Overview of Canine Babesiosis

Canine babesiosis is a tick-borne, protozoal, haemoparasitic disease that can cause varying degrees of haemolytic anaemia, splenomegaly, thrombocytopenia and fever. There are two hosts for the transmission of Babesia spp., viz. invertebrate (tick) and vertebrate host. Dogs are one among the many targets of Babesia spp., causing canine babesiosis, and now there are clinical evidences of possible vertical transmission too. Dogs of all ages can be affected with Babesia spp., but young puppies are more commonly affected. Considering advanced diagnostic techniques, for an early and specific detection of acute infections, an AgELISA that is potentially translatable to a rapid diagnostic test design is reported. Different molecular techniques used for identification and differentiation of the various species of Babesia are semi-nested PCR, reverse line blotting and PCR-restriction fragment length polymorphism analysis. Treatment consists of three components: treatment with antiprotozoal agents to eliminate the parasite, blood transfusions to treat severe anaemia and supportive care for the complications and metabolic derangements. Blood lactate concentrations can serve as a prognostic indicator in severe or complicated canine babesiosis. For prevention apart from conventional measures, vaccines against Babesia species such as B. gibsoni are currently being developed.


Introduction
Canine babesiosis is a clinically significant tick-borne disease caused by apicomplexan parasites of the genus Babesia which has been reported worldwide including India. These verity of the disease depends on multiple factors such as the type of Babesia species involved, the age and the immune status of the host [1,2]. It is caused by different Babesia species with a worldwide distribution characterised by erythrocyte destruction causing mild to severe systemic clinical manifestations [3] like varying degrees of haemolytic anaemia, splenomegaly, thrombocytopenia and fever.

Classification
Babesia organisms are frequently classified as large or small. Historically, Babesia spp. in dogs was identified by their morphological appearance in erythrocytes of blood smears (intraerythrocytic merozoite stage). Initially, all large forms measuring between 3 and 5 μm were classified as B. canis, whereas all small forms measuring 1-3 μm were designated as B. gibsoni, but molecular analysis and DNA

Zoonotic importance
Babesia species affecting dogs and/or cats are not reported to be of zoonotic importance [17]. However, dogs and cats are close companions of people and can serve as a source of infected ticks for humans [18]. Human babesiosis is a rare disease and primarily involves just two species of Babesia: Babesia divergens, a parasite of cattle in Europe, and Babesia microti that parasitizes small rodents in the USA.

Transmission
There are two hosts for transmission of Babesia spp., viz., invertebrate (tick) and vertebrate host. Dogs are one among the many targets of Babesia spp., causing canine babesiosis (formerly called canine piroplasmosis).
Hard ticks are the main vectors for Babesia spp. Species such as Rhipicephalus sanguineus, Dermacentor spp. and Haemaphysalis ellipticum 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 hexagonus [19]. Both trans-stadial and transovarial transmissions can occur, and ticks are believed to remain infective for several generations.
Babesia spp. undergoes the sexual conjugation and the sporogony portions of their life cycles inside the lumen of the intestine and then within the haemocoel of the tick. Then the sporozoites from the tick's salivary gland are transmitted to their new vertebrate host via a blood meal, and thereafter the protozoan life cycle is completed within the red blood cells by asexual replication (merogony), where the parasites appear as merozoites [20], which leaves the host cell and enters another red blood cell. This cycle continues for the entire life tenure of vertebrate host or Overview of Canine Babesiosis DOI: http://dx.doi.org /10.5772/intechopen.82243 until the host's immune system terminates the process. However, in many countries for the last one decade, Babesia gibsoni infections have been reported in the absence of tick vectors. There is now convincing evidence that these cases have arisen due to biting and fighting between infected and noninfected dogs [21,22], but these parasites are primarily transmitted through tick bites. This association arose as a by-product of the tick's adaptation to feed on blood.
The first clinical evidence of possible vertical transmission has been documented for B. canis [23] and B. microti-like spp. [24]. Although Babesia undergoes part of their life cycles in ticks, the merozoites circulating in the blood may be transmitted to a healthy host directly by blood transfusion. This scenario has been described for B. gibsoni infection [25] and by direct contact between dogs through wounds (fighting dogs), saliva or blood ingestion [21,22,26].

