feeding of cats unwilling to eat, there are several disadvantages to their long-term use, including the nasal irritation that occurs, the relative ease with which cats can (and will) remove them, and the need to use liquid enteral diets. 62 thus once the cat is deemed stable enough for general anesthesia, a long-term feeding tube solution is needed, and this typically is either an esophageal (e) tube ( figure 23 -63) or percutaneous endoscopic gastrostomy (peg) tube. 22, 62 both feeding options are generally well tolerated methods for providing long-term feeding, but e tubes have the advantage of being placed without the need for any specialized equipment, and if complications occur, they are generally easily addressed, because the most common complications are infection at the tube site or premature removal of the tube by the cat. placement of a peg tube, although relatively easy to learn to place, requires having the appropriate endoscopic equipment, and if complications occur as a result of infection or tube removal, more significant morbidity can result. because there is no advantage to placement of peg tubes easiest to use, and are an acceptable choice in most situations. finally, because many cat stomachs are volume sensitive with initiation of feeding, it is very important to start conservatively with small-volume feeding on a more frequent schedule. with prolonged fasting, the stomach volume of a cat with hepatic lipidosis may be reduced dramatically, preventing normal expansion and limiting intake to as little as 10% of normal. thus to avoid vomiting when feeding, the starting volume may have to be as small as 10 to 15 ml every 2 to 3 hours. a good rule of thumb is to start with estimation of resting energy requirement (rer) (40 to 50 kcal/kg is a good estimate of rer), and then attempt to meet 25% of rer the first day. if no problems are encountered, increase the amount to 50% of rer the second day, and so on, but during this period, keep the frequency as high as possible (feed four to six meals per day) so that the volume remains relatively small at each meal. once full rer has been achieved with multiple meals per day, the frequency of feeding can be gradually reduced to three to four meals per day. most cats will eventually tolerate three meals per day well, and some can tolerate two meals per day, but this is quite variable and should not be attempted during the first weeks of feeding. in general, most cats with hepatic lipidosis will require tube feeding for a minimum of 3 to 6 weeks before they will show interest in food and begin eating again on their own. the tube should be retained until the cat has been eating on his or her own for at least 1 week or longer and can be maintained for a longer duration if it is being used to administer medications, because cats can eat normally with the e tube in place. the other therapeutic considerations for cats diagnosed with hepatic lipidosis are directed toward dealing with the complications of the disease and reducing the oxidative stress on the liver with hepatoprotective therapy (table 2323) . in cats that are vomiting, in cats versus e tubes, placement of e tubes is advocated as the best approach for most practice situations. interested readers are referred to several recent reviews on tube placement for specific details on each method and to chapter 18. 22, 62 diet selection is the next step in treatment planning for cats with hepatic lipidosis. in contrast to the belief that animals in liver failure need lower quantities of protein to reduce the workload on the liver, cats with hepatic lipidosis actually need protein to recover. in fact, the work of biourge and coworkers showed that protein was the essential nutrient in reducing hepatic lipid accumulation, was essential to eliminate the negative nitrogen balance, and also appeared to minimize muscle catabolism. 9 further, diets high in protein can improve insulin sensitivity and assist weight loss in recovery from obesity. 8, 11 conversely, although carbohydrates are a readily available energy source, they are often associated with gastrointestinal distress (diarrhea, abdominal cramping) and hyperglycemia (secondary to the insulin resistance in place as a result of obesity and hepatic lipidosis). 2 thus diets selected for cats with hepatic lipidosis should ideally be high in protein (>40% metabolizable energy [me]) and have lower amounts of carbohydrates (<20% me), with the remaining calories coming from fat. the diets that best fit this profile are the diets formulated for diabetic cats; however, kitten food, many adult cat foods, and some of the enteral recovery diets have this high protein/low carbohydrate profile. many of the intestinal diets are not higher protein and are higher in carbohydrates, and so would not be the ideal choice. the key to using any of the foods that are not designed for use in a feeding tube is to blenderize them (and if necessary, strain the food) so that the food will easily go through the 14-or 16-g feeding tube without clogging it. enteral diets designed for use in feeding tubes are the because the primary starting point of the inflammatory disease in cats is the bile ducts (cholangitis), with inflammation extending to the hepatic parenchyma (cholangiohepatitis) only with time and severity, the term cholangitis syndrome has become the preferred terminology. the disease syndrome has been further classified by the wsava liver diseases group into one of three primary types: neutrophilic or suppurative, chronic lymphoplasmacytic (figures 23-64 and 23-65) , and lymphocytic (non-suppurative). 61 each of the forms appears to behave quite differently clinically as well as in their progression and outcome. in general, cats with the suppurative form of cch typically have an acute onset of illness, which often includes fever, anorexia, and vomiting, and they may become icteric quite quickly (figure 23-66) . 28, 52 the nonsuppurative form of cch (lymphocytic form) tends to be a more chronic condition, with affected cats showing nonspecific signs of illness that may include partial anorexia and lethargy, but the signs may wax and wane or are non-progressive. 28, 52 because of the feline pancreatic and bile duct anatomy, it is common for cats with cch to have pancreatitis and vice versa, and in some cases, cats will also have concurrent ibd; the constellation of the three conditions occurring together is called triaditis. 59 this combination is increasingly recognized in cats, and recent reports suggest from 50% to 85% of cats with one syndrome have all three diseases. 25, 51, 54, 59 at this time, the etiology of each of these syndromes and the pathogenesis is not well understood; however, the enteric microflora are presumed to play an important role in the suppurative form, and immune mechanisms are presumed to be the cause of the chronic inflammation found in the chronic nonsuppurative antiemetic therapy is beneficial, because it is imperative that the cat continues to receive some food, and vomiting will complicate this. metoclopramide is often used in cats because of its ready availability and low cost, but it is a very weak antiemetic in cats and thus may not be the best choice. in most cats, the novel nk-1 receptor antagonist maropitant has been a safe and effective choice. 32 the most commonly used antiemetics in the author's feline practice are maropitant (1 mg/kg iv, sc or through the e tube q24h), ondansetron (0.22 mg/kg iv q8-12h), or dolasetron (0.5 mg/kg iv, sc q24h). in addition to control of vomiting, all cats with hepatic lipidosis should be given vitamin k 1 (2.5 mg/cat po, sc) daily for a week, then weekly until the cat has recovered, and vitamin b 12 (cobalamin) (250 µg/cat sc) weekly for 6 weeks, then monthly until blood values are normal. 46 other vitamins may become deficient, such as some of the b vitamins and vitamin e; however, feeding is likely to rapidly replenish these deficiencies if they exist. this is also likely true of amino acid deficiencies, but supplementation of l-carnitine (250 mg/day po) may be beneficial by improving fatty acid oxidation. 10 finally, hepatoprotectant and antioxidant therapy with s-adenosylmethionine (same) (20 mg/kg po q24h) has been advocated to increase glutathione and may be beneficial in cats with hepatic lipidosis. 13, 19, 56 it is important to note that if same is given through the tube (and thus the tablets must be crushed), the dose must be increased by approximately 50% to allow for the loss of absorption from loss of the enteric coating. because drug metabolism is often impaired in cats with hepatic lipidosis, appetite stimulants, such as mirtazapine, cyproheptadine, and clonazepam, should not be used in cats because dosing and side effects can be unpredictable. benzodiazepine agonist drugs (e.g., diazepam) should be completely avoided in cats with possible lipidosis-induced hepatoencephalopathy, because they will exacerbate the signs and may cause fulminant liver failure. 2, 14 fortunately, most cats with idiopathic hepatic lipidosis that receive immediate and aggressive therapy and feeding for their disease will recover completely. cats that develop hepatic lipidosis secondary to other serious diseases (e.g., lymphoma) have a much lower chance of complete recovery and often die of their disease or its complications. the most common inflammatory liver disease in the cat is a complex syndrome with multiple subgroups of disease previously termed cholangitis/cholan giohepatitis complex (cch) but currently recognized under the terminology feline cholangitis syndrome. 61 this disease is quite variable in both its presentation and severity, and it may occur as a primary process or secondary to/ concurrent with other diseases (e.g., pancreatitis, ibd). ultrasonographic appearance of liver with lymphocytic/plasmacytic inflammation. note the varying echogenicity throughout the hepatic parenchyma; areas of hypoechogenicity likely reflect inflammatory cell infiltration. the gall bladder is distended; its shape is distorted by pressure from the transducer. (courtesy dr. randolph baral.) liver gb [10 mg/kg po q24h]), and if pancreatitis is concurrent, pain control with opioid pain relievers (e.g., buprenorphine 0.05 to 0.1 mg/kg po, sq q8-12h). 52 if culture is not possible, combination therapy with enrofloxacin (4 mg/kg po q24h) and metronidazole (5 mg/kg po q12h) is reasonable. in cats with chronic lymphoplasmacytic forms of cholangitis, management must be tailored to the individual situation and often requires therapy with either immunosuppressive doses of prednisolone (2 to 4 mg/kg po q24h) or chlorambucil (4 mg/m 2 po q2d), along with the hepatoprotectants and cholerectics, and concurrent treatment of other diseases (pancreatitis or ibd) that may be occurring. 52 the lymphocytic or lymphoplasmacytic forms of cholangitis may wax and wane in intensity over time, and may require long-term continuous or intermittent therapy to control the disease. there is no specific diet that is recommended for cats with inflammatory liver disease, but protein restriction should not be initiated unless the cat has clear evidence of severe hepatoencephalopathy. the diet should be selected based on other conditions (such as ibd), for which the diet may be more critical in the management. monitoring of serum chemistry values (especially glucose), clotting times, cobalamin levels, and pli/tli concentrations are recommended every few months, as well as careful monitoring of the cbc for all cats on chlorambucil. in all cats with chronic inflammatory liver disease, prior to initiation of immunosuppressive therapy, a careful assessment of the cat for other possible causes of inflammation should be completed (box 23-6). as in dogs, if a cat with pss can have surgical closure of the shunting vessel (ligation, placement of an ameroid constrictor, intravenous coiling), the long-term forms. however, whether or not these syndromes are related, a continuum of disease or completely different diseases remains undetermined. once a definitive diagnosis is obtained by histopathology of the liver tissue and culture of bile, treatment can be tailored to needs of the cat. cats with the more aggressive suppurative form of cholangitis often require intravenous fluid therapy, antibiotic therapy (based on results of culture whenever possible), and supportive therapy (antiemetics, vitamin k 1 , hepatoprotectants such as same [ important antioxidant and stabilizes membrane functions • n-acetylcysteine-a precursor to glutathione and antioxidant, also improves tissues oxygen delivery • ursodeoxycholic acid (tertiary bile acid)-used to replace hepatotoxic, hydrophobic bile acids and increase bile flow • silymarin (milk thistle)-a free radical scavenger and anti-inflammatory/antifibrotic agent • vitamin e-an antioxidant and antiinflammatory vitamin* although few clinical trials of these nutraceuticals have been performed in feline liver disease, a few studies have recently appeared showing that same, ursodeoxycholic acid, silymarin, and n-acetylcysteine all are hepatoprotective, have few adverse side effects, and may be beneficial in many types of liver disease in cats. 3, 26, 39, 53, 55 feline liver disease is a common problem that requires careful consideration of the presenting complaint, clinicopathologic findings, imaging results, and, if available, histopathologic interpretation to be able to provide an accurate diagnostic and therapeutic plan. a variety of insults can be responsible for liver dysfunction or failure, but hepatic lipidosis and feline cholangitis syndrome remain the most common reasons for cats to present prognosis for function and quality of life is generally very good. 5 however, even if surgical correction is anticipated, and especially if surgical correction is impossible or not completely successful, medical management of he is indicated. see table 23 -24 for the basic therapeutic approach to medical management of cats with he resulting from pss. because hepatocytes, by their position in the body between the gi tract and rest of the body, as well as their critical role in metabolism and detoxification, are uniquely susceptible to oxidative injury and reactive intermediates of metabolism, they must be able to protect themselves. the natural defenses of the liver include superoxide dismutase and glutathione, free-radical scavengers such as vitamin e and ascorbate, and other prosurvival signaling pathways that are controlled by hormones and growth factors. 56 however, in injury or overwhelming infection or inflammation, the natural defenses of the liver can be overwhelmed, and then it is essential for medicines and nutraceutical therapy to be included in the treatment plan to help reduce inflammation and fibrosis, protect against oxidant injury, and enhance bile flow. the cytoprotective agents most commonly used in liver diseases to assist in these processes (table 23 -25) are: • s-adenosylmethionine (same)-a precursor in the synthesis of glutathione and an important methyl donor to dna and proteins, is an with icterus or liver failure. therapy must be tailored to the individual, but nutritional support is critical in the management of hepatic lipidosis, and appropriate supportive therapy with hepatoprotectants may be crucial to treatment success. resorption from that space. effusion accumulation is therefore correlated to increased capillary hydrostatic pressure, widening of the oncotic pressure gradient, increased endothelial permeability, increased interstitial hydrostatic pressure, or loss of effective lymphatic drainage or a combination of these factors. 10, 23, 32 peritonitis of any cause results in vascular dilation, increased capillary permeability, and the migration of inflammatory cells into the peritoneum in response to immunomodulatory mediators. the inflamed peritoneum becomes a freely diffusible membrane, allowing a massive outpouring of fluid and plasma proteins from the circulation. 5, 30 ascites is not commonly seen in practice; one study recognized ascites in only three cats out of 1000 admissions to an american veterinary teaching hospital, 34 but the prevalence may be greater in primary care practice. in that study, dilated cardiomyopathy (dcm) was the most common disease associated with peritoneal effusion; however, dcm was diagnosed in most of these cats before 1987, when taurine deficiency was found to be a primary cause of this form of cardiomyopathy in cats. neoplasia was the most common cause after 1987. 34 feline infectious peritonitis (fip) was by far the most common cause of feline ascites diagnosed over a 10-year period at the feline centre at the university of bristol, comprising 50% of all cats with recognized ascites. 32 cats with ascites usually present with nonspecific clinical signs, such as anorexia or lethargy. the owners may present the cat because they recognize abdominal enlargement ( figure 23-67 ), but in many cases, owners perceive this as weight gain. clinicians should be aware that sudden weight gain in a chronically underweight cat may be because of fluid accumulation (which can be intrathoracic fluid if ascites is not present), particularly if muscle mass seems reduced. ascitic cats presenting subsequent to trauma may have intraabdominal hemorrhage or urinary tract rupture. fever in a young ascitic cat will often suggest fip, and cats with fip may or may not be jaundiced. presence of jugular distention or even a jugular pulse can suggest right-sided heart failure. a palpable fluid thrill can help to distinguish ascites from other causes of abdominal enlargement, such as organomegaly, abdominal masses, bladder distention, abdominal wall weakness, obesity or, occasionally, accumulations of gas within the abdominal cavity 27 (table 23-26) . recognizing a fluid thrill involves gently tapping one side of the abdominal wall with the fingers of one hand while feeling for a sensation of fluid movement the peritoneum is the serous membrane lining the abdominal cavity, as well as covering the organs of the abdomen. it comprises a single layer of squamous mesothelial cells resting on a deeper layer of loose connective tissue. the layer of peritoneum that lines the inner surface of the abdomen is called parietal peritoneum; the abdominal organs are lined by visceral peritoneum. the total surface area of the peritoneum is one to one-and-ahalf times that of the total cutaneous area of the body. 5, 30 the peritoneal cavity contains a small amount of fluid (less than 1 ml/kg body weight) that reduces friction between the abdominal organs as they slide over each other. the fluid is a pure transudate and contains solutes in the same concentration as serum (box 23-7). this fluid is absorbed from the abdominal cavity predominantly through lymphatic vessels lying beneath the mesothelial basement membrane on the surface of the diaphragm. lymphatic drainage occurs predominantly to the sternal lymph nodes. 5, 30 ascites is the abnormal effusion and accumulation of fluid in the abdominal cavity. fluid exchange across the capillary bed is determined by starling forces, that is, the balance between hydrostatic pressure, which causes transudation of fluid out of blood vessels, and the colloid osmotic pressure, which acts to retain fluid within blood vessels. the amount of peritoneal fluid is therefore determined by the balance of these, as well as vascular permeability, with excess fluid drained by the lymphatic system. accumulation of fluid within a body cavity results when the rate of filtration of fluid into a space is greater than the rate of fluid routine laboratory findings are usually nonspecific but may provide clues to the underlying cause of ascites. for example, neutrophilia may point towards septic peritonitis but can also occur with fip; most cats with hemoperitoneum are anemic at presentation 8 ; uroperitoneum often results in azotemia and electrolyte abnormalities; hypoglycemia may reflect sepsis with septic peritonitis, and a recent study recognized 83% of cases of septic peritonitis had ionized hypocalcemia 17 ; elevated liver enzymes may be associated with inflammatory, infectious or neoplastic hepatopathies including fip; elevation of serum globulins occurs in many cats with fip but can also be associated with neoplasia or septic peritonitis; and a finding of hypoalbuminemia (which can cause a pure transudate) should prompt for an assessment of urine protein : creatinine ratio to assess if there is renal protein loss. imaging may be required to confirm the presence of fluid as well as to aid in diagnosis of the underlying cause. radiographic findings can vary greatly depending on the amount of abdominal fluid present and the underlying etiology. loss of normal detail or presence of a "ground glass" appearance to the abdominal cavity is suggestive of the presence of fluid (figures 23-68 and 23-69). very young, thin or dehydrated cats may also have a loss of detail that can mimic the presence of fluid. ultrasonography of the abdomen (figures 23-70 and if a large volume effusion causes discomfort because of abdominal distention, a three-way stopcock may be used so large volumes can be drained from one puncture (figure 23-72) . however, removal of large volumes of ascitic fluid can be detrimental, because it may prevent the subsequent reabsorption of valuable protein and/or red blood cells; the resulting reduction in intraabdominal pressure may encourage further accumulation of fluid; and rapid removal of large volumes can lead to fluid shifts causing cardiovascular collapse. 32 fluid can be collected into ethylenediaminetetraacetic acid (edta) tubes (for total nucleated cell count, packed cell volume, total protein, and cytology), serum tubes (for biochemistry, such as albumin, bilirubin, creatinine, potassium, triglyceride, glucose, lactate, and lipase), sterile tubes for culture, and/or other tubes for effusion-specific tests such as pcr. samples should be prioritized according to the volume of fluid available and to the suspected underlying disease process. 10 initial assessment of fluid retrieved is made on the basis of color and protein concentration, and much information can be gleaned from this simple assessment, even before cell numbers and types are assessed. although this brief, initial assessment is useful to refine the differential diagnoses, a thorough assessment based on underlying etiology and pathophysiology is required for definitive diagnosis and therefore appropriate management (table 23-27) . ascitic fluid, classified according to its pathophysiologic cause, can be divided into transudates, modified transudates, exudates (septic or nonseptic), or effusions (chylous or hemorrhagic). 10,23 can allow the detection of even very small volumes of fluid. it also enables evaluation of the size and structure of intraabdominal organs, such as the liver and spleen, which can help determine the underlying cause of ascites. abdominocentesis confirms the presence of abdominal fluid (in cases of low-volume effusion) and assessment of the fluid is required to diagnose the underlying cause of ascites. most cats tolerate abdominocentesis without sedation and the cat can be held in a standing position or in lateral recumbency (whichever is more comfortable for the cat and familiar to the clinician). the abdomen is clipped and aseptically prepared. a 20-to 22-gauge butterfly needle may be used with a 5-to 10-ml syringe. in cases with low-volume effusion, ultrasonography can help to guide fine needle aspiration from small pockets of abdominal fluid. diagnostic peritoneal lavage can be used if ultrasound-guided aspiration is unsuccessful. for this procedure, 10 to 20 ml/kg of warmed, sterile fluid is infused into the abdomen over 2 to 5 minutes after aseptic preparation of the site. the cat is gently rolled from side to side or allowed to stand; gentle massage of the abdomen also helps distribute the fluid. the fluid is allowed to dwell for a minimum of 2 to 5 minutes before aseptic preparation is repeated before paracentesis. no attempt is made to remove all the fluid. it must be remembered that, since the recovered fluid has been diluted by this procedure, cell counts and biochemical analyses will be affected. 33 transudates are a consequence of altered fluid dynamics. protein-poor transudates (commonly referred to as pure transudates) form predominantly as a result of severe hypoalbuminemia, which causes a lowered colloid osmotic pressure. since there is no change in endothelial or mesothelial permeability, as fluid accumulates, there is no concurrent cell leakage; so, there is a decrease in the cell count through a dilutional effect. consequently, transudative effusions are typically clear and colorless. 10, 23, 32 other pathologic causes of proteinpoor transudates include cirrhosis, lymphatic obstruction, and noncirrhotic portal hypertension (presinusoidal and sinusoidal). since hypoalbuminemia is the most common cause of transudates, serum albumin concentrations must be measured to guide further diagnostics. if the serum albumin concentration is normal (or only minimally decreased), then radiographs, abdominal ultrasonography, and/or echocardiography are indicated to assess cardiac function and for urinary bladder rupture. 10 one review of feline ascitic cases found 24% of effusions were protein-poor transudates, of which 82% were the result of hepatic failure or primary renal disease. 34 modified transudates can result from increased hydrostatic pressure within the postsinusoidal vessels of the liver secondary to right-sided congestive heart failure (e.g., tricuspid insufficiency) or potentially from mass lesions (such as neoplastic masses) obstructing blood flow from the hepatic vein or caudal vena cava into the right side of the heart. the increase in hydrostatic pressure within the vessels of the liver causes a protein-rich fluid to leach out of the liver into the abdominal cavity. since cell membrane permeability does not change, cells do not accumulate in the effusion. 10 modified transudates can also result from increased vascular permeability in the early stages of an inflammatory process, in which case cellularity will be increased. modified transudates were described as the most common type of ascitic effusion identified in cats in one study, with most being resulting from neoplasia and congestive cardiac failure; however, this study partially included cases prior to 1987, when right-sided heart failure associated with dilated cardiomyopathy (dcm) was prevalent. 34 the recognition of the role of taurine deficiency in this condition and the subsequent addition of this amino acid to feline diets now means that right-sided heart failure is only rarely encountered as a cause of ascites in cats. exudates are a consequence of altered mesothelial and/ or endothelial permeability. this permeability results from a cytokine-mediated inflammatory response of any underlying cause (e.g., infectious, neoplastic, immune mediated). exudates have high protein and moderate to high cell concentrations and are classified as nonseptic or septic. exudates are often primarily composed of neutrophils. nondegenerate neutrophils (and the absence of organisms) points to a nonseptic exudate (mostly fip but also neoplasia). fip is the most common cause of exudative effusion in cats and was the most common cause of feline ascites diagnosed over a 10-year period at the feline centre at the university of bristol. 32 the presence of neoplastic cells rules in neoplasia, but the absence of such cells does not rule out this diagnosis since many cases of neoplastic ascites are not associated with exfoliated neoplastic cells. other causes of nonseptic exudates include pancreatitis, lymphocytic cholangitis, and viscus rupture, such as the gall bladder or urinary bladder. degenerate neutrophils typify septic exudates (i.e., septic peritonitis), and their presence should instigate investigation for causes of infection (mostly leakage of gastrointestinal contents). 34 chylous effusions appear as milky or pink opaque fluid, and small mature lymphocytes initially predominate in cell counts. after drainage, more macrophages and nondegenerate neutrophils may be found. chyle is typically classified as an exudate, but its physical characteristics can be consistent with a modified transudate (protein content between 25 and 40 g/l); biochemical analysis of triglyceride and cholesterol levels in the fluid are required to confirm the diagnosis. pseudochylous effusions resemble true chyle both in appearance and cytology but do not contain fat. similar conditions result in both chylous and pseudochylous effusions. chylous abdominal effusions are rarely reported in the cat and only accounted for 7% of cases of ascites in one study. 34 the described causes of chylous ascites in cats are predominantly neoplastic. in a series of nine cats, chylous ascites was associated with nonresectable abdominal neoplasia in four cases (i.e., hemangiosarcoma and paraganglioma), with intestinal and mesenteric lymphoma in two cases and lymphangiosarcoma of the abdominal wall in another. 13 one described case in a 10-year-old cat was thought to be because of fip. 28 figure 23 -71 shows an ultrasonographic image of a cat with chylous abdominal effusion associated with pancreatitis. other potential causes include right-sided congestive cardiac failure, steatitis (inflammation of fat), biliary cirrhosis, and lymphangiectasia. hemoperitoneum in companion animals is categorized as traumatic or spontaneous. traumatic hemoperitoneum is further divided into blunt causes of trauma (i.e., motor vehicle accidents and high-rise falls) and penetrating trauma (i.e., gunshot wounds and bite wounds). 8, 21 inadvertent splenic aspiration, venipuncture, or acute severe hemorrhage should be suspected if the cytology is consistent with peripheral blood including platelets but without erythrophagocytosis or if the blood clots readily. when there is no history of trauma, coagulopathy or spontaneous rupture of a vascular neoplasm should be considered. in one study of 16 feline cases of spontaneous hemoperitoneum, 12 cases (75%) were associated with hepatic pathology such as neoplasia, necrosis, and amyloidosis. 21 in another study of 65 cases of spontaneous hemoperitoneum, 46% (30 of 65) of cats had abdominal neoplasia, and 54% (35 of 65) had non-neoplastic conditions. cats with neoplasia were significantly older and had significantly lower packed cell volumes (pcvs) than cats with non-neoplastic disease. hemangiosarcoma was the most often diagnosed neoplasm (18 of 30, 60%), and the spleen was the most common location for neoplasia (11 of 30, 37%). coagulopathies (8 of 35, 23%) and hepatic necrosis (8 of 35, 23%) were the most common causes of non-neoplastic hemoperitoneum. 8 other nonneoplastic causes of hemoperitoneum include ruptured bladder, hepatic rupture secondary to hepatic amyloidosis, gastric/duodenal ulcer, hepatic hematoma, hepatitis, perinephric pseudocyst, feline infectious peritonitisinduced liver rupture, and feline infectious peritonitisinduced nephritis. 8, 21 the prognosis of cats with spontaneous hemoperitoneum is poor. in two studies, only approximately 12% of cases survived to be discharged from hospital. 8, 21 median survival time for cats that were discharged in one of those studies was 54 days (range, 5 to 1825 days). 8 feline infectious peritonitis (fip) comprised 50% of cats with recognized ascites over a 10-year period at the feline centre at the university of bristol, 32 and, as a rule of thumb, when ascites is recognized in a younger cat, fip should be considered the major rule-out. the abdominal effusion found with fip is typically straw to golden yellow (although the color can be very variable, for example, chyle may be present), may contain fibrin clots, and has a high protein concentration. the total protein content is greater than 35 g/l and often greater than 45 g/l, with globulins comprising 50% or more. 31 one study described an effusion with total protein greater than 80 g/l as 90% specific, 55% sensitive, and having a 0.78 positive predictive value to diagnose fip. 31 the rivalta test evaluates the fluid's globulin content, and was found to be very sensitive but only 80% specific; this test is performed by adding one drop of acetic acid (98%) to 5 ml of distilled water. this fluid is mixed thoroughly, and then one drop of effusion is gently placed on the surface of the mixture. if the drop stays at the top of the fluid or slowly floats to the bottom, the test is considered to be positive. this test can give inaccurate results if inappropriate technique is used or if there is a significant temperature difference between the fluid sample and the acetic acid solution. a positive rivalta test can result from lymphosarcoma, septic, or fip effusions; these can be distinguished by cytology and culture. immunofluorescence staining of coronavirus antigen in macrophages had a positive predictive value of 1.00 but a negative predictive value of 0.57. 15 the potential clinical presentations, diagnosis, and management of fip are covered in detail in chapter 33. one study found neoplasia to be the most common cause of ascites in cats, 34 and neoplasia should be considered the major rule-out in older cats with ascites. the effusion from cats with ascites resulting from neoplasia may be a modified transudate, resulting from compression of hepatic veins or the caudal vena cava, or metastases to the peritoneum; hemorrhage from neoplasia can cause hemoperitoneum; chylous effusions may result from reduced lymphatic drainage or rupture of lymphatic vessels; and raised vascular permeability caused by neoplastic infiltration can result in an exudative effusion. carcinomas, mesotheliomas, and discrete (round) cell neoplasms (e.g., lymphoma, mast cell tumors, malignant histiocytosis) exfoliate cells into effusions more readily than sarcomas, and of these, lymphosarcoma is the most common malignancy of cats. cytology of ascitic fluid reveals neoplastic cells in less than a quarter of cases; so the absence of such cells does not rule out a diagnosis of neoplasia. in these circumstances, the diagnosis may be achieved by ultrasound-guided fine-needle aspiration of affected organs, or even biopsy samples obtained at laparotomy. the specific approaches will depend on the specific neoplasia diagnosed. exudates caused by septic inflammation usually result from bacterial contamination of the peritoneal cavity secondary to gastrointestinal tract leakage or penetrating wounds associated with trauma. gastrointestinal tract leakage may occur as a result of ulceration associated with neoplasia or inflammatory disease or as a result of penetration of a sharp object ingested (such as a toothpick), it can also occur subsequent to prior abdominal surgery. 7, 9, 17, 24 primary septic peritonitis in which no apparent cause can be identified has also been described in cats. 26 septic exudates are usually yellow to tan in color, with yellow particulate matter and are foul-smelling. microscopically, the fluid is characterized by the presence of degenerate neutrophils and bacteria. bacteria are often seen intracellularly within neutrophils. the condition is associated with high morbidity and mortality rates, with survival rates reported between 32% and 80%. 7, 9, 17, 24, 26 the history and clinical signs are often vague and nonspecific but can include abdominal pain, vomiting, lethargy/depression, and anorexia. abdominal pain is an inconsistent finding, being recognized in only 62% of cats in one study 7 and 43% in another. 24 some cats may have an inappropriately low heart rate. 7, 26 hematologic and serum biochemistry findings are also inconsistent; neutrophilia with a left shift may be present, as may neutropenia or a normal neutrophil count. similarly, cats may be hypoglycemic, hyperglycemic, or normoglycemic, and they may be hypoalbuminemic. 7, 24, 26 one study recognized ionized hypocalcemia in 89% of cats with septic peritonitis at the time of diagnosis, 17 and another suggested hyperlactatemia, when present, may be associated with a poorer prognosis. 24 radiographic findings are usually typical of ascites of any cause, but presence of pneumoperitoneum in a cat that has not undergone recent surgery may suggest the presence of gas-forming bacteria or rupture of an abdominal viscus and warrants immediate surgical intervention. ultrasonography does not directly aid the diagnosis of septic peritonitis. 7 exploratory laparotomy to determine and correct an underlying problem, such as full-thickness gastrointestinal perforation (often requiring partial resection) is required, as is copious abdominal lavage with sterile, warmed fluids ( figure 23-73 ). there are no statistically significant survival differences between postsurgical primary closure, open peritoneal drainage, or closed suction drainage postsurgical lavage; however, a trend toward a higher survival rate has been seen in cats treated with primary closure. 24, 26 treatment also involves antibiotics, initially parenterally, based on culture and sensitivity findings. consistent with intestinal contents, most bacteria recognized are gram-negative aerobes, such as e. coli or enterobacter spp., but mixed infections are usually found. 7, 24 anaerobes seem more common in cats with primary septic peritonitis, 26 which perhaps suggests these cases may result from healed over-bite wounds into the abdomen. amoxicillin/ clavulanate would be an appropriate empirical choice of figure 23-73 fulminant peritonitis associated with gastrointestinal perforation. in this case, the effusion volume was low but the high degree of serosal inflammation is evident. antibiotics while awaiting sensitivity results. there are no definitive guidelines for duration of antibiotic treatment; the author uses extended treatment courses of 4 to 6 weeks. supportive care with intravenous fluids to maintain fluid and electrolyte balances is also required perioperatively. bile peritonitis is infrequently reported in cats but has been recognized in association with gunshot 20 or motor vehicle 2 trauma, with biliary obstruction from gall stones 2,22 and subsequent to percutaneous ultrasoundguided cholecystocentesis in a cat with infectious cholangitis. 4 concurrent bacterial infection was recognized in each case; this increases severity of inflammation and worsens the prognosis, although full recovery was achieved in most reported cases. 2, 4, 20 bile peritonitis has the potential to result in small-volume effusions; so, if abdominocentesis does not yield a sample of effusion but bile peritonitis is high on the differential list, then diagnostic peritoneal lavage is appropriate. since repair of or removal of the gall bladder and abdominal lavage are required, exploratory laparotomy is an appropriate means to diagnose this condition. management should be considered as for septic peritonitis of other causes. trauma, including blunt abdominal trauma, urethral catheterization, and bladder expression, is the most common cause of uroperitoneum in cats. 1 it is also recognized as a complication of ureteral surgery. 18 the bladder is the most frequent site of urine leakage after blunt abdominal trauma, whereas the urethra is most commonly injured following catheterization. cats with ruptured bladders may still have a palpable bladder and the ability to urinate. common historical complaints are anuria (53.8%) and vomiting (50%). azotemia is a common finding, and hyperkalemia is seen in around 50% of cases. drainage of urine from the peritoneal cavity seems to improve patient stabilization. morbidity and mortality depended largely on the severity of associated injuries. 1 regardless of the site of injury or the cause of uroabdomen, the first goal of treatment is patient stabilization. isotonic replacement fluids are used for initial resuscitation. treatment of hypovolemic shock, if present, is the first order of fluid therapy. after fluid resuscitation, drainage of urine from the abdomen should be established. continuous passive drainage of the urine is necessary for stabilization and allows effective diuresis to occur. indwelling catheterization of the urinary bladder is recommended to keep the bladder decompressed and reduce urine flow into the abdominal cavity in patients with bladder and proximal urethral injury. if the urethra is traumatized and a catheter cannot be placed, prepubic tube cystostomy may be necessary to achieve temporary urinary diversion. the decision to treat the uroabdomen patient surgically or conservatively should be based on the location and severity of the underlying injury, the condition of the patient at presentation, and the patient's response to initial stabilization. 1, 12 congestive heart failure has become an uncommon cause of ascites in cats since the late 1980s/early 1990s, from which time dilated cardiomyopathy has been largely eradicated. 32, 34 ascites does still result from rightsided congestive heart failure in conditions such as tricuspid insufficiency, 6 arrhythmogenic right ventricular cardiomyopathy, 16 myocardial fibrofatty infiltration, 14 or restrictive cardiomyopathy. 29, 34 concurrent pleural effusion or pulmonary edema is often, but not necessarily, present with cardiac induced ascites. a heart murmur is not necessarily noted. noting a jugular pulse or thrill is helpful diagnostically, if present. the ascitic fluid is typically a modified transudate, but a chylous effusion is also possible. cardiac diseases are covered in chapter 20. in some cases, hepatic lipidosis has been reported to cause ascites, particularly in association with pancreatitis. these cats are often hypoalbuminemic, with the possibility of intravenous fluid therapy contributing to the ascites by raising hydrostatic pressure. 11 other liver diseases which can result in ascites include lymphocytic cholangitis, 19, 25 neutrophilic cholangitis, cirrhosis, 13 necrosis, neoplasia, and suppurative cholangiohepatitis. 34 portosystemic shunts in cats rarely result in ascites, compared with dogs. 3 hypoalbuminemia and hepatic failure result in transudates; portal hypertension and cirrhosis cause higher protein ascites because of raised capillary hydrostatic pressure causing leakage of high protein lymph. hepatopathies are covered in detail elsewhere in this chapter. gold references 1. adamama-moraitou kk, rallis ts, prassinos nn et al: benign esophageal stricture in the dog and cat: a retrospective study of 20 cases chronic oesophageal foreign body in a cat use of a biodegradable self-expanding stent in the management of a benign oesophageal stricture in a cat suspected clindamycinassociated oesophageal injury in cats: five cases feline gastrointestinal foreign bodies a comparative study 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with feline infectious peritonitis restrictive cardiomyopathy in a cat with hypereosinophilic syndrome kirk's current veterinary therapy xii: small animal practice feline infectious peritonitis: a review of clinicopathological changes in 65 cases, and a critical assessment of their diagnostic value differential diagnosis of ascites in cats abdominal paracentesis and diagnostic peritoneal lavage peritoneal effusion in cats: 65 cases (1981-1997) *when present for any length of time, a pure transudate will become modified. this is particularly true of transudates that develop slowly, such as those associated with congestive heart failure or portal hypertension. modified transudates are therefore more common than pure transudates. adapted from tasker s, gunn-moore d: differential diagnosis of ascites in cats, in practice 22:472, 2000. 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 exploited for specific diagnosis and monitoring of treatment in canine leishmaniosis. high levels of l. infantum-specific antibodies are commonly observed in dogs with moderate to severe disease. controversial results have been described regarding the use of kinetics of l. infantum specific antibodies during treatment monitoring. the majority of the studies reported that antibodies often decreased slowly but remained detectable over a long period of time while older studies considered that serology was not useful for treatment monitoring. a consensus statement is that measurement of antibody levels is meaningless before 6 months of treatment. the aim of this study was to evaluate l. infantumspecific antibodies at the time of diagnosis and during treatment follow-up visits and to correlate these with the dog's clinical status. nineteen dogs were diagnosed (day 0) and followed-up during treatment (days 30, 180 and 365). the treatment protocol was a combination of meglumine antimoniate (50 mg/kg/12 h sc for 1 month) and allopurinol (10 mg/kg/12 h po for 1 year). physical examination and baseline laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis and urinary protein/creatinine ratio) were performed during all visits. leishmania infantumspecific antibodies were assessed by an end point sera dilution elisa method. the majority of dogs (n = 16) were classified as leishvet stage ii (moderate disease) at the time of diagnosis. three dogs were classified as leishvet stage iii (severe disease, n = 2) or iv (very severe disease, n = 1). results showed high and variable levels of specific antibodies at the time of diagnosis (meanaesd: 7855ae14821 elisa units (eu)). interestingly, a rapid significant reduction (p < 0.05) was observed at day 30 during treatment (mean aesd: 3503ae5980 eu). a continuing significant decrease of specific antibodies was also determined at days 180 (mean ae sd: 2139ae6829 eu) and 365 (mean aesd: 245ae108 eu). all dogs improved clinically with treatment with the exception of one dog that clinically relapsed at 6 months post-treatment and its specific antibodies level slightly increased. in conclusion, this study reports, for the first time, a rapid reduction of l. infantum specific antibodies after 30 days of treatment by an end point sera dilution elisa method associated with clinical improvement. it is important also to highlight that a marked decrease of antibody levels was also noted after 6 months and 1 year of treatment. disclosures: no disclosures to report. a broad range of clinical manifestations and immune responses have been described in canine leishmaniosis. canine l. infantum infection can manifest as a chronic subclinical infection, self-limiting disease, or non-self-limiting illness. a protective cd4+ t-cellmediated immune response characterized by production of inter-feron-gamma, il-2 and tnf-alpha is believed to be present in resistant subclinical dogs while this response seems to be diminished or absent in sick dogs. however, there are few and poorly standardized assays to evaluate this response in the dog. in addition, cellular mediated immunity has been mainly investigated in subclinical or vaccinated dogs but limited information is available in sick dogs with different degrees of disease severity. the aim of this study was to investigate l. infantum-specific cellular immunity in dogs with clinical leishmaniosis at the time of diagnosis. twenty-six dogs were diagnosed based on physical examination, routine laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis and urinary protein/creatinine ratio) and l. infantum-specific antibody levels measured by quantitative elisa. heparin whole blood was stimulated with l. infantum soluble antigen (lsa) and the mitogen concavalin a (cona) and incubated during 5 days. unstimulated whole blood from each dog was used as control. supernatants were collected and ifngamma concentration was measured with a commercial sandwich elisa. the majority of dogs (n = 16) were classified as leishvet stage ii (moderate disease). the rest of dogs were classified as stage i (n = 3), stage iii (n = 4) and stage iv (n = 2). twelve dogs (stage i, n = 2; stage iia: n = 9, stage iii: n = 1) produced ifngamma after stimulation with lsa (mean ae sd: 2137ae1526 pg/ ml) and cona (meanaesd: 6596 ae 4448 pg/ml). in contrast, 13 dogs (stage iia: n = 5; stage iib: n = 3; stage iii = n = 3; stage iv, n = 2) did not produce detectable ifn-gamma after stimulation with lsa but 12 dogs produced ifn-gamma after stimulation with cona (mean ae sd: 5665ae4923 pg/ml) while one dog was unresponsiveness to cona. no differences in ifn-gamma concentration were found between ifn-gamma producer and nonproducer dogs when blood was stimulated with cona (p = 0.6). interestingly, ifn-gamma producer sick dogs presented with a lower antibody levels (meanaesd: 1068ae1217 elisa units (eu)) when compared with ifn-gamma non-producer sick dogs (mean ae sd: 11448ae17602 eu) but differences were not statistically significant (p = 0.1). the results of this study suggest that sick dogs with a more exaggerated humoral response and a more severe disease lack l. infantum specific ifn-gamma production in stimulated blood. disclosures: no disclosures to report. canine anaplasma platys infection (capi) results in thrombocytopenia, but is considered as a subclinical disease in the united states where the disease was first identified. in europe, where the disease seems to be more severe, it has been suggested that circulating strains are more pathogenic, although co-infection with other vector-borne pathogens (vbp) may also contribute to the expression of clinical signs. however, the availability of pcrbased investigation of the impact of co-infection in naturally infected dogs is limited, compromising the assessment of their clinical significance under field conditions. the aim of the present study was to describe epidemiological and clinical features of capi under field conditions in areas endemic for several canine vbp. a study was conducted in veterinary clinics across italy, spain and portugal. sick animals were included when fitting at least 3 clinical and/or biological criteria compatible with ehrlichial disease. serological tests (snap ò 4dx, snap ò leish tests) and diagnostic pcr for ehrlichia canis, anaplasma platys, anaplasma phagocytophilum, babesia/theileria spp, hepatozoon canis and leishmania infantum detection were performed to identify the etiological agents. capi was considered on the basis of suggestive signs associated with positive pcr-based assay for anaplasma platys. among the 366 dogs included, 24 were pcr positive for a. platys. the annual incidence risk of capi was 0.02% and the probability to diagnose capi when facing 3 clinical and/or biological signs suggestive of ehrlichial disease was evaluated at 7.1%. nine dogs were mono-infected, and 11 dogs were co-infected with e.canis (3), l.infantum (2), babesia sp. (2) and h.canis (5). for 4 dogs, all tests were not performed. anorexia (58%) and weight loss (46%) were common reasons for visit. lymphadenomegaly (42%), hyperthermia and cutaneous signs (38%) were frequent findings whereas musculoskeletal disorders (25%), petechiae/ecchymosis (21%), splenomegaly (17%), dehydration and ocular lesions (8%) then epistaxis (4%) were less common. haematological abnormalities included thrombocytopenia and anaemia (81%), leucopoenia (25%) and leucocytosis (35%). a risk-analysis conducted between mono-and co-infected dogs didn't highlight significant differences except for anorexia that was significantly more frequent in mono-infected dogs. this study illustrates the magnitude of capi in the mediterranean basin and supports the existence of virulent strains in this area. co-infections were common but had a weak impact on clinical expression. these results emphasize also the importance of testing dogs for multiple vbp due to the difficulty in assigning a specific symptom or haematological abnormality to a specific vector-borne infection in endemic areas. disclosures: no disclosures to report. leptospirosis is a worldwide zoonotic disease with high mortality rate in humans and dogs. clinicopathologic changes may reflect renal disease, hepatic disease, or both causing often vomiting, polyuria/polydipsia and diarrhea. therefore, severe electrolytes and anion gap (ag) abnormalities could be expected. the aim of this cohort study was to investigate serum electrolytes and ag in dogs with natural occurring leptospirosis and to assess their prognostic values. the electronic data-base of the san marco veterinary clinic p.o.a system-plus 9.0 ò was searched between october-2004 and april-2015 for dogs with diagnosis of leptospirosis (group 1; n = 52). inclusion criteria for group 1 were consistent clinicopathologic signs and a positive microscopic agglutination test (titer≥1:1600 in vaccinated dogs, titer≥1:800 in nonvaccinated dogs or≥4-fold increase in convalescent titer) and/or a positive pcr (urine and/or blood) for leptospirosis. parameters studied were: serum electolytes (sodium, chloride, potassium), ag, and ag albumin-adjusted (ag alb-adjusted = ag + 2.5 x (3.2 -[alb] ).two control populations of randomly healthy dogs (group 2; n = 52) and sick dogs without leptospirosis (group 3; n = 52) dogs were created and matched to group 1 for age (ae6 months), sex (including sexual status) and breed. statistical differences between groups were evaluated by kruskal-wallis test and post-test analysis were performed by wilcoxon-mann-whitney. mortality relative risk (mrr) at 28 days post-admission between group 1 and group 3 was evaluated. roc curves were used to identify the best prognostic analyte. significance level for all statistical test was set at p < 0.05. serum sodium and chloride concentrations were significantly decreased in group 1 compared to group 2 and 3 (p < 0.0001 for both comparisons). serum potassium concentration was significantly decrease in group 1 compared to group 2 (p = 0.038), while no difference was present between group 1 and 3 (p = 0.466). serum ag and ag alb-adjusted were significantly increased in group 1 compared to group 2 and 3 (p < 0.0001 for all comparisons). there was a significantly increased in mortality rate in group 1 (n = 20, 38.5%) compared to group 3 (n = 5, 10.4%) (mrr = 4.0; 95% ci = 1.72-9.75). between the variables studied serum potassium (auc = 75%; p = 0.0002) and chloride (auc = 73%; p = 0.0028), ag (auc = 78%; p = 0.0002) and ag alb-adjusted (auc = 80%; p < 0.0001) were prognostic. serum electrolytes, ag, and ag alb-adjusted were significantly different in dogs with leptospirosis compared to healthy and sick dogs without leptospirosis with the exception of serum potassium that was similar between sick dogs with and without leptospirosis. between the variables studied the ag alb-adjusted resulted the best parameter in predicting death in dogs with leptospirosis. disclosures: no disclosures to report. enterococci causes urinary tract infection (uti) in companion animals and may carry important resistance genes such as for the bifunctional enzyme. furthermore, their virulence factors are seldomly reported in veterinary medicine. thus, this study aims to characterize the uropathogenic enterococci antimicrobial resistance, virulence genes and the clonallity of high-level gentamicin resistance (hlgr) enterococcus faecalis. antimicrobial susceptibility testing of 74 clinical uropathogenic enterococci isolated from dogs and cats with uti, isolated between 1999-2015, was performed by the disc diffusion method. clsi clinical breakpoints were applied. strains showing hlgr were screened for aac(6 0 )-ieaph(2 0 ')-ia and aph(2 0 ')-1d genes by pcr. e. faecalis harbouring hlgr genes were typed by multi-locus-sequencing. fifty-nine strains were further characterized by pcr for the presence of gel e (gelatinase), ace (collagen binding antigen), asa-1 (aggregation substance), and efa a (endocarditis) virulence genes. e. faecalis was the most frequently isolated (81.1%, n = 60/74) followed by enterococcus faecium (12.2%, n = 9/74). overall, antimicrobial susceptibility results were: 11.8% (n = 8/74) resistance to penicillin/ampicillin; 58.1% (n = 43/74) resistance to fluoroquinolones (enrofloxacin or ciprofloxacin); 10.0% (n = 7/70) resistance to nitrofurantoin; 11.4% (n = 8/70) resistance to chloramphenicol and 69.6% (n = 48/69) resistance to tetracycline. hlgr was detected in 14.1% (n = 9/64) enterococci, namely 7 e. faecalis and two e. faecium. all hlgr e. faecalis were aac (6 0 )-ieaph(2 0 ')-ia carriers. one e. faecium was positive for aac (6 0 )-ieaph(2 0 ')-ia whereas the other was positive for aph(2 0 ')-1d. interestingly, ampicillin-resistance was only detected in e. faecium (8 out of 9 isolates). furthermore, all hlgr e. faecium were also ampicillin-resistant. hlgr e. faecalis were found to belong to st16, st6, st35, and st59 major lineages circulating in both hospital and community settings in portugal. considering all enterococci, 64.4% (n = 38/59), 71.2% (n = 42/ 59), 32.2% (n = 19/59) and 78.0% (n = 46/59) were positive for gel e, ace, asa-1 and efa a virulence genes, respectively. uropathogenic e. faecium were only positive for ace gene (2 out of 8), thus e. faecalis had higher virulence genes frequencies. in this study we detected important human hlgr e. faecalis belonging to the clonal complex 2, such as e. faecalis st6, among uropathogens in companion animals. the presence of major clonal lineages in companion animals highlights their role as communityassociated hosts and possible reservoirs of putative human pathogenic enterococci. disclosures: conflicts of interest: c atia marques currently receives a phd grant funded by the portuguese foundation for science and technology. haemotropic mycoplasmas (haemoplasmas) can cause haemolytic anaemias in many species, including people. three feline haemoplasmas have been identified: mycoplasma haemofelis (mhf), 'candidatus mycoplasma haemominutum' (cmhm), 'candidatus mycoplasma turicensis' (cmt). mhf is considered the most pathogenic, whilst cmhm and cmt usually only cause anaemia in cats with concurrent disease or immunosuppression. the aim of this study was to estimate the prevalence of feline haemoplasmas in serbia and identify potential risk factors for infection. surplus edta blood samples from 375 cats in the belgrade region were used. for each cat, the following variables were recorded: age, health status, gender, outdoor access, presence of ectoparasites and haematological results. samples were stored at à20°c and transported to the university of bristol for haemoplasma quantitative pcr testing. serology (petchek, idexx) was performed for felv (n = 310) and fiv (n = 331) infection. statistical analysis was performed using spss; univariable associations between variables and haemoplasma status were first evaluated (v 2 for categorical variables, t-test/mann-whitney u test for continuous variables) followed by multivariable analysis. two samples were negative for internal control 28s rdna and excluded from the study. of the remaining 373 cats, 64 (17.2%) were infected with one or more haemoplasma species; 43 were singly infected (3 mhf, 31 cmhm, 9 cmt) , 16 dually infected (7 mhf/cmhm, 5 mhf/cmt, 4 cmhm/cmt) and 5 triple infected. the overall prevalences of mhf, cmhm and cmt were 5.3%, 12.6% and 6.2%, respectively. 