Schoeman_59-66.indd EPIDEMIOLOGY Babesiosis is a disease of worldwide significance and was first recognised in 1888 as a cause of fever, haemolytic anaemia, haemoglobinuria and death of cattle. Members of the genus Babesia readily para- sitize the red blood cells of dogs, causing progres- sive anaemia. Canine babesia are morphologically classified into large and small forms, both exhibiting a worldwide distribution (Table 1). Babesia canis and another novel, as yet unnamed Babesia sp. de- tected in the USA (large Babesia) and Babesia gib- soni and Babesia annae (small Ba besia) have been documented to infect dogs (Bir kenheuer, Neel, Rus- lander, Levy & Breidschwerdt 2004). Three main spe cies of large Babesia infect dogs, namely Babe- sia vogeli, B. canis and Babesia rossi. These three species are antigenically distinct, transmitted by dif- ferent vectors and differ widely in pathogenicity and geographic distribution (Uilenberg, Franssen, Perie & Spanjer 1989). Babesia vogeli is the least pathogenic. It occurs in France, Australia, Japan, Brazil, South Africa and the USA and usually causes mild disease in adult dogs, but severe disease in some puppies (Matjila, Penzhorn, Bekker, Nijhof & Jongejan 2004). Babesia canis is widespread in Europe (it affects more than 400 000 dogs per year in France alone) and Asia and is of intermediate pathogenicity. Babesia rossi occurs predominantly in southern Af ri- ca and is ostensibly the most virulent of the subspe- cies. Improved PCR techniques have lately allowed for better definition of these parasites (Matjila, Leise- witz, Jongejan & Penzhorn 2008). The smaller parasite, B. gibsoni occurs principally in the Middle East, southern Asia, Japan, North Africa, South America and is an emerging infectious dis- ease in the USA, as well as having been detected lately in Italy, Hungary and Australia (Muhlnickel et al. 2002). A more virulent subspecies of B. gibsoni has recently been identified in California (Kjemtrup, Wainwright, Miller, Penzhorn & Carreno 2006) A Babesia microti-like piroplasma, B. annae (also known as Theileria annae) has been found to be 59 Onderstepoort Journal of Veterinary Research, 76:59–66 (2009) Canine babesiosis J.P. SCHOEMAN Department of Companion Animal Clinical Studies, Faculty of Veterinary Science, University of Pretoria Private Bag X04, Onderstepoort, 0110 South Africa ABSTRACT SCHOEMAN, J.P. 2009. Canine babesiosis. Onderstepoort Journal of Veterinary Research, 76:59– 66 Babesiosis is a tick-borne disease affecting humans and many domestic and wild animals. Domestic animals showing appreciable morbidity and mortality include dogs, cats, cattle and horses. Both ca- nine and feline babesiosis are diseases characterised by haemolytic anaemia, icterus and haemo- globinuria. Canine babesiosis can range from chronic or subclinical to peracute and fatal, depending on the virulence of the species and the susceptibility of the host. This paper deals with canine babe- siosis with specific reference to pathogenesis, clinical findings, complications, diagnosis and treat- ment, as well as newly identified prognostic factors in Babesia rossi babesiosis. 60 Canine babesiosis endemic in dogs in northwest Spain (Camacho, Pal- las, Gestal, Guitan, Olmeda, Telford & Spielman 2003). Most recently another small piroplasm caus- ing a disease dubbed ‘bloody ears’ has been identi- fied in Brazil (Loretti & Barros 2005). Various species of ticks such as Rhipicephalus san- guineus, Dermacentor spp. and Haemaphysalis ellip ticum can transmit the large babesia of dogs, whereas B. gibsoni is transmitted by Haemaphysalis bispinosa and Haemaphysalis longicornis. Babesia annae is thought to be transmitted by Ixodes hex- agonus (Lobetti 2006). Both trans-stadial and trans- ovarial transmission can occur and ticks are be- lieved to remain infective for several generations. Babesia spp. can also be transmitted by blood trans- fusion. Strong circumstantial evidence exist that B. gibsoni is transmitted by dog bites (Birkenheuer, Correa, Levy & Breitschwerdt 2005), whilst trans- placental transmission from dam to offspring has recently been proven as an additional mode of transmission (Fukumoto, Suzuki, Igarashi & Xuan 2005). PATHOGENESIS AND CLINICAL SIGNS Babesia spp. cause disease mostly in young