Clinical signs and pathogenesis
Dogs of all ages can be affected with Babesia spp., but young puppies are more commonly affected. The incubation period varies from 10 to 21 days for B. canis and 14-28 days for B. gibsoni. Mortality for Babesia spp. infections ranges from 12% for B. rossi to approximately 1% for B. vogeli [19].
The most predominant feature of babesiosis in infected dogs are haemolytic anaemia and thrombocytopenia. Multiple causes like extra-and intravascular haemolysis, RBC destruction due to increased osmotic fragility, shortened life span of RBCs, erythrophagocytosis and immune-mediated destruction of RBCs because of parasitic antigens, parasite-induced membrane damage and possibly other membrane-associated antigens leads to anaemia [27][28][29]. Impaired haemoglobin function, oxidative damage, sludging and sequestration of erythrocytes also likely occur [27,28,30].
Recent study revealed about the renal involvement in babesiosis. Hypoxaemia, glomerulonephritis and haemoglobinuric nephropathy are considered possible mechanisms and supported by histological studies [31].
Pancreatitis is frequently associated with other complications and has a mortality rate of 20%. Common finding includes vomition, melaena, icterus, abdominal pain and diarrhoea. In addition, 65% of the dogs with pancreatitis also had icterus, 30% had acute respiratory distress syndrome (ARDS), 30% had immune-mediated red blood cell destruction (IMHA), and 15% had acute renal failure (ARF), while 10% had haemoconcentration, and another 10% had cerebral syndrome. It is postulated that pancreatitis is formerly described "gut" form of babesiosis [32].
The severe form of disease is characterised by marked haemolytic anaemia and acid-base abnormalities [33] with secondary multiple organ failure and complications such as ARF, hepatopathy, hypoglycaemia [34], ARDS, IMHA and cerebral pathology [35]. Small subset of dogs presents with high haematocrits (relative haemoconcentration), despite vigorous haemolysis, due to shifting of fluid from intravascular to extravascular component. These dogs are at increased risk of developing ARF or cerebral complications, as well as other organ failures [36].
The severity of the disease depends on the species of Babesia, presence of concurrent infections, age and immune status of the host. The disease presentation varies widely from peracute to chronic or even subclinical. Babesia rossi, the dominant species found in South Africa, is very virulent and causes peracute and acute disease. Most common signs include fever, anorexia, weakness, depression, pale mucous membranes, tachycardia, tachypnoea and splenomegaly. Clinical signs are because of tissue hypoxia following anaemia and a concomitant systemic inflammatory response syndrome caused by marked cytokine release [19].

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Babesia vogeli causes clinically inapparent infection in mature dogs. The parasitaemia is very low, and infection may be missed while routine examination of blood smear. Subclinical infections are common in adult dogs, but puppies tend to present with marked anaemia [37]. It 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. Anaemia is reported in majority of dogs and thrombocytopenia in all cases [38].
Babesia gibsoni infection follows either hyperacute, acute or chronic course. Among these acute course is the most common and is characterised by fever, lethargy, haemolytic anaemia, thrombocytopenia, lymphadenopathy and splenomegaly [39]. The hyperacute state is rare and is characterised by shock and extensive tissue damage. Mostly a disease of American Pit Bull and Staffordshire Bull Terriers is transmitted via dog bites [21]. In Australia and the USA, subclinical B. gibsoni infections have been reported, where they are PCR positive, but neither show clinical illness nor microscopic parasitaemia [40]. Such cases can have dire consequences if imported into non-endemic areas.
Babesia conradae is considered to be more pathogenic than B. gibsoni, resulting in higher parasitaemias and more severe anaemia [11].

Cardiac dysfunction
It's a rare complication of canine babesiosis; reported macroscopic cardiac lesions are effusions in pericardium, epicardial and endocardial haemorrhage involving one or more chambers with left ventricle being most commonly affected. Histopathological changes include necrosis, haemorrhage, fibrin micro-thrombi in the myocardium and inflammation. Lesions may be multifocal, but more generally they are limited to one area within the myocardium [41].

Consequence of canine babesiosis
Complicated babesiosis involves clinical manifestations that are not related to haemolytic disease. The most commonly documented complications include coagulopathy, ARF, ARDS, icterus and hepatopathy, haemoconcentration, immunemediated haemolytic anaemia (IMHA), pancreatitis, hypotension, myocardial pathology, cerebral babesiosis and shock. Rare complications include gastrointestinal disturbance, myalgia, ocular involvement, upper respiratory signs, necrosis of the extremities and fluid accumulation. These complications can overlap.

