Clinical management of canine babesiosis

Introduction

Clinical babesiosis is caused by hematoprotozoa of the genus Babesia. Numerous species of Babesia exist worldwide. They vary in their geographic distribution, hosts, modes of transmission, and pathogenicity. Babesia organisms parasitize erythrocytes of the definitive host, resulting in hemolytic anemia and varying degrees of polysystemic involvement.

An increased incidence of canine babesiosis has been reported in North America in the past 2 decades. Aside from the impact of this disease on morbidity and mortality of affected dogs, the zoonotic potential of babesiosis has public health implications. This review will consider the epidemiology, pathogenesis, diagnosis, and treatment of canine babesiosis with emphasis on the species present in the United States.

Epidemiology

Babesia spp. are tick-borne protozoal blood parasites with worldwide distribution. Known hosts include rodents, humans, dogs, cats, horses, and cattle. Multiple species have been documented to infect dogs: Babesia canis, Babesia gibsoni, Babesia microti, Babesia equi, Babesia conradae (California), and a large unnamed Babesia organism (North Carolina).^1–3 In North America, endemic seropositivity for B. canis has been documented over the past 50 years. Before 1990, however, only 2 cases of B. gibsoni infection were reported in the United States. Subsequently, and particularly in the last decade, an increasing number of Babesia spp. infections have been described. To date, 4 Babesia species are documented to infect dogs in North America: B. canis, B. gibsoni, B. conradae, and a large unnamed Babesia species (North Carolina) (Table 1).

B. canis is a large Babesia organism, with a piroplasm size of 3 μm × 5 μm. The intraerythrocyte piroplasm is piriform (teardrop shaped), appearing either as a singlet or in pairs. Four distinct subspecies occur: B. canis subsp. vogeli, B. canis subsp. canis, B. canis subsp. rossi, and another genetically distinct (and as yet unnamed) large piroplasm subspecies found in North Carolina.^2,3 These subspecies vary in their geographic distribution, vector, pathogenecity, and host immune response, with minimal to no cross-protective immunity occurring among subspecies. B. canis subsp. canis (moderately pathogenic) and B. canis subsp. rossi (severely pathogenic) are limited to Europe and Asia and South Africa, respectively.³B. canis subsp. vogeli is the least pathogenic strain and is found in tropical and subtropical areas of the Gulf Coast, as well as the southern, central, and southwestern United States.^2–5 Case reports document infections in Florida, North Carolina, West Virginia, Oklahoma, Texas, Mississippi, and California.^6–8 Breed susceptibility to B. canis subsp. vogeli is suspected in Greyhounds; seropositivity of up to 50% in this breed is reported in endemic areas compared with 3.8–13% seropositivity in the general canine shelter population.^6–11

B. gibsoni is the most common species of Babesia in the United States. Based on polymerase chain reaction (PCR) testing, 91% of dogs seropositive for Babesia have B. gibsoni.¹¹B. gibsoni is a small organism, measuring 1 μm × 3.2 μm. It is pleomorphic, manifesting a variety of intraerythrocytic forms; oval or signet ring shapes are most commonly described. Distribution in the United States is limited primarily to the eastern and midwestern states. However, at least one case has been reported in almost each of the 50 states.^{2,3,8,12–18} The B. gibsoni isolates reported in the eastern and midwestern states are identical to the Asian variant, B. sensu stricto.^3,12,19,20 Breed susceptibility is suggested in American Staffordshire and Pit Bull Terriers (APBT); 15–93% of B. gibsoni-seropositive dogs are APBT compared with <1% seropositivity in non-ABPT breeds. It is suspected that, in the APBT, parasite transmission may be horizontal, most notably through bite wounds, as well as vertically from dam to pups.^{5,11–14,16–18,21}

At least 3 additional small Babesia variants exist that are genotypically distinct from B. gibsoni. These include B. microti and B. equi (not reported in the United States), and a small distinct Babesia sp. reported in California.^3,19 The California species, recently named B. conradae most closely resembles Theileria annae and Babesia isolates from wildlife and humans in the western United States.^{3,16,17,19,20} It is more pathogenic than B. gibsoni, visualized in erythrocytes as a singlet most commonly, and occasionally as a tetrad or maltese-cross form.^3,14,16,19