dogs, although dogs of all ages can be affected. The incu- bation period of canine babesiosis varies from 10– 21 days for B. canis and 14–28 days for B. gibsoni. The female ticks feed on their host for about one week only and have left the host by the time disease develops. The severity of the disease depends on the species of Babesia, the presence of concurrent infections and the age and immune status of the host. The disease presentation varies widely from peracute to chronic or even subclinical. Babesia rossi, the dom- TABLE 1 Geographical spread of canine babesiosis Species of babesia Tick vector Country Endemicity Large babesia B. rossi H. elliptica South Africa Endemic B. canis Dermacentor reticularis France Endemic Spain Endemic Hungary Endemic Switzerland Small outbreaks Germany Small outbreak Belgium Three cases Netherlands Small outbreak B. vogeli R. sanguineus USA Endemic in the south, especially in kennels, but spreading Okinawa, Japan Endemic France Sample from one dog Australia Endemic South Africa Endemic Brazil Endemic Small babesia B. gibsoni – Californian isolate H. bispinosa and H. longicornis USA Endemic in the west B. gibsoni – Asian isolate USA Endemic, spreading northwards (detected in 29 states) North and East Africa Endemic Asia Endemic Australia Three Pitbull Terriers Middle East Endemic Hungary Two dogs (no PCR confirmation) Italy One case (no PCR confirmation) B. annae Spain Endemic 61 J.P. SCHOEMAN inant species found in South Africa, is very virulent and causes peracute and acute disease. Clinical signs include pale mucous membranes, de- pression, tachy cardia, tachypnoea, anorexia, weak- ness, splenomegaly and fever. It is thought that the clinical signs are the result of tissue hypoxia follow- ing the anaemia and a concomitant systemic inflam- matory response syndrome caused by marked cy- tokine release (Lobetti 2006). The pathogenesis of the anaemia is incompletely understood; intravas- cular and extravascular haemolysis take place, but other mechanisms such as poor bone marrow re- sponse are thought to play a role as well. The anae- mia is (perhaps counter-intuitively) not correlated to the degree of parasitaemia and dogs start to improve after parasiticidal treatment, even though their hae- matocrits generally drop further, before starting to rise (Jacobson, Reyers, Berry & Viljoen 1996). Some cases show additional immune-mediated break- down of red blood cells and dogs that show in-sa- line-positive red blood cell agglutination have to be carefully monitored for rapid decreases in haemat- ocrit. Mortality for Babesia spp. infections range from around 12 % for B. rossi to approxi mately 1 % for B. vogeli (Lobetti 2006). The severe form of the disease is characterized by marked haemolytic anaemia, severe acid-base ab- normalities (Leisewitz, Jacobson, De Morais & Rey- ers 2001) with frequent secondary multiple organ failure and complications such as acute renal failure (ARF), hepatopathy with marked icterus, hypogly- caemia (Keller, Jacobson, Nel, De Clerq, Thompson & Schoeman 2004), acute respiratory distress syn- drome (ARDS), cerebral pathology and additional immune-mediated red blood cell destruction (IMHA) (Jacobson 2006). A small subset of dogs presents with high haemato- crits (relative haemoconcentration), despite vigor- ous haemolysis, due to presumed shifting of fluid from the intravascular to the extravascular compo- nent. These dogs are at increased risk of develop- ing ARF or cerebral complications, as well as other organ failures (Welzl, Leisewitz, Jacobson, Vaughan- Scott & Myburgh 2001). Pancreatitis is frequently associated with other com- plications, and has a mortality rate of 20 %. Icterus, vomition, melaena, abdominal pain and diarrhoea are common findings in dogs with suspected pan- creatitis. In addition, 65 % of the dogs with pancrea- titis also had icterus, 30 % had ARDS, 30 % had IMHA, 15 % had ARF, while 10 % had haemocon- centration and another 10 % had cerebral syndrome concomitant with the pancreatitis. It is postulated that pancreatitis is the formerly described ‘gut’ form of babesiosis (Mohr, Lobetti & Van der Lugt 2000). Babesia vogeli causes a moderate, often clinically unapparent infection in mature dogs. The parasita- emia in B. vogeli also seems to be very low and as