Diagnosis
A precise and fast diagnosis and prompt treatment is required in critical situations such as hyperacute to acute phase of B. canis infection, where high mortality is generally reported.

Direct (microscopic) examination
Historically, Babesia infection in dogs was identified based on the morphologic appearance of the parasite in the erythrocyte; thus, microscopic evaluation for detecting intraerythrocytic parasites in Giemsa or Wright's stained blood smears remains the simplest, most accessible and reasonably sensitive especially during acute infections. Differentiation between large and small piroplasms is also relatively simple. Moreover, in many parts of the developing world where babesiosis is endemic, microscopy is still the only viable available option. The likelihood of spotting a piroplasm increases with proper sampling technique, viz. sampling from capillary beds (ear tip, toe nail) or examination of cells from beneath the buffy coat of a haematocrit tube [37,42] or search along the periphery of the blood smear, as parasitized red blood cells tend to marginate while making the smear. For diagnosis of large forms of Babesia (e.g. B. canis) from the majority of sick dogs, light microscopy is highly specific [2,43], but small piroplasms (B. gibsoni, B. microti-like sp.) are hard to observe by light microscopy, which has a relatively poor to moderate sensitivity [44], and expertise is needed. Moreover due to very low, often intermittent parasitaemias, identification of piroplasms in chronically infected and carrier dogs remains a significant challenge.
Diagnosis is more problematic in chronic cases of infection due to less virulent species such as B. canis and B. vogeli, where parasitaemia may be below the microscopic detection limit, and in such cases thick smears (not alcohol fixed) may be helpful in detecting the parasite.

Haematological changes
The major haematological changes include mild to moderately regenerative normocytic and normochromic anaemia, leucocytosis with normal to decreased neutrophil counts and most consistent finding thrombocytopenia which is severe in the acute phase of infection [45].

Coagulation profiles
In a recent study, it was reported that there is significantly lower mean platelet count, prolonged activated partial thromboplastin time, higher fibrinogen concentrations and D-dimer value in infected dogs as compared to healthy controls [46].

Biochemical abnormalities
Elevation of liver enzymes such as ALP, ALT and AST. Elevated serum bilirubin concentration is associated with degree and rapidity of the anaemia and accompanying hepatopathy. There will be low total serum protein and albumin level in dogs with babesiosis. Urea is disproportionately raised to creatinine, and this is probably due to increased urea production resulting from gastrointestinal haemorrhage or protein catabolism as a result of febrile inflammatory illness [47,48]. In complicated cases with renal dysfunction, there will be proportionate increase in serum urea and creatinine levels indicating decreased renal perfusion, as a result of hypovolaemia, decreased blood pressure and/or decreased myocardial function. Hypokalaemia has been reported in severely affected dog, it has not connected directly with babesiosis, and this could be attributed to decreased potassium intake. Considerable elevation of positive acute phase protein (α 1 -acid glycoprotein) has been reported in dogs with B. rossi infection, but levels do not correlate with severity of disease or outcome [49].

Metabolic abnormalities
The most common complication is hypoglycaemia and is often associated with severe anaemia, icterus, young age (<6 months) and collapse [50]. Reduced survival has been associated with hypoglycaemia (<59.4 mg/dL) and hyperlactataemia.

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Hypoglycaemia-induced central nervous system signs should not be misdiagnosed as cerebral babesiosis.

Electrocardiogram (ECG findings)
In canine babesiosis, variety of arrhythmias are reported including sinus arrest, sinoatrial block, first-and second-degree atrioventricular block, ventricular tachycardia and ventricular premature depolarizations. ECG abnormalities include prolonged QRS interval, low amplitude and notching of R waves, ST segment deviation and large T waves [41].

Urine analysis
Routine test may reveal presence of bilirubin, haemoglobin and protein in the urine. In a recent study conducted to assess renal dysfunction in Babesia rossi, infection revealed higher concentrations of urinary IgG, urinary CRP and urinary RBP suggestive of both glomerular and tubular dysfunction [51]. These can be used for early detection of Babesia-induced renal dysfunction than serum urea and creatinine and urine specific gravity. There will be minimum and variable changes in urine enzyme activity (GGT and ALP) in babesiosis, thus limiting their use as diagnostic tests [47].