Life Cycle and Transmission

Babesia spp. are most commonly transmitted through the bite of an infected tick. B. canis subsp. vogeli is transmitted via the brown dog tick, Rhipicephalus sanguineus.^2–4,22Rhipicephalus sanguineus, Dermacentor variabilis, and Ixodes ticks are the most likely vectors for B. gibsoni in the United States.^{3,14,16,23–26} The vector for the small California species is unknown but a wildlife reservoir and tick vector are suspected.³ Mechanical transmission via other biting insects and arthropods may also occur. The adult female tick is considered most important in vector transmission as transstadial and transovarial (not documented with B. gibsoni) infection occur.^3,27

Babesia organisms multiply in the definitive host, via multiple fission, to produce merozoites. Ticks are infected following ingestion of parasitized host erythrocytes. The babesial merozoites undergo a complex life cycle in the tick. During this process, multiple fission of merozoites results in the production of sporozoites (infective undeveloped cells) within the arthropod salivary glands.^3,27,28 These sporozoites are then passed via tick saliva into the host circulation. An attachment time of 2–3 days is necessary for Babesia canis transmission to occur.²⁹

In addition to tick-borne transmission, vertical, horizontal, and transmission via blood transfusion are suspected. Transplacental transmission between dam and pup was first reported in the 1970s.^21,30 Subsequently, scattered case reports document infection in pups as young as 3 days. Parasitemia is evident in all affected puppies, most lack antibody formation, and the pups display varying degrees of clinical illness.¹⁴ In one study, experimental infections resulted in stillbirths or death of all puppies within the first 6 weeks of life.³¹Babesia DNA was detected in all 5 experimental puppies at birth, confirming transplacental infection. Horizontal transmission of B. gibsoni via bite wounds is strongly suspected, occurring via blood or saliva. Greater than 60% of non-APBT species infected with B. gibsoni are male with a history of having bitten, or been bit by, an APBT.^{11,13,14,16–18,32} Clinical signs typically arise 2 weeks post-bite.³ Direct transmission via blood transfusion has been documented using Greyhound and APBT donors.^14,33–35 Disease transmission via fomites such as surgical instruments or reused needles for vaccinations is theoretically possible, but has not been documented.

Once in the definitive host, Babesia organisms attach to the erythrocyte membrane and are subsequently engulfed by endocytosis.²⁸ Within the RBC, the host membrane surrounding the parasite disintegrates granting subsequent stages of the organism direct contact with the cytoplasm. The loss of this host cell membrane is characteristic of Babesia spp. and allows for assumption of a wide of variety of shapes including piriform, oval, elongate, twisted, and folded.^28,36 Repeated binary fission occurs within the RBC, resulting in up to 16 merozoites. Pairs of merozoites are seen most commonly in B. canis and B. gibsoni infections. In contrast, the small California strain forms tetrads or maltese crosses and does not undergo binary fission.³ It is the hosts' response to these reactions within their RBCs that determines the clinical disease course.³⁶

Pathogenesis

The pathogenicity of Babesia is determined by many variables including the species and strain of Babesia organisms, and the age and immunologic response of the host. Of these, species and strain appear to be most important.³⁷

Following infection, a variable immune response is generated, dependent upon host factors. Infected erythrocytes display parasite antigens on their cell membranes that leads to antibody production, opsonization, and the removal of infected cells by the mononuclear phagocytic cells of the hemolymphatic system. Although survivors exhibit no clinical signs, they remain chronic carriers as the immune system is incapable of completely eradicating the infection.³ Immune response appears to be strain-specific; cross-protective immunity between species and strains does not occur.^38,39 Mature animals mount a more robust humoral immune response than do juveniles, as evidenced by serologic testing in animals <1 year of age. It is suspected that poor humoral response and maternal antibody protection is responsible for the lower seroprevalence seen in immature patients.^3,40,41

Splenectomized dogs demonstrate more pronounced lymphocyte immunostimulation, recruitment and activation of macrophages, hemophagocytic activity, and Kupffer cell hypertrophy than do unsplenectomized dogs infected with Babesia spp.^42–44 This leads to a more severe clinical course (as manifested by the severity of anemia, acute hepatopathy, and concurrent hemoprotozoal infections).^42–48