such the infection may frequently be missed during the routine examination of a blood smear. Subclinical infections are common in adult dogs infected with B. vogeli, but puppies tend to present with marked anaemia (Irwin & Hutchinson 1991). This is mostly a disease that is endemic in Greyhound kennels in the USA and particular care should be taken when relocating Greyhounds that can harbour subclinical infections. Babesia canis infections result in a more variable pathogenicity, intermediate between B. rossi and B. vogeli. A recent study from Italy, presumably de- scribing B. canis infection, reported anaemia in the majority of dogs and thrombocytopaenia in all cases (Furlanello, Fiorio, Caldin, Lubas & Solano-Gallego 2005). Babesia gibsoni infection may follow a hyper-acute, acute or chronic course. The acute course is the most common, and is characterized by fever, lethargy, haemolytic anaemia, thrombocytopenia, lymphade- nopathy and splenomegaly (Conrad, Thomford, Yamane, Whiting, Bosma Uno, Holshuh & Shelly 1991). The hyper-acute state is rare and is charac- terized by shock and extensive tissue damage. This is mostly a disease of American Pitbull and Staf ford- shire Bull Terriers that is putatively transmitted via dog bites (Birkenheuer et al. 2005). Dogs with sub- clinical B. gibsoni infections have been reported in Australia and the USA, where they are PCR posi- tive, but neither show microscopic parasitaemia, nor clinical illness (MacIntire, Boudreaux, West Bourne, Wright & Conrad 2002). Such cases can have dire consequences if imported into non-endemic areas. Much of the apparent discrepancy in the clinical pic- ture of B. gibsoni infections in the USA has lately been explained when distinct genotypical differenc- es have been detected between the Californian and the Asian isolate occurring in the mid-western USA. The Californian isolate seems to be a more virulent species and is found in breeds other than Pitbull Terriers (Kjemtrup et al. 2006). DIAGNOSIS Diagnosis of acute cases infected with B. canis is based on the classic clinical presentation and the 62 Canine babesiosis demonstration of the parasites within red blood cells on Diff-quick stained, thin capillary blood smears. The large babesias are typically seen as paired, 2.4 x 5 μm-sized bodies, although some red blood cells can contain up to 6–8 pyriform to round bodies. Ba- besia gibsoni, the small babesia of dogs, is typically found as single, annular bodies measuring 1 x 3.2 μm. The recognition of these small parasites require good staining technique and considerable practice, as many red blood cells in anaemic dogs are vacu- olated and pitted. Blood smears are usually taken from the ear margin. The degree of parasitaemia can differ from 0.05–10 % of counted red blood cells, depending on the virulence of the species and has been found to be higher when patients are co-in- fected with Ehrlichia spp. (Van Heerden, Reyers & Stewart 1983). Due to the virulence of B. rossi, the mere presence of parasites in one red blood cell is enough to confirm the diagnosis. In this species, as well as in B. vogeli, parasitaemia has been shown to be higher in capillary than in central blood at pres- entation (Irwin & Hutchinson 1991; Böhm, Leisewitz, Thompson & Schoeman 2006). Ironically, the con- verse has been shown when a higher parasitaemia was found in central blood rather than capillary blood in dogs experimentally infected with B. vogeli (Ewing 1966). In more chronic cases, due to less virulent species such as B. canis and B. vogeli, where parasitaemia may be below the microscopic detection limit, diag- nosis is more problematic and a presumptive diag- nosis is often based on suggestive historical find- ings, physical examination findings and positive indirect fluorescent antibody titres or PCR. Thick smears (not alcohol fixed) may be helpful in detect- ing the parasite in cases of very low parasitaemia. Another way in which to increase the likelihood of finding parasites is to search along the periphery of the blood smear, as parasitized red blood cells tend to marginate during the making of the smear. Recent or active infection can be confirmed by the demon- stration of increasing antibody titres over 2–3 weeks. An improved ELISA has recently been developed that is able