4/310 (1.3%) cats were felv infected whilst 78/331 (23.6%) were fiv infected. multivariable analysis identified significant associations between haemoplasma infection and anaemia (anaemic/non-anaemic, odds ratio (or) 2.7, ci 1.04-7.1, p = 0.041), male gender (male/female, or 4.5, ci 2.22-9.03, p < 0.0005), outdoor access (yes/no, or 5.2, ci 2.28-11.92, p < 0.0005), non-pedigree breed (non-pedigree/pedigree, or 5.5, ci 1.24-24.84, p = 0.025) and fiv positive status (positive/ negative, or 2.4, ci 1.21-4.83, p = 0.012). the overall prevalence of feline haemoplasmas in serbia (17.2%) is similar to that reported in other european countries (12.6-43.4%). cmhm was the most prevalent haemoplasma species (12.6%) in the current study, similar to other european studies (range: 9.9-41.6%). most previous studies reported that cmt infection is the least prevalent feline haemoplasma species, but in the current study the prevalence of cmt was greater than that of mhf. similarly to previous studies, the presence of anaemia, male gender, outdoor access, non-pedigree status and fiv infection were significantly associated with haemoplasma infection. disclosures: no disclosures to report. canine parvovirus type 2 (cpv-2) is a frequent digestive pathogen in dogs, responsible for high mortality rates in puppies. the control of the infection by disinfection and isolation of patients is of limited efficiency, raising questions about the contagion sources. the aim of our study was to evaluate the epidemiological role of dams in viral circulation during the reproductive period. a total of 73 bitches (mean ae standard deviation: 4.4ae1.9 years old) from one kennel were enrolled in the study. all were annually vaccinated (nobivac dhppi-lepto vaccine; msd, beaucouz e, france). 41 dams were followed from mating to whelping and 32 dams were followed from whelping until weaning. all puppies from the 32 lactating dams (n = 134) were followed since 3 until 8 weeks of age. cpv-2 fecal excretion was evaluated by real time pcr on rectal swabs [1] every 14 days during gestation (dams) and every 7 days during lactation (dams and puppies). data were analyzed through logistic regression and mixed linear models. a total of 1241 samples were collected. during pregnancy, 80% of the bitches excreted cpv2 at least once, but only one sample was above the quantification threshold (2.10 5 copies/g feces). dur-ing lactation, all bitches were found positive at least once (and 3 times in mean) and 64% went above the quantification threshold at least once. during lactation, excreted viral loads were significantly higher at d42 (5.10 8 /g feces; p = 0.001), d49 (8.10 8 /g feces; p < 0.001) and d56 (10 9 /g feces; p < 0.001) compared to the early lactation (<10 6 copies/g feces; d7 to d28). despite threshold for a clinical parvovirosis is 5.10 8 /g feces, none of the bitches expressed any symptom. in 28% of the cases, the dam excreted before her puppies. viral loads excreted by puppies were not correlated with those excreted by dams. the proportion of puppies excreting viral loads above the clinical threshold increased from d17 to d52 (from 2 to 76% per litter), with overall mortality of only 3% (4/ 134). this study demonstrates that appropriately vaccinated adult female dogs may excrete cpv2 during gestation and lactation. due to the high quantity of cpv-2 excreted, females probably represent a major source of contamination for their puppies. viral excretion by bitches after lactation until the next breeding period and by males would be interesting to follow to better understand the role of adults in cpv2 circulation. [ feline panleukopenia virus (fpv) is responsible for one of the most severe infectious diseases in cats, but only few studies have addressed factors of prognostic importance. in an earlier investigation spanning over 15 years, leukopenia, thrombocytopenia, hypoalbuminemia and hypokalemia were found associated with poor outcome. here, we aimed at identifying outcome predictors during shelter outbreaks of panleukopenia between 2011 and 2013; we limited our analysis to fresh cases treated and followed until recovery or death at the same institution. clinical records of the affected cats were reviewed and information was collected at diagnosis and during hospitalization. the data included anamnestic history, physical examination, complete blood count, biochemical profile, blood gas analysis and treatments, including types of antibiotic, antiviral, antiemetic, analgesic, crystalloid, colloid and hemoderivative administered. outcome predictors were analyzed using logistic regression and mixed-design analysis of variance. the study included 177 cats diagnosed with panleukopenia based on clinical findings and positive fecal elisa, of which 75.4% were <12 months old, and 52.5% females. clinical signs at diagnosis included lethargy (38.0%), vomiting (36.2%) and diarrhea (33.8%). at admission, median (range) leukocyte counts were 1,200/ll (0-32,000) and platelets 119,000/ll (0-949,000); 30.2% had hypoalbuminemia and 8.1% hypokalemia. treatments included administrations of amoxicillin-clavulanate (21.5%), interferon-x (39.6%) and intravenous glucose solution (50%). overall, 79.1% of the cats did not survive. lethargic cats were more likely to die (or: 6.05, ci 95%: 1.73-21.11, p < 0.01). leukocyte counts at diagnosis were not associated with outcome, but were after 3 days of hospitalization; in particular, cats alive at 3 days, which succumbed later, had leukocyte counts of 900/ll (100-20,300) whereas survivors had 7,500/ll (200-32,300) (p < 0.01). survivors were more likely to have received amoxicillin-clavulanate (or:3.26, ci95%:1.01-10.60, p < 0.05) and less likely intravenous glucose solutions (or:0.09, ci95%:0.03-0.32, p < 0.01). thrombocytopenia, hypoalbuminemia, hypokalemia and administration of interferon-x were not associated with the outcome. our results suggest that infected cats with lower leukocyte counts later during hospitalization are more likely to die despite treatment. in this study, and different from previous data, lower leukocyte counts at admission did not predict outcome, possibly due to inclusion of cats with early fpv diagnosis. administration of intravenous glucose was associated with poor outcome, perhaps because of an increased risk of sepsis; also, cats in critical conditions were more likely to receive intravenous glucose. the beneficial role of amoxicillin-clavulanate in sick cats might be due to its broad-spectrum bactericidal activity; interferon-x did not show any conspicuous effect. disclosures: no disclosures to report. according to prior studies up to 30% of cats in southern germany do not have protective antibodies against feline panleukopenia virus and thus, are likely susceptible for feline panleukopenia infection. until now, it is unknown how healthy adult cats with different antibody titers react to feline panleukopenia vaccination in the field. therefore, the aim of the study was to measure antibody titers in healthy adult cats within 28 days after feline panleukopenia vaccination. one hundred and twelve healthy adult cats were vaccinated with a rcp vaccine. before vaccination (day 0) and on days 7 and 28 antibodies against panleukopenia virus were determined by hemagglutination inhibition. in 21.3% (19/112) of the cats, no antibodies prior to vaccination were detected; 4 of these cats were vaccinated regularly. nearly one third of the cats (31.4%; 28/112) showed no antibody increase after vaccination and in 17.9% (16/112) of the cats, antibody titer decreased despite vaccination within the 28 days. however, all of these cats were likely protected by their preexisting antibody titer. in 5 cats no antibodies were detected neither prior to nor after vaccination. a large number of adult cats has no protective antibodies and is therefore at risk for feline panleukopenia virus infection. on the other hand, many other cats show high antibody titers and do not develop antibodies due to vaccination. therefore, evaluation of individual antibody status in cats and vaccination only in those cats, that have no antibodies or low titers, should be recommended. disclosures: independent study financed by merial. there was no influence on the results of the study by merial and there is no co-authorship planned with merial. the feline coronaviruses (fcov) occur as 2 pathotypes with an enigmatic, even controversial, relationship: the low virulence or nonvirulent feline enteric coronavirus (fecv) and the highly lethal feline infectious peritonitis virus (fipv). recently, sequence differences within the spike gene region encoding the putative fusion peptide were described and proposed to correlate with the mutated form of fecv (i.e. fipv) leading to the clinical presentation of feline infectious peritonitis (fip). in this presentation, the development and validation of an allelic discrimination real-time pcr typing test which can identify each mutation separately will be described. the diagnostic sensitivity and specificity will be reported from a set of 203 european clinical samples acquired from either fip confirmed cats or from healthy cats that previously tested fcov positive. of these archived samples, 187 fcov positive samples were included into the validation. from these, 13 samples did not pass quality control and 17 had virus levels that were below the limit of detection of the pcr assay. of the remaining 157 samples, 156 were typed correctly with an accuracy of 99.4%. one fip characterized sample was typed fecv (diagnostic sensitivity 98.6%) while all of the healthy cats were typed fecv (100% diagnostic specificity). to confirm that these spike gene mutations are not unique to european cats with fip, additional validation studies from us and japanese samples were conducted. the us clinical study included 68 cases, 46 from fip suspicious cases and 22 with non-fip compatible disease. the fipv realpcr biotyping assay was able to accurately differentiate between the fip or non-fip (fecv) etiologies (p < 0.0001) and did not biotype cats with confirmed non-fip disease as fipv, confirming the high diagnostic specificity of the molecular test. disclosures the aim of this study was to compare the diagnostic accuracy for fip of conventional clinico-pathological tests (routine hematology, serum protein electrophoresis, a 1 -acid glycoprotein -agpmeasurement and analysis of the effusions) with that of molecular tests such as routine pcr and pcr followed by the sequencing of the spike (s) gene. blood, effusion and tissues specimens were collected from 21 cats with symptoms imputable to fip. the in vivo examination consisted of clinico-pathological tests such as complete blood count, serum protein electrophoresis, agp measurement, cytological and biochemical examination as well as the evaluation of the sysmex dtnc of effusions, when present, and of molecular tests such as a screening pcr (directed towards the 3 0 utr region) and the pcr directed towards the s gene followed by sequencing of the amplification products in order to detect the aminoacidic substitution considered diagnostic for fip. 1 the same molecular techniques were applied to the tissues samples collected during necropsy, which also allowed to divide the cats in a fip group (13 cats) and in a non fip group (5 cats) based on histology and immunohistochemistry. the diagnostic accuracy (sensitivity, specificity, negative and positive predictive values) of each test was calculated. the best test on tissues was immunohistochemistry (sens: 92.3%; spec: 100%), while the screening pcr suffered of low sensitivity and very low specificity (sens: 92.3%; spec: 33.3%). the s gene sequencing, positive when revealing the mutated nucleotide, showed very low sensitivity (sens: 69.2; spec: 100%). on effusions, the best tests resulted the screening pcr and cytology (sens and spec: 100%) in comparison with the dtnc measurement (sens: 85.7%; spec: 100%) and the s gene sequencing (sens:42.8%; spec: 100%). in blood samples, agp measurement demonstrated the best diagnostic accuracy (sens: 81.8%; spec: 100%), while serum protein electrophoresis showed a surprisingly low sensitivity (sens: 41.7%; spec:100%). screening pcr (sens: 55.6%; spec: 100%) and s gene sequencing (sens: 33.3%; spec: 100%) proved again low accuracy, demonstrating that a negative result with these molecular tests does not allow to exclude fip. 1. chang hw, egberink hf, halpin r, spiro dj, rottier pjm "spike protein fusion peptide and feline coronavirus virulence"emerg infect dis 18 (2012) diagnosis in feline infectious peritonitis (fip) is still challenging, especially in cats without body cavity effusion. uveitis in cats with fip commonly presents without effusion, which makes a definitive confirmation of fip difficult. the aim of this study was to evaluate the diagnostic utility of an immunocytochemical (icc) assay using aqueous humor in cats suspected of having fip. samples of 26 cats with immunohistochemically confirmed fip and 13 cats that were suspected of having fip due to similar clinical or laboratory changes, but that were definitively diagnosed with another disease were examined. aqueous humor was collected post-mortem after the cats were euthanized due to their diagnosed diseases. icc analysis was carried out using an anti-feline coronavirus mouse monoclonal igg2a and an avidin-biotin complex method. sensitivity, specificity, negative and positive predictive values were determined and 95% confidence intervals (95% ci) calculated. of the 39 aqueous humor samples, 18 (16 with fip, 2 controls) revealed positive icc results. false positive icc results were obtained in 2 cats suffering from lymphoma and pulmonary adenocarcinoma. diagnostic sensitivity of the icc assay in aqueous humor was 64.0% (95% ci 42.5-82.0); diagnostic specificity was 80.0% (95% ci 44.4-97.5); the negative predictive value was 47.1% (95% ci 23.0-72.2); the positive predictive value was 88.9% (95% ci 65. 3-98.6) . unfortunately, false positive results occurred, and specificity, which is considered the most important parameter in a fatal disease like fip, was only 80.0%. positive icc results in aqueous humor should be interpreted cautiously and cannot confirm a suspicion of fip. disclosures: no disclosures to report. alanine aminotransferase (alt) level in plasma is the most commonly used indicator for hepatocellular injury in dogs. however, dogs with advanced liver disease can present with alt levels within reference range. recent studies have shown the potential of circulating micrornas as a biomarker for liver injury. hepatocyte-derived microrna-122 (mir-122) was identified to be liver specific with superior sensitivity over alt levels in mice and humans. the aim of the present study was to investigate the potential for circulating mir-122 to serve as a diagnostic serum biomarker of hepatocellular injury in dogs. hereto, liver biopsies of 46 labrador retrievers were collected. liver histology, including grade of hepatitis and stage of fibrosis, was reviewed by a boardcertified veterinary pathologist (tsgamvdi). concurrently, serum samples were collected and analyzed for alt levels and mir-122 levels. dogs were included into one of the following groups: normal liver and normal alt levels (control group), liver pathology and normal alt levels, or liver pathology and high alt levels. comparative statistics between groups were performed using the mann-whitney u test and associations between mir-122 and alt levels, grade, stage and hepatic copper concentrations were analyzed using the spearman's rank correlation coefficient. logistic regression models were used to assess the accuracy of mir-122 and alt to detect the presence of hepatocellular injury. in total, 8 dogs had normal liver histology and normal alt levels. thirtyeight dogs had liver pathology whereof only 14 dogs had increased alt levels. in the high alt group the median level of mir-122 was 57 (range, 3 -794) times higher compared to the control group (p < 0.001). even in dogs with liver pathology and normal alt levels, the median mir-122 level was 3 (range, 0.4 -35) times higher compared to the control group (p < 0.05). univariate logistic regression showed that only mir-122 and not alt level was a significant predictor for abnormal liver histology (p < 0.05). serum levels of mir-122 were positively correlated with alt levels, histological grade and stage of fibrosis (r = 0.71, r = 0.62, r = 0.48, respectively, p < 0.001 for all). this study highlights the potential of mir-122 as an early and sensitive biomarker for liver injury in dogs and is more sensitive than alt levels. early diagnosis of hepatocellular injury opens the opportunity to institute treatment in a subclinical stage of disease with a possibly more favorable outcome. disclosures: this study was financially supported by the ecvim clinical studies fund. the authors declare no further conflict of interest. the evaluation of liver fibrosis is of major importance for the management of chronic liver disease and the prediction of prognosis. although liver biopsy is the gold standard for evaluation of fibrosis, non-invasive tests enable the clinician to stage and monitor a variety of liver diseases in human medicine. as such transforming growth factor b (tgf-b) and hyaluronic acid (ha) are biomarkers of hepatic fibrogenesis, that reflect the activity of the fibrogenic and fibrinolytic process, their use has not been validated in dogs. the aim of this study was to evaluate the measurement of tgfb and ha and assess their sensibility and specificity for the monitoring of 83 dogs with different level of hepatic fibrosis. eighty three adult dogs were prospectively enrolled based on a persistent elevation of alt, with the exclusion of those with focal hepatic lesions on ultrasound examination. all dogs underwent liver biopsy and serum blood collection. lf was staged according to histopathological wsava criteria and the amount of collagen was measured through morphometric analysis. quantitative variables were expressed as meanaesd. bean plots described the relationships between variables. bivariate analysis between ha, alt and pal with lf were performed by spearman correlations. a parametric test (student test) was carried out to assess the relationship between tgfb and lf. diagnostic cut-offs were determined according to the maximum youden index [sensitivity (se) + specifity (spe) -1]. preliminary results in 42 dogs showed that 92.9% of the individuals with ha below 48 ng/ml had a density of <3.5% collagen. the serum concentration of ha was significantly (p-value = 0.01) higher in the group whose fibrosis density was ≥3.5%. tgf-b was the most sensitive marker but its specificity to diagnose dogs with more than 3.5% of collagen was quite low. the preliminary results show a potential interest of ha as a biomarker to detect liver fibrosis in dogs but the interest of the tgfb could not be demonstrated. ha combines a good sensitivity with a fair specificity. the complete results of the study will help to refine the cut-off value and to improve the diagnostic performance of ah and to evaluate the interest of tgfb. a combination of several markers would be helpful to elaborate a sensitive and specific diagnostic test for liver fibrosis. disclosures: the speaker declares a potential conflict of interest with the company echosens . echosens covers a part of the biological measurements expenses in the clinical trial associated to this abstract. primary hepatitis is a common disorder in dogs. treatments for primary hepatitis are typically symptomatic and importantly, predicting prognosis at point of diagnosis remains challenging. in contrast to human medicine, where the type of hepatitis is defined by the inciting cause, few causes of chronic hepatitis have been identified in the dog, and the majority of cases are idiopathic. systemic inflammation is well recognised in humans with liver disease. systemic inflammatory response syndrome (sirs) is the clinical expression of the action of complex intrinsic mediators of the acute phase reaction. the presence of sirs has been linked to a poor outcome in various liver diseases. the prevalence and predictive value of a sirs in dogs with primary hepatitis has not been examined in dogs with liver disease. this is surprising given the accumulating evidence that sirs is linked to the development of hepatic encephalopathy (he) in dogs with liver disease. although the pathogenesis of he is not completely understood, it has been indicated that ammonia and inflammatory cytokines play a crucial role in the development of he. he is an important cause of morbidity and mortality in patients with liver disease. the hypothesis of this study was to examine the prevalence and severity of sirs n dogs with histological confirmed primary hepatopathies. eighty dogs with primary hepatopathies (confirmed with histopathology) were included in this study. a sirs score was calculated for each (respiration rate >24 breaths per minute; heart rate >120 beats per minute; total white blood cell count <6 or >16 9 10^9/l and rectal temperature <38.1 or <39.2°c). sirs scores presented as a value from 0 to 4. patient's date of arrival in hospital and date of death were all recorded; therefore survival time (days) could be determined. sirs scoring was applied and survival time was recorded. the median survival for sirs (0-1) was 237 days, while sirs (2-4) had a median survival of 7 days (p value <0.001, log rank test). this study demonstrates that sirs is a common feature of dogs with primary hepatitis and is valuable in predicting clinical outcome. disclosures: no disclosures to report. the aim of this study was to determine the prevalence of congenital portosystemic shunts (cpss) in deerhounds, focussing on the uk and the usa, and to determine how many deerhound breeders routinely test their puppies for cpss. congenital portosystemic shunts (cpss) are over-represented in certain breeds such as irish wolfhounds, maltese and yorkshire terriers. anecdotal evidence suggests that there is also an increased prevalence in deerhounds. this has not been confirmed, however. the study was questionnaire-based, distributed online to deerhound breeders worldwide (particularly the usa and uk). in addition it was distributed by post in the uk. the questionnaire passed ethical review at the department of veterinary medicine, university of cambridge. fifty-six breeders worldwide returned questionnaires (uk 27, usa 21, other countries 8). the uk response rate was 5.4% (including postal and online responses). the prevalence of shunts was found to be 0.8% of puppies with prevalences in the uk and the usa of 1.1% and 0.4%, respectively. worldwide, 71% of breeders were found to test routinely for cpss in their puppies, while the proportions in the uk and the usa were 96% and 48%, respectively. the prevalence of cpss in uk and usa deerhounds found in this study was higher than was found for mixed-breed dogs (0.05%) in a separate study. this suggests a genetic component to the disease in deerhounds. a lower proportion of breeders rou-tinely tested for cpss in the usa compared with the uk. the prevalence of cpss was also lower in the usa. these 2 findings may be related. it would be advisable for all breeders to routinely test their puppies for cpss before sale, and to avoid breeding from affected animals. disclosures: st catharine's college, cambridge, contributed to the cost of this study. the deerhound club (uk) and the scottish deerhound club of america gave their support, helping to distribute and advertise the study. measurement of neuroendocrine markers can offer diagnostic, prognostic, and therapeutic information that cannot be obtained by clinical examination. nt-probnp is a potential biomarker for hypertensive target organ damage (tod). circulating concentrations of this biomarker are increased in human hypertensive patients with tod and with poor response to antihypertensive treatment. cats with hypertension and tod have significantly higher nt-probnp concentrations than non-hypertensive cats. the aim of this study was to investigate the utility of nt-probnp as a biomarker of hypertension, tod and efficacy of antihypertensive treatment. plasma samples from hypertensive cats seen at 2 first opinion practices were retrospectively identified. hypertension was diagnosed based on systolic blood pressure (sbp) ≥160 mmhg with evidence of hypertensive retinopathy (tod-group; n = 24) or sbp≥170 mmhg on 2 consecutive visits 1-2 weeks apart without evidence of retinal pathology (notod-group; n = 25). all cats achieved sbp control (defined as <160 mmhg) on 0.625-1.25 mg amlodipine once daily and had samples available on both a hypertensive visit and the first visit target sbp was achieved. additionally, healthy cats ≥ 9 years old (n = 25) and normotensive cats diagnosed with ckd (plasma creatinine ≥177 lmol/l in conjunction with usg<1.035; n = 25) were identified. nt-probnp concentration was measured at an external laboratory. if necessary, variables were log-transformed to meet normality of distribution. binary logistic regression was used to investigate nt-probnp as a predictor of hypertension (using the healthy and ckd cats as comparator group) and tod (using notod as comparator group). comparisons between groups and of response to treatment were performed using mann-whitney u and wilcoxon rank sum tests respectively. higher nt-probnp concentration significantly increased the probability for a cat to be hypertensive (odds ratio log(nt-probnp) = 1.7, [95% confidence interval 1.1, 2.6], p < 0.01), but could not reliably predict tod (p = 0.092). nt-probnp concentration was however significantly higher in cats with tod than in cats with no tod (295.0 [123.8, 543.5] these data suggest that increased plasma nt-probnp concentration predicts hypertensive status in cats. cats without tod have significantly lower nt-probnp concentrations at diagnosis of hypertension than cats with tod. nt-probnp concentration decreases with effective antihypertensive treatment. further studies are required to determine whether nt-probnp remains elevated in cats with poorly controlled blood pressure. disclosures: esther bijsmans's phd is funded by zoetis. hypovitaminosis d has previously been shown to be prevalent amongst dogs with protein losing enteropathy (ple). outcome is generally poor in canine ple, and there is a lack of studies identifying underlying risk factors. the hypothesis of this study was that low vitamin d 3 serum concentrations could be a risk factor for bad outcome in such patients. medical records for dogs seen at the royal veterinary college between 2005 and 2014 were reviewed to identify dogs with a diagnosis of ple confirmed by histopathology. dogs were included in the study if they had serum samples frozen within 30 minutes after sampling, had been kept at -80 degrees c until analysis, and if clinical activity scoring (cce-cai) had been recorded at the time of diagnosis. forty-three dogs were included in the study. follow-up with referring veterinarians was made to determine outcome of patients. patients were divided into 2 groups: patients deceased due to ple (poor outcome group, n = 22) and patients alive or deceased due to another disease (good outcome group, n = 21). treatments for patients were allocated to 2 groups: one group consisted of patients who were prescribed diet only and the other group received diet and immunosuppressive agents. samples were sent on dry ice to michigan state university's diagnostic center for population and animal health. ionised calcium (ica) was measured using an ion specific electrode and 25(oh)d was measured using a commercially available radio-immunoassay that has been validated for use in veterinary medicine. comparisons of outcome groups for age, ccecai, treatment, serum 25(oh)d and ica were performed using a mann-whitney u test or chi 2 . logistic regression analysis was performed to determine possible risk factors for poor outcome. results: ccecai scores, age, and ica concentrations between the 2 groups were not significantly different. there was a significantly greater number of dogs treated with food alone in the group with good outcome (13/22) than in the poor outcome group (2/21, p = 0.001). furthermore, median serum 25(oh)d concentration was significantly lower in patients with poor outcomes (16.5 nmol/l, range 0-66 nmol/l) compared to patients with good outcomes (37 nmol/l, range 6-81 nmol/l, p = 0.017). using logistical regression, 25(oh)d serum concentration was a statistically significant factor for poor outcome (p = 0.03), with an increase of 25(oh)d serum concentration reducing the odds of having a poor outcome (odds ratio = 0.96, 95% ci: 0.93-0.997). further studies are required to investigate vitamin d as a potential adjuvant therapeutic agent in ple patients. disclosures: no disclosures to report. campylobacter jejuni (cj), c. upsaliensis (cu) and c. helveticus (ch) are commonly isolated from dog and cat faeces but association with clinical signs is discordant or lacking. cj is a recognized human pathogen, cu is considered an 'emerging' pathogen and ch is not considered pathogenic despite a high level of genetic similarity. recently, the greater wax moth, galleria mellonella, was described as an animal model of disease; these invertebrates have a high degree of functional and structural homology with the mammalian innate immune system. this study aimed to evaluate the pathogenic potential of cj, cu and ch using the galleria mellonella larvae model. twelve isolates of cj, 14 of cu and 11 of ch from dogs and cats were used for the inoculation of 2490 larvae. inocula were prepared by suspending isolates in phosphate-buffered saline (pbs) from which 3 100-fold dilutions were made. each dilution was tested in duplicate sets of 10 larvae. each larva was injected with 10-15 ll into the haemocoel via the last left pro-leg using 31g insulin syringes. controls consisted of 246 pbs inoculated larvae and 267 un-inoculated larvae. survival of larvae at 37°c in a h 2enriched microaerobic atmosphere was monitored for 8 days postinjection. one subset of isolates was grown in mueller-hinton broth and used for the preparation of secretory products, and another grown on blood-agar and suspended in pbs for heat inactivation of 10 minutes at 100°c for testing of whole-cell lysates and heat-stable insoluble and soluble components. the overall median survival of larvae was 80% with cj [iqr 10-100], 100% with cu [iqr 80-100], 100% with ch [iqr 60-100], 100% with pbs [iqr 92-100] and 100% for un-inoculated larvae [iqr 100-100]. a dose-dependent association was evident for each species with larval survival being similar between a low bacterial dose and pbs. larval survival presented a consistent pattern between species for medium and high bacterial loads; cj had a higher and faster larval death rate than cu and ch (p < 0.001), but no difference was observed between cu and ch (p = 0.06). there were no significant differences between species in any of the assays with secretory products, inactivated cells and soluble/insoluble cellular components. the observations within this invertebrate disease model support a varying pathogenic potential between the species studied that appears related to the (patho)biology of the species rather than their cellular components or metabolic products. the invertebrate animal model is promising in comparative pathogenicity studies. disclosures: no disclosures to report. acute stress from medium or high duration high-intensity exercise has been reported to be associated with an increase in serum c-reactive protein (crp) concentrations, an important acute-phase reactant in dogs. however, the effect of exercise on fecal s100a12 concentration, a biomarker of intestinal inflammation has not previously been evaluated in dogs. the goal of this study was to determine if moderate intensity short duration exercise causes an increase in crp and/or s100a12 concentrations in dogs, potentially leading to misinterpretation of their results. 37 adult military working dogs (german and belgian shepherd dogs; 36 males; mean age = 4 years [1.3-7.9]) were included in the study. fecal quality, fecal s100a12, and serum crp concentrations were evaluated just before and after standardized exercise (30 minutes of bikejoring at a speed of 16 km/h). fecal quality was evaluated based on a 5-point scale (from 1: liquid to 5: dry and hard feces). fecal s100a12 and crp concentrations were assayed with previously validated elisa tests. data were analyzed with an anova test for repeated measurements (sas software). results are presented as medians and ranges. serum crp concentrations increased significantly after exercise (median before and after excercise 5 mg/l [2-12] and 6 mg/l [5-12] (p = 0.002). also, fecal s100a12 concentrations were significantly higher after exercise compared with baseline concentrations (6 ng/g [2-437] versus 4 ng/g [1-389], p = 0.043). no significant effect of exercise on fecal score was observed (4 [2.5-4.5] before and after the exercise; p = 0.482). our study demonstrates that a moderate-intensity, short-duration effort performed by healthy army dogs causes significant increases in fecal s100a12 and serum crp concentrations, as compared with baseline values, but within the respective reference intervals. therefore, a moderate exercise does not present a confounding variable in the interpretation of fecal s100a12 or serum crp concentrations in healthy dogs. disclosures: this study was performed thanks the financial support of royal canin. imaging is an integral part of the work-up of canine gastrointestinal (gi) disease. radiography and ultrasonography are noninvasive modalities that can evaluate the bowel, but many findings lack desirable sensitivity or specificity. endoscopy directly visualizes gi mucosa, but is limited by the length of the endoscope and the need for general anesthesia, advanced training and expensive equipment. ambulatory light-based imaging (ali) is a new imaging modality that utilizes high-resolution cameras, a microprocessor, and led illumination to non-invasively visualize the gastrointestinal mucosa. ali is performed by oral administration of a fully automated device the size of a pill that is propelled by peristalsis. the aim of this study was to analyze image quality and gi transit times in a series of 5 client owned dogs undergoing ali. dogs were food-restricted for 24 hours before and 8 hours after capsule administration. capsules were retrieved and images were downloaded and analyzed. video clips of 300 frames duration were obtained from the stomach; proximal, middle and distal small intestine; and proximal colon for assessment of image quality. 3 internists rated the images on a scale of 1-10 (1 = poor, 10 = excellent) based on clarity and resolution of images, and obscuration of the mucosa by fluid, bubbles or debris. scores for each region were compared using general estimating equation analysis. gastric and small intestine transit time were calculated based on visualization of passage of the capsule from the stomach to duodenum, and ileum to colon. clinical analysis of the entire video was performed by one of the authors. ali was successfully performed in 5/5 patients, with no adverse effects. average study duration was 15.7ae4.1 hours and mean image acquisition count was 22,572ae17,315. gastric and small intestinal transit times were 79.2ae39.8 minutes and 119.4ae43.7 minutes, respectively. median (range) image quality scores were 9 (8-10), 8 (6-10) and 6 (5-9), for the stomach, si and colon, respectively. image quality scores were significantly higher in the stomach and si than in the colon (p < 0.001). visualized lesions were consistent with gi ulcers (2 dogs), inflammatory bowel disease (1 dog), and bilious vomiting syndrome (1 dog). one dog receiving chronic nsaids had a normal study. ambulatory light-based imaging resulted in good to excellent image quality throughout most of the gi tract. bowel preparation should be considered to enhance visualization of the colon. ali was safe and easy to perform in ambulatory dogs, and should therefore be considered in the work-up of canine gi disease. disclosures canine pancreatitis is the most common exocrine pancreatic disorder. the prognosis of canine pancreatitis is variably and no logistic regression constructed severity scoring systems are available. four hundred and thirty nine dogs diagnosed as pancreatitis with acute onset of compatible clinical signs, a positive snap ò cpl tm test, and/or associated abdominal ultrasonographic abnormalities between january 2009 and december 2012 were presented at national taiwan university veterinary hospital (ntuvh). one hundred and three dogs hospitalized with complete medical therapy and outcomes were selected for further analysis. the 103 dogs were divided into survival (n = 61) and non-survival (n = 42) groups. forty-seven parameters including signalment, clinical signs, physical examinations, clinicopathological examination, complications and concurrent diseases were analyzed and compared between the 2 groups. logistic regression analyses were performed in this study. variables with p ≤ 0.1 were considered for further analyses. the mortality in this study was 40.8%. age, heart rate, respiratory rate, white blood cell count, albumin, bun, creatinine, potassium, presence of systemic inflammatory response syndrome (sirs) and presence of oliguria or anuria 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 dog by summing all weighting factors. the total scores of each dog ranged from 10 to 70. the severity scores in this study achieved an area under the receiver operating characteristic (auroc) of 0.871. the optimal cut-off point for discriminating outcome was 24.5 with a sensitivity of 78.6% and specificity of 90.2%, respectively. the mortality was 84.6% with a score ≥ 25, whereas 14.1% with a score ≤ 24. there was a significant difference (p < 0.001) between the 2 groups seperated by the cut-off point. the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in dogs with pancreatitis. disclosures: no disclosures to report. glucocorticoids (gcs) are known for their anti-inflammatory and immunmodulatory properties and are therefore often used in the therapy of canine inflammatory bowel disease (ibd). it was recently shown that endogenous gcs are also produced in the intestinal epithelium of men and mice and influence the gastrointestinal immune system in case of inflammatory or neoplastic conditions. thus, the aim of this project was to prove that gcs can be produced or metabolized in the canine intestinal epithelium. five healthy beagle dogs were included into this prospective study. all dogs were clinically examined, given a clinical score using the canine ibd activity index (cibdai) scoring system, also gastrointestinal endoscopy was performed. mucosal biopsy specimens from duodenum were examined histologically from a board certified pathologist using the wsava grading. biopsy incubation of 8-10 endoscopical mucosal biopsies in tissue culture medium with 3 h-labeled progesterone in the absence of any stimulation was performed. the mean age of the included dogs was 3.24+1.9 years, the mean weight was 17.8+1.8 kg. all beagle dogs had a mean clinical score of 0+0. the mean wsava scoring was 2+1.2. after 4 hours, supernatant was harvested and radioactive progesterone metabolites formed were detected using high performance liquid chromatography plus liquid scintillation counting. in all dogs the 3 h-progesterone was metabolized into various steroid species, nevertheless a local production of cortisol could not be proven. in summary, it could be shown that precursors of gcs can be metabolized by healthy canine intestinal mucosal tissue. disclosures: no disclosures to report. we studied the relationship between pancreatitis and cardiac injury in dogs and cats. previously, we validated a cardiac troponin i (ctni; vet j 185:50-7, 2010) assay for sensitive and specific detection of cardiac injury in domestic animals. we found various non-cardiac diseases of dogs and cats were associated with cardiac injury detected by serum cardiac troponin i, including some cases of pancreatitis. also, we validated the dggr-lipase assay for cost-effective, sensitive and specific detection of pancreatitis in dogs and cats (vet clin path 41: e10-11, 2012; 42:e14-15, 2013 ). herein, we tested the hypothesis that pancreatitis was associated with cardiac injury. ctni was measured by advia centaur tni-ultra assay; dggr-lipase by the randox colourimetric assay. we retrospectively analysed data from dogs and cats admitted to ucd veterinary hospital in which both ctn and lipase had been measured. upper limit of reference range for lipase in dogs is 80 u/l; we consider 80-150 indicative of mild pancreatitis, 150-500 moderate, and >500 as marked. upper limit of reference range for lipase in cats is 25. reference range for ctni is < 0.054 ug/l for dogs and cats. we consider 0.054-0.15 indicative of mild cardiac injury, 0.15-1 as moderate, and >1.0 as marked. 145 dogs and 19 cats had both lipase and ctni measured. 78 dogs had normal troponin; 113 had normal lipase and 43 had normal lipase and normal ctni. 32 dogs (22%) had pancreatitis as indicated by increased lipase. in 18(56%), pancreatitis was mild, in 9(28%) it was moderate, and in 5(16%) it was marked. 67 of 145 dogs had increased ctni: mild in 33(49%), moderate in 22(33%), and marked in 12(18%). cardiac injury in dogs with pancreatitis was absent in 28%, mild in 34%, moderate in 25%, and marked in 13%. 13 of 19 cats had normal ctn; 10 had normal lipase. 