Clinical Syndromes Associated with Babesia Infection

Anemia is the predominant clinical syndrome, the development of which is multifactorial and results in both intravascular and extravascular hemolysis. Parasitic activity directly damages the erythrocyte cell membrane, resulting in increased osmotic fragility and subsequent intravascular hemolysis.⁴⁹ In addition, indirect pathways of RBC destruction are important contributors in the pathogenicity of Babesia-induced anemia. These pathways include immune destruction secondary to the development of antierythrocyte membrane antibodies, inhibition of erythrocyte 5′-nucleiosidase, development of methemoglobinemia secondary to oxidative stress, induction of serum hemolytic proteins, and increased macrophage erythrophagocytic activity.^50–59

Immune-mediated hemolytic anemia (IMHA) may occur secondary to the inappropriate production of antierythrocyte membrane antibodies, and is assumed to occur with all Babesia spp. Elevated concentrations of antierythrocyte membrane antibodies and erythrocyte-bound immunoglobulin G have been documented in dogs infected with B. gibsoni.^60,61 Continued hemolysis, despite appropriate anti-babesial therapy, is the most prominent feature of this complication. Diagnosis requires demonstration of saline agglutination of RBCs or spherocytosis, or both.³ A positive Coombs test is not considered a reliable tool for the diagnosis of IMHA in babesiosis as 84% of canine patients infected with B. canis or B. gibsoni have positive Coombs tests.⁶²

Nucleoside inhibition contributes to the anemia. Serum from infected dogs has been shown to result in the inhibition of erythrocyte 5′-nucleiosidase activity in noninfected dogs.^57–59 The resulting accumulation of cyclic nucleotides and ribosomal DNA contributes to RBC damage, intravascular hemolysis, and progressive anemia. In addition, culture supernatant from dogs infected with B. gibsoni results in delayed morphologic maturation of reticulocytes in vitro.⁵⁹

Oxidative stress can cause lipid peroxidation and erythrocyte injury, with resultant methemoglobinemia. Methemoglobinuria, as well as elevated methemoglobin to total hemoglobin ratios have been documented in Babesia-infected dogs.^63–65 Increased macrophage production of superoxide and other reactive oxygen species has been demonstrated in B. gibsoni-infected dogs.^65,66 Studies of experimentally induced B. gibsoni infection suggest that free radical-initiated oxidative stress to the RBC is necessary for antierythrocyte membrane antibody production.^50,67 Furthermore, erythrocyte oxidation may enhance susceptibility to macrophage-mediated bone marrow phagocytosis.^56,65

Lipid peroxidation of erythrocytes also decreases RBC membrane pliability, resulting in slowed passage and further damage to the erythrocyte as it traverses capillary beds. The capillary sludging of erythrocytes, in combination with soluble parasite proteases, activate the kallikrein system leading to production of fibrinogen-like protein.³ This protein induces RBC aggregation and promotes vascular stasis, which leads to ischemia, thrombosis, and end-organ damage. The CNS, kidney, and muscle appear to be the organs most affected by the resultant tissue hypoxia.^39,68,69

Multiple organ dysfunction syndrome (MODS) can occur subsequent to infection with the more pathogenic Babesia species, particularly B. canis subsp. rossi. Documented complications include red biliary syndrome, thrombocytopenia, disseminated intravascular coagulation (DIC), acute renal failure (ARF), hepatopathy, rhabdomyolysis, noncardiogenic pulmonary edema, CNS dysfunction, pancreatitis, systemic hypotension, cardiac dysfunction, hypoglycemia, hypoxemia, and metabolic acidosis with hyperlactatemia.^{39,41,68–76} Although severe anemia and resultant cellular hypoxia contribute to these complications, it is suspected that systemic inflammatory mediators (cytokines and reactive oxygen species) generated by host tissues and inflammatory cells are largely responsible for end-organ damage.^41,68 A retrospective study of canine babesiosis in South Africa documented development of systemic inflammatory response syndrome in 87% of infected dogs.⁷¹ Of these, 52% had single-organ damage and 48% had evidence of multiple-organ dysfunction.