to distinguish between infection with B. gibsoni and the B. canis species on serology alone (Verdida, Hara et al. 2004). Some sub-clinical infec- tions require bone marrow aspirate cytology or smears prepared from red cells just below the buffy coat to demonstrate the parasite. A diagnosis should not be based solely on seropositivity, because clini- cally normal dogs in or from endemic areas can be seropositive. The high sensitivity and specificity of a newly developed PCR probe should allow the de- tection of low parasitaemias in subclinically infected cases and may be the most useful test in screening dogs newly imported into Babesia-free countries. The rest of the blood smear findings are classically those reflecting the underlying regenerative, haemo- lytic anaemia and include marked anisocytosis, poly chromasia, reticulocytosis and normoblastae- mia. It takes the bone marrow approximately 3–5 days to respond to an acute red blood cell break- down and, therefore, the blood smear findings in acute cases may appear to reflect those of a non- regenerative anaemia. Thrombocytopenia is a hall- mark of the disease, regardless of the Babesia spp. involved and is often marked, but yet petechiation or epistaxis is very rarely seen, except in cases with concomitant Ehrlichia infections (Furlanello et al. 2005; Kettner, Reyers & Miller 2003). The patho- physiology of the thrombocytopenia remains unde- termined, but sequestration or consumption is pos- tulated. Other haematological findings may include sphero- cytosis, in cases with secondary immune-mediated haemolytic anaemia and a left shift neutrophilia due to the marked systemic inflammatory response. Acute babesiosis can easily be misdiagnosed as prim ary immune-mediated haemolytic anaemia, espe cially if parasitaemias are low, only central ve- nous blood is submitted or when laboratory person- nel and clinicians are unfamiliar with the appear- ance of the parasites. In this regard a positive antiglobulin (Coombs’ test) may add to the confu- sion, since most B. canis and B. gibsoni infected dogs have been found to be positive with this test. The same diagnostic conundrum has been found in B. gibsoni-infected cases with thrombocytopenia, in the absence of detectable parasitaemia. It is impor- tant to remember that thrombocytopenia in these infections precedes the onset of anaemia and para- sitaemia. Further laboratory findings include elevation of liver enzymes such as ALP, ALT and AST—more so in the patients with marked icterus, reflecting the con- comitant hepatopathy in these cases. Serum potas- sium is often low, especially in icteric cases. Serum bilirubin concentrations are elevated, commensurate with the degree and rapidity of onset of the anaemia and the severity of the accompanying he pat opathy. Azotaemia is present in many dehydrated cases and in those with acute renal failure (Lo betti & Jacob son 2001). Urea is frequently disproportion- ately raised to creatinine and is elevated for reasons other than renal disease (De Scally, Leisewitz, Lo- betti & Thompson 2006). Urinalysis may show bili- 63 J.P. SCHOEMAN rubinuria, haemoglobinuria, proteinuria, renal tubu- lar epithelial cells and granular casts. Acid base abnormalities are common with metabolic acidosis (due to raised lactate and raised chloride ion concentration) and respiratory alkalosis being most common. Severe disease almost always caus- es mixed acid base imbalances (Leisewitz et al. 2001). TREATMENT The primary goals are to eliminate the parasite and reverse the life-threatening anaemia. Diminazene aceturate, trypan blue and imidocarb dipropionate are effective against B. canis (Table 2). Mildly to moderately affected dogs normally make an un- eventful recovery following antibabesial therapy alone. The epidemiology of the disease seems to suggest that dogs do develop a state of premunition after repeated infections. However, in non-endemic areas, it would be ideal to sterilise the infection and therefore render the dogs non-infective for ticks, in order to prevent the organism from becoming es- tablished in that country. Imidocarb dipropionate at a dose of either 7.5 mg/kg once off or 7 mg/kg given twice with a 14-day interval have been shown to sterilise the infection. Diminazene dose calculation should be meticulous