Serological diagnosis
A recent study demonstrated the opportunity of an early and specific detection of acute infections by an AgELISA that is potentially translatable to a rapid diagnostic test design and can be used in an ELISA to detect circulating Babesia antigen during acute infections and can be used to detect parasites 24-48 h before it could be detected by light microscopy [52].

Molecular diagnosis
Molecular techniques help in refining the diagnosis to the species level and thus provide a more accurate prognosis. Different molecular techniques are used for identification and differentiation of the various species of Babesia, semi-nested PCR [53] reverse line blotting [9,54] and PCR-restriction fragment length polymorphism analysis [55].
Improved PCR techniques have lately allowed for better definition of these parasites [56] and allows for a more reliable identification of the etiological agents compared to direct detection by light microscopy or serology [2]. It is more sensitive and provides an evidence of an active and ongoing infection in a clinical setting.
In addition, several genes are commonly used to discriminate among Babesia species. Typically, these include the nuclear ribosomal RNA genes [7,8] and the two internal transcribed spacers (ITS1 and ITS2) [7]. PCR DNA amplification can be a useful technique for monitoring treatment [57].

Diagnostic imaging
The most consistent findings on abdominal ultrasonography in dogs with B. canis are hepatomegaly and splenomegaly with diffuse, hypoechoic, heterogeneity and renal changes like diffuse homogenous increased cortical echogenicity and increased corticomedullary definition [58]. Dogs infected with B. rossi and B. canis having gastrointestinal signs and abdominal pain are reported to have Overview of Canine Babesiosis DOI: http://dx.doi.org /10.5772/intechopen.82243 ultrasonographic changes in the pancreases [32,59] which are found to be consistent with acute pancreatitis and included duodenal atony and peripancreatic fat hyperechogenicity.

Differential diagnosis
It includes other causes of haemolytic anaemia such as haemobartonellosis, autoimmune haemolytic anaemia, pyruvate kinase deficiency and Heinz body haemolytic anaemia. Other differentials include immune-mediated thrombocytopenia, systemic lupus erythematosus, leptospirosis, rickettsial diseases, dirofilariasis with caval syndrome, leptospirosis, zinc toxicity and neoplasia.

Therapy
Treatment for canine babesiosis consists of three components: • Treatment to eliminate the parasite • Blood transfusions to treat severe anaemia • Supportive care for the complications and metabolic derangements