6 of 19 cats had pancreatitis, severely in 3. lipase and ctni was correlated (r = 0.7) for dogs and cats. we conclude that both pancreatitis and cardiac injury, as indicated by high-sensitivity and high-specificity assays randox-dggr-lipase and centaur-ctni, respectively, are not uncommon in veterinary hospital cases. we confirm and extend our previous work. pancreatitis in dogs and cats is typically associated with cardiac injury. severities of pancreatitis and cardiac injury are correlated. for~40% of dogs and cats with pancreatitis, cardiac injury is moderate to marked. disclosures: no disclosures to report. intracellular colonization may serve as a protected niche where helicobacter spporganisms evade effective treatment, contributing to recolonization. confocal endomicroscopy (cem) is an endoscopic modality allowing in vivo gastrointestinal imaging at high resolution; and has aided real-time identification of helicobacter pylori and intracellular and mucosally associated bacterial. in dogs, non-helicobacter pylori-helicobacter (nhph) are described intracellularly. the objective of this study was to determine the utility of cem to identify nhph in dogs compared with other diagnostic modalities; and to assess its ability to identify intracellular organisms. fourteen clinically healthy dogs underwent standard gastroduodenoscopy followed by cem using topical acriflavine. images were obtained using cem at a minimum of 5 sites within the stomach. endoscopic pinch biopsies were obtained for histopathology, polymerase chain reaction (pcr) and fluorescence in situ hybridisation (fish). methodologies were compared for their sensitivity in detecting the presence and distribution of nhph and their ability to identify intracellular organisms. cem provided high quality images allowing in vivo identification ofnhph in 13 dogs, as did fish post-procedure analysis. standard histopathology identified nhph in only 11. nhph were identified within the superficial gastric mucus, and gastric pits. distribution throughout the stomach was diffuse and multi-focal. cem findings correlated with fish and pcr, however only fish enabled identification of intracellular nhph which were present in 13 of 14 dogs. cem provides in vivo histology images and is capable of identifying nhph during gastroscopy, but is unable to identify intracellular organisms using the current fluorophore protocol. nhph in the canine stomach are commonly identified intracellularly. disclosures: dr sharman has shares in optiscan imaging pty ltd. chronic enteropathies (ce) and exocrine pancreatic insufficiency (epi) can both cause hypocobalaminemia in cats. current supplementation protocols for cobalamin in cats call for repeated parenteral injections. in humans, several studies have reported equal efficacy of oral administration of cobalamin. there is also evidence that oral supplementation is effective in dogs with hypocobalaminemia. recently, it has also been reported that oral cobalamin substitution restores normocobalaminemia in healthy elderly cats. the purpose of this retrospective case series was to evaluate whether oral cobalamin supplementation can restore normocobalaminemia in hypocobalaminemic cats with chronic enteropathies. a computerized database search for cats treated at evidensia specialist animal hospital, helsingborg, sweden during 2012-2015 was performed. inclusion criteria were cats with symptoms of ce, an initial serum cobalamin concentration below 275 pmol/l (reference interval: 199-984 pmol/l) and daily oral treatment with cyanocobalamin (1 mg/tablet; ⅛-¼ tablet/cat daily). follow-up serum cobalamin concentration was measured 28 to 94 days after initiation of daily oral cobalamin supplementation. thirteen cats aged 2-14 years (median 8) of 4 different breeds met the inclusion criteria. presenting complaints included vomiting (7/13), anorexia (5/13), diarrhea (3/13), weight loss (2/13), and lethargy (2/13). increased pancreas specific lipase (spec fpl ò ) serum concentrations were reported in 3/11 cats and 4/13 had increased serum alanine transaminase activity. feline serum trypsin like immunoreactivity (ftli) was determined in 5/13 cats revealing results within the reference interval. all cats had an abdominal ultrasound, 9/13 had changes related to the gastrointestinal tract such as mild-moderate thickening of the small intestinal wall, thickening of the muscularis layer, poor definition of intestinal wall layers, and/or enlargement of the mesenterial lymph nodes, histopathology was performed in 6/13 cats, revealing small intestinal inflammation in 5 cats and small intestinal lymphoma in one. serum cobalamin increased in all cats with treatment. the concentration difference ranged from 517 to 1330 pmol/l (mean: 760 pmol/l). mean (ae standard deviation) serum cobalamin concentrations were 177 (ae49) pmol/l before and 931 (ae324) pmol/l after supplementation. this difference was statistically significant (p < 0.0001, paired t-test). our results suggest that oral cobalamin supplementation is effective in normalizing serum cobalamin concentrations in cats with various enteropathies. prospective studies are warranted comparing cellular cobalamin status in cats being treated with parenteral or oral cobalamin supplementation. disclosures: no disclosures to report. pulmonary thromboembolism (pte) is observed in dogs with idiopathic-inflammatory-bowel disease (ibd) and particularly with protein-losing enteropathy (ple). hypercoagulability has been attributed to antithrombin (at) loss although the pathogenesis is likely to be more complex. in humans, where venous thromboembolism (te) is a wellrecognised complication of crohn's disease and ulcerative colitis, the pathogenesis of te is still not completely understood. derangements in procoagulant and anticoagulant factors have been demonstrated, including increased circulating procoagulant microparticles (mps). the aim of this pilot study was to evaluate mp-procoagulant activity in the plasma of dogs with ibd and ple using a functional elisa assay (zymuphen-mp-activity, aniara). we hypothesised that all dogs with ple and a subset of dogs with ibd but without ple would have increased levels of circulating mps. the study group consisted of 11 dogs with ibd, including 4 with ple. diagnosis was based on compatible clinical and histopathology and exclusion of other causes of chronic gastrointestinal disease. ple was defined as ibd plus hypoproteinaemia (serum total protein <58 g/l) and hypoalbuminaemia (serum albumin <26 g/l). pte was diagnosed in one dog with ple, and suspected in a second. a control group comprised 8 healthy dogs undergoing blood sampling for reasons unrelated to the study including blood donor screening (n = 6) and health assessment (n = 2). dogs were considered healthy based on owner evaluation, physical examination, haematology and serum biochemistry median mp procoagulant activity in dogs with ibd was 10.38 nm (range 0.00-32.08) compared with 7.25 nm (range 0.00-70.73) in the control group. median mp activity in ple dogs was 23.16 nm (range 0.00-32.08) compared with 7.86 nm (range 2.9-21.32) in non-ple ibd dogs. using kruskal-wallis test for nonparametric data and dunn's multiple comparisons test the groups were not statistically different. interestingly, mp-procoagulant activity value in the dog with documented pte was 0.0 nm; in the dog with high clinical suspicion for pte, mp-procoagulant activity was 32.08 nm. the highest mp-procoagulant activity was detected in a healthy control dog, raising concerns for pre-analytical or sampling error. removing this measurement had no impact on statistical analysis, which remained nonsignificant. mp-procoagulant activity >10 nm is considered clinically relevant in humans. employing a similar cut-off, 2/8 of controls, 6/11 of ibd and 3/4 of ple group would be defined as having increased levels of circulating mps. further studies are required to fully evaluate the clinical relevance and diagnostic potential of mp evaluation. disclosures: no disclosures to report. the intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (ce) in dogs. while imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with ce involving the ileum and colon. the aim of the present study was to use fluorescence in situ hybridization (fish) techniques to investigate the composition and spatial organization of mucosal microbiota in endoscopic biopsies obtained from dogs with ce and controls. tissue sections from the ileum and colon from 19 dogs with inflammatory bowel disease (ibd), 6 dogs with granulomatous colitis (gc), 12 dogs with intestinal neoplasia, and 15 controls were studied by fish targeting the 16s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. the numbers of mucosal bacteria were analyzed using generalized linear models for each of the colon and ileum tissues, with spearman's rank correlation coefficients used to test the correlation between mucosal microbiota and inflammatory (cib-dai score, histopathology) indices. the ileal and colonic mucosa of healthy dogs and dogs with ce was predominantly colonized by bacteria localized to free and adherent mucus compartments. dogs with ce harbored more (p < 0.05) mucosal bacteria belonging to the clostridium-coccoides/eubacterium rectale group, bacteroides, enterobacteriaceae, and escherichia coli versus controls. within the ce group, ibd dogs had increased (p < 0.05) enterobacteriaceae and e. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. bacterial invasion with e. coli was present in the ileal and colonic mucosa of dogs with gc (p < 0.05). dogs with intestinal neoplasia had increased (p < 0.05) adherent (total bacteria, enterobacteriaceae, e. coli) and invasive (enterobacteriaceae, e. coli, and bacteroides) bacteria in biopsy specimens versus all other groups. increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity (cibdai score) in ibd dogs (p < 0.05). these results indicate that histopathologic lesions of canine ce are associated with different populations in ileal and colonic mucosal microbiota. these spatial, segment-specific structure and differential response of select bacterial groups to intestinal inflammation may be pivotal regarding the functional consequences of these alterations in the pathogenesis of canine ce. disclosures: no disclosures to report. abdominal girth is used as an indicator of human adiposity, with such measurements being made by tape measure. given concerns in precision and accuracy of repeat measurements, some tape measure designs have inbuilt mechanisms to improve consistency. although body condition scoring is the most common method of assessing adiposity in dogs, zoometric systems have also been developed requiring the use of a tape measure. however, the precision and accuracy of such zoometric measurements are not known. the aim of this study was to determine the precision and accuracy of 3 different types of tape measure for a variety of dimensional measurements. a variety of length (head, forelimb, hindlimb) and circumferential (neck, thorax, and abdomen) were made using 3 different tape measures, 2 of which were designed to improve precision (standard tape; myotape tm and gulick ii tm ). to assess intra-operator variability, 12 measurements were taken for 5 consecutive days from 4 healthy dogs; to assess inter-operator variability, 3 operators independently took 12 measurements from a group of 16 dogs of various breeds and sizes. for intra-operator comparisons, precision was good overall (coefficient of variation [cv] ≤3% for all measurements). for inter-operator comparisons, precision was more variable and, although reasonable on average (mean cv 2-5%), it varied depending upon tape measure type (p = 0.027; greatest for standard tape measure, least for gulich ii tm ), and could be highly variable for some measurements in individuals dogs (maximum cv 16% for head measurements with standard tape measure). significant differences also existed in the absolute results of circumferential measurements taken by the different tape measure types (neck p = 0.012; thorax p < 0.001; abdomen p < 0.001). finally, significant operator differences were also evident for some measurements (head p = 0.023; hindlimb p = 0.004), but not for others (forelimb p = 0.053; neck p = 0.102; thorax p = 0.073; abdomen p = 0.062). in summary, although precision for individual operators making zoometric measurements is good, significant inter-operator and tape type differences exist. these results have implications for systems using a range of zoometric measures to assess adiposity. in order to ensure precision and accuracy, it is recommended that the same operator take all measurements with the same type of tape. disclosures: the study conducted was not supported by a research grant. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. however, this method is subjective due to its sensory evaluation. therefore, the improvement of the precision of the bcs diagnosis is expected. our previous study has shown that the bcs model that we created improved the precision of the bcs diagnosis (1). however, a palpation site was not identified. a palpation site must be the site where thickness of subcutaneous fat is able to capture for measuring animal's obesity status. therefore the objective of this study was to find a remarkable body site of the changes with obesity status using ultrasonic diagnostic equipment. nine dogs which varied in the percent of body fat were used in this study. the percent of body fat was measured by a body fat analyzer for dog (kao). the image analysis of a palpation site was evaluated using echo, xario ssa-660a (toshiba) which attached to a linear probe. the measurement points were 1, 2 and 3 o'clock positions on the ribs of t6, t9 and t12. the distance (d) from skin surface to the rib was measured in the echogram. the distance (l) from scapula to ilium was measured to offset the difference in physique by dog breeds. the d/l was used to compare relative value of the quantity of fat at each measurement point. bcs of dogs which used in this study were from bcs of 2 to bcs of 4. there were no dogs in bcs of 1and bcs of 5. a statistically significant correlation was found between bcs and d/l value. the d/l value increased in order of t6, t9 and t12 in bcs of 3 and 4. this suggests that the thickness of subcutaneous fat in the chest is thicker at the head side than the tail side. also, as for the d/ l value from back to abdomen, the highest value was found at the position of 11:00 and 1:00. this tendency was the most remarkable in bcs of 4 but no difference in the d/l value was recognized in the dogs in bcs of 2. in conclusion, the position of 1:00 or 11:00 on the t6 is the suitable palpation point at the chest. ( body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. bcs has been recognized as one of the screen method of nutrition diagnosis by american animal hospital association in 2010. however, this method is subjective due to its sensory evaluation. therefore we made a bcs model to increase the precision of the bcs diagnosis and have shown the efficacy of the bcs model (1). however, the prototype model which we have reported before tended to have higher bcs than a target bcs. therefore, we improved the bcs model in this study. sixty seven dogs which varied in the bcs were used in this study. body fat percentage was measured by using a body fat analyzer for dogs (kao healthlab bif-10). the bcs model was improved by using several rubber sheets. relative hardness of stacking rubber sheets in each bcs was measured by durometer mj-dua-c2 (satotec tokyo, japan). bcs diagnosis of dogs was performed by pet owner by using the bcs model. bcs of 1 represents the most hard in the bcs model and the hardness decreased linearly and it was the lowest in 5 of bcs. these values were as expected. high correlation was recognized between bcs and body fat percentage. these results suggested the efficacy of bcs model. however, the body fat percentage in the dogs diagnosed as bcs of 1 was higher than body fat percentage which has been reported in the previous paper. there were no dogs with the body fat percentage <10% which were diagnosed as bcs of 1. we need more study in future to make clear the difference of body fat percentage between our data and date of the previous research. the completion of this bcs model will help provide the precision of nutritional diagnosis in dogs. ( in humans the metabolic syndrome (ms) is a well-recognised and extensively studied entity that comprises obesity, hypertension, dyslipidaemia, and glucose intolerance. it is associated with an increased risk of cardiovascular diseases and diabetes. recently, human ms criteria were adapted for dogs to define the condition of obesity-related metabolic dysfunction (ormd). it was observed that ormd was associated with increased circulating insulin and decreased adiponectin concentrations, suggesting that in dogs, as in humans, there are links between obesity, ormd, and associated diseases, although pathogenetic mechanisms and health significance for dogs remain unknown. the main aim of the present study was to compare plasma proteomes of obese dogs with and without ormd, so as to investigate the mechanisms associated with canine ormd and their possible significance in the health status. eight obese dogs referred for weight management at the royal canin weight management clinic, university of liverpool participated in the study. clinical assessments included physical examination, body condition scoring, blood pressure measurement and routine clinicopathological analysis. surplus plasma was used in proteomic analysis. samples were first treated with proteominer for thedepletion of high-abundance proteins and subsequently analysed by using2-de dige methodology. of the 8 dogs in the study, 4 dogs had ormd and 4 dogs did not. image analysis and further statistical analysis allowed identification of 8 spots with differential expression concentration between dogs with and without ormd. among the 8 spots, 3 were over-expressed and 5 were down-expressed in dogs with ormd than in dogs that did not presented ormd. although the results of the present study are preliminary and still the identification of the spots is up to be performed, the observed datareveal that dogs with ormd present alterations in their plasma proteomes that could be responsible for the development of ormd-related pathologies. disclosures: the study was funded by waltham. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. vb is an employee of royal canin and pjm is an employee of wal-tham. the aim of the study was to assess the diagnostic value and the discrimination potential between the normal heart size and microcardia or cardiomegaly of a method which calculates the cardiothoracic ratio (ctr) using area measurement, compared to the vertebral heart scale method (vhs) used as reference for the cardiac size, in dogs. one hundred-nine dog x-rays were accepted into study. the patients belonged to small and medium size breeds, 47 were males and 62 females with age between 1 and 17 years. the analogic xrays were scanned and transferred to a computer where the vhs and ctr was calculated for each patient with a commercial software and the data was collected and processed in a statystical analysis software. the patients were distributed into groups by respiratory phase and heart size. there was a low correlation between the vhs and ctr (r 2 = 0.650), but statistically significant (p?0.01). a good correlation was obtained between vhs and ctr in microcardia, normal heart size and cardiomegaly groups (p < 0.01). furthermore, between the ctr in dogs with microcardia and those with normal cardiac size, as well as between ctr in dogs with normal cardiac size and those with cardiomegaly, a significantly statistic difference (p < 0.05), respectively (p < 0.01), was obtained. among the groups distributed by respiratory phase and vhs, a statistically significant difference was obtained only between normal cardiac size and cardiomegaly during inspiratory phase groups (p > 0.01). for the x-rays taken in inspiratory phase, a cutoff of 31.31 had a sensitivity of 80% and a specificity of 75% for diagnosing cardiomegaly. the ctr can be considered a valid method being able to discriminate between the patients with microcardia and cardiomegaly from those with normal heart size. moreover, it was found that a ctr over the cutoff of 31.31, mesured during inspiratory phase is a good predictor for cardiomegaly. key words: cardiac, cardio-thoracic ratio, dog, x-ray. the canine cardiac conduction system is modified by anatomical and functional adaptations of the maternal heart during gestation. however, it is not clear if these changes persist or are modified after parturition. therefore, the aim of this study was to describe canine electrocardiographic features during the course of normal puerperium. twenty healthy pure-bred, 2-5 (3.85ae0.16) year-old, weighing 1.5-6 kg (3.55ae0.26) bitches were included in this study. all the animals whelped healthy puppies at term which were weaned on day 60 after parturition (day 0). all the dogs were electrochardiographically evaluated on days à3, 3, 10, 17, 24, 38, 52 and 80. mean electrical axis (mea; degrees), p wave amplitude (pa; mv) and duration (pd; ms), p-r interval (pr; ms), qrs complex amplitude (qrsa; mv) and duration (qrsd; ms), q-t interval (qt; ms), and s-t segment (st; mv) were calculated at 50 mm/s of velocity. the rr interval immediately preceding each complex was recorded and qt interval was corrected (qtc) by van de water formula [qtc = qt-0.087(rr-1000)]. later, lead ii was recorded at 25 mm/sec to analyze heart rate (hr; bpm) and cardiac rhythm (cr; normal sinus rhythm or sinus arrythmia). values of hr, mea, pa, pd, pr, qrsa, qrsd, qt, rr and qtc were analyzed by anova for repeated measures followed by tukey test. cardiac rhythm was analyzed by chi square test (spss 17.0, spss inc. chicago, il, usa). p < 0.05 was considered significant. during the study period, hr (p < 0.01) and qtc (p < 0.01) progressively decreased, while rr (p < 0.01) and pa increased (p < 0.01). qrs complex amplitude diminished in the second week after parturition and then increased during the following weeks (p < 0.01). mean electrical axis shifted to the right during this period (p < 0.01). on day à3, most of the bitches presented normal sinus rhythm in contrast with day 3, in which most of the bitches presented sinus arrhythmia (p < 0.01). from day 10 onward, all the bitches showed sinus arrhythmia. p wave duration, pr, qrsd, qt and st remained unchanged during puerperium. it is concluded that most electrophysiological adaptive changes of canine gestation reverted during normal puerperium. the pre-sent study contributes to the understanding of canine cardiac physiology during this reproductive stage. disclosures: no disclosures to report. esvc-p-3 echocardiographic assessment of pregnant queens. p.g. blanco, r. rodr ıguez, a. carranza, a. rube, r. vercellini, p.r. batista, m. t ortora, c. gobello. national university of la plata, la plata, argentina cardiovascular adaptation during gestation guarantees an appropriate development of the fetuses and maternal cardiovascular maladaptation is highly correlated with adverse pregnancy outcome. while, the hemodynamic changes occurring during canine pregnancy have been described there is scarce information concerning maternal cardiac variations during feline gestation. thus, the aim of this study was to describe cardiac morphology and systolic function variations during normal feline pregnancy. eighteen pregnant queens were echocardiographically evaluated (toshiba nemio xg, japan, 10 mhz transducer) every 10 days from day 0 (defined as day of mating) to parturition. left ventricular dimensions were measured in the short axis view, during mmode tracing. shortening fraction was calculated as (lvdd -lvds)/lvdd x 100 to assess systolic function. stroke volume (ml) was calculated as the product of the velocity time integral (measured by pulsed-wave doppler) and the cross-sectional area of the aorta. cardiac output (l/min) was calculated as the product of stroke volume and heart rate (bpm) derived from electrocardiographic monitoring. uterine artery resistance index (ri) was obtained by doppler ultrasound. all the parameters were analyzed by repeated measures anova. all the queens delivered healthy kittens at term. throughout the study period, interventricular septum in diastole (p < 0.01) and systole (p < 0.01) and left ventricular diameter in diastole (p < 0.01) augmented during gestation. shortening fraction (p < 0.01), cardiac output (p < 0.01) and maternal heart rate (p < 0.01) also increased up to parturition. conversely, uterine artery resistance index decreased in the same period (p < 0.01). it is concluded that cardiac structure and function varied during normal pregnancy in these queens. cardiac eccentric hypertrophy, systolic function and cardiac output increases appear to be the consequences of the hemodynamic modifications occurring during pregnancy. the assessment of maternal cardiovascular function may prove a useful screening tool to detect pregnancy complications in feline reproduction. disclosures: no disclosures to report. tricuspid annular plane systolic excursion (tapse) is an echocardiographic measure that allows to assess right ventricular systolic function. it has been described reference values for tapse in normal adult dogs, but there is no reference to influence of age in tapse in dogs. this influence has been reported in humans. thus, the goal of this study is to determine the reproducibility of the measure tapse in normal dogs and to determine the relationship between tapse and age in healthy beagle dogs. tapse was measured from an m-mode recording of the lateral aspect of the tricuspid valve annulus obtained through a left parasternal apical 4-chamber view. tapse values were averaged from measurements on 5 consecutive beats during sinus rhythm. the measurements were recorded by 2 different persons (c-v, a.; m-m f.) with different grade of experience in canine echocardiography studies.. all patients had a complete 2-dimensional and doppler study using an envisor chd (philips ò ) ultrasound system. twenty-three healthy beagles were used. the study was approved by the ethical committee of veterinary medicine service of las palmas de gran canaria university (spain) and it was carried out in accordance with the current european legislation on animal protection. these dogs were divided in 3 different groups according to age: group 1 included 12 dogs under 4 years, group 2 included 3 dogs between 4 and 10 years, group 3 included eight dogs older than 10 years. we analyzed differences between groups using non-parametric (kruskal wallis and wilcoxon scores (rank sums)) test. there were no differences with respect to sex. dogs in group 1 presented higher tapse values than group 2 or 3 (1.33ae0.26 cm versus 0.98ae0.14 cm versus 1.13ae0.18 cm; p < 0.05). statistic intra-observer and inter-observer agreement using the intraclass correlation coefficient was 0.99 (p < 0.05). this study showed that tapse measurement is easily obtainable with a standard echocardiography system, and has adequate interobserver agreement. this study showed higher values of tapse in normal young dogs with respect to older dogs. these results are similar to the results obtained in humans, and could reflect a less effective right ventricle with age. the values presented should be taken with caution due to the relatively small number of patients included. it may also be necessary to validate results in future studies with a second independent sample of dogs of other races. disclosures: no disclosures to report. tetralogy of fallot (tof) is a congenital heart disease characterized by 4 abnormalities, i.e., pulmonic stenosis, ventricular septal defect (vsd), aortic overriding and secondary right ventricular hypertrophy, caused by anterior deviation and abnormal septation of the conal septum during the embryonic period. few studies have reported the hemodynamic consequences and clinical outcome of tof in small animals. the objective of this retrospective study was therefore to document the epidemiological, clinical, echo-doppler findings, and survival, in a canine and feline population with tof. the case records of animals diagnosed with tof by combined use of echocardiography and doppler examination were reviewed (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) . tetralogy of fallot was identified in 31 animals (15 dogs, 16 cats). the most commonly represented breeds were terriers for dogs (7/15, 46.7%), and domestic shorthair for cats (12/16, 75.0%). most included animals (28/31, 90.3%) were clinically affected at the time of diagnosis. pulmonic stenosis was characterized by a variable systolic doppler-derived pressure gradient both in dogs (median [range] 106 mmhg ) and cats (109 mmhg ), and associated with hypoplasia of the pulmonary trunk in one third of the cases (35.7%). most vsd were large, with a median vsd:aorta ratio of 0.60 [0.35-1.02] in dogs and 0.59 [0.18-1.15] in cats. median age at death from cardiac cause was 23.4 months [3.5-92.9 ] without significant difference between dogs and cats (p = 0.298). these results suggest that in both cats and dogs tof-related death occurs predominantly in young adult animals with major hemodynamic consequences at the time of diagnosis. disclosures: no disclosures to report. the aim of this study was to assess whether and how radiographic and echocardiographic cardiovascular variables differ across age bands of healthy cats. a cohort of 98 clinically healthy cats were categorized into 3 groups: adolescent-adult (0.6-6 years; n = 45), middle-aged (7-10 years; n = 28), and geriatric (11-17 years; n = 25). all cats underwent a full physical examination, a complete blood count, routine biochemical profile, a baseline serum total thyroxine concentration, auscultation, non-invasive blood pressure measurements, thoracic radiography, electrocardiography, and echocardiography. cats with hypertension, hyperthyroidism, cardiac, or renal disease were excluded from the study. body weight, body condition score, systolic blood pressure, heart rate, and all echocardiographic indices were similar across the 3 groups. the mean (ae standard deviation [sd]) vertebral heart scale (vhs) value obtained for the geriatric group (7.7 ae 0.6) was significantly greater than that obtained for the adolescent-adult group (7.4 ae 0.4; p = 0.018). the mean ratio of the distance between the cardiac base and dorsal sternum to thoracic cavity height at the point of the cardiac base was significantly less in the middle-aged (0.64 ae 0.04) and geriatric (0.61 ae 0.05) groups than in the adolescent-adult group (0.69 ae 0.04; both p < 0.001). the mean angle between the cardiac long axis and the body axis was significantly smaller in middle-aged (40.4 ae 7.7°) and geriatric cats (40.0 ae 7.9°) than in adolescent-adult cats (53.1 ae 7.3°; both p < 0.001). the mean angle between the cardiac long axis and the sternum of middle-aged (36.8 ae 6.6°) and geriatric cats (36.2 ae 7.9°) was significantly smaller than that in adolescentadult cats (42.6 ae 5.7°; p = 0.006 and p = 0.002, respectively). additionally, the degree of undulation of the thoracic aorta correlated positively with age (r 2 = 0.307, p = 0.003). these findings suggest that differences in the horizontal alignment of the heart, thoracic-aorta undulation, and vhs in healthy geriatric cats, relative to observations in younger cats, can be considered to be agerelated. disclosures: no disclosures to report. the aim of this study was to investigate the presence of pulmonary hypertension (ph) in young cats affected by single or mixed lungworm infections. twenty-three cats infected with lungworms were examined at the veterinary teaching hospital of teramo, italy, in 2013-2014. animals underwent to a complete physical examination and to 2-or 3-views radiographic analysis of the thorax. a minimum database (i.e. cbc, serum biochemistry, serology for fiv antibody and felv antigen) was obtained for each patient. nine cats were excluded for concomitant diseases, while 14 cats were included in the study. microscopic identification of parasites was confirmed by molecular tests and all cats received an anthelmintic treatment. a single infection by aelurostrongylus abstrusus was diagnosed in 11 cats, while 3 cats had a troglostrongylus brevior infection either alone or in combination with a. abstrusus. transthoracic echocardiography was performed using an ultrasound unit with a 5 mhz phased array transducer. no structural abnormalities of the tricuspid valve and sign of pulmonary stenosis were detected. the 2-dimensional and m-mode echocardiography showed a cardiac involvement in 3 cats. one cat, infected by a. abstrusus and t. brevior showed a mild systolic tricuspid regurgitant jet with color doppler of 1.64 m/sec, while another a. abstrusus-infected cat, had mild tr of 2.2 m/sec with a mean paps of 29 mmhg which resolved within 4 weeks after therapy. one cat diagnosed with troglostrongylosis, showed a marked right-sided cardiac enlargement of 6 mm, and a large systolic tricuspid regurgitant jet with a tr peak velocity of 3.1 m/s recorded at continuous-wave doppler via a color doppler echocardiography. the minimum pressure difference between the right ventricle and the right atrium was estimated 38 mmhg and the paps was at least 48 mmhg. the echocardiographic and doppler evidence of mild ph persisted at further examination performed until 3 months after diagnosis. ph is rare in cats, despite cases of reversible ph are known in cat aelurostrongylosis. in this study the first case of irreversible ph infection in a cat affected by t. brevior is presented and this finding further supports the high pathogenicity of troglostrongylosis, especially in young patients. in cats with lungworm infection, possible cardiovascular complications must be taken into account and these infections should be always considered in the differential diagnosis in cats with cardiorespiratory signs. disclosures: no disclosures to report. although uncommonly assessed in veterinary cardiology, a right ventricular (rv) function has been shown to be an important prognostic determinant of many congenital and acquired heart diseases in human patients. our group has already demonstrated that 2-dimensional (2d) color tissue doppler imaging provides a noninvasive evaluation of systolic and diastolic rv function in the awake dog with adequate repeatability and reproducibility. b however, other noninvasive ultrasound imaging variables reflecting rv function need to be further investigated, particularly in correlation with pulmonary arterial pressure (pap) values and left ventricular (lv) function. the aim of this prospective study was therefore to assess several indices of systolic and diastolic rv function using conventional echocardiography and speckle tracking echocardiography (ste) in 104 healthy awake dogs of different breeds with documented systolic pap (spap) and lv function (lv ejection fraction and global lv systolic strain assessed using the simpson's derived method of disks and ste, respectively). imaging rv tested variables included tricuspid annular plane systolic excursion (tapse), right fractional area change (rfac, %), ste longitudinal systolic strain of the rv free wall (rvfw, %) and of the whole rv (i.e., global rv strain, %), ste longitudinal systolic strain rate (sr, s à1 ) and diastolic early:late sr ratio. additionally, 2d-guided m-mode ventricular measurements included the end-diastolic rv:lv diameter ratio (rvdd: lvdd) and the end-systolic rvfw:lvfw ratio. correlations between imaging variables were calculated by using spearman's correlation coefficients. means of age and body weight (ae sd; range) of the study population were 4.3 years (ae2.6; 0.6-11.6) and 20.4 kg (ae10.7; 3.3-49.0), respectively. no correlations were found between rv morphological variables (i.e., rvdd:lvdd and rvfw:lvfw ratios) and all indices of systolic and diastolic rv function. global rv strain (meanaesd = 26.4ae3.8%) and rvfw strain (31.9ae6.2%) were positively correlated (p < 0.01) with rfac (50.6ae10.5%, r = 0.36 and r = 0.32, respectively), and negatively correlated (p < 0.05) with spap (17.4ae7.0 mmhg [7.0-30.0], r = à0.21 and r = à0.24, respectively). spap was also negatively correlated with the tapse:body weight ratio and systolic sr (r = à0.31 and à0.34 respectively, p < 0.01). there was no correlation between indices of lv function and ste indices of rv function, and no correlation either between ste rv indices of systolic function and the diastolic early:late sr ratio. in conclusion, ste provides a rapid and non-invasive evaluation of rv function that may be used for clinical investigations in canine cardiology. doppler-derived +dp/dt and -dp/dt from mitral regurgitation are considered indexes for assessment of systolic and diastolicfunction respectively, that have less load dependence than the ejection phase indexes. this study aimed to determine correlation between doppler-deriveddp/dt and other systolic and diastolic echocardiographic indexes, and if they can be used to identify dogs with and without remodeling, with or without congestive heart failure (chf) and for evaluation of chronic mitral valve disease (cmvd) severity. fifty-seven dogs with cmvd (stages b1, b2, c+d) were included prospectively in an observational cross-sectional clinical study and distributed in groups regarding the presence of remodeling and chf, to evaluate+dp/dt and -dp/dt, and distributed according to tdi-diastolic pattern tocompare -dp/dt. group c+d (2142 mmhg/s, p 25 -p 75 = 2023-2456)had +dp/dt significantly lower compared to b1 (2865 mmhg/s, p 25 -p 75 = 2383-3308)and b2 (2721 mmhg/s, p 25 -p 75 = 2241-3186) (p = 0.0023). groupc+d also had lower -dp/dt, compared to b1 (968.5 mmhg/sae266.8 and 1198 mmhg/sae165.7; p = 0.0115). dogs with chf compared to those without chf, presented lower +dp/dt(2142 mmhg/s, p 25 -p 75 = 2023-2456; 2858 mmhg/s, p 25 -p 75 = 2299-3241; p = 0.0007) and -dp/dt (968.5 mmhg/sae266.8; 1155 mmhg/sae199.0; p = 0.0041). regardingdiastolic function, -dp/dt was lower for the restrictive pattern group (769.7 mmhg/sae124.1)compared to those without diastolic dysfunction, (1132 mmhgae204.0), delayedrelaxation (1229 mmhgae186.9) and pseudonormal patterns (1107 mmhgae223.4) (p < 0.0001).when +dp/dt<1800 mmhg/s, the post-test chance for the dog with cmvd to havechf is twice the chance than not having it. for -dp/dt<800 mmhg/s theposttest chance of having chf is 8 times higher than not having it. in conclusion, doppler-derived +dp/dt and-dp/dt may contribute respectively, for systolic and diastolic assessment ofdogs with cmvd. disclosures: no disclosures to report. pulmonic stenosis (ps) is one of the most common congenital heart defects seen in veterinarycardiology practice. pulmonary balloon valvuloplasty (pbv) is considered to be the treatment of choice for dogs withsevere stenosis. whether dogs with moderate stenosis benefit from pbv remains unclear, and variables such as degree of hypertrophy, valve morphology, amount of tricuspid insufficiency and presence or absence of clinical signs aregenerally used when recommendations are made to pet owners. in this study we report the effect of valve type on pbv outcome in 110 dogs treated at 3 different academic speciality cardiology practices. baseline echocardiographic images were evaluated at each institution and valve morphology was classified as either type a (n = 78, 137.96 mmhg, range 53-278) or type b (n = 33, 153.72 mmhg, range 81-300) and 'no' (n = 87, 140 mmhg, range 53-279) or 'yes' (n = 24, 151 mmhg, range 87-300) for presence of pulmonary annular hypoplasia when diameter was compared to aortic annulus. twenty four hours following pbv both type a (56 mmhg, range 17-210) and type b (78 mmhg, range 25-197) valves had significant reduction in gradient compared to baseline (p < 0.0001). this reduction remained significant at 30 days (a: 77 mmhg, range 22-193; b: 60 mmhg, range 30-116; p < 0.0001 for both). dogs with annular hypoplasia (65 mmhg, range 25-197) and without annular hypoplasia (69 mmhg range 17-210) had a significant reduction in gradient 24 hours post pbv. it remained significant at 30 days (with annular hypoplasia: 77 mmhg, range 30-116; without annular hypoplasia: 61 mmhg, range 22-193; p < 0.0001 for both). when comparing to baseline, considering valve type, there was no significant difference in percent reduction in gradient for type a versus type b valves at both the 24-hour (a: 58%, range 17-88; b: 48%, range 12-82; p = 0.1014) and 30-day (a: 43%, range 23-89; b: 58%, range 33-81; p = 0.0544) recheck evaluation time points. additionally, there was no significant difference in gradient reduction when looking only at whether or not there was annular hypoplasia at 24 hours (yes: 57%, range 24-78; no: 49%, range 17-89; p = 0.2673) and 30 days (yes: 48%, range 25-81; no: 47%, range 22-89; p = 0.4695). in conclusion, classification of dogs with ps according to valve type and annulus morphology did not help predict the 30-day response to pbv. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is the most common feline inherited cardiac disease and it is a major cause of morbidity and mortality. the osservatorio italiano hcm felina was formed in 2008 by a network of clinicians, geneticists and breeders, to monitor and study hcm in italian cats. since april 2008, 1308 adult cats, belonging to various breeds, including maine coon, siberian, norwegian forest cats, ragdoll, sphynx, british sh, birmans and others have been prospectively enrolled. recheck evaluations were performed in 287 cats. each cat underwent a clinical examination, echocardiography, and blood collection for genetic testing (when appropriate) and storage in the italian feline bio-bank. the disease status was defined by echocardiography according to established guidelines (left ventricular diastolic wall thickness <5.5 mm = hcm negative, =5.5 but <6 mm = hcm equivocal; =6 mm = hcm positive). the prevalence of hcm in the population was 6% (74 cats); equivocal diagnoses were conferred on 4% (57 cats). these prevalences did not differ between breeds. the prevalence of hcm in the italian feline population was lower compared to those reported by other investigators. evaluation of data from the entire population demonstrated that left ventricular end-diastolic wall thicknesses and aortic diameter showed a weak positive correlation with body weight (p < 0.0001, r 2 <0.12 for all variables), suggesting that weight-dependent limits on wall thickness should be considered in cats as is currently practiced in dogs. the lower prevalence of hcm in italian cat breeds compared with those examined elsewhere might be explained by different criteria for determining presence or absence of disease, differences in ages at which the subjects were examined, or a selection bias by breeders in presenting cats they consider 'normal'. disclosures: no disclosures to report. chronic mitral valve disease is by far the most common cardiovascular disease in dogs. the disease is caused by myxomatous degeneration of the mitral valve leaflets and, in approximately 30% of cases, it's accompanied by degeneration of the tricuspid valve. it is also described in previous studies that approximately 14% of affected dogs also have evidence of associated pulmonary arterial hypertension. the prevalence of the disease is higher in small breed dogs (under 20kg), although large breeds can also be affected and it occurs more frequently in males than in females. the present study aims to characterize the disease in a population of dogs in portugal. we retrospectively reviewed the medical records of dogs presented to hospital veterin ario do porto, with an echocardiographic diagnosis of canine chronic mitral valve disease, during a period of 13 years. from this records, 542 cases were identified, from which 331 (61.1%) were males and 211 (38.9%) were females. most of the dogs were mixed breed (215) and 48 different breeds of dogs were represented. the poodle was by far the most represented breed (n = 101; 39.7%), followed by english cocker spaniel (18.6%), yorkshire terrier (2.8%), boxer (2.6%), epagneul breton (2.6%), dalmatian (2.4%), pekingese (2.4%), labrador retriever (2%) and portuguese podengo (1.8%). all other breeds represented 16.2% of the population. regarding weight, 79.8% of the dogs (n = 395) weighted <20 kg, with a mean body weight of 13.45 kg (range 1.6-62 kg). the mean age at diagnosis was 11.34 years old. we also observed that 42.1% of the dogs (n = 278) had concomitant degeneration of the tricuspid valve and 19.4% (n = 105) pulmonary arterial hypertension (ph). we categorized these dogs according to the severity of ph, in mild ph if they had a doppler echocardiography derived systolic pulmonary arterial pressure (spap) of 30-50 mm/hg, moderate ph (spap 51-75 mm/hg) and severe ph (spap > 75 mm/hg). we found that 72.7% (n = 72) of dogs had mild ph; 19.2% (n = 19) moderate ph and 8.1% (n = 8) severe ph. as described in previous studies, the disease affects mainly males and small breed dogs, with a breed distribution that reflects the canine population in the country, including very including very popular large breed dogs in portugal, as the boxer and labrador. both the presence of concomitant tricuspid valve disease and ph had a higher prevalence in our study than previously described. disclosures: no disclosures to report. in people anemia is frequent in patients with heart failure (hf) and it is associated with poor outcomes. the most likely pathogenic factors include iron deficiency, chronic kidney disease (ckd), and cytokine production, although other factors may contribute. little is known about the prevalence of anemia in dog with cardiovascular disease. the aim of this retrospective study was to define the prevalence of anemia (hct ≤ 37%) in dogs with mitral valve disease (mvd) and to investigate associated risk factors (age, weight, azotemia, hf, iris/acvim class). medical records of dogs presented at the cardiology service, divet, university of milan (january 2003 -march 2015) were retrospectively evaluated. dogs with mvd with complete physical, thoracic and echocardiographic examinations, and serum biochemical panel, including serum creatinine (scr), were included in the study. dogs with other heart or systemic diseases, except ckd, or neoplasm were excluded. statistical analysis was performed using jmp 12.0 (sas institute). a p value <0.05 was considered significant. two hundred and ninety dogs (161 males/129 females), 11.6ae2.9 years of age, 12.5ae9.2 kg of body weight fulfilled the inclusion criteria. the 22% of males and the 30% of females were neutered. the most represented breeds were mongrel (40%), miniature poodle (12%), york shire terrier (7%), and cavalier king charles (5%). dogs were 29% b1, 13% b2, 54% c and 4% d acvim class. while the 72% of the dogs were normoazotemic (scr <1.4 mg/dl), 13.5% were staged in iris 2, 13% in iris 3 and 1.5% in iris 4. the prevalence of anemia in dogs with mvd was 17% (50/290): 40 showed mild (30≤ hct ≤37%) and 10 moderate (20≤ hct ≤29%) anemia. sixteen dogs were in b1, 5 in b2, 27 in c and 2 in d acvim class; 34 were normoazotemic (68%). anemic dogs showed a significant higher scr. normoazotemic dog showed significant higher hb, hct and rbc both in the overall population and in the anemic group. in the overall population dogs in different iris class showed statistically different hb, hct and rbc and hb was significantly lower in decompensated hf dogs. in conclusion although a relationship between anemia and azotemia/ckd was documented in our study, it is important to emphasize that most of the anemic dog were normoazotemic: anemia is not an exclusive finding of cardiorenal syndrome and should be considered as possible complication in dogs with mvd alone. disclosures: no disclosures to report. the objective of this study was to evaluate left atrial (la) function by left atrial total fractional area change (la-factotal) and left atrial ejection fraction (laef) in dogs affected with chronic mitral valve disease (cmvd) naturally acquired with and without congestive heart failure (chf). our hypothesis was that la-factotal and laef decrease with severity of cmvd. eighty dogs were included in a prospective observational cross-section clinical study, grouped according to cmvd severity based on echocardiographic evaluation and clinical signs. the dogs were equally distributed in each group: a, b1, b2 and c, according to american college of veterinary internal medicine staging system. indicators of la function were calculated with the following equations: la-factotal = 100 9 (lamaximum area -laminimum area)/lamaximum area, measured by apical 4 view; and laef = 100 x (lamaximum volume -laminimum volume)/lamaximum volume, by biplane area-length method from the left apical 4 and 2chamber views. la-factotal showed lower values (p < 0.0001) in group c (31.88%, p25-75% = 26.47-41.12) compared with groups a (52.75%, p25-75% = 48.08-56.07), b1 (48.38%, p25-75% = 42.57-51.91) and b2 (46.15%, p25-75% = 41.17-50). group c had lower laef (40.69%, p25-75% = 34.89-52.09) than groups a (68.12%, p25-75% = 64.96-69.91), b1 (58.72%, p25-75% = 52.25-64.60) and b2 (56.98%, p25-75% = 52.08-61) (p < 0.0001). left atrial function, assessed by la-factotal and laef, was reduced in dogs with cmvd and chf compared with healthy and asymptomatic cmvd groups. disclosures: no disclosures to report. recurrent episodes of heart and/or kidney failure are considered one of the causes leading to worsening heart/renal functions in human patients. the aim of this prospective study was to assess the influence of heart/kidney worsening on elected parameters of heart/kidney function in dogs affected by mitral valve disease (mvd). between july 2012 and may 2013, dogs affected by mvd in acvim class b2 and without comorbidities were included in the study group. the control group was constituted by healthy dogs, matched with the cases for age (older than 6 years) and gender. all the dogs underwent physical examination, thorax radiography, ecg, echocardiography, systemic blood pressure assessment, a complete blood count, serum biochemical analysis, including assessment of serum creatinine (scr), serum urea nitrogen (urea) and glycaemia (gly) and urine analysis with urine protein/creatinine ratio (upc). dogs were re-evaluated every 6-month until october 2014. statistical analysis was performed using ibm spss statistics 20 (p value significant if <0.05). twenty-one dogs affected by mvd (cases) were included and 20 healthy dogs (controls) were randomly selected among the eligible population. the 33% of cases experienced at least one episode of congestive heart failure (chf), but none of these patients developed chronic kidney disease (ckd). the 14% of cases developed ckd while remaining in acvim class b2. no dogs in the control group developed ckd or mvd. correlations between worsening renal function (wrf -scr elevation ≥0.3 mg/dl or 25% from baseline), furosemide administration, upc levels, radiographic parameters of heart enlargement and echocardiographic parameter were investigated. only a statistically significant difference in iris class between the groups according to wrf and in the echocardiographic parameter left atrium to aortic root (la/ao) according to furosemide amount were observed. both these results were expected. none of the cases included experienced renal damage (wrf or iris class change or upc change) concomitant to episodes of chf. the persistence of normal renal condition regardless of chf events and therapy administration was unexpected. in conclusion, experiencing chf seems not to directly affect renal function. to authors' opinion, the use of wrf, better than single scr and urea levels, may be useful in the long term management of aged patients affected by mvd. however, the small number of cases included in this study represents a great limit. we consider this work a pilot study. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is a primary myocardial disease characterized by inappropriate thickening of the myocardium in absence of other causes of hypertrophy including hypertension, hyperthyroidism, aortic stenosis and acromegaly. it is also the most common heart disease in cats. hcm presents a wide variety of clinical sings depending on the severity and location of the hypertrophy. cats affected with hcm have a mean age of 5.5-6.5 years old at the time of the diagnosis however this disease can affect cats as young as 3 months although this later age is unusual hcm is a heterogeneous disease both in terms of phenotypic degree of hypertrophy and clinical outcome. hallmark histopathological hallmarks lesions of hcm are myocyte disarray, small coronary arteriosclerosis and interstitial fibrosis replacement in order to confirm hcm echocardiography has to be made. primary hypertrophy diagnosis is made based on the presence of ventricular hypertrophy, symmetric or asymmetric, in the absence of systemic disorders. the purpose of this study was to assess the prevalence of hcm in a feline population. in order to achieve this goal echocardiograms were made in all cats older of 6 years clinically asymptomatic with or without cardiac murmur. all echocardiograms were made according to the guidelines of the acvim published in 1993. diagnosis of ventricular hypertrophy was made from the right parasternal window using the b mode to measure the diameter of the lvfw and the ivs in diastole. cats with more than 6 mm of wall thickness measured t4, bun, crea, blood pressure. only cats within the normal limits of the later parameters were considered hcm positive. total number of cats in this study was 150 cats 89 male and 61 female. from this population 94 had no defined breed, 37 were persian, 6 maine coon, 4 norwegian woods, 8 siamese, 1 chartreaux. no murmour was detected in 64 (42.7%) cats, s3 or s4 was detected in 9 (6%) cats and differente degree of murmour was detected in 77 (51.3%) cats. hypertrophy was detected in 69 cats. from this cats 41 (59.4%) were diagnosed as hcm, 28 (40.6%) cats were excluded either because of lack of values of t4 and or because they had high values of blood pressure, t4 levels or crea. in this study 27.3% of the population had hcm. the epidemiological and phenotype distribution is highly variable. the average age at diagnosis of hcm in this study was 11.33 years. disclosures: no disclosures to report. mildly increased concentrations of crp are associated with cardiovascular disease in humans and dogs. it is not known whether increased concentrations of crp are associated with myxomatous mitral valve disease (mmvd) in dogs, or rather its sequel, congestive heart failure (chf). the aim of this study was to investigate whether serum concentrations of crp, determined using a novel automated canine-specific high-sensitivity crp assay (gentian hscrp), were associated with severity of mmvd and certain clinical variables in dogs. the study included 188 client-owned dogs with different severities of mmvd. disease severity was determined by medical history, physical examination, echocardiography and response to diuretic therapy. dogs were allocated into groups based on acvim consensus statement guidelines (group a (n = 62), group b1 (n = 55), group b2 (n = 35) and group c (n = 36)). data were analysed using descriptive statistics and multiple regression analysis. dogs with chf (group c) had significantly higher serum crp concentrations (2.65 mg/l, [1.09; 5.09]) (median, [quartile 1; quartile 3]) compared to dogs in groups a (0.96 mg/l, [<0.50; 1.82]) (p = 0.0004), b1 (0.80 mg/l, [<0.50; 1.73]) (p < 0.0001) and b2 (0.53 mg/l, [<0.50; 1.26]) (p < 0.0001). other measures of disease severity including left atrial to aortic root ratio and left ventricular end-diastolic diameter normalized for body weight were positively correlated with serum crp concentration. in conclusion, slightly higher serum crp concentrations were found in dogs with chf whereas the severity of asymptomatic mmvd showed limited association with serum crp concentrations. disclosures: no disclosures to report. medetomidine is a a 2 -agonist widely employed for sedation in dogs but its use is discouraged in cardiac patients even those suffering from myxomatous mitral valve disease (mmvd). however, only one investigation was previously conducted in a wide rangeregarding the class of the disease -of mmvd patients, reporting a general safety of that protocol. the present study was focused just on class b2 of mmvd, with the aim to provide more detailed information on the cardiovascular effects of medetomidine in such patients, by the analysis of clinical and instrumental parameters suggestive of disease severity or congestive heart failure. dogs weighing <15 kg, needing a soft clinical procedure and showing a systolic apical heart murmur were screened and selected for the study if la/ao < 1.6. the sedative protocol consisted in an iv injection of 30 lg/kg medetomidine antagonized, after the clinical procedure, by an im injection of the recommended dose of atipamezole. clinical parameters, echocardiographic variables, thoracic radiographs and oscillometric blood pressure measurements were collected at baseline (t0), 30 minutes after medetomidine administration (t1) and 3 hours after atipamezole injection (t2). of 13 dogs screened, 8 were definitively enrolled. at t1 a significative decrease in the right parasternal regurgitant jet area (rp-arj/laa), peak velocity of mitral regurgitation and shortening fraction was observed along with an increase in lvids (p < 0.05). left parasternal arj/laa decreased without reaching statistical significance but showing a high correlation with rp-arj/laa (r = 0.7). interestingly, la/ao changed only mildly and never reached a value > 1.6. the other echocardiographic variables did not show a particular trend. systolic blood pressure showed values at the upper physiologic limit at t0, lower values than t0 at t1, and an increase above the initial value at t2 but without significance. thoracic radiographs were evocative of heart enlargement without pulmonary venous congestion or pulmonary oedema both at t0 and t2. respiratory rate did not change between t0 and t2. the degree of sedation was optimal during the clinical procedure in all cases. sedation with 30 lg/kg medetomidine is safe in dogs suffering from mmvd in stage b2 (la/ao < 1.6). the decrease observed in peak velocity and color-doppler appearance of mitral regurgitation at t1 could be due to a reduction of both myocardial contractility and systolic blood pressure, by a lowering of sympathetic activity via baroreceptors stimulation. disclosures: no disclosures to report. in this retrospective study were included a total of 25 clientowned dogs, undergoing transarterial occlusion of pda with mreye ò flipper detachable embolization coil (n = 7), amplatz â canine duct occluder (acdo; n = 16) and amplatzer â vascular plug (n = 2). device size selection was based on pda dimensions assessed by transesophageal echocardiography (tee) in 10 cases and transthoracic echocardiography (tte) in 15 cases. angiography was performed during the procedure to assess the success of the occlusion, and it confirmed complete occlusion in 20 dogs and a trivial residual flow in 5 dogs. the following day, transthoracic color-doppler echocardiography revealed that complete ductal closure was achieved in all dogs. the procedure was hemodynamically successful, as evidenced, by a reduction in indexed left ventricular internal diameter in diastole (lvidd; p < 0.01), fractional shortening (fs; p < 0.01) and left atrial to aortic ratio (la: ao; p < 0.001) within 24 hours after procedure. four months after surgery, indexed lvidd was significantly reduced (p = 0.03) and la:ao remained constant. secondary complications included pulmonary arterial embolization of an acdo and a late rotation of an amplatzer â vascular plug resulting in an increased flow through the pda. the dog with the rotated device required subsequent surgical ligation of the pda. at this time, 23 dogs were reported to be alive and the other 2 dogs were lost to follow up. only one dog remained on congestive heart failure therapy after the pda occlusion. we can conclude that pda occlusion using an acdo for dogs with more than 3 kg and a transarterial coil embolization for dogs with <3 kg had a high rate of immediate complete occlusion. pda occlusion using those devices proved to be a safe and effective therapeutic method for pda in dogs. disclosures: no disclosures to report. echocardiographic evaluation of the right pulmonary artery distensibility index (rpad index) was recently described as a valuable method for early detection and severity evaluation of pulmonary arterial hypertension in dogs. rpad index is calculated as the percentage change in diameter of the right pulmonary artery (rpa) between systole and diastole, obtained by m-mode echocardiography from the right parasternal long axis view. the aim of this study was to compare the rpad index obtained by 2 different echocardiographic views in dogs. the study design was a prospective, multicenter, observational study. forty-five clientowned dogs from different breeds were included: 31 dogs with heart disease and 14 healthy dogs. two different right parasternal views, long axis (rpla) and short axis (rpsa), were used to measure the rpad index. from the rpla view (method 1) and rpsa view (method 2) a short axis and a long axis image were respectively optimized for the right pulmonary artery. the rpad index was calculated by m-mode as the percentage change in diameter of the right pulmonary artery: [(systolic diameter -diastolic diameter)/ systolic diameter]*100. measurements were done off-line as an average of 5 consecutive cardiac cycles by a single investigator blinded to the dogs' diagnosis. a pearson and a bland-altman test were used to assess correlation and agreement between the 2 methods, respectively. intra-and inter-observer measurement variability was quantified by average coefficient of variation (cv). level of significance was set at p < 0.05. m-mode evaluation of the rpad index was satisfactorily obtained by both methods in all dogs. pearson test showed a strong positive linear correlation between the values of rpad index obtained from both methods (r 2 = 0.9346, p < 0.0001). bland-altman test showed a good agreement between the 2 methods in estimating rpad index (bias = 0.51%, sd = 2.96%, 95% limits of agreement = à5.30, 6.33%). the mean difference between the 2 methods was 0.51% (95% confidence interval = à0.35; 1.35). intra-and inter-observer measurement variability was clinically acceptable (cv<10%).the study showed a good agreement between short axis and long axis m-mode evaluation of rpa. both methods can be used interchangeably to evaluate rpad index. further studies are needed to evaluate the rpad index in a larger population of healthy dogs and the diagnostic and prognostic role of this echocardiographic parameter in dogs with different types of pulmonary hypertension. disclosures: no disclosures to report. this retrospective study (march 2005 to october 2014) includes 11 client-owned great pyrenees diagnosed with hypoadrenocorticism. the medical records of dogs with a diagnosis of naturally occurring hypoadrenocorticism were reviewed, with an emphasis on great pyrenees' record. the prevalence of hypoadrenocorticism in the studied population, as well as the prevalence per breed, was calculated. data collected included breed, clinical signs, laboratory findings, age at diagnosis, treatment, and cause of the death. one hundred dogs were diagnosed with naturally occurring hypoadrenocorticism, representing 0.38% of the overall canine population studied. thirty-five breeds were represented, with a prevalence per breed varying between 0.17% and 9.73%. a high prevalence was observed in west highland white terriers (4.66%), great danes (1.87%), standard poodles (1.76%), saint-bernards (1.72%) and jack russell terriers (1.48%). out of 114 gp presented during that period of time, 9.73% (n = 11) were diagnosed with hypoadrenocorticism. median age at diagnosis was 4.71 years (range: 0.39 to 11.07) in dogs with hypoadrenocorticism, and 3.51 years (1.02 to 8.21) in gp. the main reason for presentation of the addisonian gp was lethargy (n = 7) and anorexia (n = 5). clinical findings included hypotension (n = 7), poor body condition (n = 3), and heart murmur (n = 3). the majority (n = 9) had serum electrolytes abnormalities, with a na:k ratio ranging from 15.2 to 22.45. other major laboratory findings included azotemia (n = 8), anemia (n = 7) and the absence of a stress leukogram (n = 5). the majority (n = 9) received fludrocortisone, with prednisone as needed. one gp was euthanized at time of diagnosis. great pyrenees diagnosed with hypoadrenocortism were overrepresented in the studied population, with a prevalence of hypoadrenocorticism in our gp population of 9.73%. therefore, an inherited susceptibility can be suspected. reason for presentation and clinical signs were nonspecific, and similar to what is reported in other breed. in human medicine, haemoglobin a1c (hba1c), a form of glycosylated haemoglobin, is used as the standard measure of average glycaemic control over 2 to 3 months. the measurement of hba1c in dogs has been previously demonstrated however high pressure liquid chromatography techniques are too technically difficult for routine use and other methods are no longer available. the objective of this study was to assess the use of latex immunoagglutination inhibition using a monoclonal antibody for the measurement of hba1c in dogs, using the siemens dca vantage ò . repeatability was assessed by measuring 4 samples 5 times within 45 minutes. the effect of storage on edta anticoagulated samples was examined by measuring 3 samples stored at 4°c every day for up to 5 days. storage was further assessed by freezing 5 samples and measuring them at 0, 4 and 8 weeks. the machine was then used to compare the hba1c values in 3 groups of dogs with diabetes mellitus (group 1, n = 16), hyperadrenocorticism (group 2, n = 5) or non-diabetic/cushingoid hospitalised patients (group 3, n = 23). differences in the groups were examined for significance using a kruskal-wallis analysis of variance. the reference range of hba1c has been previously calculated to be 3.7-5.6% (17 -38 mmol/mol) and values of 4.9 ->13% (30 ->119 mmol/mol) are seen in diabetic animals using high pressure liquid chromatography. the median coefficient of variation for the repeatability study was found to be 5% (range 3% to 6%). it was possible to store samples at 4°c for up to 5 days (median cv% = 3%, range 2% to 3%) and at à20°c for at least 8 weeks (median cv% = 6%, range 4% to 9%). the median hba1c concentrations were group 1; 5.6% (38 mmol/mol), group 2; 3.4% (14.2 mmol/mol) and group 3; 3.3% (13 mmol/mol). group 1 was significantly different from the other 2 groups using kruskal-wallis analysis of variance. in conclusion, the latex immunoagglutination method was repeatable for the measurement of hba1c in dogs. in addition, hba1c in canine edta anticoagulated samples were stable at 4°c for up to 5 days and, if frozen, could be stored for at least 8 weeks without significant sample deterioration. the assay provides the expected results in dogs with and without abnormalities of glycaemic control. disclosures: the siemens dca vantage was provided on loan from siemens, as well as the cartridges used on this machine to run all samples in the study. a. wehner 1 , r. anderson 1 , s. reese 2 , k. hartmann 1 . 1 clinic of small animal medicine, munich, germany, 2 institute of veterinary anatomy, histology and embryology, munich, germany measurement of total thyroxine (t4) is often the first diagnostic step in the work up of thyroid disease in dogs and cats. blood samples are routinely sent to a reference laboratory causing a delay in testing which might impact the results. the aim of this study was to validate an enzyme fluorescence assay (elfa) as an inhouse method to measure t4 in dogs and cats. t4 was measured in sera of 162 dogs and 88 cats by 2 methods, an enzyme immunoassay (eia) and an enzyme fluorescence assay (elfa). the eia served as the standard method to which the elfa results were compared. the elfa was performed with the minividas automated analyser (biom erieux, craponne, france) according to the manufactors instructions. coefficient of variation (cv) of the elfa in dogs sera was 5.8% and of the eia 6-9.5%, respectively. cv of the elfa in cats sera was 0.7-3.4% and of the eia 7.6-15.7%, respectively. overall bias of the elfa in dogs was 1.4%; however up to à26.7% in lower t4 ranges. maximal bias of the elfa in cats was 6.9%. correlation of both methods was linear only in cats. using bland altman plots limits of agreement were -74-72% in dogs and -58-72% in cats. cohen 0 s kappa revealed only slight agreement between both methods in dogs, but a good to very good agreement in cats. the elfa is a fast method with a high precision and can be recommended to measure t4 in cats, but cannot be recommended for dogs. disclosures: no disclosures to report. dysfunctions of the central nervous system (cns) are the most frequent causes of neurological disorders in dogs. our study aimed to find (1) if some cns diseases could be associated with a selected group of common microbial or viral pathogens in dogs and (2) if cns diseases have any characteristic profile with regard to 2 parameters, c-reactive protein (crp) and iga, that are reported to be potentially useful but unspecific markers of cns diseases. we analysed 210 cerebrospinal fluid samples obtained from dogs with varying neurological signs between june and november 2014. real-time pcr was employed with probes for toxoplasma gondii, neospora caninum, anaplasma phagocytophilum and canine distemper virus. igg-antibodies to tickborne encephalitis virus (tbe) were assayed and iga titres were measured using elisa, while crp concentrations were determined by immunoturbidimetric assay. the dogs had a median age of 4 years (range: 0.5-14) and comprised 60 breeds most frequently involving chihuahuas, labrador retrievers, bernese mountain dogs and boxers. gender distribution was 22.4% female, 12.9% spayed bitches, 42.8% male, 15.7% neutered male, and 6.2% non-identified. none of the cases were pcr-positive for toxoplasma gondii or canine distemper virus. one dog was positive for anaplasma phagocytophilum and another for neospora caninum. antibodies to tbe virus were within the borderline range in 6/210 dogs. the 210 dogs could be divided into 2 age groups: 60 (=28.6%) for young dogs (<2 years, median 1 year) and 150 (=71.4%) for older dogs (≥2 years, median 6 years). igahigh (>0.1 mg/dl) cases represented 95% and 90% for young and older dogs, respectively. crp-high (>1.0 mg/l) cases were almost half and equal: 51.7% and 56.0% in young and older dogs, respectively. compared with older dogs, young dogs had higher levels of crp (p = 0.022) and iga (p = 0.054). within the iga-high cohorts, crp-high and crp-low cases distributed almost equally (46.7% versus 48.3%) in young dogs but disproportionate (52.0% versus 38.0%) in older dogs. there were no [iga-low/crp-low] cases in young dogs but 6.0% in older dogs. our present data suggest that (1) canine cns disorders were largely characterized by high iga and particularly in young dogs (2) inflammatory types (crp-high) were almost equal in both groups and (3) although the significance remains yet to be determined, pathogens like anaplasma phagocytophilum and neospora caninum could be detected in a few cases of canine cns disorders. disclosures: the authors breu d and guthardt j are employed at laboklin gmbh & co kg, germany. m€ uller, e is owner/manager of the laboklin gmbh & co kg. internet is a potential source of medical information for pet owners. therefore, it could play an indirect but important role in the veterinary practice. this survey assesses the online search behaviour of french pet owners for their pets' health and its influence on a veterinary consultation. in april 2013, 260 french pet owners coming in a veterinary teaching hospital for a medical or a surgical consultation were surveyed. 239 (91.9%) owners fulfilled the questionnaire on a voluntary basis. the survey contained 26 questions dealing with 3 topics: the online search behaviour of owners for their pets' health, their perception of the information found online and the internet's influence on a consultation and on the veterinarian/client relationship.73.6% of owners use the internet to obtain information on their pets' health. among them, 32.6% use it rarely and 28.9% occasionally. they mainly look for information on a disease (51.1%), a symptom (51.1%), a breed (50%) or a nutritional advice (48.9%). 79.7% of owners try to verify the accuracy of the information found, most often by questioning their veterinarian (82.8%). few owners (15.4%) think that online information is always trustworthy. most of the research (81.9%) is randomly made, websites being found through search engines. the majority of pet owners (81.3%) aren't aware of any health certification label for websites. internet enables certain pet owners to feel more at ease with their pets' health care: they ask more questions to their veterinarian (88.9%) and feel more involved in medical choices (55.6%). 37.9% of owners consider that the internet can positively impact their relationship with the veterinarian. relief is the most common (73.6%) emotional response to online research for medical information. however, 58.8% of owners feel overwhelmed by the amount of information found, 56% are confused and 35.2% frightened by the serious or graphic nature of the information found online. this study emphasizes the frequent but measured use of the internet by french pet owners for their pets' health. they seem to consider information found on the net with a critical mind. unexpectedly, it appears that the internet could be an ally for veterinarians by promoting exchanges between the clients and the veterinarian and by improving compliance with the care project. disclosures: no disclosures to report. sinonasal aspergillosis is a well-known and described fungal infection of the sinonasal cavities in dogs. topical treatment either with enilconazole or itraconazole infusion administered surgically or endoscopically are effective. the use of 1% clotrimazole cream have been described in a surgical setting after itraconazole infusion by sissener et al. in 2006. the aim of the work was to report the effectiveness of the use of topical 1% clotrimazole cream as the only treatment for sinonasal aspergillosis in dogs. inclusion criteria were a full medical record with radiological and endoscopical imaging, record of clotrimazole discharge after instillation and endoscopic control between 60 and 90 days after procedure. the 1% clotrimazole cream was applied through catheters placed under direct endoscopic vision after fungal plaques removal. nine dogs were included. three dogs were mixed breed dogs, 2 dogs golden retriever, one dog a german shepherd and one old english sheepdog, one bull terrier and one cane corso. six dogs were male (one neutered) and 3 female (one intact). mean age was 6.8 years. main clinical signs were muco-purulent discharge (8), pain at sinonasal palpation (8), nasal planum alterations (6), epistaxis (3) . nasal discharge was bilateral in 5 dogs. mean duration of clinical signs was 1.5 months. main radiological findings were turbinates lysis (9), frontal sinus empyema (8), frontal bone thickening (3) and frontal bone lysis (4). rhinoscopy disclosed lysis and remodelling of the nasal turbinates (9), easy access to the frontal sinus (7), septum lysis (5), bilateral sinonasal aspergillosis (2), monolateral nasal aspergillosis (2), monolateral sinonasal aspergillosis and contralateral nasal aspergillosis (2), monolateral frontal aspergillosis (3) . main duration of nasal cream discharge was 3.5 days. all dogs underwent endoscopic control between 60 and 90 days after the procedure. seven dogs were disease free; 2 dogs had persistent fungal plaques and underwent a second treatment. success rate was 77.8%. success rate of this study is comparable to other studies with larger and smaller case series. endoscopic removal of the fungal plaques can be time consuming and topical administration of either enilconazole or itraconazole require an additional hour. the catheter placement and the 1% clotrimazole cream application lasted 5 minutes for each cavity in the dogs here reported. the use of 1% clotrimazole cream as the only treatment for sinonasal aspergillosis needs further evaluation on a larger case series. disclosures: no disclosures to report. few studies exist on euthanasia in small animal practice. however, such an act belongs to veterinary procedures, more or less frequently depending of the kind of practice, and may deeply impact both owners and veterinarians. we intended to study practical, ethical and psychological aspects of euthanasia of dogs and cats among french veterinary practitioners. from october 2014 to february 2015, an on-line 79-item questionnaire on small animals' euthanasia, addressing practical aspects of euthanasia, communication with the owners, ethical problems, owners' and veterinarians' perceptions, was emailed via professional associations and networks. results were analyzed using commercial software (sphinx iq ò and excel ò ). 2770 french veterinarians practicing small animal medicine completed the questionnaire, representing >20% of this population. euthanasia occurs rarely at home. over 85% of veterinarians propose the owners some time alone with their pet, and to stay during euthanasia, performed most commonly by intravenous injection (91.4%) mainly after sedation/anesthesia (95.9%). ninety nine percent of veterinarians consider communication, including description of events' sequence, and disposal of the body, as important. estimated minimum communication time required varies from 5-15 to 15-30 minutes. following euthanasia, 64.4% are often thanked by the owners. most veterinarians (>85%) have refused a euthanasia, considered unjustified, or had their own suggestion of euthanasia rejected. reasons for such a suggestion include intractable pain (98.7%), non-acceptable complications (79.9%), financial considerations (44.1%) or animal considered dangerous (71.6%). veterinarians think most owners (63.9%) experience some sense of guilt during euthanasia. themselves perceive euthanasia most commonly as relief of animal's suffering (81.7%) and part of veterinary practice (64%), less frequently as a defeat (22.6%). almost all veterinarians have experienced emotionally challenging euthanasia, and 72.4% estimate that practicing euthanasia influences their perception of death. practical (74%) and psychological (48.4%) aspects of euthanasia have been discussed in most teams. veterinarians' gender influences euthanasia management, mostly concerning some communicational and practical aspects. euthanasia is definitely not an ordinary veterinary act, neither for the owner nor for the veterinarian. therefore, this act must be performed with as much care and communication as possible. disclosures: no disclosures to report. canine pancytopenia is associated with a range of intra-marrow or extra-marrow causes, including though not limited to, infectious agents, drugs, toxins and neoplasms. there is currently limited information regarding the clinicopathological features of the underlying causes or the prognosis in pancytopenic dogs. the objective of this retrospective study was to better define the spectrum of diseases associated with canine pancytopenia, to establish possible clinicopathological discriminators for the common causes and to investigate if the severity of pancytopenia or the underlying disease were associated with the clinical outcome (death or survival). medical records of dogs with a comprehensive diagnostic investigation admitted in a veterinary teaching hospital were retrospectively reviewed. pancytopenia was defined by a hematocrit (hct) <37% (<30% for dogs <5 months of age), white blood cell counts (wbc) <6,000/ll and platelet counts (plt) <200,000/ll. a control group of 238 dogs without any evidence of blood cytopenia(s) was also established. in total, 119 pancytopenic dogs were studied. bone marrow aspiration cytology was examined in 42 cases and aplasia of all hematopoietic lineages was observed in 22 (52.4%) dogs. the most common diagnoses included monocytic ehrlichiosis (cme) (n = 43), leishmaniosis (canl) (n = 28), parvoviral enteritis (pe) (n = 19), and concurrent cme and canl (n = 12). mixed breed dogs were more likely to develop pancytopenia as compared to purebreds and pancytopenic dogs tended to be younger than the controls (conditional dependent logistic regression model, p = 0.013 and p = 0.001, respectively). among the most common diseases associated with pancytopenia, the mean wbc counts were significantly lower in dogs with cme and pe compared to dogs with canl (one way anova with bonferroni test for multiple comparisons, p = 0.004 and p = 0.03, respectively), while plt counts were significantly lower in cme compared to canl (p<0.0001) or pe (p < 0.0001). total protein concentrations were significantly lower in dogs with pe compared to cme (p < 0.0001) and canl (p < 0.0001). using a univariable logistic regression analysis model, no association was established between the underlying disease and the final outcome. however, higher hct (by at least one percentage unit), wbc (by at least 1,000/ll) and plt (by at least 10,000/ll) values tended to significantly increase the odds of survival (p = 0.025, p < 0.0001 and p = 0.006, respectively). in the present study, cme, canl and pe were the major causes of canine pancytopenia. potentially useful diagnostic indicators included severe leucopenia (cme, pe), thrombocytopenia (cme) and hypoproteinemia (pe). disclosures: no disclosures to report. laryngeal masses (lm) usually produce air flow limitation during inspiration, expiration or transiently during subsegments of both breathing phases. feline bronchial diseases (fbd) have predominantly expiratory flow restrictions. barometric whole-body plethysmography (bwbp) is a non-invasive pulmonary function test (pft) that allows a dynamic study of breathing patterns widely used to evaluate lower airway responsiveness. the objective of this preliminary study was to evaluate if there were significant differences in respiratory rate [rr ( bwbp detects both upper and lower airways diseases because any site of airway obstruction will result in increased pressure changes associated with breathing. nevertheless our results suggest that there are significant differences in tv(p = 0.0001), ti (p = 0.0001), pef(p = 0.0001), eep(p = 0.003), penh(p = 0.0001) and pau(p = 0.0001) between lm and fbd cats. no other significant difference in bwbp parameters was found. upper airway obstructions have been previously evaluated in cats by using pft (mckieman, 1993 , lin, 2014 but in authors' knowledgement this is the first study designed to compare upper and lower airway obstructions by using bwbp. attending our results, there is the evidence that bwbp can help characterize mechanical dysfunction of the airways in cats with lm obstruction. however we must keep in mind some limits of this study as the low number of animals, individual variability in breathing pattern and to have the chance of doing bronchoreactivity tests. disclosures: no disclosures to report. the median lifespan of domestic dogs has been estimated to 9-12 years, but little is known about risk factors for mortality in aged dogs: most mortality studies in dogs have been carried out among diseased dogs (renal or heart diseases, cancers, post-operative death). to determine which characteristics are associated with mortality in a priori healthy aged dogs, a prospective cohort study has been conducted in 116 guide dogs, followed from a systematic geriatric examination (ge) to either (all-cause) death or cut-off date (july 16 th , 2013) . survival analyses (kaplan-meier estimators, log-rank tests, and multivariate cox proportional hazards models) were used to assess the associations with time to death. median age at ge was 8.9 years, all dogs were neutered, and 50% were female. the majority of dogs were golden retriever (n = 48) and labrador retriever (n = 27). among these 116 dogs, 16% were obese, 47% presented skin nodules and 90% used bus as transportation. a total of 76 dogs died during follow-up, leading to a median survival time from ge of 4.4 years. after adjustment for demographic and biochemical variables (age, sex, total proteins, cholesterol and alp), an increased alanine amionotransferase level (≥102 ui/l; adjusted hazard ratio [ahr], 6.0), presenting skin nodules (ahr, 2.3), and not being a labrador (ahr, 3.3) were independently associated with a shorter time to death (p < 0.05). public transportation tended to be associated with mortality (ahr, 3.0; p = 0.06), highlighting the importance of environment in mortality. neither sex nor other biochemical parameters were significantly associated with mortality. the alanine amionotransferase level and the presence of skin nodules seem predictors of mortality in senior guide dogs, mostly labrador, golden, or mixed breed of labrador/golden. the impact of environment, in particular urban environment, on mortality needs further investigation. studies in other breeds and pets are also necessary to generalize these results. disclosures: sara hoummady received a grant from mp labo for his phd work about dog aging and marc blondot work at the paris guide dog school. laryngeal dysfunction is most commonly associated with aspiration pneumonia, however its role in other lower airway diseases has not been investigated. laryngoscopic and bronchoscopic findings in dogs examined by the author between 2001 and 2014 were evaluated for the presence or absence of laryngeal abnormalities. dogs that presented for evaluation of inspiratory difficulty or panting were excluded from analysis. clinical diagnoses of inflammatory airway disease, airway collapse, airway infection, eosinophilic bronchopneumopathy or a combination of these disorders were obtained through bronchoscopy and bronchoalveolar lavage fluid analysis. detection rates for laryngeal abnormalities were compared among disease groups using chi square analysis and fisher's exact test, with significance set at p < 0.05. a total of 138 dogs were evaluated and varied in age between 4 months to 15.5 years (median 8 years). weight ranged from 1.5-63.4 kg (median 13 kg), with 31 dogs <5 kg, 28 dogs from 5.1-9.9 kg, 24 dogs from 10-20 kg, 45 dogs from 20.1-40 kg, and 9 dogs >40 kg. laryngeal hyperemia or swelling was found in 73/138 dogs (53%), and detection rate did not differ among disease processes. laryngeal function was considered suspect in 59/138 cases, prompting administration of doxapram, which normalized function in 30/59 dogs. laryngeal paresis or paralysis was reported in a total of 26/ 138 dogs (19%). a substantial number of dogs with chronic cough displayed evidence of abnormal laryngeal structure or function, suggesting that a complete laryngoscopic examination should be performed in all dogs evaluated for cough. disclosures: member of the feline advisory board, speaker honoraria for international, national, and regional continuing education meetings. bronchiectasis is a poorly characterized disease in dogs identified by airway dilatation on radiographs, computed tomography, bronchoscopy, or histopathology. little is known about underlying disease processes associated with bronchiectasis in dogs. medical records from dogs presented to uc davis were searched for identification of bronchiectasis. underlying disease processes and clinical diagnoses were obtained through review of the history, physical examination, respiratory endoscopy and bronchoalveolar lavage fluid analysis and microbiology. historical reports, results of imaging, bronchoscopy and fluid analysis, and scrutiny of pathologic and clinical diagnoses were comprehensively evaluated to identify the most likely underlying disease process associated with bronchiectasis. between 2003 and 2014, bronchiectasis was diagnosed in 86/621 dogs (14%) that had bronchoscopy performed. dogs ranged in age from 0.5 to 14 years (median 10 years) with 1/85 dogs < 6 months, 16/85 dogs (19%) 1-5 years, 37/85 dogs (43%) 5.1-10 years of age and 31/85 dogs (37%) over 10 years of age. dog breeds affected more than once included 6 labrador retrievers, 5 cocker spaniels, 4 golden retrievers and 4 standard poodles. duration of cough ranged from 3 days to 10 years (median 6 months). underlying disease processes included pneumonia in 45/86 (52%) dogs, inflammatory airway disease in 24/86 (28%) dogs, and eosinophilic bronchopneumopathy in 10/86 (12%) dogs. twenty-three of 85 dogs (27%) had positive bacterial cultures, with isolation of streptococcus (n = 6) and enteric species (n = 5) most commonly. this study found that bronchiectasis often occurs in older, large breed dogs with infectious or inflammatory pneumonia. disclosures: johnson: feline advisory board, speaker honoraria. chronic respiratory disease, often characterized by a chronic cough, is common in dogs. the purpose of this study was to determine the etiology of chronic respiratory disease in dogs that were presented with persistent and chronic coughing. a retrospective study of 126 client-owned dogs with signs of persistent and chronic lower respiratory disease, that underwent bronchoscopy together with either an endotracheal wash (etw) or broncho-alveolar lavage (bal), was performed. all dogs were evaluated by means of full clinical examination, hematology and serum biochemistry analyses, survey thoracic radiographs, echocardiography and ecg (if indicated), and bronchoscopy with cytological analysis and aerobic culture of etw or bal fluid. an etw was performed in 112/126 (89%) dogs while a bal was performed in 15/126 (11%) dogs. a positive aerobic bacterial culture was identified in 42/126 (33%) of submitted etw/bal fluid samples. most commonly isolated bacteria included mycoplasma sp. (24%), bordetella bronchiseptica (24%) and pseudomonas aeruginosa (12%). a definitive diagnosis was made in 118/126 cases (93.6%). chronic bronchitis was the most common diagnosis (37.3%), median age 8 years; followed by airway tracheal collapse or bronchomalacia (23%), median age 11 years,; and primary bacterial infections (15.8%), median age 3 years. less common etiologies identified included neoplasia (5.5%), median age 14 years; parasitic infections (4.8%), median age 7 years; and eosinophilic bronchopneumopathy (3.2%), median age 6 years. rare etiologies identified included primary pulmonary hypertension, primary ciliary dyskinesia, excitement-induced cough, and obesity. myxodematous mitral valve disease was found concurrently in 12/126 (9.5%) dogs. this study concluded that by using a structured combination of survey thoracic radiography, bronchoscopy and etw or bal with cytology and culture, a diagnosis could be made in the majority of dogs with chronic respiratory disease. disclosures: no disclosures to report. canine sterile steroid-responsive lymphadenitis (cssrl) is an uncommon cause of lymphadenomegaly. diagnosis is one of exclusion after extensive investigations exclude infectious, inflammatory or neoplastic aetiologies. resolution of clinical signs occurs with corticosteroids. this retrospective study aimed to further define characteristics, progression and treatment regimens. cases were recruited from 5 uk referral centres between 2009-2015. diagnostic investigations in each case excluded other potential causes of lymphadenomegaly. thirty-six dogs were diagnosed with cssrl from lymph node cytology and/or histopathology. eighteen breeds were represented, of which 16 were spaniels. english springer spaniels (ess) accounted for 10 cases along with cocker spaniels (4), cavalier king charles spaniels (2) and border collies (3) clinical presentation varied between dogs. clinical signs of pyrexia (77%), lethargy (70%) and anorexia (41.6%) were the most common. other signs included cough, tachypnoea, dyspnoea, dysphagia, vomiting, diarrhoea, neck or spinal pain, abdominal pain, joint effusion or dermatologic signs. median referral time was 24 days (ess 25, cockers 33 and border collies 8 days). twenty-two animals were pyrexic at presentation (mean 39.8°c, range 39.1-40.9°c). thirty-one animals presented with peripheral lymphadenomegaly, but 5 animals displayed only internal lymph node enlargement. cytology was performed in 30/36 cases; neutrophilic lymphadenitis (20), followed by granulomatous (5), pyogranulomatous (5) and reactive hyperplasia (4). histopathology was performed in 22/36 cases documenting neutrophilic (5), pyogranulomatous (9) or granulomatous (2) lymphadenitis. lymph node culture or staining (gram, pas, zn) was performed in 17 and 18 animals respectively, all of which were negative. prednisolone was administered in all cases (dose range 0.5-3 mg/kg daily). 24 animals were initiated therapy at 1 mg/kg q 12 hours. mean treatment length was 18 weeks. 16 dogs relapsed throughout the study period (9 ess, 3 cockers, 1 ckcs, border collie, lurcher and beagle). 7 ess relapsed within 18 months of diagnosis. median relapse time was 26 weeks. this study documents dogs with cssrl in the uk suggesting an over-representation in spaniel breeds (particularly ess), with females and young dogs typically affected. cytologic and histopathologic examination confirmed sterile lymphadenitis with animals showing marked and rapid clinical improvement with corticosteroids. disclosures: no disclosures to report. brachycephalic dogs have unique upper respiratory anatomy with abnormal breathing patterns similar to those in humans with obstructive sleep apnea syndrome (osas). oxidative stress in the body represents the imbalance between the production of reactive oxidative species and the ability of antioxidant defense mechanisms to detoxify the reactive intermediates. oxidative stress is involved in the pathogenesis of many diseases, including hemolytic anemia, atherosclerosis, tissue reperfusion injury and has also carcinogenic potential. several studies have clearly shown an association between obstructive sleep apnea syndrome in humans and oxidative stress, but detailed underlying pathomechanism remains unclear. due to the consideration of brachycephalic dogs as an animal model of human osas, this study was designed to evaluate oxidative stress (paraoxonase type-1 activity; pon1 and total antioxidant status; tac) in brachycephalic dogs with brachycephalic airways obstructive syndrome (baos) before and after surgery compared to control dogs. this study was conducted on 37 dogs with baos and 34 control dogs. twenty out of 37 baos dogs were evaluated 1-2 months after surgical correction. mean values of tac and pon1 in different studied groups were as follows: control dogs (tac: 0.614; pon1:2.53), baos dogs (tac: 0.233; pon1: 2.438), baos dogs post-surgery (tac:0.177; pon1: 2.705) a statistically significant difference on tac levels is observed between dogs with baos and control dogs (p < 0.05). no statistically significant differences were observed in pon1 and tac levels before and after surgery. on the other hand, no differences have been observed between pon1 and tac levels in baos dogs according type of brachycephalic breed, grade of respiratory and digestive signs or presence of everted ventricular laryngeals. the results of our study showed a statistically significant difference in tac values between control and dogs with baos, confirming the oxidative stress previously described in humans. even that human patients with osas can partially reverse their increase in oxidative stress by using a nasal continuous positive airway pressure treatment, in dogs with baos no differences were observed before and 1 month after surgical treatment. baos surgical treatment is not useful to reduce pon-1 or tac levels, probably because baos does not induce such an evident inflammatory process in dogs as in human patients with osas. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). the cipf course varies greatly among dogs from rapid deterioration to slowly progressive forms and the survival time from onset of clinical signs ranges from a few months to several years. in human ipf, increased chemokine (cc-motif) ligand 2 (ccl2) concentrations in bronchoalveolar lavage fluid (balf) are indicative of a poor outcome and serum concentrations are correlated with clinical parameters of lung function. in dogs, serum and balf ccl2 concentrations were shown to be elevated in whwts with cipf compared with healthy whwts. the aim of the present study was to investigate whether serum ccl2 concentrations measured in whwts with cipf at diagnosis (1) can be used as an indicator of prognosis and (2) correlate with lung function parameters. ccl2 concentrations were determined by elisa (canine ccl2 quantikine elisa kit, r&d systems) in the serum of 60 whwts suspected of cipf (median age 11.7 years, range 5.7 -14.5), for which a follow-up was available (median follow-up time 8.6 months, range 0 -71.8). serum sampling extended from may 2007 to january 2015. cipf diagnosis was confirmed by thoracic high resolution computed tomography, lung histopathology, or both examinations in 17, 6 and 27 whwts respectively. kaplan-meier analysis was conducted to investigate differences in survival times according to serum ccl2 concentrations at diagnosis. spearman analysis was used to assess correlations between serum ccl2 concentrations and lung function parameters, namely the distance walked in the 6-minute walking test (6mwd) and the arterial partial pressure of oxygen (po 2 ). among the 60 cipf whwts included, 31 died or were euthanized for cipf-related reason, 12 died or were euthanized for non-cipf-related reason and 17 were still alive at the end of the study. the median survival of whwts with cipf-related death or euthanasia was 6.4 (range 0.4 -71.9) months from diagnosis. serum ccl2 concentrations above 700 pg/ml were significantly associated with a shorter survival time in whwts affected with cipf (p = 0.02). a weak negative correlation was found between serum ccl2 concentrations and the 6mwd (r = à0.382, p = 0.03, n = 31), while no correlation was observed with arterial po 2 values (n = 49). in conclusion, serum ccl2 concentration provides prognostic information in whwts suffering from cipf, while this marker is weakly correlated with the clinically lung function parameters available in the present study. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). this study was intended to (1) describe thoracic high-resolution computed tomography (t-hrct) findings obtained in cipf dogs under general anesthesia (ga) using the glossary of the fleischner society and (2) compare images obtained under ga (t-hrct ga ) with those obtained under sedation (t-hrct s ). t-hrct images from 11 whwts with cipf and 9 control whwts were retrospectively reviewed by 3 observers in consensus. specific t-hrct features were assessed and graded for each lung lobe (0 = absence, 1 = mild, 2 = moderate and 3 = severe). a global score was then calculated. the khi² test with the threshold 5% was used for the statistical analysis. ground glass opacity (ggo) was observed in all cipf whwts and in 5/9 of controls (p = 0.013). in controls, ggo was mild and localised mainly in cranial lobes. in cipf whwts, ggo was mild, moderate or severe in 2, 4 and 5 dogs respectively, without lobe predilection. consolidation was observed in 5/11 cipf whwts but not in controls (p = 0.020) and was mild (3/5) to moderate (2/5). a mosaic pattern, suggestive of air trapping, was noticed in 8/11 cipf whwts but not in controls (p = 0.001) and was mild, moderate or severe in 3, 2 and 3 whwts respectively, without lobe predilection. nodules were present in 3/11 cipf whwts but not in controls. reticulation, subpleural bands and parenchymal bands were noticed in 1, 1, and 3/11 cipf whwts respectively. honeycombing, emphysema, pleural effusion and pleural thickening were never observed. bronchial wall thickening and mild bronchiectasis were present in 6/11 and 3/11 cipf whwts respectively but not in controls (p = 0.008 and p = 0.09). the overall t-hrct s quality was good in 10/17 examinations compared with 16/20 for t-hrct ga (p = 0.160). the presence of motion artefacts was higher for t-hrct s (p < 0.001), but were most frequently graded as mild (p < 0.001). t-hrct s allowed identification of a mosaic pattern in 2 additional cipf whwts, while consolidation could not be identified in 2 others. there was no difference in identification or gradation for the other features between t-hrct ga and t-hrct s . in conclusion, ggo, consolidation, mosaic pattern and bronchial wall thickening are the main t-hrct features of cipf in whwts. honeycombing, the major feature of ipf in humans, was never observed, which suggests a different pathophysiology between the 2 entities. t-hrct s images are in accordance with t-hrct ga and can be used for cipf diagnosis. disclosures: no disclosures to report. literature on mesenterial lymphadenitis (lad) or mesenterial lymph node abscesses (lab) in small animals is scarce. case files from 2005 to 2014 were searched for dogs with the diagnosis of mesenterial lad/lab based on cytology and/or histopathology. of 24 cases identified, 5 had to be excluded due to incomplete data. the remaining dogs were of mixed breed (n = 6), small munsterlander (n = 4) and one each of other breeds. nine dogs were male and 10 female. median age was 47 months (range . diagnosis was based on fine needle aspiration (fna; n = 9), histopathology (n = 1) or both (n = 9). significant findings in the dogs' history included gastrointestinal signs (n = 2), 1 puppy whose mother had mastitis, bite wounds/abscesses of the skin (n = 2), pulmonic stenosis (n = 1) and orthopaedic diseases (n = 3). most common presenting complaints were lethargy (n = 13), hyperthermia (n = 12), diarrhoea (n = 7), vomiting/nausea (n = 6), inappetence/anorexia (n = 5), back/abdominal pain (n = 4) and lameness (n = 2). diagnostic tests performed included haematology/serum biochemistry (n = 19), thoracic (n = 12) and abdominal (n = 7) radiographs, abdominal ultrasound (n = 19), ct/mri (n = 3), fnas of other organs (n = 7), urinalysis (n = 12) with culture (n = 7), coagulation profiles (n = 5), orthopaedic radiographs (n = 2), cpl (n = 5), blood cultures (n = 1), and csf/joint taps (n = 2). dogs were retrospectively divided into group a (n = 9): dogs with no other disease than lad/lab ("idiopathic") and group b (n = 10): dogs with other diseases diagnosed simultaneously. these included neoplasia (carcinoma n = 1, lymphoma n = 2, leiomysarcoma n = 1, histiocytic neoplasia n = 1, prostate carcinoma n = 1), gastroenteritis (n = 3), presumed pancreatitis (n = 2), purulent monoarthritis (n = 1), purulent hepatitis/ splenitis (n = 2), fungal infection at a distant site (n = 1), and mycobacteriosis (n = 1). eleven dogs received surgical treatment and antibiotics, and 8 dogs conservative medical management consisting of supportive treatment and antibiotics. all dogs were discharged alive. dogs in group a were hospitalized longer (mean 8 days, sd 3.4) than dogs in group b (mean 3 days, sd 2.2) (p = 0.004). the median follow-up time was 67 days (4-784 days). there was no difference in pretreatment with antibiotics or anti-inflammatories between groups. t-tests or kruskall-wallis tests showed that dogs in group a were borderline significantly younger (p = 0.052), had significantly higher respiratory rate (p = 0.004), rectal temperature (p = 0.007), monocyte count (p = 0.014) and crp concentration (p = 0.027) than dogs in group b. the small munsterlander had an odds ratio of 32 over other breeds to suffer from lad/lab. in conclusion, idiopathic mesenterial lad/lab was seen in young dogs with hyperthermia and gastrointestinal signs. diagnosis of purulent lad/lab on fna does not exclude the presence of another underlying pathogenesis. disclosures: no disclosures to report. dogs were included, if they showed respiratory signs (<14 days) consistent with cird and if non-infectious respiratory conditions could be excluded. nasal and pharyngeal swabs were taken from 61 dogs with cird and tested for respiratory pathogens, including canine parainfluenza virus (cpiv), canine adenovirus (cav), canines distemper virus (cdv), canine herpes virus (chv), canine respiratory coronavirus (crcov), canines influenza virus (civ), and bordetella bronchiseptica (b. bronchiseptica) by polymerase chain reaction. results of clinical and laboratory data were correlated with the underlying pathogen using fisher's exact test and chi-square test (p≤0.05). cpiv was detected in 23, crcov in 6, and b. bronchiseptica in 48 dogs; 23 patients showed infections with more than one pathogen. there was no significant difference for age and gender distribution between the 3 groups; however, dogs infected with cpiv more likely originated from a shelter (p = 0.037). when clinical data were compared, there was no significant difference for the parameters depression, fever, cough, nasal discharge, dyspnoea, and abnormal lung sounds. furthermore, there was no significant difference regarding abnormalities of erythrocytes, platelets, leukocytes, and differential count between groups. the study shows that in dogs with cird clinical and laboratory parameters cannot indicate the underlying pathogen. furthermore, diseases severity does not seem to depend on the infectious organism involved. disclosures: no disclosures to report. breed related predisposition to bacterial bronchopneumonia (bp) has been reported in irish wolfhounds (iwhs). underlying factors are unknown, however immune deficit, ciliary dysfunction and aspiration have been suggested as predisposing factors. the purpose of this prospective study was to evaluate lymphocyte subpopulations in iwhs with one or more previous bp and compare results to elderly iwhs without any previous bacterial respiratory infections. additionally, healthy dogs of other breeds were included as controls. previous bp was confirmed in 11 iwhs (median age 5.1 years, interquartile range 2.2-7.0 years). healthy iwhs (n = 13, 6.8, 6.3-8.2 years) or dogs of other breeds (n = 15, 5.5, 3.5-6.3 years) had no history or findings suggestive of previous bp. edta blood samples, collected from all dogs when they were healthy, were stained with fluorescent mouse anti dog cd3, cd4, cd8, cd21 and mhc class ii antibodies (abd serotec ò ) and flow cytometry analysis was performed with bd facsaria ò ii cell sorter and facsdiva ò software. statistical comparison between groups and the effect of age was studied using analysis of covariance (ancova) models. the number of leucocytes did not differ significantly between groups. the total numbers of lymphocytes and numbers of major lymphocyte subpopulations (b-cells, cd4+ and cd8+ t-cells) were significantly lower in healthy iwhs and iwhs with previous bp compared to dogs of other breeds (lymphocytes p < 0.001 and p < 0.001; b-cells p = 0.013 and p = 0.005; cd4+ t-cells p = 0.026 and p = 0.029; cd8+ t-cells p < 0.001 and p = 0.001 respectively). percentage and number of mhc class ii+ non-b lymphocytes was significantly higher in both iwh groups than in dogs of other breeds (p < 0.001 in all comparisons). lymphocyte numbers and subpopulations did not differ significantly in healthy iwhs compared to iwhs with previous bp. an age-related decline in the total number of lymphocytes (p = 0.015), t-cells (p = 0.007), cd4+ t-cells (p = 0.007) and mhc class ii+ non bcells (p < 0.001) was noticed only in the group of iwhs with previous bp. these preliminary results indicate that iwhs may have significantly lower numbers of lymphocytes, b-cells as well as cd4+ and cd8+ t-cells than dogs of other breeds. further studies are needed to determine whether these alterations represent a breed related phenomenon or are connected to the predisposition to bp. an age-related decline in lymphocyte, total t-cell and cd4+ t-cell numbers was detected in iwhs with previous bp. in humans, age related changes in cd4+ t-cells have been associated with increased susceptibility to infections. disclosures: no disclosures to report. feline chronic kidney disease (ckd) is a common feature of ageing cats. angiotensin-converting enzyme inhibitors (acei) are recommended to treat hypertension associated with ckd to limit target-organ damage and especially glomerular hypertension. in addition, the international renal interest society (iris) guidelines recommends the prescription of an acei in patients with ckd and proteinuria. to our knowledge no study has demonstrated the effects of long term administration of acei in a client owned population of cats suffering from ckd on glomerular filtration rate (gfr). the aim of the study was to evaluate the effect of an acei (imidapril, prilium ò , v etoquinol sa) on the gfr of cats suffering from naturally occurring chronic kidney disease (ckd) over 12 months. sixty-six cats presenting with clinical and biological signs of ckd were enrolled by 20 european investigators and followed up till 24 months in this randomized blinded study. thirty-two cats provided suitable