due to its low therapeutic in- dex, especially in puppies and the drug should not be repeated within an interval of shorter than a 3- week period (Miller, Swan, Lobetti & Jacobson 2005). The absolute minimum interval might be longer in certain breeds and individuals, which appear to show a propensity to develop severe cerebral toxicity with classic cerebellar sulci haemorrhages. In the author’s experience this complication is fortunately quite rare. Trypan blue is one of the oldest drugs, but is still used in some parts of the world. Neither diminazene, nor trypan blue is capable of sterilising the infection and their use in non-endemic areas is therefore dis- couraged. The first treatment that has been shown to be effec- tive against B. gibsoni is a combination of atova- quone and azithromycin (Birkenheuer, Levy & Breit- schwerdt 2004). These parasites are very difficult to clear with conventional therapy and dogs usually become chronic carriers or present with recurrent episodes of acute babesiosis. Markedly anaemic animals or those with any of the abovementioned complications require a variety of supportive treatments, depending on the severity of the case and range from single to multiple blood transfusions, corticosteroid drug administration for the concomitant immune-mediated red blood cell destruction, aggressive colloidal and crystalloid fluid support with added electrolytes such as potassium chloride and diuretics in cases with acute renal fail- ure. Assisted respiration is frequently necessary for cases with pulmonary oedema due to acute respira- tory distress syndrome. Cross matching is not necessary for the first transfu- sion in dogs, because dogs do not have naturally occurring allo-antibodies. To further minimize de- layed transfusion reactions or the risk of sensitising patients that might invariably require future transfu- sions in endemic areas, only DEA 1.1 and 1.2 neg- ative donors are used. Whole blood can safely be used in cases of canine babesiosis. The decision to administer a blood transfusion is a multi-facetted one and is not determined solely by the patient’s haematocrit, but rather by the clinical condition of the animal and the rapidity of onset of the anaemia. TABLE 2 Drugs used in the treatment of babesiosis Babesia spp. Drug Dose Route of administration Duration of therapy B. canis Diminazene aceturate (Berenil®) 3.5 mg/kg IM/SC One dose B. canis Trypan blue (Trypan blue®) 10 mg/kg Strictly IV One dose followed by imidocarb or diminazene 1 week later B. canis Imidocarb dipropionate (Imizol®) 7 mg/kg IM Two doses, 14 days apart B. gibsoni Atovaquone and Azithromycin 13.3 mg/kg q 8 h 10 mg/kg q 24 h PO PO 10 consecutive days B. felis Primaquin phosphate 0.5 mg/kg IM Daily for 1–3 days 64 Canine babesiosis As such, dogs with haematocrits as low as 0.07 ℓ/ℓ can survive without transfusion, whereas some dogs with haematocrits as high as 0.20 ℓ/ℓ have to be transfused. Given the stringent criteria for donor se- lection and the significant owner and veterinarian effort needed to procure safe blood, it should be con sidered a precious resource. Due to this and other equally cogent arguments, the use of compo- nent therapy (i.e. red blood cells or plasma) has gained favour, allowing a single unit of blood to meet the needs of several patients. Acute canine babesi- osis cases are not necessarily volume depleted and mostly require oxygen-carrying capacity only (in some cases with ARF or ARDS volume expansion may actually be harmful), and thus benefit most from the use of packed red blood cells. Blood is initially transfused slowly (2 mℓ/kg/h) for the first 30–60 min, while observing the patients for transfusion reactions, such as a sudden rise in body temperature and/or respiratory rate and lip and ear pinna swelling. PROGNOSIS The prognosis is generally quite good, with approxi- mately 85–90 % of cases surviving the illness, de- pending on the level of care and the Babesia spp. Involved (Keller et al. 2004; Schoeman, Rees & Herrtage 2007). The use of blood transfusions has a major impact on survival in severely anaemia ani- mals. Cases with haemoconcentrated babesiosis and cases developing acute renal failure, acute res- piratory distress syndrome or cerebral babesiosis have the worst prognosis and mortality