Main drugs and drug combinations used in the antiprotozoal treatment of babesiosis
a. Imidocarb dipropionate: It is an aromatic diamidine and is recommended to be used as 6.6 mg/kg intramuscularly (IM) or subcutaneously (SC) with a repeated dose in 2 weeks in dogs. Among many of the proposed mechanisms of action includes blockage of the entry of inositol into erythrocytes containing Babesia, resulting in starvation of the parasite [60], interference with production [61] or combination with DNA in susceptible Babesia species, causing nucleic acid damage and inhibition of cellular repair and replication [62] ( Table 1). It is approved for treatment of different forms of Babesia spp. [39,57,63]. The adverse effects of this medication include pain during injection and cholinergic effects such as salivation, drooling, nasal drip or vomiting which can be mitigated by premedicating with atropine at 0.05 mg/kg. Additionally some less frequent adverse effects are panting, restlessness, diarrhoea, renal tubular or hepatic necrosis and injection site inflammation and more rarely ulceration, which usually heals within days to weeks.
b. Diminazene aceturate: It is 4,4′-(diazoamino) dibenzamidine diaceturate which is widely used in tropical countries as a first-line agent for the treatment of Babesia gibsoni infection of dogs, usually as an intramuscular injection of 3.5 mg/kg. Although diminazene aceturate has anti-Babesia activity, it often fails to eliminate B. gibsoni from affected dogs and a relapse may occur. Furthermore, diminazene has a narrow clinical safety margin and can induce fatal nervous complications after 24-48 h of overdose. Clinical signs associated with diminazene toxicity are depression or stupor, continuous vocalisation, ataxia, opisthotonos, extensor rigidity, nystagmus and seizures [3]. There are reported toxicity such as acute CNS signs including ataxia, nystagmus and occasional seizures in dogs administered with one recommended intramuscular dose (3.5 mg/kg) of diminazene for treatment of babesiosis [64].
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Combination therapies
a. Atovaquone and azithromycin: The first treatment that has been shown to be effective against B. gibsoni is a combination of an analog of ubiquinone, atovaquone (ATV) and a macrolide antibiotic, azithromycin (AZM) [57]. The use of ATV alone inhibits the growth of Babesia spp. and is presumed to act through blocking protozoan mitochondrial electron transport causing inhibition of pyrimidine and ATP synthesis [62]. Azithromycin acts by inhibiting the translation of mRNA and bacterial protein synthesis by binding to the 50S subunit of the prokaryote ribosome. It exerts its antiprotozoal effects by specifically acting on apicoplasts, a non-photosynthetic plastid organelles with a limited genome and found in apicomplexan parasites [65] including Babesia spp. [66]. In addition, the simultaneous use of ATV and AZM produces an additive or synergistic therapeutic effect, while the single use of each drug tends to result in a relapse of signs. Moreover possible emergence of drugresistant variants of B. gibsoni can occur after the use of ATV alone, and these variants might be caused by mutations in the cytochrome b (CYTb) gene, resulting in amino acid substitutions at the putative ATV binding site [67]. Further, the results are not consistent based on molecular analysis [68].
b. Buparvaquone and azithromycin: Buparvaquone is a hydroxynaphthoquinone antiprotozoal drug-related to atovaquone and parvaquone. Buparvaquone (Butalex ® ) is used in the treatment of bovine theileriosis [69]. It is also used to treat B. vulpes infection in dogs at an off-label dose of 5 mg/kg IM twice 48 h apart, in combination with azithromycin at 10 mg/kgPO once daily for 10 days [63]. Buparvaquone's mode of action on piroplasms is probably comparable to atovaquone's.
c. Combination therapy of clindamycin (CLDM), metronidazole (MNZ) and doxycycline (DOXY) is an efficacious alternative treatment strategy for B. gibsoni infection [70]. However, this treatment takes a relatively long time to show its therapeutic effect [71].

Prognostic indicators
In a recent study, it was suggested that lactate as a prognostic indicator with mean lactate in complicated cases of canine babesiosis wherein the blood lactate concentration in non-survivors (145 mg/dL) was higher than in survivors (13.8 mg/ dL). Pretreatment hyperlactataemia (>45 mg/dL) and subsequent serial lactate concentrations that failed to return to normal reference range (persistently >40 mg/ dL) indicate poor prognosis [75].

Prevention
The most effective preventive measures practised worldwide are regular control of the tick vectors by routinely dipping or spraying pets or using tick collars or spot-on preparations. As it takes a minimum of 48 hours for Babesia transmission, so regular examination of dogs for the presence of any ticks and to remove it soon after they attach is important. The merozoites circulating in the blood may be transmitted to a healthy host directly by blood transfusion, so blood donors should be screened negative for babesiosis, preferably by polymerase chain reaction. Moreover, it is also reported that B. gibsoni can be transmitted by transfer of blood during dog fighting, which should be prevented. In Europe a vaccine is available against B. canis with a reported efficacy of 70-100% [76]. More recently a bivalent vaccine called Pirodog ® (Merial) derived from soluble parasite antigens from B. canis and B. rossi obtained from culture media supernatant has been shown to reduce duration and severity of clinical signs [77]. Although vaccination against canine babesiosis does not prevent infection, it does seem to block the initiation of pathologic processes involved in the pathogenesis of the disease [78]. This vaccine can be administered from 5 months of age and requires annual revaccination but does not cross-protect against other Babesia species. Vaccines against other Babesia species such as B. gibsoni are currently being developed including recombinant antigen and DNA vaccines [79][80][81]. Although dogs can be vaccinated, the level of protection is highly variable, which might be due to genetic diversity of B. canis strains.

Conclusion
The spectrum of Babesia pathogens that infect dogs is gradually being elucidated with the aid of new molecular techniques and meticulous clinical investigation. Species of Babesia that cannot be distinguished morphologically cause diverse diseases and are transmitted by different vector ticks. Nonvector transmission by blood transfusion and directly from dog to dog is of special concern and could be responsible for the spread of infection to areas that were previously non-endemic. Correctly identifying the infectious agent is important for treatment planning and prognosis.