data for gfr analysis; 17 animals received imidapril at the dosage of 0.5 mg/kg/day and 15 received placebo. animals with no available gfr value on day0 or with no data after day0 were excluded as well as animals for which the iohexol clearance could not be determined. follow up was censored after 12 months due to small sample sizes for statistical comparisons. on day0, month3, month6 and month12, cats were sampled 30, 60 and 120 minutes after intravenous administration of 647 mg of iohexol. gfr was based on determination of the iohexol clearance which was calculated with the phoenix ò winnonlin 6.3 software. statistical analyses were performed with sas/stat 9.2 software. as gfr were not available on each time point for a given animal, the 2 treatment groups were compared on each gfr determination point using an ancova (analysis of covariance) with the gfr determined on day0 as the covariate. on d0, gfr were 1.53ae0.68 and 1.68ae0.60 ml/kg/min in the imidapril and placebo group, respectively. a significant statistical difference (1.62ae0.63 versus 1.24ae0.59 ml/kg/min in the imidapril and placebo group respectively, p = 0.029) was observed in favor of a higher gfr in the imidapril treated animals on month6. higher gfr were also observed in the imidapril group on mon-th3 and month12 but not significantly different from the placebo group. daily long term imidapril treatment compared to placebo, may be an effective treatment to slow the progression of renal failure in cats with naturally occurring chronic kidney disease. disclosures: authors are employees of vetoquinol. there is a concern over the potential of cytotoxic drugs which could harm exposed workers. the speciality of oncology of the ecvim-ca published guidelines in order to prevent that risk. however few data exist for evaluation of the real risk of occupationnal exposure. this concern was the aim of our study. biomarkers used were vincristine and cyclophosphamide. surface samples were collected in the consultation room and ward dedicated for chemotherapy. samples were collected with wet filter paper on 10 9 10 cm surfaces, or objects (computers for instance). samples were analysed by liquid chromatography and mass spectrometric method.the limit of quantification was 0.2 ng/ 100 cm 2 (or 0.2 g/object) for vincristine and 0.02 ng/100 cm 2 (or 0.02 g/object) for cyclophosphamide. samples in consultation room were collected after treating 2 dogs with vincristine and after cleaning. samples in dedicated ward were collected after cleaning and after 2-days stay of treated dogs. 18 aeras/objects were tested in consultation room; 15 in dedicated ward for vincristine; 9 in ward for cyclophosphamide. after treatment of dogs in consultation room, traces of vincristine were detected on the floor and the laboratory bench top (0.47 to 0.50 ng/100 cm 2 ). moreover, the surface of auscultation table and extern side of gloves were contaminated after preparation and administration of vincristine (respectively 0.90, 314 and 510 ng/objet). after cleaning, 32% of samples in consultation room were positive. traces of vincristine were detected on the floor or objects (wall otoscope. . .:0.47 to 0.49 ng/100 cm 2 ). after cleaning, 40% of samples in ward were positive. traces of vincristine were detected on the floor and objects (boxes, wastebin, bowls. . .: 0.48 to 0.64 ng/objet) after cleaning and animals treatment. cyclophosphamide were detected on all areas tested (0.04 a 2.23 ng/objet). despite protective guidelines to avoid spread of cytotoxic drug, environemental exposure was demonstrated. however contaminations were limited and show that handling procedures of cytotoxic drugs are well controlled. most-elevated contamination level for vincristin were noticed on extern side of gloves depsite of using appropriate material. workers should pay a particular attention for gloves withdrawal to limit exposure. small amounts of vincristine were found in inapropriate places: computeur mouse,. . . even if there is few of those residues, we thought about people working on a regular basis in this room for other activities than chemotherapy., and we decided to adapt our clinical practices. evaluation of occupational exposure to cytotoxic drugs is an important step to prevent incidents and heigt awarness of nursing staff to apply those good clinical practices. disclosures: no disclosures to report. the tumor-associated inflammatory response had the effect of enhancing mammary tumorigenesis, helping incipient neoplasias to acquire hallmark capabilities, both in human and dogs. t-cells migration to the tumor site and the following activation may be the essential requirement for their promoting effect on tumors. in human breast cancer the signaling pathways of c-kit have been described as possibly being involved in differentiation, migration, survival, and maturation of t-cells and other inflammatory cells into tumor sites. in order to clarify this subject in canine mammary tumors (cmt), 80 malignant neoplasms were studied by using immunohistochemistry comparing the intratumoral cd3+ tlymphocytes and c-kit expression together with vegf, microvessel density (mvd) and clinicopathological characteristics of tumor aggressiveness. cd3+ t cells and high c-kit immunoexpression revealed a positive and statistically significant correlation with vegf (r = 0.503, p < 0.0001; r = 0.284, p = 0.023 for cd3 and c-kit respectively) and cd31 (r = 0.654, p < 0.0001; r = 0.365, p = 0.003 for cd3 and c-kit respectively). a statistically significant association (p = 0.039) and a positive correlation (r = 0.263, p = 0.039) between cd3+ t-lymphocytes and c-kit was also observed. tumors with high c-kit expression showed higher counts of cd3+ t-cells. the mvd of high cd3/vegf tumors was significantly more elevated (p < 0.0001). a similar association was observed for high c-kit/vegf tumors (p < 0.001). in this study high cd3/vegf, high c-kit/vegf and high cd3/c-kit tumors were statistically significantly associated with elevated grade of malignancy (p < 0.0001 for cd3/vegf, c-kit/vegf and cd3/ckit), presence of neoplastic intravascular emboli (p < 0.0001 for cd3/vegf and cd3/c-kit; p = 0.002 for c-kit/vegf) and presence of lymph node metastasis (p < 0.0001 for cd3/vegf, c-kit/ vegf and cd3/c-kit). tumors with high cd3/vegf (p = 0.006), high c-kit/vegf (p < 0.0001) and high cd3/c-kit (p = 0.002) expression were associated with shorter os time. inter-estingly the group of tumors with high c-kit/vegf retained their significance by multivariate analysis arising as independent predictor of os. results of this study suggest that t-lymphocytes may share common signaling pathways with c-kit and vegf in cmt progression and may contribute to increased angiogenesis, aggression and shorter os in these tumors. disclosures: no disclosures to report. lgl lymphoma, but no comparisons were made with cats with other diseases or other forms of feline lymphoma. therefore, the objective of this study was to assess differences in prevalence, signalment (breed, sex, and age), physical exam findings (body weight, body condition score, body temperature, heart rate, respiratory rate, and systolic blood pressure), and felv/ fiv status between cats with lgl lymphoma (group 1) and all other type of feline lymphoma (group 2). the electronic data-base of the san marco veterinary clinic was searched between january-2007 and december-2014 for cats with a cytological or histopathological diagnosis of lymphoma. differences between groups were assessed by t-test, mann whitney, pearson-chi square, pearson chi square yates corrected, and fisher's exact test. during the study period 176 out of 3858 sick cats seen at the clinic were diagnosed with lymphoma (group 1: n = 32; group 2: n = 144). the prevalence of all type of feline lymphoma between 2007 and 2014 compared to sick cats did not change over time ranging from 3.7% to 6.5% per year (p = 0.42; overall prevalence 4.5%, 95% ci 3.8-5.1). the lymphoma lgl prevalence between 2007 and 2014 compared to all types of lymphoma did not change over time ranging from 11.1% to 30.0% per year (p = 0.73; overall prevalence 17.9%, 95% ci 12.2-23.6). among the variables studied, only sex (group 1: 19 [59.4%] females, 13 [40.6%] males; group 2: 54 [37.5%] females, 90 [62.5%] males; p = 0.023) and age (group 1: 126ae34 months; group 2: 110ae57 months; p = 0.037) were significantly different between groups. sixteen out of 32 cats with lgl lymphoma were tested for their felv/fiv status resulting all felv-and one (6.3%) fiv+. seventy-four out of 144 cats with all other types of lymphoma were tested for their felv/fiv status resulting 28 (37.8%) felv+ and 13 (17.6%) fiv+. five cats (6.6%) were both felv+/fiv+. prevalence of felv infection was significantly lower (p = 0.002) in group 1 compared to group 2. there was no difference in prevalence of fiv infection between groups. lymphoma lgl affects more females and older cats compared to all other type of feline lymphoma. opposite to all other type of lymphoma, and in accordance to previous litterature information, felv+ status does not play a role in the pathogenesis of feline lgl lymphoma. disclosures: no disclosures to report. the activity of t regulatory cells (tregs) is known to be closely associated with the expression of foxp3 transcription factor. foxp3 regulatory t cells (foxp3treg) are a distinct group of t lymphocytes that have immunosuppressive properties. normally this cells work for prevention of harmful autoimmune responses, however can also interfere with beneficial immune responses such as anti-tumor immunity. in human breast cancer these cells play a crucial role in tumor progression. in canine mammary tumors (cmt) there are only a few studies and this topic are not welldocumented. in this study we included 80 malignant cmt and studied, by immunohistochemistry, the intratumoral foxp3 expression together with vascular endothelial growth factor (vegf), microvessel density (mvd, by cd31 antibody) and several clinicopathological characteristics. abundant foxp3treg cells was statistically associated with presence of tumor necrosis (p = 0.004), nuclear grade (p = 0.001), poor differentiation of tumors (p < 0.0001), high mitotic grade (p < 0.0001), high histological grade of malignancy (p < 0.0001), presence of neoplastic intravascular emboli (p < 0.0001) and presence of lymph node metastasis (p < 0.0001). intratumoral foxp3 levels were correlated with the levels of vegf (r = 0.427; p < 0.0001) and intratumoral mvd (r = 0.520; p < 0.0001). additionally tumors with abundant foxp3treg cells were associated with shorter overall survival (os) time (p = 0.0001). results suggest that treg cells play a role in cmt progression and may contribute to increased angiogenesis and aggression in these tumors. the association of intratumoral foxp3 expression with shorter os of animals suggests a utility of treg cells activity as a prognostic marker. disclosures: no disclosures to report. cyprus is an island state in the eastern mediterranean basin. no epidemiological studies have yet been performed on infectious agents in cats from cyprus. the aim of this study was to determine the prevalence of several infectious agents, including some vector-borne infections, in cats from cyprus. surplus edta-blood and serum samples were recruited from 176 cypriot cats, from which signalment and lifestyle characteristics were recorded. dna was extracted and real-time quantitative polymerase chain reaction (qpcr) assays were used to detect haemplasmas(mycoplasma haemofelis, 'candidatus mycoplasma haemominutum' and 'candidatus mycoplasma turicensis'), leishmania spp.and bartonella henselae. conventional pcr assays were used to detect ehrlichia/anaplasma spp. samples yielding positive results for leishmania spp. or ehrlichia/anaplasma spp. underwent further characterisation (sequencing). elisas were performed for the detection of l. infantum antibodies, feline leukaemia virus (felv) antigen and feline immunodeficiency virus (fiv) antibodies. statistical analysis was performed using spss for the assessment of any associations between variables and infectious agents. of the 176 samples extracted, 2 were excluded due to failure of ≥ one internal control pcr. of the remaining 174 samples, 46 (26.4%) were positive by pcr for haemoplasma including 13 (7.5%) for m. haemofelis, 36 (20.7%) for 'ca. m. haemominutum' and 12 (6.9%) for 'ca. m. turicensis'. nineteen (10.9%) were positive for b. henselae. one cat (0.6%) was pcr positive for ehrlichia/anaplasma spp. and sequencing revealed identity with anaplasma platys. leishmania spp. dna was detected in 6 of the 174 (3.4%) cats; sequencing revealed l. infantum in 4 of these cases. l. infantum serology was positive in 7 of the 162 cats tested (4.3%). only one cat was positive for both leishmania pcr and serology. of the 166 cats that underwent retroviral serology, 11 (6.6%) were felv and 30 (18.4%) were fiv positive. statistical analysis identified several significant associations (p < 0.05) including the following; haemoplasma infection and both outdoor access and feral-shelter cat origin, felv or fiv infection and both anaemia and feral-shelter cat origin. this study documents, for the first time, the presence of haemoplasmas, l. infantum, b. henselae, a. platys, felv and fiv in the feline population of cyprus. the prevalence of haemoplasma, fiv and b. henselae infections were among the highest reported in cats from mediterranean countries, while that of leishmania spp. was similar. this is the second report of a. platys infection in a cat from a mediterranean country. disclosures: no disclosures to report. bartonella species (spp.) are zoonotic pathogens, and infections in cats are common. prevalence in cats from southern germany is still unknown. the aim of this study was to determine the prevalence of bartonella spp. dna in blood of cats in southern germany and to evaluate associations between bartonella bacteremia, housing conditions, feline immunodeficiency virus (fiv), and feline leukemia virus (felv) status, including progressive, regressive, and abortive felv infection. blood samples of 479 cats that were presented to different veterinary clinics in southern germany for various reasons were tested for bartonella spp. dna using a previously published conventional polymerase chain reaction (pcr) targeting a fragment of the 16s-23s rrna intergenic spacer region. for statistical analysis, fisher's exact test was used. prevalence rate of bartonella spp. bacteremia was 2.5% (12/479; ci: 0.01% -0.04%). b. henselae was amplified in 11 of these cats. one cat was positive for b. clarridgeiae dna. most of the infected cats were clinically healthy, but half of the cats had thrombocytopenia, potentially caused by their bartonella spp. infection. there was no significantly higher risk to be infected with bartonella spp. when living mainly outdoors or being fiv-or felv-infected. prevalence of bartonella spp. bacteremia is low in southern german cats, but there is still a risk of human bartonella infection associated with cat ownership. most clinical changes of the bartonella spp.-infected cats were related to other diseases. however, thrombocytopenia was common and further studies are required to define its potential clinical relevance. disclosures: no disclosures to report. ante-mortem diagnosis of feline infectious peritonitis (fip) is still challenging. the aim of this study was to evaluate sensitivity and specificity of a 'combined reverse transcription nested polymerase chain reaction (rt-npcr) and sequencing approach', detecting mutations at 2 different nucleotide positions within the spike gene, that previously were shown to correlate with the fip phenotype. the study population consisted of 64 cats with confirmed fip and a defined control group of 63 cats for which fip was considered an important differential diagnosis, but that were definitively diagnosed with other diseases. blood and/or effusion samples were examined for feline coronavirus (fcov) rna by rt-npcr and, if positive, nucleotide positions 23531 and 23537 were sequenced for nucleotide transitions. sensitivity, specificity, negative and posi-tive predictive values were determined and 95% confidence intervals (95% ci) calculated. rt-npcr detected fcov in 38 cats in blood (n = 3) and/or effusion (n = 36); all of them had fip. one of the mutations of interest was found in 2/3 of the pcr-positive blood samples and in 32/36 of the pcr-positive effusion samples. diagnostic specificity of the 'combined rt-npcr and sequencing approach' was 100% in blood (95% ci 83.9-100.0) and effusion (95% ci 93.0-100.0). diagnostic sensitivity was 6.5% (95% ci 0.8-21.4) in blood and 65.3% (95% ci 50.4-78.3) in effusion. a positive test result therefore confirms a suspicion of fip. a negative result, however, cannot be used to rule out fip, especially if only blood is available. therefore, if no effusion is present, diagnosis of fip still remains challenging. disclosures: dr. elisabeth mueller is the managing director of laboklin gmbh & co.kg. dr. karola weider is employed at laboklin gmbh & co.kg. this laboratory offered the 'combined rt-npcr and sequencing approach' on a commercial basis and performed the testing in this study. feline infectious peritonitis (fip) is a viral disease caused by the virulent strain of feline coronavirus (fipv). the disease can appear under 2 clinical forms, dry or effusive, both leading to a fatal outcome. diagnosis was based on histopathologic lesions on necropsy until the recent discovery of mutations associated with the fipv strain in the 3c and spike (s) genes. our main goal was to detail the distribution of 3c or s gene mutations in different biological samples of cats suffering from fip. this was a retrospective, observational study of 33 cats showing clinical signs compatible with fip. ten out of 33 cats were of pure breed. 57.7% were males and 42.3% females. median age was 36.5 months at presentation. the clinical presentation, pathologic findings and virologic data were reviewed. according to clinical signs, 11 cats were classified with a dry form and 22 with a wet form of fip. the main clinical signs included dehydration, hyperthermia, icterus, abnormal abdominal palpation, neurological and ocular disorders. when possible blood, fecal material, effusion, fine needle aspiration (fna) from relevant organs or a combination of these, was recovered from each cat. feline coronavirus (fcov) was first researched by rt-pcr, then the 3c and part of the s genes were sequenced to determine the eventual presence of mutations. among the 11 dry cases, fcov was detected in 2/8 blood samples, 3/8 fecal samples and fna (11/11). among the 22 wet cases, 9/15 blood samples, 12/14 fecal samples and all effusion samples (21/21) were positive for the presence of fcov. 3c mutations were never found in fecal samples but were found in 6/10 effusion samples and in 8/8 fna. s mutations were detected in 4/9 fecal samples, 8/9 fna and 14/15 effusion samples. for 3 cats, no mutation, neither in 3c or s genes was identified despite the confirmation of fip by necropsy. s gene mutation is more frequently observed than 3c gene despite in 2 cases where only 3c mutations were identified. moreover the presence of strains harbouring s mutation in feces has never been described before and could suggest the possible diffusion of fipv among feline population. in conclusion, viral diagnosis of fip based on rt-pcr sequencing in effusion and fna samples is essential. rt-pcr resulting from blood samples should be carefully interpreted because of high risk of missing fipv. finally, searching for mutations in both s and 3c genes is recommended. disclosures: no disclosures to report. methicillin resistant staphylococcus aureus (mrsa) has recently become a great concern for pet animals' disease and zoonotic infection. mrsa strains transfer between pet animals and humans could occur. the aim of the present study was to determine the occurrence of mrsa in 34 household dogs. from january to june 2012, clinical samples were collected from 34 dogs, patients of the veterinary teaching hospital of the department of veterinary sciences of messina (italy), affected by several diseases of various origins. all samples were processed by bacteriological conventional methods for isolation and identification. all strains were tested for phenotypic susceptibility to oxacillin and were subjected to a pcr protocol for the detection of meca gene. strains carrying the gene were considered methicillin resistant (mrs). lastly, on both mrs and methicillin sensitive (mss) strains, kirby-bauer disk diffusion susceptibility testing were performed to highlight resistance profiles using 44 molecules belonging to the main classes of antimicrobials used in veterinary practice. strains resistant to at least one molecule of 3 or more classes of antibiotics were considered multidrug-resistant (mdr). the statistical analysis of the results was made using the z-test by a primer ò software. forty staphylococcus spp. strains were isolated, belonging to 12 species. the most frequently isolated microorganisms were staphylococcus aureus with 14 isolations (35%) and staphylococcus pseudintermedius with 11 isolations (27.5%), followed by staphylococcus epidermidis with 4 isolations (10%) and staphylococcus cohnii and staphylococcus warneri, both with 2 isolations (5%). a single isolation (2.5%) was obtained for each of the species staphylococcus chromogenes, staphylococcus haemolyticus, staphylococcus lentus, staphylococcus lugdunensis, staphylococcus saprophyticus, staphylococcus simulans and staphylococcus sciuri. thirteen (32.5%) strains of staphylococcus spp. were phenotypically resistant to oxacillin and 3 staphylococcus aureus (7.5%; n.2 from pyoderma, n.1 from exudative pleural effusion) were positive for the meca gene. all 40 strains of staphylococcus spp. were mdr. our results showed the presence of mrsa and multidrug-resistant staphylococcal strains in household dogs. a lack of correspondence between antimicrobial susceptibility tests and molecular methods was found in the present study. disclosures: no disclosures to report. a fourth haemoplasma called 'candidatus m. haematoparvun-like' (cmhp) was latter identified in cats. the only published study in chile was carried out in 30 cats, with prevalences by pcr of 3.3% to mhf, and 10% to cmhm.the aim of this study was to perform molecular detection of haemoplasmas in cats from valdivia, southern chile. blood samples were taken from 384 cats and used for haemoplasmas dna detection by quantitative real time pcr (qpcr) at universidad austral de chile. qpcr protocol was based on detection of feline dna targeting 28s housekeeping gene and mycoplasma spp. 16s rrna gene (universal primers, my16sf forward, my16sr1 y my16sr2, both reverse) by sybr green method. the melting temperature (tm) analysis allowed identifying the infecting mycoplasma species (mhf, cmhm, cmt). it was not posible to identify haemoplasmas species on co infected cats, so a second qpcr specie specific protocol was applied on these samples. second qpcr protocol was based on 16s rrna gene, with specific primers to detect mhf, cmhm, cmt and cmhp. all samples (384/384) were positive to 28s gene, proving presence of cat dna. from the 384 cats, 15.1% (58/384) were positive to haemoplasmas, where 7.8% (30/384) corresponded to cmhm (tm 73.5-75.0°c), 4.4% (17/ 384) to mhf (tm 75.0-76.5°c), 2% (4/384) to cmt (tm 76.5-78.0°c) and 1.8% (7/384) to co infections. associations between cmhm+mhf, cmhm+cmt, cmhm+cmhp and cmhm+mhf+ cmhp were detected on co infected animals. these results agree with those found in previous reports from chile, europe, eua and brazil, where cmhm is the most prevalent species and co infections are less frequent. valdivia cats are infected by 4 different haemoplasma species and cmt and cmhp are reported for the first time in chile.founding: did uach, project s-2014-25 disclosures: no disclosures to report. clinical manifestations of canine visceral leishmaniasis (canl) are non-specific and include progressive weight loss, anemia, lymphadenomegaly, hepatosplenomegaly, dermatological, renal and ocular alterations. cardiac lesions resulting in clinical signs has been scarcely described in dogs with vl, and the presence of the parasite in the cardiac tissue has been involved in few reports. accordingly, the present study aimed to evaluate histopathological abnormalities in cardiac tissue from dogs naturally infected by leishmania infantum chagasi. a total of 20 dogs were evaluated. all dogs were symptomatic but no one presented clinical signs of cardiac involvement. in compliance with a federal law and under the owners' signed consent, all dogs were submitted to euthanasia and comprehensive post-mortem evaluation. samples from right atrium free wall (ra), right ventricle free wall (rv), interventricular septum (ivs) and left ventricle free wall (lv) were collected and evaluated. tissue samples were fixed in formalin, embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin (he) and anti-leishmania immunohistochemistry was also performed. the study was approved by the ethics committee in animal experimentation and animal welfare (protocol number 0463/2013). histopathological changes were observed in at least one of the 4 evaluated cardiac regions in 75% (15/20) of the dogs. the most frequent cardiac injury was an inflammatory reaction, characterized by the presence of mononuclear cell infiltrate in different degrees. of the evaluated regions, ra was the one with the highest incidence of histopathological changes, observed in 80% (12/15) of the animals, followed by rv, lv and siv, affected in 73.3% (11/15), 66.7% (10/15) and 53.3% (8/15) of the dogs, respectively. immunohistochemistry revealed amastigotes in the cardiac tissue in 70% (14/20) of the dogs. a positive correlation was found between cardiac lesions and the presence of amastigotes in the myocardium (p < 0.05). disclosures: no disclosures to report. canine leishmaniosis is a life threatening zoonotic disease. the combination of meglumine antimoniate and allopurinol is consid-ered the most effective therapy for canine leishmaniosis and constitutes the first line protocol against this disease. allopurinol is a parasitostatic drug used in long-term to maintain low parasite loads and to avoid clinical relapses. traditionally, allopurinol is considered a very safe drug in the dog. however, some reports indicate that xanthinuria and xanthine urolithiasis is produced after prolonged therapy with allopurinol in the dog. the aim of this prospective study was to evaluate the prevalence of urinary adverse effects of allopurinol treatment (10 mg/kg/bid/po) in dogs with leishmaniosis. diagnosis was made by compatible clinicopathological abnormalitites with leishmaniosis and high leishmania infantum-specific antibody levels assessed by quantitative elisa. once leishmaniosis was diagnosed, a close follow-up (day 0, 30, 90, 180 and 360 during treatment) including physical examination, baseline laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis, urinary protein/creatinine ratio) and abdominal ultrasound was performed. in our preliminary results, 13 dogs were included. dogs did not present any urinary abnormalities based on biochemistry profile, urinalysis and abdominal ultrasound at the time of diagnosis. four out of 13 presented xanthinuria (day-30 (n = 3), day-90 (n = 4), and day-180 (n = 4)). two out of 13 dogs presented renal mineralization at day-90 of treatment. two out of 13 dogs presented bladder urolithiasis since day-90 of treatment. xanthinuria was presented initially in all dogs that developed renal mineralization or bladder urolithiasis. dogs with renal mineralization and urolithiasis were treated with a restricted protein diet and, so far, they did not develop renal disease. the present study describes early xanthinuria, renal mineralization and urolithiasis as adverse effects due to chronic allopurinol treatment in dogs with leishmaniosis. neither mineral analysis nor renal biopsy was performed to confirm the origin of these lesions, but no urinary abnormality was present before allopurinol treatment was instituted. a thorough monitoring of dogs treated against leishmaniosis combined with urinalysis and abdominal ultrasound should be performed to evaluate urinary adverse effects and to help in the clinical management of these adverse effects. disclosures: no disclosures to report. giardia duodenalis is one of the most important gastrointestinal parasites in dogs and cats with a zoonotic potential. in germany the prevalence in dogs and cats reaches up to 29% and 24%, respectively. genotypes of 2 genetic assemblages of the parasites infect humans (assemblages a and b) and other mammals including small animals. in contrast, parasites of the assemblages c and d are specific for dogs, assemblage f for cats. objectives of the study were to analyse the prevalence, potential epidemiological risk factors and symptoms of g. duodenalis infections in dogs and cats. to detect g. duodenalis, feces from dogs and cats was analysed with an elisa technique. after dna extraction real time pcr as well as multi-locus sequence typing was performed for the following gene loci: triosephosphate isomerase-, glutamate dehydrogenase-, beta-giardin-gene, ssu rrna. with a questionnaire possible epidemiological risk factors were evaluated. statistical analyses were performed using spss 21 (odds ratio, kolmogorow-smirnow test, spearman correlation). fecal samples of 618 dogs and 156 cats were collected over a time period of 23 months. the elisa test was positive in 101/618 dogs and 10/156 cats. 67 of 101 giardia positive dogs and 9 of 10 positive cats had gastrointestinal signs. genotyping was successful in 54 of 101 dog samples and were assigned to assemblages as follows: assemblage a (n = 12), a/c (n = 2), a/d (n = 4), b (n = 2), b/d (n = 1), c (n = 7), c/d (n = 2), d (n = 24). only one of 10 positive cat samples could be genotyped and was atypically identified as assemblage d. significant correlations between giardia infection and age, clinical signs, deworming status and staying abroad were found. in this monocenter study a prevalence rate of 16.3% in dogs and 6.4% in cats was detected, which is in good accordance with previous studies. the study further highlights a high rate (34%) of asymptomatically g. duodenalis infected animals. as potential zoonotic assemblages were detected, transmission of giardia from small animals to humans (and vice versa) cannot be excluded. especially young and not dewormed animals had a higher prevalence. disclosures: no disclosures to report. canine parvoviral enteritis remains a common cause of morbidity and mortality in young dogs. the goal of this study was to document a large cohort of affected dogs and analyze several factors as possible predictors of fatal outcome. medical records were retrospectively searched for dogs with parvoviral enteritis diagnosed with a positive fecal antigenic test or a fecal pcr. dogs were included only if the medical records were complete. the population was compared to the reference population of the hospital on the same time period with chi square tests and several factors were analyzed as possible predictors of death with a logistic regression. one hundred and fourty seven cases were included. seventy percent of the dogs were non vaccinated puppies under the age of 6 months. intact females and rottweiler, american staffordshire terrier and french beauce shepherd dogs were over-represented. clinical signs such as vomiting, diarrhea and dehydration were present in 92.7%, 86.4% and 70.1% of the dogs respectively. hyperthermia, anemia and leucopenia were observed in 17.8%, 26.5% and 36. 1% of dogs respectively. the majority of the affected dogs were hospitalized for 3 to 6 days and the mortality rate was 14.3% (21/147 dogs). hypoglycemia at admission was observed in 11/81 (13.6%) dogs in which blood glucose was measured and was the only risk factor associated with death (p < 0.05). in this study, a predisposition of rottweiler, american staffordshire terrier and french beauce shepherd dogs was observed and hypoglycemia at admission was the only predictor of fatal outcome. disclosures: no disclosures to report. canine parvovirus (cpv) infections in dogs remain widespread around the world and still represent a major health threat in puppies. all vaccine manufacturers include this component in their core vaccination package, recommending 2 injections at 3-5 weeks interval from 7 to 8 weeks of age. despite broad vaccination coverage, number of reports suggesting lack of efficacy in vaccinated dogs have been reported, which implicate vaccines belonging to all major manufacturers. these cases are usually considered as being linked to the interference with maternal antibodies (matab), able to persist beyond 12 weeks of age, which has led most expert groups to recommend a third vaccination around 16 weeks of age. persistence of matab actually represents a major issue when immunizing puppies against parvovirosis. indeed, matab titres higher than 1/40 in the haemaglutination inhibition (hi) test can still inhibit vaccine uptake whereas such titres do not prevent field virus infection. in contrast, hi titres higher than 1/80 to 1/120 are usually considered as protective against disease and virus excretion. this "immunity gap"is therefore a critical period for the puppy and the outcome of the vaccination. in order to evaluate the impact of residual mda on the efficacy of a standard primary vaccination protocol, we performed a vaccination field trial with serological follow-up. 88 puppies from 7 to 24 weeks of age presented at veterinary practice received 2 injections at 4 weeks interval. serology was performed by elisa before (at d0/v1), during (at d28/v2) and after (d42) vaccination. average maternal antibodies titres were strongly correlated with the age of the puppy at primary vaccination, remaining at vaccine inhibiting level until~10 week of age. average titres increased significantly after 1st injection of primary vaccination in most groups and in all groups after the 2nd injection of primary vaccination. individual variability remained significant: vaccine uptake was inversely and strongly correlated to the pre-vaccinal matab titre at vaccination. 7 out of 88 puppies (8%) didn't seroconvert, despite vaccination complying to the recommended schedule. vaccination was started in such dogs between 8.5 and 10.5 weeks and completed between 12.5 and 14.5 weeks and average initial matab titre was 2.4 log10 compared to 1.6 for the general population. in conclusion, this trial supports the recommendation of an additional injection of primary vaccination at 16 weeks, especially in areas of high parvovirus prevalence / pressure, where high levels of matab are likely to be transferred to puppies. disclosures: all authors are employees of merial. (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) . 5.3% of the dogs were pcr positive. the aim of our study was to evaluate the usefulness of determining serum level of c-reactive protein (crp) in dogs naturally infected with bacterium a. phagocytophilum as a possible indicator of the clinical phase of the disease. pcr and/or ifa positive dogs with clinical presentation and/or thrombocytopenia were included in the study. based on the results, the dogs were divided into 4 groups: pcr positive dogs; ifa positive (subdivided according to titer level from 1:128 ->1:2048) and pcr negative dogs; positive control group -pcr and ifa negative dogs with clinical signs and/or thrombocytopenia; negative control group -clinically healthy, pcr and ifa negative dogs. serum level of crp was determined using lifeassays ò canine crp (lifeassays, lund, sveden) . an elevated concentration of crp (> 35 mg/l) was determined in pcr positive and ifa positive dogs with an ifa titer ≥ 1:2048 and coincides with the presence of clinical signs (most commonly general clinical signs, elevated body temperature, gastrointestinal problems) and/or mild (14.3%) or severe (71%) thrombocytopenia. the assessment of crp concentration, in correlation with certain clinical alterations and thrombocytopenia, suggests that crp concentration is elevated in the acute phase of the disease and is in correlation with the aforementioned changes therefore can serve as an additional diagnostic parameter. the crp concentration in ifa positive dogs, regardless of ifa titer levels, and with present clinical signs and thrombocytopenia is higher (above detectable level, > 10 mg/l) than in dogs without clinical signs or laboratory alterations, which may speak in favor of reinfection or reactivation of a persistent infection at least in cases when no other cause of inflammation can be found. specific treatment would therefore be reasonable in such cases, especially in cases of rising crp concentration. disclosures: no disclosures to report. gallbladder agenesis is a very rare cause of elevated liver enzymes in dogs. in this study, we evaluated the features of 15 dogs [6 males (3 castrated) and 9 females (2 spayed)] with suspected gallbladder agenesis on ultrasonography. five different breeds were included: chihuahua (n = 9), toy poodle (3), german shepherd (1), jack russell terrier (1), and shiba dog (1). the median age was 1.9 (0.7-7.4) years. ten dogs were asymptomatic, while the other 5 dogs showed decreased appetite (3), vomiting (3), ascites (2), seizure (1), and diarrhea (1). all dogs showed elevated liver enzymes, with high alanine aminotransferase levels (median, 306 u/l; 38-1374 u/l) in 13 dogs and high gamma-glutamyl transpeptidase levels (median, 12 u/l; 3-19 u/l) in 11. gallbladder agenesis was confirmed using laparoscopy in 12 dogs and laparotomy in 3. liver biopsy samples were obtained from all dogs. additional computed tomography cholangiography was performed for 12 dogs using a 16-slice multidetector computed tomography (mdct) scanner following the intravenous administration of contrast medium (meglumine iotroxate). the obtained images were analyzed on a workstation, and they revealed an absent gallbladder in 9 dogs and a vestigial gallbladder in 3. the common bile duct was dilated in 5 dogs. for all dogs, laparotomy or laparoscopy was used to visualize the gallbladder and liver abnormalities, including malformed lobes and surface irregularities. acquired portal systemic collaterals were visually confirmed in 5 dogs, who also exhibited hypoplasia of the portal vein on histological examination. in conclusion, most animals with gallbladder agenesis were asymptomatic in our study, indicating a good long-term prognosis. however, symptoms associated with portal hypertension must be monitored in animals with primary portal vein hypoplasia. disclosures: no disclosures to report. assessment of systolic arterial blood pressure (sap) is an important tool in small animal internal medicine practice, especially with diseases or clinical conditions that can cause hypertension or hypotension. the doppler method is noninvasive and has several advantages compared to oscillometric method. there are few studies about the effect of body position on sap in conscious dogs. the hypothesis was that animal positioning during measurement alters sap values. the study design was prospective and randomized regarding order of positioning measurements. one hundred and twenty client-owned, conscious, healthy or sick adult dogs, weighing up to 10 kg were included. sap was recorded by doppler ultrasonography following american college of veterinary internal medicine consensus statement with animals positioned in sternal recumbency, right lateral recumbency and with the dog laying down on owner 0 s lap. the order of body position was raffled at the time of measurement. five consecutive measurements on each body position were performed always on left forelimb and the average was calculated. sap values were higher in sternal recumbency (153 mmhg, p 25%-75% = 134-176.5; p < 0.0126) compared to those obtained on the owner 0 s lap (139 mmhg, p 25%-75% =125.5-159.8), and both were similar to right lateral recumbency (141 mmhg, p 25%-75% =125-159). these results suggest that sap measurement obtained on owner 0 s lap or right lateral recumbency can be used on clinic routine, but sap measurement obtained on sternal recumbency should be avoided, because such measures may be overestimated. disclosures: no disclosures to report. canine patients may be presented for blood pressure (bp) assessment when clinical diseases associated with systemic hypertension (ht) are suspected but not confirmed; this population may encompass patients that have normal bp, true ht or situational ht. the clinician's aim is to identify animals with ht reliably, while minimizing false positives. this prospective study investigated the repeatability of duplicate within-visit systolic bp assessments (sbp1 and sbp2) in consecutive canine patients presented for bp assessment in the small animal clinic (91 duplicate sbp recorded from 70 dogs) and in a control group of healthy dogs (37 duplicate sbp obtained from 19 control dogs), resulting in 128 duplicate measurements for analysis. doppler methods were used for 16 duplicate assessments and oscillometric methods were used for 112 duplicate assessments; cuff size/location were consistent within any dog. sbp ≤ 160 mmhg was considered normal (nml); sbp > 160 mmhg was considered abnormal (abn). median (range) elapsed time between duplicate readings was 30 (5-310) minutes; 75% of sbp2 were obtained within 80 minutes of sbp1. there was no correlation between elapsed time and change in sbp (p = 0.40). 70% of sbp2 were equal to or lower than sbp1; median decrease was 18 (0no dog with sbp1> 200 mmhg (n = 15) had nml sbp2. more dogs with abn sbp1 were panting (26/71 scored, 37%) compared to the group with dogs with nml sbp1 (6/44 scored, 14%, p = 0.02). sbp2 of dogs that stopped panting (14/23, 61%) tended to decrease (p = 0.16). within-visit repeatability of bp diagnosis was good in dogs with nml sbp1, but apparent false positive diagnoses of ht occurred in 37% of dogs with abn sbp1. sbp1 > 200 mmhg was repeatable in all dogs. panting may be associated with increased measured sbp by these methods. duplicate within-day measurements may help identify false positive ht diagnoses in dogs with initial sbp measurements > 160 mmhg. disclosures: no disclosures to report. hypovitaminosis d has previously been shown to be prevalent amongst dogs with protein losing enteropathy (ple). outcome is generally poor in canine ple, and there is a lack of studies identifying underlying risk factors. the hypothesis of this study was that low vitamin d 3 serum concentrations could be a risk factor for bad outcome in such patients. medical records for dogs seen at the royal veterinary college between 2005 and 2014 were reviewed to identify dogs with a diagnosis of ple confirmed by histopathology. dogs were included in the study if they had serum samples frozen within 30 minute after sampling, had been kept at à80 degrees c until analysis, and if clinical activity scoring (cce-cai) had been recorded at the time of diagnosis. forty-three dogs were included in the study. follow-up with referring veterinarians was made to determine outcome of patients. patients were divided into two groups: patients deceased due to ple (poor outcome group, n = 22) and patients alive or deceased due to another disease (good outcome group, n = 21). treatments for patients were allocated to two groups: one group consisted of patients who were prescribed diet only and the other group received diet and immunosuppressive agents. samples were sent on dry ice to michigan state university's diagnostic center for population and animal health. ionised calcium (ica) was measured using an ion specific electrode and 25(oh)d was measured using a commercially available radio-immunoassay that has been validated for use in veterinary medicine. comparisons of outcome groups for age, ccecai, treatment, serum 25(oh)d and ica were performed using a mann-whitney u test or chi 2 . logistic regression analysis was performed to determine possible risk factors for poor outcome. results: ccecai scores, age, and ica concentrations between the two groups were not significantly different. there was a significantly greater number of dogs treated with food alone in the group with good outcome (13/22) than in the poor outcome group (2/21, p = 0.001). furthermore, median serum 25(oh)d concentration was significantly lower in patients with poor outcomes (16.5 nmol/l, range 0-66 nmol/l) compared to patients with good outcomes (37 nmol/l, range 6-81 nmol/l, p = 0.017). using logistical regression, 25(oh)d serum concentration was a statistically significant factor for poor outcome (p = 0.03), with an increase of 25(oh)d serum concentration reducing the odds of having a poor outcome (odds ratio = 0.96, 95% ci: 0.93-0.997). further studies are required to investigate vitamin d as a potential adjuvant therapeutic agent in ple patients. disclosures: no disclosures to report. campylobacter jejuni (cj), c. upsaliensis (cu) and c. helveticus (ch) are commonly isolated from dog and cat faeces but association with clinical signs is discordant or lacking. cj is a recognized human pathogen, cu is considered an "emerging" pathogen and ch is not considered pathogenic despite a high level of genetic similarity. recently, the greater wax moth, galleria mellonella, was described as an animal model of disease; these invertebrates have a high degree of functional and structural homology with the mammalian innate immune system. this study aimed to evaluate the pathogenic potential of cj, cu and ch using the galleria mellonella larvae model. twelve isolates of cj, 14 of cu and 11 of ch from dogs and cats were used for the inoculation of 2490 larvae. inocula were prepared by suspending isolates in phosphate-buffered saline (pbs) from which three 100-fold dilutions were made. each dilution was tested in duplicate sets of 10 larvae. each larva was injected with 10-15 ll into the haemocoel via the last left pro-leg using 31g insulin syringes. controls consisted of 246 pbs inoculated larvae and 267 un-inoculated larvae. survival of larvae at 37°c in a h 2enriched microaerobic atmosphere was monitored for 8 days postinjection. one subset of isolates was grown in mueller-hinton broth and used for the preparation of secretory products, and another grown on blood-agar and suspended in pbs for heat inactivation of 10 minutes at 100°c for testing of whole-cell lysates and heat-stable insoluble and soluble components. the overall median survival of larvae was 80% with cj [iqr 10-100], 100% with cu [iqr 80-100], 100% with ch [iqr 60-100], 100% with pbs [iqr 92-100] and 100% for un-inoculated larvae [iqr 100-100]. a dose-dependent association was evident for each species with larval survival being similar between a low bacterial dose and pbs. larval survival presented a consistent pattern between species for medium and high bacterial loads; cj had a higher and faster larval death rate than cu and ch (p < 0.001), but no difference was observed between cu and ch (p = 0.06). there were no significant differences between species in any of the assays with secretory products, inactivated cells and soluble/insoluble cellular components. the observations within this invertebrate disease model support a varying pathogenic potential between the species studied that appears related to the (patho)biology of the species rather than their cellular components or metabolic products. the invertebrate animal model is promising in comparative pathogenicity studies. disclosures: no disclosures to report. a. grellet 1 , s. dubois 1 , a. feugier 1 , c. girardet 2 , s. magnan 2 , v. andr eo 2 , g. trombini 2 , c.a. boehringer 1 , j. suchodolski 3 , j. steiner 3 . 1 royal canin, aimargues, france, 2 veterinary department of the french army health service, suippes, france, 3 gastrointestinal laboratory, texas a&m university, college station, tx, usa acute stress from medium or high duration high-intensity exercise has been reported to be associated with an increase in serum c-reactive protein (crp) concentrations, an important acute-phase reactant in dogs. however, the effect of exercise on fecal s100a12 concentration, a biomarker of intestinal inflammation has not previously been evaluated in dogs. the goal of this study was to determine if moderate intensity short duration exercise causes an increase in crp and/or s100a12 concentrations in dogs, potentially leading to misinterpretation of their results. thirty-seven adult military working dogs (german and belgian shepherd dogs; 36 males; mean age = 4 years [1.3-7.9]) were included in the study. fecal quality, fecal s100a12, and serum crp concentrations were evaluated just before and after standardized exercise (30 minutes of bikejoring at a speed of 16 km/h). fecal quality was evaluated based on a 5-point scale (from 1: liquid to 5: dry and hard feces). fecal s100a12 and crp concentrations were assayed with previously validated elisa tests. data were analyzed with an anova test for repeated measurements (sas software). results are presented as medians and ranges. serum crp concentrations increased significantly after exercise (median before and after excercise 5 mg/l [2-12] and 6 mg/l [5-12] (p = 0.002). also, fecal s100a12 concentrations were significantly higher after exercise compared with baseline concentrations (6 ng/g [2-437] vs. 4 ng/g [1-389], p = 0.043). no significant effect of exercise on fecal score was observed (4 [2.5-4.5] before and after the exercise; p = 0.482). our study demonstrates that a moderate-intensity, short-duration effort performed by healthy army dogs causes significant increases in fecal s100a12 and serum crp concentrations, as compared with baseline values, but within the respective reference intervals. therefore, a moderate exercise does not present a confounding variable in the interpretation of fecal s100a12 or serum crp concentrations in healthy dogs. disclosures: this study was performed thanks the financial support of royal canin. imaging is an integral part of the work-up of canine gastrointestinal (gi) disease. radiography and ultrasonography are noninvasive modalities that can evaluate the bowel, but many findings lack desirable sensitivity or specificity. endoscopy directly visualizes gi mucosa, but is limited by the length of the endoscope and the need for general anesthesia, advanced training and expensive equipment. ambulatory light-based imaging (ali) is a new imaging modality that utilizes high-resolution cameras, a microprocessor, and led illumination to non-invasively visualize the gastrointestinal mucosa. ali is performed by oral administration of a fully automated device the size of a pill that is propelled by peristalsis. the aim of this study was to analyze image quality and gi transit times in a series of five client owned dogs undergoing ali. dogs were food-restricted for 24 hour before and 8 hour after capsule administration. capsules were retrieved and images were downloaded and analyzed. video clips of 300 frames duration were obtained from the stomach; proximal, middle and distal small intestine; and proximal colon for assessment of image quality. 3 internists rated the images on a scale of 1-10 (1 = poor, 10 = ex-cellent) based on clarity and resolution of images, and obscuration of the mucosa by fluid, bubbles or debris. scores for each region were compared using general estimating equation analysis. gastric and small intestine transit time were calculated based on visualization of passage of the capsule from the stomach to duodenum, and ileum to colon. clinical analysis of the entire video was performed by one of the authors. ali was successfully performed in 5/5 patients, with no adverse effects. average study duration was 15.7 ae 4.1 hour and mean image acquisition count was 22.572 ae 17.315. gastric and small intestinal transit times were 79.2 ae 39.8 minute and 119.4 ae 43.7 minute, respectively. median (range) image quality scores were 9 (8-10), 8 (6-10) and 6 (5-9), for the stomach, si and colon, respectively. image quality scores were significantly higher in the stomach and si than in the colon (p < 0.001). visualized lesions were consistent with gi ulcers (2 dogs), inflammatory bowel disease (1 dog), and bilious vomiting syndrome (1 dog). one dog receiving chronic nsaids had a normal study. ambulatory light-based imaging resulted in good to excellent image quality throughout most of the gi tract. bowel preparation should be considered to enhance visualization of the colon. ali was safe and easy to perform in ambulatory dogs, and should therefore be considered in the work-up of canine gi disease. disclosures: drs. hardy and solomon are employed by infiniti medical. escg-p-6 establishment of a severity scoring system for outcome prediction in dogs with pancre-atitis. p.c. liu 1 , f.r. wu 2 , y.j. lee 3 , b.l. su 3 . 