can be great- er than 50 %—in some cases approaching 100 %, despite intensive, technically advanced interven- tions (Welzl et al. 2001). Lactate concentrations of > 5 mmol/ℓ on admission are associated with increased mortality, but lactate > 2.5 mmol/ℓ and/or increase after admission and/or failure to decrease to < 50 % of admission value, is strongly predictive of death. Lactate persistently > 4.4 mmol/ℓ strongly predicts death and lactate > 4.4 mmol/ℓ at 24 h after admission correctly pre- dicts death in every case. Hyperlactataemia is pres- ent in approximately half of hospitalised cases. Extremely high values (> 10 mmol/ℓ) are not uncom- mon. Lac tate correlates negatively with glucose. Hyperlactataemia is also positively associated with clinical collapse (Nel, Lobetti, Keller & Thompson 2004). Acute renal failure (ARF) is rare. Renal involvement in babesiosis is diagnosed on the basis of persist- ently elevated creatinine despite appropriate fluid therapy. Renal involvement in babesiosis resem- bles the functional renal failure of sepsis. Oliguria is an ominous sign (Lobetti & Jacobson 2001). Hypotension is common in babesiosis and is asso- ciated with clinical collapse (Jacobson, Lobetti & Vaughan-Scott 2000). Clinical collapse is also asso- ciated with hypoglycaemia (Keller et al. 2004). This appears to be a very simple and useful prognostic factor (Böhm et al. 2006). A small study, comparing 3 dogs that died naturally with 31 other dogs, showed higher cardiac tropon- in I values in the non-survivors. This suggested that high cardiac troponin I values are associated with a poor outcome. From these results, cTnI also corre- lated with disease severity (Lobetti, Dvir & Pearson 2002). Hypoglycaemia (glucose < 3.3 mmol/ℓ) is present in c. 20 % of cases. Hypoglycaemia is associated with stupor and collapse. Miosis disappears after dex- trose infusion. Dogs that are collapsed (non-ambu- latory) at presentation have an 18-fold increased risk for hypoglycaemia. Pups less than 6 months of age are at risk of developing hypoglycaemia. The risk is slightly increased in dogs with severe anae- mia, icterus or vomition. Response to treatment with intravenous dextrose is sometimes dramatic. Hypo- glycaemia is an important differential diagnosis for cerebral babesiosis. When glycaemic status is tak- en into account, the incidence of true cerebral babe- siosis is very low (Keller et al. 2004). Timing, source of blood (venous versus capillary) and counting method cause large variability in para- site counts. A significant positive correlation between high parasitemia and mortality has recently been demonstrated. There is also a significant positive correlation between capillary and venous parasita- emia, with capillary parasitaemias being consist- ently higher than venous ones, making the capillary the preferential site for obtaining diagnostic smears (Böhm et al. 2006). At presentation to the hospital, dogs that died had a significantly higher median cortisol concentration (482 nmol/ℓ) than other admitted dogs that survived (150 nmol/ℓ). Conversely, the total and free thyrox- ine concentrations in all but one of the dogs that died were below the limit of detection and signifi- cantly lower (2.7 nmol/ℓ and 0.12 pmol/ℓ, respec- tively) than in other admitted dogs that survived (7.4 nmol/ℓ and 0.4 pmol/ℓ, respectively). Six of 11 dogs (55 %) with a serum cortisol above 400 nmol/ℓ died, whereas all dogs with admission serum cortisol con- centrations less than 260 nmol/ℓ survived (Schoe- 65 J.P. SCHOEMAN man et al. 2007). In addition, dogs that died also had increased ACTH-stimulated cortisol concentra- tions, indicating that acute up-regulation of adrenal function is associated with poor outcome (Schoeman & Herrtage 2008). Outcome is not affected by systemic inflammatory response syndrome (SIRS) and multiple organ dys- function syndrome positive status, nor the number of organs involved, but rather by the organ system affected, such as the lungs, kidneys or brain (Welzl et al. 2001). PREVENTION Regular control of the tick vectors by routinely dip- ping or spraying pets or using tick collars or spot-on preparations is the only effective way of preventing this disease in most parts of the world. 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