1 graduate institute of veterinary medicine, national taiwan university, taipei, taiwan, 2 national taiwan university veterinary hospital, national taiwan university, taipei, taiwan, 3 institute of veterinary clinical sciences, national taiwan university, taipei, taiwan canine pancreatitis is the most common exocrine pancreatic disorder. the prognosis of canine pancreatitis is variably and no logistic regression constructed severity scoring systems are available. four hundred and thirty nine dogs diagnosed as pancreatitis with acute onset of compatible clinical signs, a positive snap ò cpl[trademark] test, and/or associated abdominal ultrasonographic abnormalities between january 2009 and december 2012 were presented at national taiwan university veterinary hospital (ntuvh). one hundred and three dogs hospitalized with complete medical therapy and outcomes were selected for further analysis. the 103 dogs were divided into survival (n = 61) and non-survival (n = 42) groups. forty-seven parameters including signalment, clinical signs, physical examinations, clinicopathological examination, complications and concurrent diseases were analyzed and compared between the two groups. logistic regression analyses were performed in this study. variables with p ≤ 0.1 were considered for further analyses. the mortality in this study was 40.8%. age, heart rate, respiratory rate, white blood cell count, albumin, bun, creatinine, potassium, presence of systemic inflammatory response syndrome (sirs) and presence of oliguria or anuria 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 dog by summing all weighting factors. the total scores of each dog ranged from 10 to 70. the severity scores in this study achieved an area under the receiver operating characteristic (auroc) of 0.871. the optimal cut-off point for discriminating outcome was 24.5 with a sensitivity of 78.6% and specificity of 90.2%, respectively. the mortality was 84.6% with a score ≥25, whereas 14.1% with a score ≤24. there was a significant difference (p < 0.001) between the two groups seperated by the cut-off point. the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in dogs with pancreatitis. disclosures: no disclosures to report. glucocorticoids (gcs) are known for their anti-inflammatory and immunmodulatory properties and are therefore often used in the therapy of canine inflammatory bowel disease (ibd). it was recently shown that endogenous gcs are also produced in the intestinal epithelium of men and mice and influence the gastrointestinal immune system in case of inflammatory or neoplastic conditions. thus, the aim of this project was to prove that gcs can be produced or metabolized in the canine intestinal epithelium. five healthy beagle dogs were included into this prospective study. all dogs were clinically examined, given a clinical score using the canine ibd activity index (cibdai) scoring system, also gastrointestinal endoscopy was performed. mucosal biopsy specimens from duodenum were examined histologically from a board certified pathologist using the wsava grading. biopsy incubation of 8-10 endoscopical mucosal biopsies in tissue culture medium with 3 h-labeled progesterone in the absence of any stimulation was performed. the mean age of the included dogs was 3.24 + 1.9 years, the mean weight was 17.8 + 1.8 kg. all beagle dogs had a mean clinical score of 0 + 0. the mean wsava scoring was 2 + 1.2. after 4 hours, supernatant was harvested and radioactive progesterone metabolites formed were detected using high performance liquid chromatography plus liquid scintillation counting. in all dogs the 3 h-progesterone was metabolized into various steroid species, nevertheless a local production of cortisol could not be proven. in summary, it could be shown that precursors of gcs can be metabolized by healthy canine intestinal mucosal tissue. disclosures: no disclosures to report. we studied the relationship between pancreatitis and cardiac injury in dogs and cats. previously, we validated a cardiac troponin i (ctni; vet j 185:50-7, 2010) assay for sensitive and specific detection of cardiac injury in domestic animals. we found various non-cardiac diseases of dogs and cats were associated with cardiac injury detected by serum cardiac troponin i, including some cases of pancreatitis. also, we validated the dggr-lipase assay for cost-effective, sensitive and specific detection of pancreatitis in dogs and cats (vet clin path 41:e10-11, 2012; 42:e14-15, 2013 ). herein, we tested the hypothesis that pancreatitis was associated with cardiac injury. ctni was measured by advia centaur tni-ultra assay; dggr-lipase by the randox colourimetric assay. we retrospectively analysed data from dogs and cats admitted to ucd veterinary hospital in which both ctn and lipase had been measured. upper limit of reference range for lipase in dogs is 80 u/l; we consider 80-150 indicative of mild pancreatitis, 150-500 moderate, and >500 as marked. upper limit of reference range for lipase in cats is 25. reference range for ctni is <0.054 ug/l for dogs and cats. we consider 0.054-0.15 indicative of mild cardiac injury, 0.15-1 as moderate, and >1.0 as marked. 145 dogs and 19 cats had both lipase and ctni measured. seventy-eight dogs had normal troponin; 113 had normal lipase and 43 had normal lipase and normal ctni. 32 dogs (22%) had pancreatitis as indicated by increased lipase. in 18(56%), pancreatitis was mild, in 9(28%) it was moderate, and in 5(16%) it was marked. sixty-seven of 145 dogs had increased ctni: mild in 33(49%), moderate in 22 (33%), and marked in 12(18%). cardiac injury in dogs with pancreatitis was absent in 28%, mild in 34%, moderate in 25%, and marked in 13%. 13 of 19 cats had normal ctn; 10 had normal lipase. 6 of 19 cats had pancreatitis, severely in 3. lipase and ctni was correlated (r = 0.7) for dogs and cats. we conclude that both pancreatitis and cardiac injury, as indicated by high-sensitivity and high-specificity assays randox-dggr-lipase and centaur-ctni, respectively, are not uncommon in veterinary hospital cases. we confirm and extend our previous work. pancreatitis in dogs and cats is typically associated with cardiac injury. severities of pancreatitis and cardiac injury are correlated. for~40% of dogs and cats with pancreatitis, cardiac injury is moderate to marked. disclosures: no disclosures to report. intracellular colonization may serve as a protected niche where helicobacter spporganisms evade effective treatment, contributing to recolonization. confocal endomicroscopy (cem) is an endoscopic modality allowing in vivo gastrointestinal imaging at high resolution; and has aided real-time identification of helicobacter pylori and intracellular and mucosally associated bacterial. in dogs, non-helicobacter pylori-helicobacter (nhph) are described intracellularly. the objective of this study was to determine the utility of cem to identify nhph in dogs compared with other diagnostic modalities; and to assess its ability to identify intracellular organisms. fourteen clinically healthy dogs underwent standard gastroduodenoscopy followed by cem using topical acriflavine. images were obtained using cem at a minimum of five sites within the stomach. endoscopic pinch biopsies were obtained for histopathology, polymerase chain reaction (pcr) and fluorescence in situ hybridisation (fish). methodologies were compared for their sensitivity in detecting the presence and distribution of nhph and their ability to identify intracellular organisms. cem provided high quality images allowing in vivo identification ofnhph in 13 dogs, as did fish post-procedure analysis. standard histopathology identified nhph in only 11. nhph were identified within the superficial gastric mucus, and gastric pits. distribution throughout the stomach was diffuse and multi-focal. cem findings correlated with fish and pcr, however only fish enabled identification of intracellular nhph which were present in 13 of 14 dogs. cem provides in vivo histology images and is capable of identifying nhph during gastroscopy, but is unable to identify intracellular organisms using the current fluorophore protocol. nhph in the canine stomach are commonly identified intracellularly. disclosures: dr sharman has shares in optiscan imaging pty ltd. chronic enteropathies (ce) and exocrine pancreatic insufficiency (epi) can both cause hypocobalaminemia in cats. current supplementation protocols for cobalamin in cats call for repeated parenteral injections. in humans, several studies have reported equal efficacy of oral administration of cobalamin. there is also evidence that oral supplementation is effective in dogs with hypocobalaminemia. recently, it has also been reported that oral cobalamin substitution restores normocobalaminemia in healthy elderly cats. the purpose of this retrospective case series was to evaluate whether oral cobalamin supplementation can restore normocobalaminemia in hypocobalaminemic cats with chronic enteropathies. a computerized database search for cats treated at evidensia specialist animal hospital, helsingborg, sweden during 2012-2015 was performed. inclusion criteria were cats with symptoms of ce, an initial serum cobalamin concentration below 275 pmol/l (reference interval: 199-984 pmol/l) and daily oral treatment with cyanocobalamin (1 mg/tablet; ⅛-¼ tablet/cat daily). follow-up serum cobalamin concentration was measured 28-94 days after initiation of daily oral cobalamin supplementation. thirteen cats aged 2-14 years (median 8) of 4 different breeds met the inclusion criteria. presenting complaints included vomiting (7/13), anorexia (5/13), diarrhea (3/13), weight loss (2/13), and lethargy (2/13). increased pancreas specific lipase (spec fpl ò ) serum concentrations were reported in 3/11 cats and 4/13 had increased serum alanine transaminase activity. feline serum trypsin like immunoreactivity (ftli) was determined in 5/13 cats revealing results within the reference interval. all cats had an abdominal ultrasound, 9/13 had changes related to the gastrointestinal tract such as mild-moderate thickening of the small intestinal wall, thickening of the muscularis layer, poor definition of intestinal wall layers, and/or enlargement of the mesenterial lymph nodes, histopathology was performed in 6/13 cats, revealing small intestinal inflammation in five cats and small intestinal lymphoma in one. serum cobalamin increased in all cats with treatment. the concentration difference ranged from 517 to 1330 pmol/l (mean: 760 pmol/l). mean (aestandard deviation) serum cobalamin concentrations were 177 (ae49) pmol/l before and 931 (ae324) pmol/l after supplementation. this difference was statistically significant (p < 0.0001, paired t-test). our results suggest that oral cobalamin supplementation is effective in normalizing serum cobalamin concentrations in cats with various enteropathies. prospective studies are warranted comparing cellular cobalamin status in cats being treated with parenteral or oral cobalamin supplementation. disclosures: no disclosures to report. pulmonary thromboembolism (pte) is observed in dogs with idiopathic-inflammatory-bowel disease (ibd) and particularly with protein-losing enteropathy (ple). hypercoagulability has been attributed to antithrombin (at) loss although the pathogenesis is likely to be more complex. in humans, where venous thromboembolism (te) is a wellrecognised complication of crohn's disease and ulcerative colitis, the pathogenesis of te is still not completely understood. derangements in procoagulant and anticoagulant factors have been demonstrated, including increased circulating procoagulant microparticles (mps). the aim of this pilot study was to evaluate mp-procoagulant activity in the plasma of dogs with ibd and ple using a functional elisa assay (zymuphen-mp-activity, aniara). we hypothesised that all dogs with ple and a subset of dogs with ibd but without ple would have increased levels of circulating mps. the study group consisted of 11 dogs with ibd, including 4 with ple. diagnosis was based on compatible clinical and histopathology and exclusion of other causes of chronic gastrointestinal disease. ple was defined as ibd plus hypoproteinaemia (serum total protein <58 g/l) and hypoalbuminaemia (serum albumin <26 g/l). pte was diagnosed in one dog with ple, and suspected in a second. a control group comprised 8 healthy dogs undergoing blood sampling for reasons unrelated to the study including blood donor screening (n = 6) and health assessment (n = 2). dogs were considered healthy based on owner evaluation, physical examination, haematology and serum biochemistry. median mp procoagulant activity in dogs with ibd was 10.38 nm (range 0.00-32.08) compared with 7.25 nm (range 0.00-70.73) in the control group. median mp activity in ple dogs was 23.16 nm (range 0.00-32.08) compared with 7.86 nm (range 2.9-21.32) in non-ple ibd dogs. using kruskal-wallis test for nonparametric data and dunn's multiple comparisons test the groups were not statistically different. interestingly, mp-procoagulant activity value in the dog with documented pte was 0.0 nm; in the dog with high clinical suspicion for pte, mp-procoagulant activity was 32.08 nm. the highest mp-procoagulant activity was detected in a healthy control dog, raising concerns for pre-analytical or sampling error. removing this measurement had no impact on statistical analysis, which remained nonsignificant. mp-procoagulant activity >10 nm is considered clinically relevant in humans. employing a similar cut-off, 2/8 of controls, 6/11 of ibd and 3/4 of ple group would be defined as having increased levels of circulating mps. further studies are required to fully evaluate the clinical relevance and diagnostic potential of mp evaluation. disclosures: no disclosures to report. the intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (ce) in dogs. while imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with ce involving the ileum and colon. the aim of the present study was to use fluorescence in situ hybridization (fish) techniques to investigate the composition and spatial organization of mucosal microbiota in endoscopic biopsies obtained from dogs with ce and controls. tissue sections from the ileum and colon from 19 dogs with inflammatory bowel disease (ibd), 6 dogs with granulomatous colitis (gc), 12 dogs with intestinal neoplasia, and 15 controls were studied by fish targeting the 16s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. the numbers of mucosal bacteria were analyzed using generalized linear models for each of the colon and ileum tissues, with spearman's rank correlation coefficients used to test the correlation between mucosal microbiota and inflammatory (cib-dai score, histopathology) indices. the ileal and colonic mucosa of healthy dogs and dogs with ce was predominantly colonized by bacteria localized to free and adherent mucus compartments. dogs with ce harbored more (p < 0.05) mucosal bacteria belonging to the clostridium-coccoides/eubacterium rectale group, bacteroides, enterobacteriaceae, and escherichia coli versus controls. within the ce group, ibd dogs had increased (p < 0.05) enterobacteriaceae and e. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. bacterial invasion with e. coli was present in the ileal and colonic mucosa of dogs with gc (p < 0.05). dogs with intestinal neoplasia had increased (p < 0.05) adherent (total bacteria, enterobacteriaceae, e. coli) and invasive (enterobacteriaceae, e. coli, and bacteroides) bacteria in biopsy specimens versus all other groups. increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity (cibdai score) in ibd dogs (p < 0.05). these results indicate that histopathologic lesions of canine ce are associated with different populations in ileal and colonic mucosal microbiota. these spatial, segment-specific structure and differential response of select bacterial groups to intestinal inflammation may be pivotal regarding the functional consequences of these alterations in the pathogenesis of canine ce. disclosures: no disclosures to report. abdominal girth is used as an indicator of human adiposity, with such measurements being made by tape measure. given concerns in precision and accuracy of repeat measurements, some tape measure designs have inbuilt mechanisms to improve consistency. although body condition scoring is the most common method of assessing adiposity in dogs, zoometric systems have also been developed requiring the use of a tape measure. however, the precision and accuracy of such zoometric measurements are not known. the aim of this study was to determine the precision and accuracy of 3 different types of tape measure for a variety of dimensional measurements. a variety of length (head, forelimb, hindlimb) and circumferential (neck, thorax, and abdomen) were made using three different tape measures, two of which were designed to improve precision (standard tape; myotape tm and gulick ii tm ). to assess intra-operator variability, 12 measurements were taken for 5 consecutive days from 4 healthy dogs; to assess inter-operator variability, 3 operators independently took 12 measurements from a group of 16 dogs of various breeds and sizes. for intra-operator comparisons, precision was good overall (coefficient of variation [cv] ≤3% for all measurements). for interoperator comparisons, precision was more variable and, although reasonable on average (mean cv 2-5%), it varied depending upon tape measure type (p = 0.027; greatest for standard tape measure, least for gulich ii tm ), and could be highly variable for some measurements in individuals dogs (maximum cv 16% for head measurements with standard tape measure). significant differences also existed in the absolute results of circumferential measurements taken by the different tape measure types (neck p = 0.012; thorax p < 0.001; abdomen p < 0.001). finally, significant operator differences were also evident for some measurements (head p = 0.023; hindlimb p = 0.004), but not for others (forelimb p = 0.053; neck p = 0.102; thorax p = 0.073; abdomen p = 0.062). in summary, although precision for individual operators making zoometric measurements is good, significant inter-operator and tape type differences exist. these results have implications for systems using a range of zoometric measures to assess adiposity. in order to ensure precision and accuracy, it is recommended that the same operator take all measurements with the same type of tape. disclosures: the study conducted was not supported by a research grant. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. koizumi 1 , m. noda 1 , c. shimokawa 1 , a. kusumi 2 , t. kobayashi 1 , t. watari 3 , k. otsuji 1 . 1 teikyo university of science, tokyo, japan, 2 grace animal hospital, tokyo, japan, 3 nihon university, fujisawa, japan body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. however, this method is subjective due to its sensory evaluation. therefore, the improvement of the precision of the bcs diagnosis is expected. our previous study has shown that the bcs model that we created improved the precision of the bcs diagnosis (1). however, a palpation site was not identified. a palpation site must be the site where thickness of subcutaneous fat is able to capture for measuring animal's obesity status. therefore the objective of this study was to find a remarkable body site of the changes with obesity status using ultrasonic diagnostic equipment. nine dogs which varied in the percent of body fat were used in this study. the percent of body fat was measured by a body fat analyzer for dog (kao). the image analysis of a palpation site was evaluated using echo, xario ssa-660a (toshiba) which attached to a linear probe. the measurement points were 1, 2 and 3 o'clock positions on the ribs of t6, t9 and t12. the distance (d) from skin surface to the rib was measured in the echogram. the distance (l) from scapula to ilium was measured to offset the difference in physique by dog breeds. the d/l was used to compare relative value of the quantity of fat at each measurement point. bcs of dogs which used in this study were from bcs of 2 to bcs of 4. there were no dogs in bcs of 1 and bcs of 5. a statistically significant correlation was found between bcs and d/l value. the d/l value increased in order of t6, t9 and t12 in bcs of 3 and 4. this suggests that the thickness of subcutaneous fat in the chest is thicker at the head side than the tail side. also, as for the d/ l value from back to abdomen, the highest value was found at the position of 11:00 and 1:00. this tendency was the most remarkable in bcs of 4 but no difference in the d/l value was recognized in the dogs in bcs of 2. in conclusion, the position of 1:00 or 11:00 on the t6 is the suitable palpation point at the chest. (1) k. otsuji, m. suzuki, n. furukawa, n. kobayashi, a. koizumi, a. kusumi, t. kobayashi efficacy of the body condition score (bcs) model in the bcs diagnosis wsava proceeding p481, 2014 disclosures: no disclosures to report. body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. bcs has been recognized as one of the screen method of nutrition diagnosis by american animal hospital association in 2010. however, this method is subjective due to its sensory evaluation. therefore we made a bcs model to increase the precision of the bcs diagnosis and have shown the efficacy of the bcs model (1). however, the prototype model which we have reported before tended to have higher bcs than a target bcs. therefore, we improved the bcs model in this study. sixty seven dogs which varied in the bcs were used in this study. body fat percentage was measured by using a body fat analyzer for dogs (kao healthlab bif-10). the bcs model was improved by using several rubber sheets. relative hardness of stacking rubber sheets in each bcs was measured by durometer mj-dua-c2 (satotec tokyo, japan). bcs diagnosis of dogs was performed by pet owner by using the bcs model. bcs of 1 represents the most hard in the bcs model and the hardness decreased linearly and it was the lowest in 5 of bcs. these values were as expected. high correlation was recognized between bcs and body fat percentage. these results suggested the efficacy of bcs model. however, the body fat percentage in the dogs diagnosed as bcs of 1 was higher than body fat percentage which has been reported in the previous paper. there were no dogs with the body fat percentage <10% which were diagnosed as bcs of 1. we need more study in future to make clear the difference of body fat percentage between our data and date of the previous research. the completion of this bcs model will help provide the precision of nutritional diagnosis in dogs. ( in humans the metabolic syndrome (ms) is a well-recognised and extensively studied entity that comprises obesity, hypertension, dyslipidaemia, and glucose intolerance. it is associated with an increased risk of cardiovascular diseases and diabetes. recently, human ms criteria were adapted for dogs to define the condition of obesity-related metabolic dysfunction (ormd). it was observed that ormd was associated with increased circulating insulin and decreased adiponectin concentrations, suggesting that in dogs, as in humans, there are links between obesity, ormd, and associated diseases, although pathogenetic mechanisms and health significance for dogs remain unknown. the main aim of the present study was to compare plasma proteomes of obese dogs with and without ormd, so as to investigate the mechanisms associated with canine ormd and their possible significance in the health status. eight obese dogs referred for weight management at the royal canin weight management clinic, university of liverpool participated in the study. clinical assessments included physical examination, body condition scoring, blood pressure measurement and routine clinicopathological analysis. surplus plasma was used in proteomic analysis. samples were first treated with proteominer for thedepletion of high-abundance proteins and subsequently analysed by using2-de dige methodology. of the eight dogs in the study, 4 dogs had ormd and 4 dogs did not. image analysis and further statistical analysis allowed identification of 8 spots with differential expression concentration between dogs with and without ormd. among the 8 spots, 3 were over-expressed and 5 were down-expressed in dogs with ormd than in dogs that did not presented ormd. although the results of the present study are preliminary and still the identification of the spots is up to be performed, the observed datareveal that dogs with ormd present alterations in their plasma proteomes that could be responsible for the development of ormd-related pathologies. disclosures: the study was funded by waltham. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. vb is an employee of royal canin and pjm is an employee of wal-tham. the aim of the study was to assess the diagnostic value and the discrimination potential between the normal heart size and microcardia or cardiomegaly of a method which calculates the cardiothoracic ratio (ctr) using area measurement, compared to the vertebral heart scale method (vhs) used as reference for the cardiac size, in dogs. one hundred-nine dog x-rays were accepted into study. the patients belonged to small and medium size breeds, fourty-seven were males and sixty-two females with age between 1 and 17 years. the analogic x-rays were scanned and transferred to a computer where the vhs and ctr was calculated for each patient with a commercial software and the data was collected and processed in a statystical analysis software. the patients were distributed into groups by respiratory phase and heart size. there was a low correlation between the vhs and ctr (r 2 = 0.650), but statistically significant (p?0.01). a good correlation was obtained between vhs and ctr in microcardia, normal heart size and cardiomegaly groups (p < 0.01). furthermore, between the ctr in dogs with microcardia and those with normal cardiac size, as well as between ctr in dogs with normal cardiac size and those with cardiomegaly, a significantly statistic difference (p < 0.05), respectively (p < 0.01), was obtained. among the groups distributed by respiratory phase and vhs, a statistically significant difference was obtained only between normal cardiac size and cardiomegaly during inspiratory phase groups (p > 0.01). for the x-rays taken in inspiratory phase, a cutoff of 31.31 had a sensitivity of 80% and a specificity of 75% for diagnosing cardiomegaly. the ctr can be considered a valid method being able to discriminate between the patients with microcardia and cardiomegaly from those with normal heart size. moreover, it was found that a ctr over the cutoff of 31.31, mesured during inspiratory phase is a good predictor for cardiomegaly. key words: cardiac, cardio-thoracic ratio, dog, x-ray. disclosures: no disclosures to report. the canine cardiac conduction system is modified by anatomical and functional adaptations of the maternal heart during gestation. however, it is not clear if these changes persist or are modified after parturition. therefore, the aim of this study was to describe canine electrocardiographic features during the course of normal puerperium. twenty healthy pure-bred, 2-5 (3.85 ae 0.16) year-old, weighing 1.5-6 kg (3.55 ae 0.26) bitches were included in this study. all the animals whelped healthy puppies at term which were weaned on day 60 after parturition (day 0). all the dogs were electrochardiographically evaluated on days à3, 3, 10, 17, 24, 38, 52 and 80. mean electrical axis (mea; degrees), p wave amplitude (pa; mv) and duration (pd; ms), p-r interval (pr; ms), qrs complex amplitude (qrsa; mv) and duration (qrsd; ms), q-t interval (qt; ms), and s-t segment (st; mv) were calculated at 50 mm/s of velocity. the rr interval immediately preceding each complex was recorded and qt interval was corrected (qtc) by van de water formula [qtc = qt-0.087(rr-1000)]. later, lead ii was recorded at 25 mm/s to analyze heart rate (hr; bpm) and cardiac rhythm (cr; normal sinus rhythm or sinus arrythmia). values of hr, mea, pa, pd, pr, qrsa, qrsd, qt, rr and qtc were analyzed by anova for repeated measures followed by tukey test. cardiac rhythm was analyzed by chi square test (spss 17.0, spss inc. chicago, il, usa). p < 0.05 was considered significant. during the study period, hr (p < 0.01) and qtc (p < 0.01) progressively decreased, while rr (p < 0.01) and pa increased (p < 0.01). qrs complex amplitude diminished in the second week after parturition and then increased during the following weeks (p < 0.01). mean electrical axis shifted to the right during this period (p < 0.01). on day à3, most of the bitches presented normal sinus rhythm in contrast with day 3, in which most of the bitches presented sinus arrhythmia (p < 0.01). from day 10 onward, all the bitches showed sinus arrhythmia. p wave duration, pr, qrsd, qt and st remained unchanged during puerperium. it is concluded that most electrophysiological adaptive changes of canine gestation reverted during normal puerperium. the present study contributes to the understanding of canine cardiac physiology during this reproductive stage. disclosures: no disclosures to report. esvc-p-3 echocardiographic assessment of pregnant queens. p.g. blanco, r. rodr ıguez, a. carranza, a. rube, r. vercellini, p.r. batista, m. t ortora, c. gobello. national university of la plata, la plata, argentina cardiovascular adaptation during gestation guarantees an appropriate development of the fetuses and maternal cardiovascular maladaptation is highly correlated with adverse pregnancy outcome. while, the hemodynamic changes occurring during canine pregnancy have been described there is scarce information concerning maternal cardiac variations during feline gestation. thus, the aim of this study was to describe cardiac morphology and systolic function variations during normal feline pregnancy. eighteen pregnant queens were echocardiographically evaluated (toshiba nemio xg, japan, 10 mhz transducer) every 10 days from day 0 (defined as day of mating) to parturition. left ventricular dimensions were measured in the short axis view, during mmode tracing. shortening fraction was calculated as (lvdd à lvds)/lvdd 9 100 to assess systolic function. stroke volume (ml) was calculated as the product of the velocity time integral (measured by pulsed-wave doppler) and the cross-sectional area of the aorta. cardiac output (l/minute) was calculated as the product of stroke volume and heart rate (bpm) derived from electrocardiographic monitoring. uterine artery resistance index (ri) was obtained by doppler ultrasound. all the parameters were analyzed by repeated measures anova. all the queens delivered healthy kittens at term. throughout the study period, interventricular septum in diastole (p < 0.01) and systole (p < 0.01) and left ventricular diameter in diastole (p < 0.01) augmented during gestation. shortening fraction (p < 0.01), cardiac output (p < 0.01) and maternal heart rate (p < 0.01) also increased up to parturition. conversely, uterine artery resistance index decreased in the same period (p < 0.01). it is concluded that cardiac structure and function varied during normal pregnancy in these queens. cardiac eccentric hypertrophy, systolic function and cardiac output increases appear to be the consequences of the hemodynamic modifications occurring during pregnancy. the assessment of maternal cardiovascular function may prove a useful screening tool to detect pregnancy complications in feline reproduction. disclosures: no disclosures to report. tricuspid annular plane systolic excursion (tapse) is an echocardiographic measure that allows to assess right ventricular systolic function. it has been described reference values for tapse in normal adult dogs, but there is no reference to influence of age in tapse in dogs. this influence has been reported in humans. thus, the goal of this study is to determine the reproducibility of the measure tapse in normal dogs and to determine the relationship between tapse and age in healthy beagle dogs. tapse was measured from an m-mode recording of the lateral aspect of the tricuspid valve annulus obtained through a left parasternal apical 4-chamber view. tapse values were averaged from measurements on 5 consecutive beats during sinus rhythm. the measurements were recorded by two different persons (c-v, a.; m-m f.) with different grade of experience in canine echocardiography studies. all patients had a complete two-dimensional and doppler study using an envisor chd (philips ò ) ultrasound system. twenty-three healthy beagles were used. the study was approved by the ethical committee of veterinary medicine service of las palmas de gran canaria university (spain) and it was carried out in accordance with the current european legislation on animal protection. these dogs were divided in three different groups according to age: group 1 included twelve dogs under 4 years, group 2 included three dogs between 4 and 10 years, group 3 included eight dogs older than 10 years. we analyzed differences between groups using non-parametric (kruskal wallis and wilcoxon scores [rank sums]) test. there were no differences with respect to sex. dogs in group 1 presented higher tapse values than group 2 or 3 (1.33 ae 0.26 cm vs 0.98 ae 0.14 cm vs 1.13 ae 0.18 cm; p < 0.05). statistic intra-observer and inter-observer agreement using the intraclass correlation coefficient was 0.99 (p < 0.05). this study showed that tapse measurement is easily obtainable with a standard echocardiography system, and has adequate interobserver agreement. this study showed higher values of tapse in normal young dogs with respect to older dogs. these results are similar to the results obtained in humans, and could reflect a less effective right ventricle with age. the values presented should be taken with caution due to the relatively small number of patients included. it may also be necessary to validate results in future studies with a second independent sample of dogs of other races. disclosures: no disclosures to report. tetralogy of fallot (tof) is a congenital heart disease characterized by 4 abnormalities, i.e., pulmonic stenosis, ventricular septal defect (vsd), aortic overriding and secondary right ventricular hypertrophy, caused by anterior deviation and abnormal septation of the conal septum during the embryonic period. few studies have reported the hemodynamic consequences and clinical outcome of tof in small animals. the objective of this retrospective study was therefore to document the epidemiological, clinical, echo-doppler findings, and survival, in a canine and feline population with tof. the case records of animals diagnosed with tof by combined use of echocardiography and doppler examination were reviewed (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) . tetralogy of fallot was identified in 31 animals (15 dogs, 16 cats). the most commonly represented breeds were terriers for dogs (7/15, 46.7%), and domestic shorthair for cats (12/16, 75.0%). most included animals (28/31, 90.3%) were clinically affected at the time of diagnosis. pulmonic stenosis was characterized by a variable systolic doppler-derived pressure gradient both in dogs (median [range] 106 mmhg ) and cats (109 mmhg ), and associated with hypoplasia of the pulmonary trunk in one third of the cases (35.7%). most vsd were large, with a median vsd: aorta ratio of 0.60 [0.35-1.02] in dogs and 0.59 [0.18-1.15] in cats. median age at death from cardiac cause was 23.4 months [3.5-92.9 ] without significant difference between dogs and cats (p = 0.298). these results suggest that in both cats and dogs tof-related death occurs predominantly in young adult animals with major hemodynamic consequences at the time of diagnosis. disclosures: no disclosures to report. the aim of this study was to assess whether and how radiographic and echocardiographic cardiovascular variables differ across age bands of healthy cats. a cohort of 98 clinically healthy cats were categorized into three groups: adolescent-adult (0.6-6 years; n = 45), middle-aged (7-10 years; n = 28), and geriatric (11-17 years; n = 25). all cats underwent a full physical examination, a complete blood count, routine biochemical profile, a baseline serum total thyroxine concentration, auscultation, noninvasive blood pressure measurements, thoracic radiography, electrocardiography, and echocardiography. cats with hypertension, hyperthyroidism, cardiac, or renal disease were excluded from the study. body weight, body condition score, systolic blood pressure, heart rate, and all echocardiographic indices were similar across the three groups. the mean (aestandard deviation [sd] ) vertebral heart scale (vhs) value obtained for the geriatric group (7.7 ae 0.6) was significantly greater than that obtained for the adolescent-adult group (7.4 ae 0.4; p = 0.018). the mean ratio of the distance between the cardiac base and dorsal sternum to thoracic cavity height at the point of the cardiac base was significantly less in the middle-aged (0.64 ae 0.04) and geriatric (0.61 ae 0.05) groups than in the adolescent-adult group (0.69 ae 0.04; both p < 0.001). the mean angle between the cardiac long axis and the body axis was significantly smaller in middle-aged (40.4 ae 7.7°) and geriatric cats (40.0 ae 7.9°) than in adolescent-adult cats (53.1 ae 7.3°; both p < .001). the mean angle between the cardiac long axis and the sternum of middle-aged (36.8 ae 6.6°) and geriatric cats (36.2 ae 7.9°) was significantly smaller than that in adolescent-adult cats (42.6 ae 5.7°; p = 0.006 and p = 0.002, respectively). additionally, the degree of undulation of the thoracic aorta correlated positively with age (r 2 = 0.307, p = 0.003). these findings suggest that differences in the horizontal alignment of the heart, thoracic-aorta undulation, and vhs in healthy geriatric cats, relative to observations in younger cats, can be considered to be age-related. disclosures: no disclosures to report. the aim of this study was to investigate the presence of pulmonary hypertension (ph) in young cats affected by single or mixed lungworm infections. twenty-three cats infected with lungworms were examined at the veterinary teaching hospital of teramo, italy, in 2013 -2014 . animals underwent to a complete physical examination and to two-or three-views radiographic analysis of the thorax. a minimum database (i.e. cbc, serum biochemistry, serology for fiv antibody and felv antigen) was obtained for each patient. nine cats were excluded for concomitant diseases, while 14 cats were included in the study. microscopic identification of parasites was confirmed by molecular tests and all cats received an anthelmintic treatment. a single infection by aelurostrongylus abstrusus was diagnosed in eleven cats, while three cats had a troglostrongylus brevior infection either alone or in combination with a. abstrusus. transthoracic echocardiography was performed using an ultrasound unit with a 5 mhz phased array transducer. no structural abnormalities of the tricuspid valve and sign of pulmonary stenosis were detected. the two-dimensional and m-mode echocardiography showed a cardiac involvement in three cats. one cat, infected by a. abstrusus and t. brevior showed a mild systolic tricuspid regurgitant jet with color doppler of 1.64 m/s, while another a. abstrusus-infected cat, had mild tr of 2.2 m/s with a mean paps of 29 mmhg which resolved within 4 weeks after therapy. one cat diagnosed with troglostrongylosis, showed a marked right-sided cardiac enlargement of 6 mm, and a large systolic tricuspid regurgitant jet with a tr peak velocity of 3.1 m/s recorded at continuous-wave doppler via a color doppler echocardiography. the minimum pressure difference between the right ventricle and the right atrium was estimated 38 mmhg and the paps was at least 48 mmhg. the echocardiographic and doppler evidence of mild ph persisted at further examination performed until 3 months after diagnosis. ph is rare in cats, despite cases of reversible ph are known in cat aelurostrongylosis. in this study the first case of irreversible ph infection in a cat affected by t. brevior is presented and this finding further supports the high pathogenicity of troglostrongylosis, especially in young patients. in cats with lungworm infection, possible cardiovascular complications must be taken into account and these infections should be always considered in the differential diagnosis in cats with cardiorespiratory signs. disclosures: no disclosures to report. ventricular function by conventional echocardiography and speckle tracking imag-ing although uncommonly assessed in veterinary cardiology, a right ventricular (rv) function has been shown to be an important prognostic determinant of many congenital and acquired heart diseases in human patients. our group has already demonstrated that two-dimensional (2d) color tissue doppler imaging provides a non-invasive evaluation of systolic and diastolic rv function in the awake dog with adequate repeatability and reproducibility. b however, other noninvasive ultrasound imaging variables reflecting rv function need to be further investigated, particularly in correlation with pulmonary arterial pressure (pap) values and left ventricular (lv) function. the aim of this prospective study was therefore to assess several indices of systolic and diastolic rv function using conventional echocardiography and speckle tracking echocardiography (ste) in 104 healthy awake dogs of different breeds with documented systolic pap (spap) and lv function (lv ejection fraction and global lv systolic strain assessed using the simpson's derived method of disks and ste, respectively). imaging rv tested variables included tricuspid annular plane systolic excursion (tapse), right fractional area change (rfac, %), ste longitudinal systolic strain of the rv free wall (rvfw, %) and of the whole rv (i.e., global rv strain, %), ste longitudinal systolic strain rate (sr, s à1 ) and diastolic early:late sr ratio. additionally, 2d-guided m-mode ventricular measurements included the end-diastolic rv:lv diameter ratio (rvdd: lvdd) and the end-systolic rvfw:lvfw ratio. correlations between imaging variables were calculated by using spearman's correlation coefficients. means of age and body weight (aesd; range) of the study population were 4.3 years (ae2.6; 0.6-11.6) and 20.4 kg (ae10.7; 3.3-49.0), respectively. no correlations were found between rv morphological variables (i.e., rvdd:lvdd and rvfw:lvfw ratios) and all indices of systolic and diastolic rv function. global rv strain (mean ae sd = 26.4 ae 3.8%) and rvfw strain (31.9 ae 6.2%) were positively correlated (p < 0.01) with rfac (50.6 ae 10.5%, r = 0.36 and r = 0.32, respectively), and negatively correlated (p < 0.05) with spap (17.4 ae 7.0 mmhg [7.0-30.0], r = à0.21 and r = à0.24, respectively). spap was also negatively correlated with the tapse:body weight ratio and systolic sr (r = à0.31 and à0.34 respectively, p < 0.01). there was no correlation between indices of lv function and ste indices of rv function, and no correlation either between ste rv indices of systolic function and the diastolic early:late sr ratio. in conclusion, ste provides a rapid and non-invasive evaluation of rv function that may be used for clinical investigations in canine cardiology. doppler-derived +dp/dt and -dp/dt from mitralregurgitation are considered indexes for assessment of systolic and diastolicfunction respectively, that have less load dependence than the ejection phaseindexes. this study aimed to determine correlation between doppler-deriveddp/dt and other systolic and diastolic echocardiographic indexes, and if theycan be used to identify dogs with and without remodeling, with or withoutcongestive heart failure (chf) and for evaluation of chronic mitral valvedisease (cmvd) severity. fifty-seven dogs with cmvd (stages b1, b2, c+d) wereincluded prospectively in an observational cross-sectional clinical study anddistributed in groups regarding the presence of remodeling and chf, to evaluate+dp/dt and -dp/dt, and distributed according to tdi-diastolic pattern tocompare àdp/dt. group c+d (2142 mmhg/s, p 25 -p 75 = 2023-2456) had +dp/dt significantly lower compared to b1 (2865 mmhg/s, p 25 -p 75 = 2383-3308) and b2 (2721 mmhg/s, p 25 -p 75 = 2241-3186) (p = 0.0023). groupc+d also had lower àdp/dt, compared to b1 (968.5 mmhg/s ae 266.8 and 1198 mmhg/s ae 165.7; p = 0.0115). dogs with chf compared to those without chf, presented lower +dp/dt (2142 mmhg/s, p 25 -p 75 = 2023-2456; 2858 mmhg/s, p 25 -p 75 = 2299-3241; p = 0.0007) and àdp/dt (968.5 mmhg/ s ae 266.8; 1155 mmhg/s ae 199.0; p = 0.0041). regardingdiastolic function, àdp/dt was lower for the restrictive pattern group (769.7 mmhg/s ae 124.1) compared to those without diastolic dysfunction, (1132 mmhg ae 204.0), delayedrelaxation (1229 mmhg ae 186.9) and pseudonormal patterns (1107 mmhg ae 223.4) (p < 0.0001).when +dp/dt<1800 mmhg/s, the post-test chance for the dog with cmvd to havechf is twice the chance than not having it. for àdp/dt<800 mmhg/s theposttest chance of having chf is eight times higher than not having it. in conclusion, doppler-derived +dp/dt and àdp/dt may contribute respectively, for systolic and diastolic assessment ofdogs with cmvd. disclosures: no disclosures to report. pulmonic stenosis (ps) is one of the most common congenital heart defects seen in veterinarycardiology practice. pulmonary balloon valvuloplasty (pbv) is considered to be the treatment of choice for dogs withsevere stenosis. whether dogs with moderate stenosis benefit from pbv remains unclear, and variables such as degree of hypertrophy, valve morphology, amount of tricuspid insufficiency and presence or absence of clinical signs aregenerally used when recommendations are made to pet owners. in this study we report the effect of valve type on pbv outcome in 110 dogs treated at three different academic speciality cardiology practices. baseline echocardiographic images were evaluated at each institution and valve morphology was classified as either type a (n = 78, 137.96 mmhg, range 53-278) or type b (n = 33, 153.72 mmhg, range 81-300) and "no" (n = 87, 140 mmhg, range 53-279) or "yes" (n = 24, 151 mmhg, range 87-300) for presence of pulmonary annular hypoplasia when diameter was compared to aortic annulus. twenty-four hours following pbv both type a (56 mmhg, range 17-210) and type b (78 mmhg, range 25-197) valves had significant reduction in gradient compared to baseline (p < 0.0001). this reduction remained significant at 30 days (a: 77 mmhg, range 22-193; b: 60 mmhg, range 30-116; p < 0.0001 for both). dogs with annular hypoplasia (65 mmhg, and without annular hypoplasia (69 mmhg range 17-210) had a significant reduction in gradient 24 hours post pbv. it remained significant at 30 days (with annular hypoplasia: 77 mmhg, range 30-116; without annular hypoplasia: 61 mmhg, range 22-193; p < 0.0001 for both). when comparing to baseline, considering valve type, there was no significant difference in percent reduction in gradient for type a versus type b valves at both the 24-hour (a: 58%, range 17-88; b: 48%, range 12-82; p = 0.1014) and 30-day (a: 43%, range 23-89; b: 58%, range 33-81; p = 0.0544) recheck evaluation time points. additionally, there was no significant difference in gradient reduction when looking only at whether or not there was annular hypoplasia at 24 hours (yes: 57%, range 24-78; no: 49%, range 17-89; p = 0.2673) and 30 days (yes: 48%, range 25-81; no: 47%, range 22-89; p = 0.4695). in conclusion, classification of dogs with ps according to valve type and annulus morphology did not help predict the 30-day response to pbv. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is the most common feline inherited cardiac disease and it is a major cause of morbidity and mortality. the osservatorio italiano hcm felina was formed in 2008 by a network of clinicians, geneticists and breeders, to monitor and study hcm in italian cats. since april 2008, 1308 adult cats, belonging to various breeds, including maine coon, siberian, norwegian forest cats, ragdoll, sphynx, british sh, birmans and others have been prospectively enrolled. recheck evaluations were performed in 287 cats. each cat underwent a clinical examination, echocardiography, and blood collection for genetic testing (when appropriate) and storage in the italian feline bio-bank. the disease status was defined by echocardiography according to established guidelines (left ventricular diastolic wall thickness <5.5 mm = hcm negative, =5.5 but <6 mm = hcm equivocal; =6 mm = hcm positive). the prevalence of hcm in the population was 6% (74 cats); equivocal diagnoses were conferred on 4% (57 cats). these prevalences did not differ between breeds. the prevalence of hcm in the italian feline population was lower compared to those reported by other investigators. evaluation of data from the entire population demonstrated that left ventricular end-diastolic wall thicknesses and aortic diameter showed a weak positive correlation with body weight (p < 0.0001, r 2 < 0.12 for all variables), suggesting that weight-dependent limits on wall thickness should be considered in cats as is currently practiced in dogs. the lower prevalence of hcm in italian cat breeds compared with those examined elsewhere might be explained by different criteria for determining presence or absence of disease, differences in ages at which the subjects were examined, or a selection bias by breeders in presenting cats they consider "normal". disclosures: no disclosures to report. chronic mitral valve disease is by far the most common cardiovascular disease in dogs. the disease is caused by myxomatous degeneration of the mitral valve leaflets and, in approximately 30% of cases, it's accompanied by degeneration of the tricuspid valve. it is also described in previous studies that approximately 14% of affected dogs also have evidence of associated pulmonary arterial hypertension. the prevalence of the disease is higher in small breed dogs (under 20 kg), although large breeds can also be affected and it occurs more frequently in males than in females. the present study aims to characterize the disease in a population of dogs in portugal. we retrospectively reviewed the medical records of dogs presented to hospital veterin ario do porto, with an echocardiographic diagnosis of canine chronic mitral valve disease, during a period of 13 years. from this records, 542 cases were identified, from which 331 (61.1%) were males and 211 (38.9%) were females. most of the dogs were mixed breed (215) and 48 different breeds of dogs were represented. the poodle was by far the most represented breed (n = 101; 39.7%), followed by english cocker spaniel (18.6%), yorkshire terrier (2.8%), boxer (2.6%), epagneul breton (2.6%), dalmatian (2.4%), pekingese (2.4%), labrador retriever (2%) and portuguese podengo (1.8%). all other breeds represented 16.2% of the population. regarding weight, 79.8% of the dogs (n = 395) weighted <20 kg, with a mean body weight of 13.45 kg (range 1.6-62 kg). the mean age at diagnosis was 11.34 years old. we also observed that 42.1% of the dogs (n = 278) had concomitant degeneration of the tricuspid valve and 19.4% (n = 105) pulmonary arterial hypertension (ph). we categorized these dogs according to the severity of ph, in mild ph if they had a doppler echocardiography derived systolic pulmonary arterial pressure (spap) of 30-50 mm/hg, moderate ph (spap 51-75 mm/hg) and severe ph (spap >75 mm/hg). we found that 72.7% (n = 72) of dogs had mild ph; 19.2% (n = 19) moderate ph and 8.1% (n = 8) severe ph. as described in previous studies, the disease affects mainly males and small breed dogs, with a breed distribution that reflects the canine population in the country, including very including very popular large breed dogs in portugal, as the boxer and labrador. both the presence of concomitant tricuspid valve disease and ph had a higher prevalence in our study than previously described. disclosures: no disclosures to report. in people anemia is frequent in patients with heart failure (hf) and it is associated with poor outcomes. the most likely pathogenic factors include iron deficiency, chronic kidney disease (ckd), and cytokine production, although other factors may contribute. little is known about the prevalence of anemia in dog with cardiovascular disease. the aim of this retrospective study was to define the prevalence of anemia (hct ≤37%) in dogs with mitral valve disease (mvd) and to investigate associated risk factors (age, weight, azotemia, hf, iris/acvim class). medical records of dogs presented at the cardiology service, divet, university of milan (january 2003-march 2015) were retrospectively evaluated. dogs with mvd with complete physical, thoracic and echocardiographic examinations, and serum biochemical panel, including serum creatinine (scr), were included in the study. dogs with other heart or systemic diseases, except ckd, or neoplasm were excluded. statistical analysis was performed using jmp 12.0 (sas institute). a p value < 0.05 was considered significant. two hundred and ninety dogs (161 males/129 females), 11.6 ae 2.9 years of age, 12.5 ae 9.2 kg of body weight fulfilled the inclusion criteria. the 22% of males and the 30% of females were neutered. the most represented breeds were mongrel (40%), miniature poodle (12%), york shire terrier (7%), and cavalier king charles (5%). dogs were 29% b1, 13% b2, 54% c and 4% d acvim class. while the 72% of the dogs were normoazotemic (scr <1.4 mg/dl), 13.5% were staged in iris 2, 13% in iris 3 and 1.5% in iris 4. the prevalence of anemia in dogs with mvd was 17% (50/290): 40 showed mild (30≤ hct ≤37%) and 10 moderate (20≤ hct ≤29%) anemia. sixteen dogs were in b1, 5 in b2, 27 in c and 2 in d acvim class; thirty-four were normoazotemic (68%). anemic dogs showed a significant higher scr. normoazotemic dog showed significant higher hb, hct and rbc both in the overall population and in the anemic group. in the overall population dogs in different iris class showed statistically different hb, hct and rbc and hb was significantly lower in decompensated hf dogs. in conclusion although a relationship between anemia and azotemia/ckd was documented in our study, it is important to emphasize that most of the anemic dog were normoazotemic: anemia is not an exclusive finding of cardiorenal syndrome and should be considered as possible complication in dogs with mvd alone. disclosures: no disclosures to report. the objective of this study was to evaluate left atrial (la) function by left atrial total fractional area change (la-factotal) and left atrial ejection fraction (laef) in dogs affected with chronic mitral valve disease (cmvd) naturally acquired with and without congestive heart failure (chf). our hypothesis was that la-factotal and laef decrease with severity of cmvd. eighty dogs were included in a prospective observational cross-section clinical study, grouped according to cmvd severity based on echocardiographic evaluation and clinical signs. the dogs were equally distributed in each group: a, b1, b2 and c, according to american college of veterinary internal medicine staging system. indicators of la function were calculated with the following equations: la-factotal = 100 9 (lamaximum area àlaminimum area)/lamaximum area, measured by apical four view; and laef = 100 9 (lamaximum volume à laminimum volume)/ lamaximum volume, by biplane area-length method from the left apical four and two-chamber views. la-factotal showed lower values (p < 0.0001) in group c (31.88%, p25%-75% = 26.47-41.12) compared with groups a (52.75%, p25%-75% = 48.08-56.07), b1 (48.38%, p25%-75% = 42.57-51.91) and b2 (46.15%, p25%-75% = 41.17-50). group c had lower laef (40.69%, p25%-75% = 34.89-52.09) than groups a (68.12%, p25%-75% = 64.96-69.91), b1 (58.72%, p25%-75% = 52.25-64.60) and b2 (56.98%, p25%-75% = 52.08-61) (p < 0.0001). left atrial function, assessed by la-factotal and laef, was reduced in dogs with cmvd and chf compared with healthy and asymptomatic cmvd groups. disclosures: no disclosures to report. recurrent episodes of heart and/or kidney failure are considered one of the causes leading to worsening heart/renal functions in human patients. the aim of this prospective study was to assess the influence of heart/kidney worsening on elected parameters of heart/kidney function in dogs affected by mitral valve disease (mvd). between july 2012 and may 2013, dogs affected by mvd in acvim class b2 and without comorbidities were included in the study group. the control group was constituted by healthy dogs, matched with the cases for age (older than 6 years) and gender. all the dogs underwent physical examination, thorax radiography, ecg, echocardiography, systemic blood pressure assessment, a complete blood count, serum biochemical analysis, including assessment of serum creatinine (scr), serum urea nitrogen (urea) and glycaemia (gly) and urine analysis with urine protein/creatinine ratio (upc). dogs were re-evaluated every 6-month until october 2014. statistical analysis was performed using ibm spss statistics 20 (p value significant if <0.05). twenty-one dogs affected by mvd (cases) were included and 20 healthy dogs (controls) were randomly selected among the eligible population. the 33% of cases experienced at least one episode of congestive heart failure (chf), but none of these patients developed chronic kidney disease (ckd). the 14% of cases developed ckd while remaining in acvim class b2. no dogs in the control group developed ckd or mvd. correlations between worsening renal function (wrf -scr elevation ≥0.3 mg/dl or 25% from baseline), furosemide administration, upc levels, radiographic parameters of heart enlargement and echocardiographic parameter were investigated. only a statistically significant difference in iris class between the groups according to wrf and in the echocardiographic parameter left atrium to aortic root (la/ao) according to furosemide amount were observed. both these results were expected. none of the cases included experienced renal damage (wrf or iris class change or upc change) concomitant to episodes of chf. the persistence of normal renal condition regardless of chf events and therapy administration was unexpected. in conclusion, experiencing chf seems not to directly affect renal function. to authors' opinion, the use of wrf, better than single scr and urea levels, may be useful in the long term management of aged patients affected by mvd. however, the small number of cases included in this study represents a great limit. we consider this work a pilot study. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is a primary myocardial disease characterized by inappropriate thickening of the myocardium in absence of other causes of hypertrophy including hypertension, hyperthyroidism, aortic stenosis and acromegaly. it is also the most common heart disease in cats. hcm presents a wide variety of clinical sings depending on the severity and location of the hypertrophy. cats affected with hcm have a mean age of 5.5-6.5 years old at the time of the diagnosis however this disease can affect cats as young as 3 months although this later age is unusual hcm is a heterogeneous disease both in terms of phenotypic degree of hypertrophy and clinical outcome. hallmark histopathological hallmarks lesions of hcm are myocyte disarray, small coronary arteriosclerosis and interstitial fibrosis replacement in order to confirm hcm echocardiography has to be made. primary hypertrophy diagnosis is made based on the presence of ventricular hypertrophy, symmetric or asymmetric, in the absence of systemic disorders. the purpose of this study was to assess the prevalence of hcm in a feline population. in order to achieve this goal echocardiograms were made in all cats older of 6 years clinically asymptomatic with or without cardiac murmur. all echocardiograms were made according to the guidelines of the acvim published in 1993. diagnosis of ventricular hypertrophy was made from the right parasternal window using the b mode to measure the diameter of the lvfw and the ivs in diastole. cats with more than 6 mm of wall thickness measured t4, bun, crea, blood pressure. only cats within the normal limits of the later parameters were considered hcm positive. total number of cats in this study was 150 cats 89 male and 61 female. from this population 94 had no defined breed, 37 were persian, 6 maine coon, 4 norwegian woods, 8 siamese, 1 chartreaux. no murmour was detected in 64 (42.7%) cats, s3 or s4 was detected in 9 (6%) cats and differente degree of murmour was detected in 77 (51.3%) cats. hypertrophy was detected in 69 cats. from this cats 41 (59.4%) were diagnosed as hcm, 28 (40.6%) cats were excluded either because of lack of values of t4 and or because they had high values of blood pressure, t4 levels or crea. in this study 27.3% of the population had hcm. the epidemiological and phenotype distribution is highly variable. the average age at diagnosis of hcm in this study was 11.33 years. disclosures: no disclosures to report. mildly increased concentrations of crp are associated with cardiovascular disease in humans and dogs. it is not known whether increased concentrations of crp are associated with myxomatous mitral valve disease (mmvd) in dogs, or rather its sequel, congestive heart failure (chf). the aim of this study was to investigate whether serum concentrations of crp, determined using a novel automated canine-specific high-sensitivity crp assay (gentian hscrp), were associated with severity of mmvd and certain clinical variables in dogs. the study included 188 client-owned dogs with different severities of mmvd. disease severity was determined by medical history, physical examination, echocardiography and response to diuretic therapy. dogs were allocated into groups based on acvim consensus statement guidelines ( in conclusion, slightly higher serum crp concentrations were found in dogs with chf whereas the severity of asymptomatic mmvd showed limited association with serum crp concentrations. disclosures: no disclosures to report. medetomidine is a a 2 -agonist widely employed for sedation in dogs but its use is discouraged in cardiac patients even those suffering from myxomatous mitral valve disease (mmvd). however, only one investigation was previously conducted in a wide rangeregarding the class of the diseaseof mmvd patients, reporting a general safety of that protocol. the present study was focused just on class b2 of mmvd, with the aim to provide more detailed information on the cardiovascular effects of medetomidine in such patients, by the analysis of clinical and instrumental parameters suggestive of disease severity or congestive heart failure. dogs weighing <15 kg, needing a soft clinical procedure and showing a systolic apical heart murmur were screened and selected for the study if la/ao <1.6. the sedative protocol consisted in an iv injection of 30 lg/kg medetomidine antagonized, after the clinical procedure, by an im injection of the recommended dose of atipamezole. clinical parameters, echocardiographic variables, thoracic radiographs and oscillometric blood pressure measurements were collected at baseline (t0), 30 minutes after medeto-midine administration (t1) and 3 hours after atipamezole injection (t2). of 13 dogs screened, 8 were definitively enrolled. at t1 a significative decrease in the right parasternal regurgitant jet area (rp-arj/laa), peak velocity of mitral regurgitation and shortening fraction was observed along with an increase in lvids (p < 0.05). left parasternal arj/laa decreased without reaching statistical significance but showing a high correlation with rp-arj/laa (r = 0.7). interestingly, la/ao changed only mildly and never reached a value >1.6. the other echocardiographic variables did not show a particular trend. systolic blood pressure showed values at the upper physiologic limit at t0, lower values than t0 at t1, and an increase above the initial value at t2 but without significance. thoracic radiographs were evocative of heart enlargement without pulmonary venous congestion or pulmonary oedema both at t0 and t2. respiratory rate did not change between t0 and t2. the degree of sedation was optimal during the clinical procedure in all cases. sedation with 30 lg/kg medetomidine is safe in dogs suffering from mmvd in stage b2 (la/ao <1.6). the decrease observed in peak velocity and color-doppler appearance of mitral regurgitation at t1 could be due to a reduction of both myocardial contractility and systolic blood pressure, by a lowering of sympathetic activity via baroreceptors stimulation. disclosures: no disclosures to report. in this retrospective study were included a total of 25 clientowned dogs, undergoing transarterial occlusion of pda with mreye ò flipper detachable embolization coil (n = 7), amplatz â canine duct occluder (acdo; n = 16) and amplatzer â vascular plug (n = 2). device size selection was based on pda dimensions assessed by transesophageal echocardiography (tee) in 10 cases and transthoracic echocardiography (tte) in 15 cases. angiography was performed during the procedure to assess the success of the occlusion, and it confirmed complete occlusion in 20 dogs and a trivial residual flow in 5 dogs. the following day, transthoracic color-doppler echocardiography revealed that complete ductal closure was achieved in all dogs. the procedure was hemodynamically successful, as evidenced, by a reduction in indexed left ventricular internal diameter in diastole (lvidd; p < 0.01), fractional shortening (fs; p < 0.01) and left atrial to aortic ratio (la: ao; p < 0.001) within 24 hours after procedure. four months after surgery, indexed lvidd was significantly reduced (p = 0.03) and la:ao remained constant. secondary complications included pulmonary arterial embolization of an acdo and a late rotation of an amplatzer â vascular plug resulting in an increased flow through the pda. the dog with the rotated device required subsequent surgical ligation of the pda. at this time, 23 dogs were reported to be alive and the other 2 dogs were lost to follow up. only one dog remained on congestive heart failure therapy after the pda occlusion. we can conclude that pda occlusion using an acdo for dogs with more than 3 kg and a transarterial coil embolization for dogs with <3 kg had a high rate of immediate complete occlusion. pda occlusion using those devices proved to be a safe and effective therapeutic method for pda in dogs. disclosures: no disclosures to report. echocardiographic evaluation of the right pulmonary artery distensibility index (rpad index) was recently described as a valuable method for early detection and severity evaluation of pulmonary arterial hypertension in dogs. rpad index is calculated as the percentage change in diameter of the right pulmonary artery (rpa) between systole and diastole, obtained by m-mode echocardiography from the right parasternal long axis view. the aim of this study was to compare the rpad index obtained by two different echocardiographic views in dogs. the study design was a prospective, multicenter, observational study. forty-five client-owned dogs from different breeds were included: 31 dogs with heart disease and 14 healthy dogs. two different right parasternal views, long axis (rpla) and short axis (rpsa), were used to measure the rpad index. from the rpla view (method 1) and rpsa view (method 2) a short axis and a long axis image were respectively optimized for the right pulmonary artery. the rpad index was calculated by m-mode as the percentage change in diameter of the right pulmonary artery: [(systolic diameter à diastolic diameter)/systolic diameter]*100. measurements were done off-line as an average of 5 consecutive cardiac cycles by a single investigator blinded to the dogs' diagnosis. a pearson and a bland-altman test were used to assess correlation and agreement between the 2 methods, respectively. intra-and inter-observer measurement variability was quantified by average coefficient of variation (cv). level of significance was set at p < 0.05. m-mode evaluation of the rpad index was satisfactorily obtained by both methods in all dogs. pearson test showed a strong positive linear correlation between the values of rpad index obtained from both methods (r 2 = 0.9346, p < 0.0001). bland-altman test showed a good agreement between the 2 methods in estimating rpad index (bias = 0.51%, sd = 2.96%, 95% limits of agreement = à5.30, 6.33%). the mean difference between the two methods was 0.51% (95% confidence interval = à0.35; 1.35). intra-and inter-observer measurement variability was clinically acceptable (cv<10%).the study showed a good agreement between short axis and long axis m-mode evaluation of rpa. both methods can be used interchangeably to evaluate rpad index. further studies are needed to evaluate the rpad index in a larger population of healthy dogs and the diagnostic and prognostic role of this echocardiographic parameter in dogs with different types of pulmonary hypertension. disclosures: no disclosures to report. naturally occurring hypoadrenocorticism (addison's disease) is an uncommon illness. its prevalence in the general canine population is estimated between 0.06 and 0.28%. certain breeds appear to have an increased risk for developing hypoadrenocorticism, including bearded collie, standard poodle, portuguese water dog and nova scotia duck tolling retriever, with reported prevalence of 9.4, 8.6, 1.5 and 1.4%, respectively. the objective is to evaluate the prevalence and clinical features of naturally occurring hypoadrenocorticism in great pyrenees (gp) presented at the centre hospitalier universitaire v et erinaire (chuv) of the university of montreal. this retrospective study (march 2005 to october 2014) includes 11 client-owned great pyrenees diagnosed with hypoadrenocorticism. the medical records of dogs with a diagnosis of naturally occurring hypoadrenocorticism were reviewed, with an emphasis on great pyrenees' record. the prevalence of hypoad-renocorticism in the studied population, as well as the prevalence per breed, was calculated. data collected included breed, clinical signs, laboratory findings, age at diagnosis, treatment, and cause of the death. one hundred dogs were diagnosed with naturally occurring hypoadrenocorticism, representing 0.38% of the overall canine population studied. thirty-five breeds were represented, with a prevalence per breed varying between 0.17% and 9.73%. a high prevalence was observed in west highland white terriers (4.66%), great danes (1.87%), standard poodles (1.76%), saint-bernards (1.72%) and jack russell terriers (1.48%). out of 114 gp presented during that period of time, 9.73% (n = 11) were diagnosed with hypoadrenocorticism. median age at diagnosis was 4.71 years (range: 0.39 to 11.07) in dogs with hypoadrenocorticism, and 3.51 years (1.02-8.21) in gp. the main reason for presentation of the addisonian gp was lethargy (n = 7) and anorexia (n = 5). clinical findings included hypotension (n = 7), poor body condition (n = 3), and heart murmur (n = 3). the majority (n = 9) had serum electrolytes abnormalities, with a na:k ratio ranging from 15.2 to 22.45. other major laboratory findings included azotemia (n = 8), anemia (n = 7) and the absence of a stress leukogram (n = 5). the majority (n = 9) received fludrocortisone, with prednisone as needed. one gp was euthanized at time of diagnosis. great pyrenees diagnosed with hypoadrenocortism were overrepresented in the studied population, with a prevalence of hypoadrenocorticism in our gp population of 9.73%. therefore, an inherited susceptibility can be suspected. reason for presentation and clinical signs were nonspecific, and similar to what is reported in other breed. in human medicine, haemoglobin a1c (hba1c), a form of glycosylated haemoglobin, is used as the standard measure of average glycaemic control over 2-3 months. the measurement of hba1c in dogs has been previously demonstrated however high pressure liquid chromatography techniques are too technically difficult for routine use and other methods are no longer available. the objective of this study was to assess the use of latex immunoagglutination inhibition using a monoclonal antibody for the measurement of hba1c in dogs, using the siemens dca vantage ò . repeatability was assessed by measuring 4 samples 5 times within 45 minutes. the effect of storage on edta anticoagulated samples was examined by measuring 3 samples stored at 4°c every day for up to 5 days. storage was further assessed by freezing 5 samples and measuring them at 0, 4 and 8 weeks. the machine was then used to compare the hba1c values in 3 groups of dogs with diabetes mellitus (group 1, n = 16), hyperadrenocorticism (group 2, n = 5) or non-diabetic/cushingoid hospitalised patients (group 3, n = 23). differences in the groups were examined for significance using a kruskal-wallis analysis of variance. the reference range of hba1c has been previously calculated to be 3.7-5.6% (17-38 mmol/mol) and values of 4.9 to >13% (30 to >119 mmol/mol) are seen in diabetic animals using high pressure liquid chromatography. the median coefficient of variation for the repeatability study was found to be 5% (range 3-6%). it was possible to store samples at 4°c for up to 5 days (median cv% = 3%, range 2-3%) and at à20°c for at least 8 weeks (median cv% = 6%, range 4-9%). the median hba1c concentrations were group 1; 5.6% (38 mmol/mol), group 2; 3.4% (14.2 mmol/mol) and group 3; 3.3% (13 mmol/mol). group 1 was significantly different from the other two groups using kruskal-wallis analysis of variance. in conclusion, the latex immunoagglutination method was repeatable for the measurement of hba1c in dogs. in addition, hba1c in canine edta anticoagulated samples were stable at 4°c for up to 5 days and, if frozen, could be stored for at least 8 weeks without significant sample deterioration. the assay provides the expected results in dogs with and without abnormalities of glycaemic control. disclosures: the siemens dca vantage was provided on loan from siemens, as well as the cartridges used on this machine to run all samples in the study. a. wehner 1 , r. anderson 1 , s. reese 2 , k. hartmann 1 . 1 clinic of small animal medicine, munich, germany, 2 institute of veterinary anatomy, histology and embryology, munich, germany measurement of total thyroxine (t4) is often the first diagnostic step in the work up of thyroid disease in dogs and cats. blood samples are routinely sent to a reference laboratory causing a delay in testing which might impact the results. the aim of this study was to validate an enzyme fluorescence assay (elfa) as an inhouse method to measure t4 in dogs and cats. t4 was measured in sera of 162 dogs and 88 cats by two methods, an enzyme immunoassay (eia) and an enzyme fluorescence assay (elfa). the eia served as the standard method to which the elfa results were compared. the elfa was performed with the minividas automated analyser (biom erieux, craponne, france) according to the manufactors instructions. coefficient of variation (cv) of the elfa in dogs sera was 5.8% and of the eia 6-9.5%, respectively. cv of the elfa in cats sera was 0.7-3.4% and of the eia 7.6-15.7%, respectively. overall bias of the elfa in dogs was 1.4%; however up to à26.7% in lower t4 ranges. maximal bias of the elfa in cats was 6.9%. correlation of both methods was linear only in cats. using bland altman plots limits of agreement were à74 to 72% in dogs and à58 to 72% in cats. cohen 0 s kappa revealed only slight agreement between both methods in dogs, but a good to very good agreement in cats. the elfa is a fast method with a high precision and can be recommended to measure t4 in cats, but cannot be recommended for dogs. disclosures: no disclosures to report. dysfunctions of the central nervous system (cns) are the most frequent causes of neurological disorders in dogs. our study aimed to find (1) if some cns diseases could be associated with a selected group of common microbial or viral pathogens in dogs and (2) if cns diseases have any characteristic profile with regard to two parameters, c-reactive protein (crp) and iga, that are reported to be potentially useful but unspecific markers of cns diseases. we analysed 210 cerebrospinal fluid samples obtained from dogs with varying neurological signs between june and november 2014. real-time pcr was employed with probes for toxoplasma gondii, neospora caninum, anaplasma phagocytophilum and canine distemper virus. igg-antibodies to tickborne encephalitis virus (tbe) were assayed and iga titres were measured using elisa, while crp concentrations were determined by immunoturbidimetric assay. the dogs had a median age of 4 years (range: 0.5-14) and comprised 60 breeds most frequently involving chihuahuas, labrador retrievers, bernese mountain dogs and boxers. gender distribution was 22.4% female, 12.9% spayed bitches, 42.8% male, 15.7% neutered male, and 6.2% non-identified. none of the cases were pcr-positive for toxoplasma gondii or canine distemper virus. one dog was positive for anaplasma phagocytophilum and another for neospora caninum. antibodies to tbe virus were within the borderline range in 6/210 dogs. the 210 dogs could be divided into two age groups: 60 (=28.6%) for young dogs (<2 years, median 1 year) and 150 (=71.4%) for older dogs (≥2 years, median 6 years). igahigh (>0.1 mg/dl) cases represented 95% and 90% for young and older dogs, respectively. crp-high (>1.0 mg/l) cases were almost half and equal: 51.7% and 56.0% in young and older dogs, respectively. compared with older dogs, young dogs had higher levels of crp (p = 0.022) and iga (p = 0.054). within the iga-high cohorts, crp-high and crp-low cases distributed almost equally (46.7% versus 48.3%) in young dogs but disproportionate (52.0% versus 38.0%) in older dogs. there were no [iga-low/crp-low] cases in young dogs but 6.0% in older dogs. our present data sug-gest that (1) canine cns disorders were largely characterized by high iga and particularly in young dogs (2) inflammatory types (crp-high) were almost equal in both groups and (3) internet is a potential source of medical information for pet owners. therefore, it could play an indirect but important role in the veterinary practice. this survey assesses the online search behaviour of french pet owners for their pets' health and its influence on a veterinary consultation. in april 2013, 260 french pet owners coming in a veterinary teaching hospital for a medical or a surgical consultation were surveyed. 239 (91.9%) owners fulfilled the questionnaire on a voluntary basis. the survey contained 26 questions dealing with three topics: the online search behaviour of owners for their pets' health, their perception of the information found online and the internet's influence on a consultation and on the veterinarian/client relationship.73.6% of owners use the internet to obtain information on their pets' health. among them, 32.6% use it rarely and 28.9% occasionally. they mainly look for information on a disease (51.1%), a symptom (51.1%), a breed (50%) or a nutritional advice (48.9%). 79.7% of owners try to verify the accuracy of the information found, most often by questioning their veterinarian (82.8%). few owners (15.4%) think that online information is always trustworthy. most of the research (81.9%) is randomly made, websites being found through search engines. the majority of pet owners (81.3%) aren't aware of any health certification label for websites. internet enables certain pet owners to feel more at ease with their pets' health care: they ask more questions to their veterinarian (88.9%) and feel more involved in medical choices (55.6%). 37.9% of owners consider that the internet can positively impact their relationship with the veterinarian. relief is the most common (73.6%) emotional response to online research for medical information. however, 58.8% of owners feel overwhelmed by the amount of information found, 56% are confused and 35.2% frightened by the serious or graphic nature of the information found online. this study emphasizes the frequent but measured use of the internet by french pet owners for their pets' health. they seem to consider information found on the net with a critical mind. unexpectedly, it appears that the internet could be an ally for veterinarians by promoting exchanges between the clients and the veterinarian and by improving compliance with the care project. disclosures: no disclosures to report. sinonasal aspergillosis is a well-known and described fungal infection of the sinonasal cavities in dogs. topical treatment either with enilconazole or itraconazole infusion administered surgically or endoscopically are effective. the use of 1% clotrimazole cream have been described in a surgical setting after itraconazole infusion by sissener et al. in 2006. the aim of the work was to report the effectiveness of the use of topical 1% clotrimazole cream as the only treatment for sinonasal aspergillosis in dogs. inclusion criteria were a full medical record with radiological and endoscopical imaging, record of clotrimazole discharge after instil-lation and endoscopic control between 60 and 90 days after procedure. the 1% clotrimazole cream was applied through catheters placed under direct endoscopic vision after fungal plaques removal. nine dogs were included. three dogs were mixed breed dogs, two dogs golden retriever, one dog a german shepherd and one old english sheepdog, one bull terrier and one cane corso. six dogs were male (one neutered) and three female (one intact). mean age was 6.8 years. main clinical signs were muco-purulent discharge (8), pain at sinonasal palpation (8), nasal planum alterations (6), epistaxis (3) . nasal discharge was bilateral in 5 dogs. mean duration of clinical signs was 1.5 months. main radiological findings were turbinates lysis (9), frontal sinus empyema (8), frontal bone thickening (3) and frontal bone lysis (4). rhinoscopy disclosed lysis and remodelling of the nasal turbinates (9), easy access to the frontal sinus (7), septum lysis (5), bilateral sinonasal aspergillosis (2), monolateral nasal aspergillosis (2), monolateral sinonasal aspergillosis and contralateral nasal aspergillosis (2), monolateral frontal aspergillosis (3). main duration of nasal cream discharge was 3.5 days. all dogs underwent endoscopic control between 60 and 90 days after the procedure. seven dogs were disease free; two dogs had persistent fungal plaques and underwent a second treatment. success rate was 77.8%. success rate of this study is comparable to other studies with larger and smaller case series. endoscopic removal of the fungal plaques can be time consuming and topical administration of either enilconazole or itraconazole require an additional hour. the catheter placement and the 1% clotrimazole cream application lasted 5 minutes for each cavity in the dogs here reported. the use of 1% clotrimazole cream as the only treatment for sinonasal aspergillosis needs further evaluation on a larger case series. disclosures: no disclosures to report. few studies exist on euthanasia in small animal practice. however, such an act belongs to veterinary procedures, more or less frequently depending of the kind of practice, and may deeply impact both owners and veterinarians. we intended to study practical, ethical and psychological aspects of euthanasia of dogs and cats among french veterinary practitioners. from october 2014 to february 2015, an on-line 79-item questionnaire on small animals' euthanasia, addressing practical aspects of euthanasia, communication with the owners, ethical problems, owners' and veterinarians' perceptions, was emailed via professional associations and networks. results were analyzed using commercial software (sphinx iq ò and excel ò ). 2770 french veterinarians practicing small animal medicine completed the questionnaire, representing >20% of this population. euthanasia occurs rarely at home. over 85% of veterinarians propose the owners some time alone with their pet, and to stay during euthanasia, performed most commonly by intravenous injection (91.4%) mainly after sedation/anesthesia (95.9%). ninety nine percent of veterinarians consider communication, including description of events' sequence, and disposal of the body, as important. estimated minimum communication time required varies from 5-15 to 15-30 minute. following euthanasia, 64.4% are often thanked by the owners. most veterinarians (>85%) have refused a euthanasia, considered unjustified, or had their own suggestion of euthanasia rejected. reasons for such a suggestion include intractable pain (98.7%), non-acceptable complications (79.9%), financial considerations (44.1%) or animal considered dangerous (71.6%). veterinarians think most owners (63.9%) experience some sense of guilt during euthanasia. themselves perceive euthanasia most commonly as relief of animal's suffering (81.7%) and part of veterinary practice (64%), less frequently as a defeat (22.6%). almost all veterinarians have experienced emotionally challenging euthanasia, and 72.4% estimate that practicing euthanasia influences their perception of death. practical (74%) and psychological (48.4%) aspects of euthanasia have been discussed in most teams. veterinarians' gender influences euthanasia management, mostly concerning some communicational and practical aspects. euthanasia is definitely not an ordinary veterinary act, neither for the owner nor for the veterinarian. therefore, this act must be performed with as much care and communication as possible. disclosures: no disclosures to report. canine pancytopenia is associated with a range of intra-marrow or extra-marrow causes, including though not limited to, infectious agents, drugs, toxins and neoplasms. there is currently limited information regarding the clinicopathological features of the underlying causes or the prognosis in pancytopenic dogs. the objective of this retrospective study was to better define the spectrum of diseases associated with canine pancytopenia, to establish possible clinicopathological discriminators for the common causes and to investigate if the severity of pancytopenia or the underlying disease were associated with the clinical outcome (death or survival). medical records of dogs with a comprehensive diagnostic investigation admitted in a veterinary teaching hospital were retrospectively reviewed. pancytopenia was defined by a hematocrit (hct) <37% (<30% for dogs <5 months of age), white blood cell counts (wbc) <6.000/ll and platelet counts (plt) <200,000/ll. a control group of 238 dogs without any evidence of blood cytopenia(s) was also established. in total, 119 pancytopenic dogs were studied. bone marrow aspiration cytology was examined in 42 cases and aplasia of all hematopoietic lineages was observed in 22 (52.4%) dogs. the most common diagnoses included monocytic ehrlichiosis (cme) (n = 43), leishmaniosis (canl) (n = 28), parvoviral enteritis (pe) (n = 19), and concurrent cme and canl (n = 12). mixed breed dogs were more likely to develop pancytopenia as compared to purebreds and pancytopenic dogs tended to be younger than the controls (conditional dependent logistic regression model, p = 0.013 and p = 0.001, respectively). among the most common diseases associated with pancytopenia, the mean wbc counts were significantly lower in dogs with cme and pe compared to dogs with canl (one way anova with bonferroni test for multiple comparisons, p = 0.004 and p = 0.03, respectively), while plt counts were significantly lower in cme compared to canl (p < 0.0001) or pe (p < 0.0001). total protein concentrations were significantly lower in dogs with pe compared to cme (p < 0.0001) and canl (p < 0.0001). using a univariable logistic regression analysis model, no association was established between the underlying disease and the final outcome. however, higher hct (by at least one percentage unit), wbc (by at least 1000/ll) and plt (by at least 10,000/ll) values tended to significantly increase the odds of survival (p = 0.025, p < 0.0001 and p = 0.006, respectively). in the present study, cme, canl and pe were the major causes of canine pancytopenia. potentially useful diagnostic indicators included severe leucopenia (cme, pe), thrombocytopenia (cme) and hypoproteinemia (pe). disclosures: no disclosures to report. bwbp detects both upper and lower airways diseases because any site of airway obstruction will result in increased pressure changes associated with breathing. nevertheless our results suggest that there are significant differences in tv (p = 0.0001), ti (p = 0.0001), pef (p = 0.0001), eep (p = 0.003), penh (p = 0.0001) and pau (p = 0.0001) between lm and fbd cats. no other significant difference in bwbp parameters was found. upper airway obstructions have been previously evaluated in cats by using pft (mckieman, 1993 , lin, 2014 but in authors' knowledgement this is the first study designed to compare upper and lower airway obstructions by using bwbp. attending our results, there is the evidence that bwbp can help characterize mechanical dysfunction of the airways in cats with lm obstruction. however we must keep in mind some limits of this study as the low number of animals, individual variability in breathing pattern and to have the chance of doing bronchoreactivity tests. disclosures: no disclosures to report. the median lifespan of domestic dogs has been estimated to 9-12 years, but little is known about risk factors for mortality in aged dogs: most mortality studies in dogs have been carried out among diseased dogs (renal or heart diseases, cancers, post-operative death). to determine which characteristics are associated with mortality in a priori healthy aged dogs, a prospective cohort study has been conducted in 116 guide dogs, followed from a systematic geriatric examination (ge) to either (all-cause) death or cut-off date (july 16th, 2013). survival analyses (kaplan-meier estimators, log-rank tests, and multivariate cox proportional hazards models) were used to assess the associations with time to death. median age at ge was 8.9 years, all dogs were neutered, and 50% were female. the majority of dogs were golden retriever (n = 48) and labrador retriever (n = 27). among these 116 dogs, 16% were obese, 47% presented skin nodules and 90% used bus as transportation. a total of 76 dogs died during follow-up, leading to a median survival time from ge of 4.4 years. after adjustment for demographic and biochemical variables (age, sex, total proteins, cholesterol and alp), an increased alanine amionotransferase level (≥102 ui/l; adjusted hazard ratio [ahr], 6.0), presenting skin nodules (ahr, 2.3), and not being a labrador (ahr, 3.3) were independently associated with a shorter time to death (p < 0.05). public transportation tended to be associated with mortality (ahr, 3.0; p = 0.06), highlighting the importance of environment in mortality. neither sex nor other biochemical parameters were significantly associated with mortality. the alanine amionotransferase level and the presence of skin nodules seem predictors of mortality in senior guide dogs, mostly labrador, golden, or mixed breed of labrador/golden. the impact of environment, in particular urban environment, on mortality needs further investigation. studies in other breeds and pets are also necessary to generalize these results. disclosures: sara hoummady received a grant from mp labo for his phd work about dog aging and marc blondot work at the paris guide dog school. laryngeal dysfunction is most commonly associated with aspiration pneumonia, however its role in other lower airway diseases has not been investigated. laryngoscopic and bronchoscopic findings in dogs examined by the author between 2001 and 2014 were evaluated for the presence or absence of laryngeal abnormalities. dogs that presented for evaluation of inspiratory difficulty or panting were excluded from analysis. clinical diagnoses of inflammatory airway disease, airway collapse, airway infection, eosinophilic bronchopneumopathy or a combination of these disorders were obtained through bronchoscopy and bronchoalveolar lavage fluid analysis. detection rates for laryngeal abnormalities were compared among disease groups using chi square analysis and fisher's exact test, with significance set at p < 0.05. a total of 138 dogs were evaluated and varied in age between 4 months to 15.5 years (median 8 years). weight ranged from 1.5 to 63.4 kg (median 13 kg), with 31 dogs <5 kg, 28 dogs from 5.1-9.9 kg, 24 dogs from 10-20 kg, 45 dogs from 20.1-40 kg, and 9 dogs >40 kg. laryngeal hyperemia or swelling was found in 73/138 dogs (53%), and detection rate did not differ among disease processes. laryngeal function was considered suspect in 59/138 cases, prompting administration of doxapram, which normalized function in 30/59 dogs. laryngeal paresis or paralysis was reported in a total of 26/ 138 dogs (19%). a substantial number of dogs with chronic cough displayed evidence of abnormal laryngeal structure or function, suggesting that a complete laryngoscopic examination should be performed in all dogs evaluated for cough. disclosures: member of the feline advisory board, speaker honoraria for international, national, and regional continuing education meetings. bronchiectasis is a poorly characterized disease in dogs identified by airway dilatation on radiographs, computed tomography, bronchoscopy, or histopathology. little is known about underlying disease processes associated with bronchiectasis in dogs. medical records from dogs presented to uc davis were searched for identification of bronchiectasis. underlying disease processes and clinical diagnoses were obtained through review of the history, physical examination, respiratory endoscopy and bronchoalveolar lavage fluid analysis and microbiology. historical reports, results of imaging, bronchoscopy and fluid analysis, and scrutiny of pathologic and clinical diagnoses were comprehensively evaluated to identify the most likely underlying disease process associated with bronchiectasis. between 2003 and 2014, bronchiectasis was diagnosed in 86/621 dogs (14%) that had bronchoscopy performed. dogs ranged in age from 0.5 to 14 years (median 10 years) with 1/85 dogs <6 months, 16/85 dogs (19%) 1-5 years, 37/85 dogs (43%) 5.1-10 years of age and 31/85 dogs (37%) over 10 years of age. dog breeds affected more than once included 6 labrador retrievers, 5 cocker spaniels, 4 golden retrievers and 4 standard poodles. duration of cough ranged from 3 days to 10 years (median 6 months). underlying disease processes included pneumonia in 45/86 (52%) dogs, inflammatory airway disease in 24/86 (28%) dogs, and eosinophilic bronchopneumopathy in 10/86 (12%) dogs. twenty-three of 85 dogs (27%) had positive bacterial cultures, with isolation of streptococcus (n = 6) and enteric species (n = 5) most commonly. this study found that bronchiectasis often occurs in older, large breed dogs with infectious or inflammatory pneumonia. disclosures: johnson: feline advisory board, speaker honoraria. chronic respiratory disease, often characterized by a chronic cough, is common in dogs. the purpose of this study was to determine the etiology of chronic respiratory disease in dogs that were presented with persistent and chronic coughing. a retrospective study of 126 client-owned dogs with signs of persistent and chronic lower respiratory disease, that underwent bronchoscopy together with either an endotracheal wash (etw) or broncho-alveolar lavage (bal), was performed. all dogs were evaluated by means of full clinical examination, hematology and serum biochemistry analyses, survey thoracic radiographs, echocardiography and ecg (if indicated), and bronchoscopy with cytological analysis and aerobic culture of etw or bal fluid. an etw was performed in 112/126 (89%) dogs while a bal was performed in 15/126 (11%) dogs. a positive aerobic bacterial culture was identified in 42/126 (33%) of submitted etw/bal fluid samples. most commonly isolated bacteria included mycoplasma sp. (24%), bordetella bronchiseptica (24%) and pseudomonas aeruginosa (12%). a definitive diagnosis was made in 118/126 cases (93.6%). chronic bronchitis was the most common diagnosis (37.3%), median age 8 years; followed by airway tracheal collapse or bronchomalacia (23%), median age 11 years,; and primary bacterial infections (15.8%), median age 3 years. less common etiologies identified included neoplasia (5.5%), median age 14 years; parasitic infections (4.8%), median age 7 years; and eosinophilic bronchopneumopathy (3.2%), median age 6 years. rare etiologies identified included primary pulmonary hypertension, primary ciliary dyskinesia, excitement-induced cough, and obesity. myxodematous mitral valve disease was found concurrently in 12/126 (9.5%) dogs. this study concluded that by using a structured combination of survey thoracic radiography, bronchoscopy and etw or bal with cytology and culture, a diagnosis could be made in the majority of dogs with chronic respiratory disease. disclosures: no disclosures to report. canine sterile steroid-responsive lymphadenitis (cssrl) is an uncommon cause of lymphadenomegaly. diagnosis is one of exclu-brachycephalic dogs have unique upper respiratory anatomy with abnormal breathing patterns similar to those in humans with obstructive sleep apnea syndrome (osas). oxidative stress in the body represents the imbalance between the production of reactive oxidative species and the ability of antioxidant defense mechanisms to detoxify the reactive intermediates. oxidative stress is involved in the pathogenesis of many diseases, including hemolytic anemia, atherosclerosis, tissue reperfusion injury and has also carcinogenic potential. several studies have clearly shown an association between obstructive sleep apnea syndrome in humans and oxidative stress, but detailed underlying pathomechanism remains unclear. due to the consideration of brachycephalic dogs as an animal model of human osas, this study was designed to evaluate oxidative stress (paraoxonase type-1 activity; pon1 and total antioxidant status; tac) in brachycephalic dogs with brachycephalic airways obstructive syndrome (baos) before and after surgery compared to control dogs. this study was conducted on 37 dogs with baos and 34 control dogs. twenty out of 37 baos dogs were evaluated 1-2 months after surgical correction. mean values of tac and pon1 in different studied groups were as follows: control dogs (tac: 0.614; pon1:2.53), baos dogs (tac: 0.233; pon1: 2.438), baos dogs post-surgery (tac:0.177; pon1: 2.705) a statistically significant difference on tac levels is observed between dogs with baos and control dogs (p < 0.05). no statistically significant differences were observed in pon1 and tac levels before and after surgery. on the other hand, no differences have been observed between pon1 and tac levels in baos dogs according type of brachycephalic breed, grade of respiratory and digestive signs or presence of everted ventricular laryngeals. the results of our study showed a statistically significant difference in tac values between control and dogs with baos, confirming the oxidative stress previously described in humans. even that human patients with osas can partially reverse their increase in oxidative stress by using a nasal continuous positive airway pressure treatment, in dogs with baos no differences were observed before and 1 month after surgical treatment. baos surgical treatment is not useful to reduce pon-1 or tac levels, probably because baos does not induce such an evident inflammatory process in dogs as in human patients with osas. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). the cipf course varies greatly among dogs from rapid deterioration to slowly progressive forms and the survival time from onset of clinical signs ranges from a few months to several years. in human ipf, increased chemokine (cc-motif) ligand 2 (ccl2) concentrations in bronchoalveolar lavage fluid (balf) are indicative of a poor outcome and serum concentrations are correlated with clinical parameters of lung function. in dogs, serum and balf ccl2 concentrations were shown to be elevated in whwts with cipf compared with healthy whwts. the aim of the present study was to investigate whether serum ccl2 concentrations measured in whwts with cipf at diagnosis (1) can be used as an indicator of prognosis and (2) correlate with lung function parameters. ccl2 concentrations were determined by elisa (canine ccl2 quantikine elisa kit, r&d systems) in the serum of 60 whwts suspected of cipf (median age 11.7 years, range 5.7-14.5), for which a follow-up was available (median follow-up time 8.6 months, range 0-71.8). serum sampling extended from may 2007 to january 2015. cipf diagnosis was confirmed by thoracic high resolution computed tomography, lung histopathology, or both examinations in 17, 6 and 27 whwts respectively. kaplan-meier analysis was conducted to investigate differences in survival times according to serum ccl2 concentrations at diagnosis. spearman analysis was used to assess correlations between serum ccl2 concentrations and lung function parameters, namely the distance walked in the 6-minute walking test (6mwd) and the arterial partial pressure of oxygen (po 2 ). among the 60 cipf whwts included, 31 died or were euthanized for cipf-related reason, 12 died or were euthanized for non-cipf-related reason and 17 were still alive at the end of the study. the median survival of whwts with cipf-related death or euthanasia was 6.4 (range 0.4-71.9) months from diagnosis. serum ccl2 concentrations above 700 pg/ml were significantly associated with a shorter survival time in whwts affected with cipf (p = 0.02). a weak negative correlation was found between serum ccl2 concentrations and the 6mwd (r = à0.382, p = 0.03, n = 31), while no correlation was observed with arterial po 2 values (n = 49). in conclusion, serum ccl2 concentration provides prognostic information in whwts suffering from cipf, while this marker is weakly correlated with the clinically lung function parameters available in the present study. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). this study was intended to (1) describe thoracic high-resolution computed tomography (t-hrct) findings obtained in cipf dogs under general anesthesia (ga) using the glossary of the fleischner society and (2) compare images obtained under ga (t-hrct ga ) with those obtained under sedation (t-hrct s ). t-hrct images from 11 whwts with cipf and 9 control whwts were retrospectively reviewed by three observers in consensus. specific t-hrct features were assessed and graded for each lung lobe (0 = absence, 1 = mild, 2 = moderate and 3 = severe). a global score was then calculated. the khi² test with the threshold 5% was used for the statistical analysis. ground glass opacity (ggo) was observed in all cipf whwts and in 5/9 of controls (p = 0.013). in controls, ggo was mild and localised mainly in cranial lobes. in cipf whwts, ggo was mild, moderate or severe in 2, 4 and 5 dogs respectively, without lobe predilection. consolidation was observed in 5/11 cipf whwts but not in controls (p = 0.020) and was mild (3/5) to moderate (2/5). a mosaic pattern, suggestive of air trapping, was noticed in 8/11 cipf whwts but not in controls (p = 0.001) and was mild, moderate or severe in 3, 2 and 3 whwts respectively, without lobe predilection. nodules were present in 3/11 cipf whwts but not in controls. reticulation, subpleural bands and parenchymal bands were noticed in 1, 1, and 3/11 cipf whwts respectively. honeycombing, emphysema, pleural effusion and pleural thickening were never observed. bronchial wall thickening and mild bronchiectasis were present in 6/11 and 3/11 cipf whwts respectively but not in controls (p = 0.008 and p = 0.09). the overall t-hrct s quality was good in 10/17 examinations compared with 16/20 for t-hrct ga (p = 0.160). the presence of motion artefacts was higher for t-hrct s (p < 0.001), but were most frequently graded as mild (p < 0.001). t-hrct s allowed identification of a mosaic pattern in 2 additional cipf whwts, while consolidation could not be identified in 2 others. there was no difference in identification or gradation for the other features between t-hrct ga and t-hrct s . in conclusion, ggo, consolidation, mosaic pattern and bronchial wall thickening are the main t-hrct features of cipf in whwts. honeycombing, the major feature of ipf in humans, was never observed, which suggests a different pathophysiology between the two entities. t-hrct s images are in accordance with t-hrct ga and can be used for cipf diagnosis. disclosures: no disclosures to report. literature on mesenterial lymphadenitis (lad) or mesenterial lymph node abscesses (lab) in small animals is scarce. case files from 2005 to 2014 were searched for dogs with the diagnosis of mesenterial lad/lab based on cytology and/or histopathology. of 24 cases identified, 5 had to be excluded due to incomplete data. the remaining dogs were of mixed breed (n = 6), small munsterlander (n = 4) and one each of other breeds. nine dogs were male and 10 female. median age was 47 months (range . diagnosis was based on fine needle aspiration (fna; n = 9), histopathology (n = 1) or both (n = 9). significant findings in the dogs' history included gastrointestinal signs (n = 2), 1 puppy whose mother had mastitis, bite wounds/abscesses of the skin (n = 2), pulmonic stenosis (n = 1) and orthopaedic diseases (n = 3). most common presenting complaints were lethargy (n = 13), hyperthermia (n = 12), diarrhoea (n = 7), vomiting/nausea (n = 6), inappetence/anorexia (n = 5), back/abdominal pain (n = 4) and lameness (n = 2). diagnostic tests performed included haematology/serum biochemistry (n = 19), thoracic (n = 12) and abdominal (n = 7) radiographs, abdominal ultrasound (n = 19), ct/mri (n = 3), fnas of other organs (n = 7), urinalysis (n = 12) with culture (n = 7), coagulation profiles (n = 5), orthopaedic radiographs (n = 2), cpl (n = 5), blood cultures (n = 1), and csf/joint taps (n = 2). dogs were retrospectively divided into group a (n = 9): dogs with no other disease than lad/lab ("idiopathic") and group b (n = 10): dogs with other diseases diagnosed simultaneously. these included neoplasia (carcinoma n = 1, lymphoma n = 2, leiomysarcoma n = 1, histiocytic neoplasia n = 1, prostate carcinoma n = 1), gastroenteritis (n = 3), presumed pancreatitis (n = 2), purulent monoarthritis (n = 1), purulent hepatitis/ splenitis (n = 2), fungal infection at a distant site (n = 1), and mycobacteriosis (n = 1). eleven dogs received surgical treatment and antibiotics, and 8 dogs conservative medical management consisting of supportive treatment and antibiotics. all dogs were discharged alive. dogs in group a were hospitalized longer (mean 8 days, sd 3.4) than dogs in group b (mean 3 days, sd 2.2) (p = 0.004). the median follow-up time was 67 days (4-784 days). there was no difference in pretreatment with antibiotics or anti-inflammatories between groups. t-tests or kruskall-wallis tests showed that dogs in group a were borderline significantly younger (p = 0.052), had significantly higher respiratory rate (p = 0.004), rectal temperature (p = 0.007), monocyte count (p = 0.014) and crp concentration (p = 0.027) than dogs in group b. the small munsterlander had an odds ratio of 32 over other breeds to suffer from lad/lab. in conclusion, idiopathic mesenterial lad/lab was seen in young dogs with hyperthermia and gastrointestinal signs. diagnosis of purulent lad/lab on fna does not exclude the presence of another underlying pathogenesis. disclosures: no disclosures to report. canine infectious respiratory disease (cird) is a disease complex caused by different viral and bacterial pathogens. aim of the study was to evaluate clinical and laboratory factors associated with different infectious agents. dogs were included, if they showed respiratory signs (<14 days) consistent with cird and if non-infectious respiratory conditions could be excluded. nasal and pharyngeal swabs were taken from 61 dogs with cird and tested for respiratory pathogens, including canine parainfluenza virus (cpiv), canine adenovirus (cav), canines distemper virus (cdv), canine herpes virus (chv), canine respiratory coronavirus (crcov), canines influenza virus (civ), and bordetella bronchiseptica (b. bronchiseptica) by polymerase chain reaction. results of clinical and laboratory data were correlated with the underlying pathogen using fisher's exact test and chi-square test (p ≤ 0.05). cpiv was detected in 23, crcov in six, and b. bronchiseptica in 48 dogs; 23 patients showed infections with more than one pathogen. there was no significant difference for age and gender distribution between the three groups; however, dogs infected with cpiv more likely originated from a shelter (p = 0.037). when clinical data were compared, there was no significant difference for the parameters depression, fever, cough, nasal discharge, dyspnoea, and abnormal lung sounds. furthermore, there was no sig-nificant difference regarding abnormalities of erythrocytes, platelets, leukocytes, and differential count between groups. the study shows that in dogs with cird clinical and laboratory parameters cannot indicate the underlying pathogen. furthermore, diseases severity does not seem to depend on the infectious organism involved. disclosures: no disclosures to report. breed related predisposition to bacterial bronchopneumonia (bp) has been reported in irish wolfhounds (iwhs). underlying factors are unknown, however immune deficit, ciliary dysfunction and aspiration have been suggested as predisposing factors. the purpose of this prospective study was to evaluate lymphocyte subpopulations in iwhs with one or more previous bp and compare results to elderly iwhs without any previous bacterial respiratory infections. additionally, healthy dogs of other breeds were included as controls. previous bp was confirmed in 11 iwhs (median age 5.1 years, interquartile range 2.2-7.0 years). healthy iwhs (n = 13, 6.8, 6.3-8.2 years) or dogs of other breeds (n = 15, 5.5, 3.5-6.3 years) had no history or findings suggestive of previous bp. edta blood samples, collected from all dogs when they were healthy, were stained with fluorescent mouse anti dog cd3, cd4, cd8, cd21 and mhc class ii antibodies (abd serotec ò ) and flow cytometry analysis was performed with bd facsaria ò ii cell sorter and facsdiva ò software. statistical comparison between groups and the effect of age was studied using analysis of covariance (ancova) models. the number of leucocytes did not differ significantly between groups. the total numbers of lymphocytes and numbers of major lymphocyte subpopulations (b-cells, cd4+ and cd8+ t-cells) were significantly lower in healthy iwhs and iwhs with previous bp compared to dogs of other breeds (lymphocytes p < 0.001 and p < 0.001; b-cells p = 0.013 and p = 0.005; cd4+ t-cells p = 0.026 and p = 0.029; cd8+ t-cells p < 0.001 and p = 0.001 respectively). percentage and number of mhc class ii+ non-b lymphocytes was significantly higher in both iwh groups than in dogs of other breeds (p < 0.001 in all comparisons). lymphocyte numbers and subpopulations did not differ significantly in healthy iwhs compared to iwhs with previous bp. an age-related decline in the total number of lymphocytes (p = 0.015), t-cells (p = 0.007), cd4+ t-cells (p = 0.007) and mhc class ii+ non bcells (p < 0.001) was noticed only in the group of iwhs with previous bp. these preliminary results indicate that iwhs may have significantly lower numbers of lymphocytes, b-cells as well as cd4+ and cd8+ t-cells than dogs of other breeds. further studies are needed to determine whether these alterations represent a breed related phenomenon or are connected to the predisposition to bp. an age-related decline in lymphocyte, total t-cell and cd4+ t-cell numbers was detected in iwhs with previous bp. in humans, age related changes in cd4+ t-cells have been associated with increased susceptibility to infections. disclosures: no disclosures to report. feline chronic kidney disease (ckd) is a common feature of ageing cats. angiotensin-converting enzyme inhibitors (acei) are recommended to treat hypertension associated with ckd to limit target-organ damage and especially glomerular hypertension. in addition, the international renal interest society (iris) guidelines recommends the prescription of an acei in patients with ckd and proteinuria. to our knowledge no study has demonstrated the effects of long term administration of acei in a client owned population of cats suffering from ckd on glomerular filtration rate (gfr). the aim of the study was to evaluate the effect of an acei (imidapril, prilium ò , v etoquinol sa) on the gfr of cats suffering from naturally occurring chronic kidney disease (ckd) over 12 months. sixty-six cats presenting with clinical and biological signs of ckd were enrolled by 20 european investigators and followed up till 24 months in this randomized blinded study. thirty-two cats provided suitable data for gfr analysis; 17 animals received imidapril at the dosage of 0.5 mg/kg/d and 15 received placebo. animals with no available gfr value on day 0 or with no data after day 0 were excluded as well as animals for which the iohexol clearance could not be determined. follow up was censored after 12 months due to small sample sizes for statistical comparisons. on day 0, month 3, month 6 and month 12, cats were sampled 30, 60 and 120 minutes after intravenous administration of 647 mg of iohexol. gfr was based on determination of the iohexol clearance which was calculated with the phoenix ò winnonlin 6.3 software. statistical analyses were performed with sas/stat 9.2 software. as gfr were not available on each time point for a given animal, the two treatment groups were compared on each gfr determination point using an ancova (analysis of covariance) with the gfr determined on day 0 as the covariate. on d0, gfr were 1.53 ae 0.68 and 1.68 ae 0.60 ml/kg/min in the imidapril and placebo group, respectively. a significant statistical difference (1.62 ae 0.63 versus 1.24 ae 0.59 ml/kg/min in the imidapril and placebo group respectively, p = 0.029) was observed in favor of a higher gfr in the imidapril treated animals on month 6. higher gfr were also observed in the imidapril group on month 3 and month 12 but not significantly different from the placebo group. daily long term imidapril treatment compared to placebo, may be an effective treatment to slow the progression of renal failure in cats with naturally occurring chronic kidney disease. disclosures: authors are employees of vetoquinol. there is a concern over the potential of cytotoxic drugs which could harm exposed workers. the speciality of oncology of the ecvim-ca published guidelines in order to prevent that risk. however few data exist for evaluation of the real risk of occupationnal exposure. this concern was the aim of our study. biomarkers used were vincristine and cyclophosphamide. surface samples were collected in the consultation room and ward dedicated for chemotherapy. samples were collected with wet filter paper on 10 9 10 cm surfaces, or objects (computers for instance). samples were analysed by liquid chromatography and mass spectrometric method.the limit of quantification was 0.2 ng/ 100 cm 2 (or 0.2 g/object) for vincristine and 0.02 ng/100 cm 2 (or 0.02 g/object) for cyclophosphamide. samples in consultation room were collected after treating 2 dogs with vincristine and after cleaning. samples in dedicated ward were collected after cleaning and after 2-days stay of treated dogs. 18 aeras/objects were tested in consultation room; 15 in dedicated ward for vincristine; 9 in ward for cyclophosphamide. after treatment of dogs in consultation room, traces of vincristine were detected on the floor and the laboratory bench top (0.47-0.50 ng/100 cm 2 ). moreover, the surface of auscultation table and extern side of gloves were contaminated after preparation and administration of vincristine (respectively 0.90, 314 and 510 ng/objet). after cleaning, 32% of samples in consultation room were positive. traces of vincristine were detected on the floor or objects (wall otoscope: 0.47-0.49 ng/100 cm 2 ). after cleaning, 40% of samples in ward were positive. traces of vincristine were detected on the floor and objects (boxes, wastebin, bowls: 0.48-0.64 ng/objet) after cleaning and animals treatment. cyclophosphamide were detected on all areas tested (0.04 a 2.23 ng/objet). despite protective guidelines to avoid spread of cytotoxic drug, environemental exposure was demonstrated. however contaminations were limited and show that handling procedures of cytotoxic drugs are well controlled. most-elevated contamination level for vincristin were noticed on extern side of gloves depsite of using appropriate material. workers should pay a particular attention for gloves withdrawal to limit exposure. small amounts of vincristine were found in inapropriate places: computeur mouse. even if there is few of those residues, we thought about people working on a regular basis in this room for other activities than chemotherapy, and we decided to adapt our clinical practices. evaluation of occupational exposure to cytotoxic drugs is an important step to prevent incidents and heigt awarness of nursing staff to apply those good clinical practices. disclosures: no disclosures to report. the tumor-associated inflammatory response had the effect of enhancing mammary tumorigenesis, helping incipient neoplasias to acquire hallmark capabilities, both in human and dogs. t-cells migration to the tumor site and the following activation may be the essential requirement for their promoting effect on tumors. in human breast cancer the signaling pathways of c-kit have been described as possibly being involved in differentiation, migration, survival, and maturation of t-cells and other inflammatory cells into tumor sites. in order to clarify this subject in canine mammary tumors (cmt), 80 malignant neoplasms were studied by using immunohistochemistry comparing the intratumoral cd3+ tlymphocytes and c-kit expression together with vegf, microvessel density (mvd) and clinicopathological characteristics of tumor aggressiveness. cd3+ t cells and high c-kit immunoexpression revealed a positive and statistically significant correlation with vegf (r = 0.503, p < 0.0001; r = 0.284, p = 0.023 for cd3 and c-kit respectively) and cd31 (r = 0.654, p < 0.0001; r = 0.365, p = 0.003 for cd3 and c-kit respectively). a statistically significant association (p = 0.039) and a positive correlation (r = 0.263, p = 0.039) between cd3+ t-lymphocytes and c-kit was also observed. tumors with high c-kit expression showed higher counts of cd3+ t-cells. the mvd of high cd3/vegf tumors was significantly more elevated (p < 0.0001). a similar association was observed for high c-kit/vegf tumors (p < 0.001). in this study high cd3/vegf, high c-kit/vegf and high cd3/c-kit tumors were statistically significantly associated with elevated grade of malignancy (p < 0.0001 for cd3/vegf, c-kit/vegf and cd3/ckit), presence of neoplastic intravascular emboli (p < 0.0001 for cd3/vegf and cd3/c-kit; p = 0.002 for c-kit/vegf) and presence of lymph node metastasis (p < 0.0001 for cd3/vegf, c-kit/ vegf and cd3/c-kit). tumors with high cd3/vegf (p = 0.006), high c-kit/vegf (p < 0.0001) and high cd3/c-kit (p = 0.002) expression were associated with shorter os time. interestingly the group of tumors with high c-kit/vegf retained their significance by multivariate analysis arising as independent predictor of os. results of this study suggest that t-lymphocytes may share common signaling pathways with c-kit and vegf in cmt progression and may contribute to increased angiogenesis, aggression and shorter os in these tumors. disclosures: no disclosures to report. few epidemiological data have been published on feline large granular lymphocyte (lgl) lymphoma. a recent study (krick et al. 2008 ) described clinicopathologic features in cats with lgl lymphoma, but no comparisons were made with cats with other diseases or other forms of feline lymphoma. therefore, the objective of this study was to assess differences in prevalence, signalment (breed, sex, and age), physical exam findings (body weight, body condition score, body temperature, heart rate, respiratory rate, and systolic blood pressure), and felv/ fiv status between cats with lgl lymphoma (group 1) and all other type of feline lymphoma (group 2). the electronic data-base of the san marco veterinary clinic was searched between january-2007 and december-2014 for cats with a cytological or histopathological diagnosis of lymphoma. differences between groups were assessed by t-test, mann whitney, pearson-chi square, pearson chi square yates corrected, and fisher's exact test. during the study period 176 out of 3858 sick cats seen at the clinic were diagnosed with lymphoma (group 1: n = 32; group 2: n = 144). the prevalence of all type of feline lymphoma between 2007 and 2014 compared to sick cats did not change over time ranging from 3.7% to 6.5% per year (p = 0.42; overall prevalence 4.5%, 95% ci 3.8-5.1). the lymphoma lgl prevalence between 2007 and 2014 compared to all types of lymphoma did not change over time ranging from 11.1% to 30.0% per year (p = 0.73; overall prevalence 17.9%, 95% ci 12.2-23.6). among the variables studied, only sex (group 1: 19 [59.4%] females, 13 [40.6%] males; group 2: 54 [37.5%] females, 90 [62.5%] males; p = 0.023) and age (group 1: 126 ae 34 months; group 2: 110 ae 57 months; p = 0.037) were significantly different between groups. sixteen out of 32 cats with lgl lymphoma were tested for their felv/fiv status resulting all felv-and one (6.3%) fiv+. seventy-four out of 144 cats with all other types of lymphoma were tested for their felv/fiv status resulting 28 (37.8%) felv+ and 13 (17.6%) fiv+. five cats (6.6%) were both felv+/fiv+. prevalence of felv infection was significantly lower (p = 0.002) in group 1 compared to group 2. there was no difference in prevalence of fiv infection between groups. lymphoma lgl affects more females and older cats compared to all other type of feline lymphoma. opposite to all other type of lymphoma, and in accordance to previous litterature information, felv+ status does not play a role in the pathogenesis of feline lgl lymphoma. disclosures: no disclosures to report. the activity of t regulatory cells (tregs) is known to be closely associated with the expression of foxp3 transcription factor. foxp3 regulatory t cells (foxp3treg) are a distinct group of t lymphocytes that have immunosuppressive properties. normally this cells work for prevention of harmful autoimmune responses, however can also interfere with beneficial immune responses such as anti-tumor immunity. in human breast cancer these cells play a crucial role in tumor progression. in canine mammary tumors (cmt) there are only a few studies and this topic are not welldocumented. in this study we included 80 malignant cmt and studied, by immunohistochemistry, the intratumoral foxp3 expression together with vascular endothelial growth factor (vegf), microvessel density (mvd, by cd31 antibody) and several clinicopathological characteristics. abundant foxp3treg cells was statistically associated with presence of tumor necrosis (p = 0.004), nuclear grade (p = 0.001), poor differentiation of tumors (p < 0.0001), high mitotic grade (p < 0.0001), high histological grade of malignancy (p < 0.0001), presence of neoplastic intravascular emboli (p < 0.0001) and presence of lymph node metastasis (p < 0.0001). intratumoral foxp3 levels were correlated with the levels of vegf (r = 0.427; p < 0.0001) and intratumoral mvd (r = 0.520; p < 0.0001). additionally tumors with abundant foxp3treg cells were associated with shorter overall survival (os) time (p = 0.0001). results suggest that treg cells play a role in cmt progression and may contribute to increased angiogenesis and aggression in these tumors. the association of intratumoral foxp3 expression with shorter os of animals suggests a utility of treg cells activity as a prognostic marker. disclosures: no disclosures to report. clinical manifestations of canine visceral leishmaniasis (canl) are non-specific and include progressive weight loss, anemia, lymphadenomegaly, hepatosplenomegaly, dermatological, renal and ocular alterations. cardiac lesions resulting in clinical signs has been scarcely described in dogs with vl, and the presence of the parasite in the cardiac tissue has been involved in few reports. accordingly, the present study aimed to evaluate histopathological abnormalities in cardiac tissue from dogs naturally infected by leishmania infantum chagasi. a total of 20 dogs were evaluated. all dogs were symptomatic but no one presented clinical signs of cardiac involvement. in compliance with a federal law and under the owners' signed consent, all dogs were submitted to euthanasia and comprehensive post-mortem evaluation. samples from right atrium free wall (ra), right ventricle free wall (rv), interventricular septum (ivs) and left ventricle free wall (lv) were collected and evaluated. tissue samples were fixed in formalin, embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin (he) and anti-leishmania immunohistochemistry was also performed. the study was approved by the ethics committee in animal experimentation and animal welfare (protocol number 0463/2013). histopathological changes were observed in at least one of the four evaluated cardiac regions in 75% (15/20) of the dogs. the most frequent cardiac injury was an inflammatory reaction, characterized by the presence of mononuclear cell infiltrate in different degrees. of the evaluated regions, ra was the one with the highest incidence of histopathological changes, observed in 80% (12/15) of the animals, followed by rv, lv and siv, affected in 73.3% (11/15), 66.7% (10/15) and 53.3% (8/15) of the dogs, respectively. immunohistochemistry revealed amastigotes in the cardiac tissue in 70% (14/20) of the dogs. a positive correlation was found between cardiac lesions and the presence of amastigotes in the myocardium (p < 0.05). disclosures: no disclosures to report. canine leishmaniosis is a life threatening zoonotic disease. the combination of meglumine antimoniate and allopurinol is considered the most effective therapy for canine leishmaniosis and constitutes the first line protocol against this disease. allopurinol is a parasitostatic drug used in long-term to maintain low parasite loads and to avoid clinical relapses. traditionally, allopurinol is considered a very safe drug in the dog. however, some reports indicate that xanthinuria and xanthine urolithiasis is produced after prolonged therapy with allopurinol in the dog. the aim of this prospective study was to evaluate the prevalence of urinary adverse effects of allopurinol treatment (10 mg/kg/bid/po) in dogs with leishmaniosis. diagnosis was made by compatible clini-copathological abnormalitites with leishmaniosis and high leishmania infantum-specific antibody levels assessed by quantitative elisa. once leishmaniosis was diagnosed, a close follow-up (day 0, 30, 90, 180 and 360 during treatment) including physical examination, baseline laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis, urinary protein/creatinine ratio) and abdominal ultrasound was performed. in our preliminary results, 13 dogs were included. dogs did not present any urinary abnormalities based on biochemistry profile, urinalysis and abdominal ultrasound at the time of diagnosis. four out of 13 presented xanthinuria (day-30 [n = 3], day-90 [n = 4], and day-180 [n = 4]). two out of 13 dogs presented renal mineralization at day-90 of treatment. two out of 13 dogs presented bladder urolithiasis since day-90 of treatment. xanthinuria was presented initially in all dogs that developed renal mineralization or bladder urolithiasis. dogs with renal mineralization and urolithiasis were treated with a restricted protein diet and, so far, they did not develop renal disease. the present study describes early xanthinuria, renal mineralization and urolithiasis as adverse effects due to chronic allopurinol treatment in dogs with leishmaniosis. neither mineral analysis nor renal biopsy was performed to confirm the origin of these lesions, but no urinary abnormality was present before allopurinol treatment was instituted. a thorough monitoring of dogs treated against leishmaniosis combined with urinalysis and abdominal ultrasound should be performed to evaluate urinary adverse effects and to help in the clinical management of these adverse effects. disclosures: no disclosures to report. giardia duodenalis is one of the most important gastrointestinal parasites in dogs and cats with a zoonotic potential. in germany the prevalence in dogs and cats reaches up to 29% and 24%, respectively. genotypes of two genetic assemblages of the parasites infect humans (assemblages a and b) and other mammals including small animals. in contrast, parasites of the assemblages c and d are specific for dogs, assemblage f for cats. objectives of the study were to analyse the prevalence, potential epidemiological risk factors and symptoms of g. duodenalis infections in dogs and cats. to detect g. duodenalis, feces from dogs and cats was analysed with an elisa technique. after dna extraction real time pcr as well as multi-locus sequence typing was performed for the following gene loci: triosephosphate isomerase-, glutamate dehydrogenase-, beta-giardin-gene, ssu rrna. with a questionnaire possible epidemiological risk factors were evaluated. statistical analyses were performed using spss 21 (odds ratio, kolmogorow-smirnow test, spearman correlation). fecal samples of 618 dogs and 156 cats were collected over a time period of 23 months. the elisa test was positive in 101/618 dogs and 10/156 cats. sixty-seven of 101 giardia positive dogs and 9 of 10 positive cats had gastrointestinal signs. genotyping was successful in 54 of 101 dog samples and were assigned to assemblages as follows: assemblage a (n = 12), a/c (n = 2), a/d (n = 4), b (n = 2), b/d (n = 1), c (n = 7), c/d (n = 2), d (n = 24). only one of 10 positive cat samples could be genotyped and was atypically identified as assemblage d. significant correlations between giardia infection and age, clinical signs, deworming status and staying abroad were found. in this monocenter study a prevalence rate of 16.3% in dogs and 6.4% in cats was detected, which is in good accordance with previous studies. the study further highlights a high rate (34%) of asymptomatically g. duodenalis infected animals. as potential zoonotic assemblages were detected, transmission of giardia from small animals to humans (and vice versa) cannot be excluded. especially young and not dewormed animals had a higher prevalence. disclosures: no disclosures to report. canine parvoviral enteritis remains a common cause of morbidity and mortality in young dogs. the goal of this study was to document a large cohort of affected dogs and analyze several factors as possible predictors of fatal outcome. medical records were retrospectively searched for dogs with parvoviral enteritis diagnosed with a positive fecal antigenic test or a fecal pcr. dogs were included only if the medical records were complete. the population was compared to the reference population of the hospital on the same time period with chi square tests and several factors were analyzed as possible predictors of death with a logistic regression. one hundred and forty seven cases were included. seventy percent of the dogs were non vaccinated puppies under the age of 6 months. intact females and rottweiler, american staffordshire terrier and french beauce shepherd dogs were over-represented. clinical signs such as vomiting, diarrhea and dehydration were present in 92.7%, 86.4% and 70.1% of the dogs respectively. hyperthermia, anemia and leucopenia were observed in 17.8%, 26.5% and 36. 1% of dogs respectively. the majority of the affected dogs were hospitalized for 3-6 days and the mortality rate was 14.3% (21/147 dogs). hypoglycemia at admission was observed in 11/81 (13.6%) dogs in which blood glucose was measured and was the only risk factor associated with death (p < 0.05). in this study, a predisposition of rottweiler, american staffordshire terrier and french beauce shepherd dogs was observed and hypoglycemia at admission was the only predictor of fatal outcome. disclosures: no disclosures to report. canine parvovirus (cpv) infections in dogs remain widespread around the world and still represent a major health threat in puppies. all vaccine manufacturers include this component in their core vaccination package, recommending two injections at 3-5 weeks interval from 7 to 8 weeks of age. despite broad vaccination coverage, number of reports suggesting lack of efficacy in vaccinated dogs have been reported, which implicate vaccines belonging to all major manufacturers. these cases are usually considered as being linked to the interference with maternal antibodies (matab), able to persist beyond 12 weeks of age, which has led most expert groups to recommend a third vaccination around 16 weeks of age. persistence of matab actually represents a major issue when immunizing puppies against parvovirosis. indeed, matab titres higher than 1/40 in the haemaglutination inhibition (hi) test can still inhibit vaccine uptake whereas such titres do not prevent field virus infection. in contrast, hi titres higher than 1/80 to 1/120 are usually considered as protective against disease and virus excretion. this "immunity gap"is therefore a critical period for the puppy and the outcome of the vaccination. in order to evaluate the impact of residual mda on the efficacy of a standard primary vaccination protocol, we performed a vaccination field trial with serological follow-up. eighty-eight puppies from 7 to 24 weeks of age presented at veterinary practice received 2 injections at 4 weeks interval. serology was performed by elisa before (at d0/v1), during (at d28/v2) and after (d42) vaccination. average maternal antibodies titres were strongly correlated with the age of the puppy at primary vaccination, remaining at vaccine inhibiting level until~10 week of age. average titres increased significantly after 1st injection of primary vaccination in most groups and in all groups after the 2nd injection of primary vaccination. individual variability remained significant: vaccine uptake was inversely and strongly correlated to the pre-vaccinal matab titre at vaccination. seven out of 88 puppies (8%) didn't seroconvert, despite vaccination complying to the recommended schedule. vaccination was started in such dogs between 8.5 and 10.5 weeks and completed between 12.5 and 14.5 weeks and average initial matab titre was 2.4 log10 compared to 1.6 for the general population. in conclusion, this trial supports the recommendation of an additional injection of primary vaccination at 16 weeks, especially in areas of high parvovirus prevalence / pressure, where high levels of matab are likely to be transferred to puppies. disclosures: all authors are employees of merial. (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) . 5.3% of the dogs were pcr positive. the aim of our study was to evaluate the usefulness of determining serum level of c-reactive protein (crp) in dogs naturally infected with bacterium a. phagocytophilum as a possible indicator of the clinical phase of the disease. pcr and/or ifa positive dogs with clinical presentation and/or thrombocytopenia were included in the study. based on the results, the dogs were divided into 4 groups: pcr positive dogs; ifa positive (subdivided according to titer level from 1:128 to >1:2048) and pcr negative dogs; positive control group -pcr and ifa negative dogs with clinical signs and/or thrombocytopenia; negative control groupclinically healthy, pcr and ifa negative dogs. serum level of crp was determined using lifeassays ò canine crp (lifeassays, lund, sweden). an elevated concentration of crp (>35 mg/l) was determined in pcr positive and ifa positive dogs with an ifa titer ≥1:2048 and coincides with the presence of clinical signs (most commonly general clinical signs, elevated body temperature, gastrointestinal problems) and/or mild (14.3%) or severe (71%) thrombocytopenia. the assessment of crp concentration, in correlation with certain clinical alterations and thrombocytopenia, suggests that crp concentration is elevated in the acute phase of the disease and is in correlation with the aforementioned changes therefore can serve as an additional diagnostic parameter. the crp concentration in ifa positive dogs, regardless of ifa titer levels, and with present clinical signs and thrombocytopenia is higher (above detectable level, >10 mg/l) than in dogs without clinical signs or laboratory alterations, which may speak in favor of reinfection or reactivation of a persistent infection at least in cases when no other cause of inflammation can be found. specific treatment would therefore be reasonable in such cases, especially in cases of rising crp concentration. disclosures: no disclosures to report. gallbladder agenesis is a very rare cause of elevated liver enzymes in dogs. in this study, we evaluated the features of 15 dogs [six males (three castrated) and nine females (two spayed)] with suspected gallbladder agenesis on ultrasonography. five different breeds were included: chihuahua (n = 9), toy poodle (3), german shepherd (1), jack russell terrier (1), and shiba dog (1). the median age was 1.9 (0.7-7.4) years. ten dogs were asymptomatic, while the other five dogs showed decreased appetite (3), vomiting (3), ascites (2), seizure (1), and diarrhea (1). all dogs showed elevated liver enzymes, with high alanine aminotransferase levels (median, 306 u/l; 38-1374 u/l) in 13 dogs and high gamma-glutamyl transpeptidase levels (median, 12 u/l; 3-19 u/l) in 11. gallbladder agenesis was confirmed using laparoscopy in 12 dogs and laparotomy in three. liver biopsy samples were obtained from all dogs. additional computed tomography cholangiography was performed for 12 dogs using a 16-slice multidetector computed tomography (mdct) scanner following the intravenous administration of contrast medium (meglumine iotroxate). the obtained images were analyzed on a workstation, and they revealed an absent gallbladder in nine dogs and a vestigial gallbladder in three. the common bile duct was dilated in five dogs. for all dogs, laparotomy or laparoscopy was used to visualize the gallbladder and liver abnormalities, including malformed lobes and surface irregularities. acquired portal systemic collaterals were visually confirmed in five dogs, who also exhibited hypoplasia of the portal vein on histological examination. in conclusion, most animals with gallbladder agenesis were asymptomatic in our study, indicating a good long-term prognosis. however, symptoms associated with portal hypertension must be monitored in animals with primary portal vein hypoplasia. disclosures: no disclosures to report. assessment of systolic arterial blood pressure (sap) is an important tool in small animal internal medicine practice, especially with diseases or clinical conditions that can cause hypertension or hypotension. the doppler method is noninvasive and has several advantages compared to oscillometric method. there are few studies about the effect of body position on sap in conscious dogs. the hypothesis was that animal positioning during measurement alters sap values. the study design was prospective and randomized regarding order of positioning measurements. one hundred and twenty client-owned, conscious, healthy or sick adult dogs, weighing up to 10 kg were included. sap was recorded by doppler ultrasonography following american college of veterinary internal medicine consensus statement with animals positioned in sternal recumbency, right lateral recumbency and with the dog laying down on owner 0 s lap. the order of body position was raffled at the time of measurement. five consecutive measurements on each body position were performed always on left forelimb and the average was calculated. sap values were higher in sternal recumbency (153 mmhg, p 25%-75% = 134-176.5; p < 0.0126) compared to those obtained on the owner 0 s lap (139 mmhg, p 25%-75% = 125.5-159.8), and both were similar to right lateral recumbency (141 mmhg, p 25%-75% = 125-159). these results suggest that sap measurement obtained on owner 0 s lap or right lateral recumbency can be used on clinic routine, but sap measurement obtained on sternal recumbency should be avoided, because such measures may be overestimated. disclosures: no disclosures to report. canine patients may be presented for blood pressure (bp) assessment when clinical diseases associated with systemic hypertension (ht) are suspected but not confirmed; this population may encompass patients that have normal bp, true ht or situational ht. the clinician's aim is to identify animals with ht reliably, while minimizing false positives. this prospective study investigated the repeatability of duplicate within-visit systolic bp assessments (sbp1 and sbp2) in consecutive canine patients presented for bp assessment in the small animal clinic (91 duplicate sbp recorded from 70 dogs) and in a control group of healthy dogs (37 duplicate sbp obtained from 19 control dogs), resulting in 128 duplicate measurements for analysis. doppler methods were used for 16 duplicate assessments and oscillometric methods were used for 112 duplicate assessments; cuff size/location were consistent within any dog. sbp ≤160 mmhg was considered normal (nml); sbp >160 mmhg was considered abnormal (abn). median (range) elapsed time between duplicate readings was 30 (5-310) minutes; 75% of sbp2 were obtained within 80 minutes of sbp1. there was no correlation between elapsed time and change in sbp (p = 0.40). 70% of sbp2 were equal to or lower than sbp1; median decrease was 18 (0no dog with sbp1 >200 mmhg (n = 15) had nml sbp2. more dogs with abn sbp1 were panting (26/71 scored, 37%) compared to the group with dogs with nml sbp1 (6/44 scored, 14%, p = 0.02). sbp2 of dogs that stopped panting (14/23, 61%) tended to decrease (p = 0.16). within-visit repeatability of bp diagnosis was good in dogs with nml sbp1, but apparent false positive diagnoses of ht occurred in 37% of dogs with abn sbp1. sbp1 >200 mmhg was repeatable in all dogs. panting may be associated with increased measured sbp by these methods. duplicate within-day measurements may help identify false positive ht diagnoses in dogs with initial sbp measurements >160 mmhg. disclosures: no disclosures to report. median age at presentation was 4 years. ess, cocker spaniels, ckcs and border collies presented at 4.4, 2.5, 2.75 and 2.5 years respectively. females were over-represented with 22/36 females (15/36 fn and 7/36 fe) and 14 males (10/36 mn and 4/36 me). 60% of the ess cases presented were fn dogs. clinical presentation varied between dogs. clinical signs of pyrexia (77%), lethargy (70%) and anorexia (41.6%) were the most common. other signs included cough, tachypnoea, dyspnoea, dysphagia, vomiting, diarrhoea, neck or spinal pain, abdominal pain, joint effusion or dermatologic signs thirty-one animals presented with peripheral lymphadenomegaly, but five animals displayed only internal lymph node enlargement. cytology was performed in 30/ 36 cases pyogranulomatous (9) or granulomatous (2) lymphadenitis. lymph node culture or staining (gram, pas, zn) was performed in 17 and 18 animals respectively, all of which were negative twenty-four animals were initiated therapy at 1 mg/kg q 12 hours. mean treatment length was 18 weeks median relapse time was 26 weeks. this study documents dogs with cssrl in the uk suggesting an over-representation in spaniel breeds (particularly ess), with females and young dogs typically affected. cytologic and histopathologic examination confirmed sterile lymphadenitis with animals showing marked and rapid clinical improvement pastor 2 . 1 hospital clinic veterinari animal medicine and surgery department, faculty of veterinary medicine of murcia, murcia, spain if only blood is available. therefore, if no effusion is present, diagnosis of fip still remains challenging. disclosures: dr. elisabeth mueller is the managing director of laboklin gmbh & co.kg. dr. karola weider is employed at laboklin gmbh & co.kg. this laboratory offered the "combined rt-npcr and sequencing approach which samples are the most relevant for analysis? a retrospective study of 33 cases from maisons alfort, france, 2 umr 1161 virologie inra-enva-anses cyprus is an island state in the eastern mediterranean basin. no epidemiological studies have yet been performed on infectious agents in cats from cyprus. the aim of this study was to determine the prevalence of several infectious agents, including some vector-borne infections, in cats from cyprus.surplus edta-blood and serum samples were recruited from 176 cypriot cats, from which signalment and lifestyle characteristics were recorded. dna was extracted and real-time quantitative polymerase chain reaction (qpcr) assays were used to detect haemplasmas (mycoplasma haemofelis, "candidatus mycoplasma haemominutum" and "candidatus mycoplasma turicensis"), leishmania spp.and bartonella henselae. conventional pcr assays were used to detect ehrlichia/anaplasma spp. samples yielding positive results for leishmania spp. or ehrlichia/anaplasma spp. underwent further characterisation (sequencing). elisas were performed for the detection of l. infantum antibodies, feline leukaemia virus (felv) antigen and feline immunodeficiency virus (fiv) antibodies. statistical analysis was performed using spss for the assessment of any associations between variables and infectious agents.of the 176 samples extracted, 2 were excluded due to failure of ≥one internal control pcr. of the remaining 174 samples, 46 (26.4%) were positive by pcr for haemoplasma including 13 (7.5%) for m. haemofelis, 36 (20.7%) for "ca. m. haemominutum" and 12 (6.9%) for "ca. m. turicensis". nineteen (10.9%) were positive for b. henselae. one cat (0.6%) was pcr positive for ehrlichia/anaplasma spp. and sequencing revealed identity with anaplasma platys. leishmania spp. dna was detected in 6 of the 174 (3.4%) cats; sequencing revealed l. infantum in 4 of these cases. l. infantum serology was positive in 7 of the 162 cats tested (4.3%). only one cat was positive for both leishmania pcr and serology. of the 166 cats that underwent retroviral serology, 11 (6.6%) were felv and 30 (18.4%) were fiv positive.statistical analysis identified several significant associations (p < 0.05) including the following; haemoplasma infection and both outdoor access and feral-shelter cat origin, felv or fiv infection and both anaemia and feral-shelter cat origin.this study documents, for the first time, the presence of haemoplasmas, l. infantum, b. henselae, a. platys, felv and fiv in the feline population of cyprus. the prevalence of haemoplasma, fiv and b. henselae infections were among the highest reported in cats from mediterranean countries, while that of leishmania spp. was similar. this is the second report of a. platys infection in a cat from a mediterranean country.disclosures: no disclosures to report. bartonella species (spp.) are zoonotic pathogens, and infections in cats are common. prevalence in cats from southern germany is still unknown. the aim of this study was to determine the prevalence of bartonella spp. dna in blood of cats in southern germany and to evaluate associations between bartonella bacteremia, housing conditions, feline immunodeficiency virus (fiv), and feline leukemia virus (felv) status, including progressive, regressive, and abortive felv infection.blood samples of 479 cats that were presented to different veterinary clinics in southern germany for various reasons were tested for bartonella spp. dna using a previously published conventional polymerase chain reaction (pcr) targeting a fragment of the 16s-23s rrna intergenic spacer region. for statistical analysis, fisher's exact test was used.prevalence rate of bartonella spp. bacteremia was 2.5% (12/479; ci: 0.01%-0.04%). b. henselae was amplified in eleven of these cats. one cat was positive for b. clarridgeiae dna. most of the infected cats were clinically healthy, but half of the cats had thrombocytopenia, potentially caused by their bartonella spp. infection. there was no significantly higher risk to be infected with bartonella spp. when living mainly outdoors or being fiv-or felv-infected.prevalence of bartonella spp. bacteremia is low in southern german cats, but there is still a risk of human bartonella infection associated with cat ownership. most clinical changes of the bartonella spp.-infected cats were related to other diseases. however, thrombocytopenia was common and further studies are required to define its potential clinical relevance.disclosures: no disclosures to report. ante-mortem diagnosis of feline infectious peritonitis (fip) is still challenging. the aim of this study was to evaluate sensitivity and specificity of a "combined reverse transcription nested polymerase chain reaction (rt-npcr) and sequencing approach", detecting mutations at two different nucleotide positions within the spike gene, that previously were shown to correlate with the fip phenotype. the study population consisted of 64 cats with confirmed fip and a defined control group of 63 cats for which fip was considered an important differential diagnosis, but that were definitively diagnosed with other diseases. blood and/or effusion samples were examined for feline coronavirus (fcov) rna by rt-npcr and, if positive, nucleotide positions 23531 and 23537 were sequenced for nucleotide transitions. sensitivity, specificity, negative and positive predictive values were determined and 95% confidence intervals (95% ci) calculated.rt-npcr detected fcov in 38 cats in blood (n = 3) and/or effusion (n = 36); all of them had fip. one of the mutations of interest was found in 2/3 of the pcr-positive blood samples and in 32/36 of the pcr-positive effusion samples. diagnostic specificity of the "combined rt-npcr and sequencing approach" was 100% in blood (95% ci 83.9-100.0) and effusion (95% ci 93.0-100.0). diagnostic sensitivity was 6.5% (95% ci 0.8-21.4) in blood and 65.3% (95% ci 50.4-78.3) in effusion.a positive test result therefore confirms a suspicion of fip. a negative result, however, cannot be used to rule out fip, especially feline infectious peritonitis (fip) is a viral disease caused by the virulent strain of feline coronavirus (fipv). the disease can appear under two clinical forms, dry or effusive, both leading to a fatal outcome. diagnosis was based on histopathologic lesions on necropsy until the recent discovery of mutations associated with the fipv strain in the 3c and spike (s) genes. our main goal was to detail the distribution of 3c or s gene mutations in different biological samples of cats suffering from fip.this was a retrospective, observational study of 33 cats showing clinical signs compatible with fip. ten out of 33 cats were of pure breed. 57.7% were males and 42.3% females. median age was 36.5 months at presentation. the clinical presentation, pathologic findings and virologic data were reviewed. according to clinical signs, 11 cats were classified with a dry form and 22 with a wet form of fip. the main clinical signs included dehydration, hyperthermia, icterus, abnormal abdominal palpation, neurological and ocular disorders.when possible blood, fecal material, effusion, fine needle aspiration (fna) from relevant organs or a combination of these, was recovered from each cat. feline coronavirus (fcov) was first researched by rt-pcr, then the 3c and part of the s genes were sequenced to determine the eventual presence of mutations.among the 11 dry cases, fcov was detected in 2/8 blood samples, 3/8 fecal samples and fna (11/11). among the 22 wet cases, 9/15 blood samples, 12/14 fecal samples and all effusion samples (21/21) were positive for the presence of fcov. 3c mutations were never found in fecal samples but were found in 6/10 effusion samples and in 8/8 fna. s mutations were detected in 4/9 fecal samples, 8/9 fna and 14/15 effusion samples. for three cats, no mutation, neither in 3c or s genes was identified despite the confirmation of fip by necropsy.s gene mutation is more frequently observed than 3c gene despite in two cases where only 3c mutations were identified. moreover the presence of strains harbouring s mutation in feces has never been described before and could suggest the possible diffusion of fipv among feline population.in conclusion, viral diagnosis of fip based on rt-pcr sequencing in effusion and fna samples is essential. rt-pcr resulting from blood samples should be carefully interpreted because of high risk of missing fipv.finally, searching for mutations in both s and 3c genes is recommended.disclosures: no disclosures to report. methicillin resistant staphylococcus aureus (mrsa) has recently become a great concern for pet animals' disease and zoonotic infection. mrsa strains transfer between pet animals and humans could occur. the aim of the present study was to determine the occurrence of mrsa in 34 household dogs.from january to june 2012, clinical samples were collected from 34 dogs, patients of the veterinary teaching hospital of the department of veterinary sciences of messina (italy), affected by several diseases of various origins. all samples were processed by bacteriological conventional methods for isolation and identification. all strains were tested for phenotypic susceptibility to oxacillin and were subjected to a pcr protocol for the detection of meca gene. strains carrying the gene were considered methicillin resistant (mrs). lastly, on both mrs and methicillin sensitive (mss) strains, kirby-bauer disk diffusion susceptibility testing were performed to highlight resistance profiles using 44 molecules belonging to the main classes of antimicrobials used in veterinary practice. strains resistant to at least one molecule of three or more classes of antibiotics were considered multidrug-resistant (mdr). the statistical analysis of the results was made using the z-test by a primer ò software.forty staphylococcus spp. strains were isolated, belonging to 12 species. the most frequently isolated microorganisms were staphylococcus aureus with 14 isolations (35%) and staphylococcus pseudintermedius with 11 isolations (27.5%), followed by staphylococcus epidermidis with 4 isolations (10%) and staphylococcus cohnii and staphylococcus warneri, both with 2 isolations (5%). a single isolation (2.5%) was obtained for each of the species staphylococcus chromogenes, staphylococcus haemolyticus, staphylococcus lentus, staphylococcus lugdunensis, staphylococcus saprophyticus, staphylococcus simulans and staphylococcus sciuri. thirteen (32.5%) strains of staphylococcus spp. were phenotypically resistant to oxacillin and three staphylococcus aureus (7.5%; n.2 from pyoderma, n.1 from exudative pleural effusion) were positive for the meca gene. all 40 strains of staphylococcus spp. were mdr. our results showed the presence of mrsa and multidrug-resistant staphylococcal strains in household dogs. a lack of correspondence between antimicrobial susceptibility tests and molecular methods was found in the present study.disclosures: no disclosures to report. haemotropic mycoplasmas (haemoplasmas) are bacteria that infect domestic cats. approximately 25% of ill cats have haemoplasma infection. three main feline haemoplasma species have been detected worldwide; mycoplasma haemofelis (mhf), "candidatus m. haemominutum" (cmhm) and "candidatus m. turicensis" (cmt). a fourth haemoplasma called "candidatus m. haematoparvun-like" (cmhp) was latter identified in cats. the only published study in chile was carried out in 30 cats, with prevalences by pcr of 3.3% to mhf, and 10% to cmhm.the aim of this study was to perform molecular detection of haemoplasmas in cats from valdivia, southern chile. blood samples were taken from 384 cats and used for haemoplasmas dna detection by quantitative real time pcr (qpcr) at universidad austral de chile. qpcr protocol was based on detection of feline dna targeting 28s housekeeping gene and mycoplasma spp. 16s rrna gene (universal primers, my16sf forward, my16sr1 y my16sr2, both reverse) by sybr green method. the melting temperature (tm) analysis allowed identifying the infecting mycoplasma species (mhf, cmhm, cmt). it was not posible to identify haemoplasmas species on co infected cats, so a second qpcr specie specific protocol was applied on these samples. second qpcr protocol was based on 16s rrna gene, with specific primers to detect mhf, cmhm, cmt and cmhp. all samples (384/384) were positive to 28s gene, proving presence of cat dna. from the 384 cats, 15.1% (58/384) were positive to haemoplasmas, where 7.8% (30/384) corresponded to cmhm (tm 73.5-75.0°c), 4.4% (17/384) to mhf (tm 75.0-76.5°c), 2% (4/384) to cmt (tm 76.5-78.0°c) and 1.8% (7/384) to co infections. associations between cmhm+mhf, cmhm+cmt, cmhm+cmhp and cmhm+mhf+cmhp were detected on co infected animals. these results agree with those found in previous