A paradoxical phenomenon of hemoconcentration in conjunction with severe intravascular hemolysis has been reported, referred to as red biliary syndrome.³ Systemic inflammation leads to vasculitis, increased capillary permeability, and the extravasation of intravascular fluids. Concurrent hypoalbuminemia contributes to the fluid shift, and hemoconcentration ensues. This phenomenon typically occurs in conjunction with other complications and is associated with a significantly higher mortality rate than seen with anemia alone.⁷³

Thrombocytopenia can occur in combination with other hematologic abnormalities or as a singular entity, and may be transient or persistent.^3,77 It is commonly seen in both B. canis subsp. rossi and B. gibsoni infection and is the most consistently reported hemostatic abnormality.³ Coagulopathic consumption in association with DIC is postulated. Splenic platelet sequestration, or immune-mediated platelet destruction, or both may be contributory.

Traditional hemostatic testing (prothrombin time, activated partial thromboplastin time, platelet count, and fibrinogen concentration) has detected up to a 20% incidence of DIC in canine babesiosis. D-dimers were elevated in 50% of these patients.⁷⁸ Prothrombin times and activated partial thromboplastin times were within reference intervals in up to 80% of dogs infected with B. canis in Italy and Spain. In spite of this, parasite-induced inflammation is thought to lead to activation of the intrinsic coagulation system, resulting in subclinical microthrombosis and platelet consumption.^3,78,79 Hyperfibrinogenemia is exhibited by infected dogs supporting the role of an inflammatory response, while documentation of a normal bone marrow response with adequate platelet precursors is consistent with platelet consumption in these patients.^78,79 Endothelial damage induced by hemolysis and hypoxia, interaction of parasitized erythrocytes with the endothelium, and activation of the coagulation cascade secondary to an acute phase response may also contribute to alterations in coagulation.^78,80,81

ARF, although uncommon, is a devastating consequence of complicated Babesia infection, and has been associated with up to a 5-fold increased risk of death.^71,73,74 Most notably, azotemia is commonly seen in B. microti-like infections and is the only factor shown to significantly correlate with mortality.⁸² The pathogenesis of ARF is multifactorial. An experimental study documented roles for renal hypoxia and hemoglobinuria in the development of renal tubular damage.⁶⁹ In this model, renal hypoxia resulted in a larger degree of tubular necrosis with a decrease in glomerular filtration rate than did hemoglobinuria, suggesting a more pathogenic role of hypoxia on the nephron. Histologic renal examination of dogs with B. canis revealed necrosis and detachment of tubular epithelial cells from the basement membrane, most consistent with hypoxic damage.⁸³ Postulated causes of renal hypoxia include anemia, capillary sludging, and systemic hypotension with compensatory renal vasoconstriction. Immune-mediated development of membranoproliferative glomerulonephritis may also contribute to renal damage; multifocal deposits of immunoglobulin M have been demonstrated in glomeruli of infected patients via immunohistopathologic testing.⁸⁴

BUN measurement is affected by intravascular hemolysis, and is therefore an unreliable marker of renal dysfunction in this patient population. Serum creatinine concentration is unaffected by hemolysis and remains a useful diagnostic tool.⁸⁵ The presence of casts, renal tubular cells, and proteinuria is commonly seen on urinalysis. While this is suggestive of renal damage, it is not predictive of ensuing renal dysfunction. The degree of proteinuria appears to correlate with the severity of the systemic disease rather than the degree of renal damage.^86,87

Icterus and elevated hepatic enzymes occur frequently. Although the associated hemolytic anemia contributes to hyperbilirubinemia, it is not the sole cause. Centrilobular hepatitis is the most commonly documented and severe histopathologic change in babesial infection, and is most likely the result of hypoxic liver damage.⁸⁴ Kupffer cell hypertrophy and increased numbers of CD3+ lymphocytes and macrophages have been demonstrated, suggesting that immune-mediated inflammation likely plays a contributing role.⁸⁴ Although histopathologic and laboratory evidence of hepatic damage is common, hepatopathy is not reported to significantly affect outcome.⁷¹

Rhabdomyolysis can complicate babesiosis. It is characterized by muscle pain, tremors, pigmenturia, and elevated concentrations of myoglobin, creatinine kinase, and alanine aminotrasferase.^71,73 Histopathologic findings include muscle necrosis and hemorrhage.⁶⁸ Capillary sludging and the production of inflammatory cytokines and nitric oxide appear to play an important role.^39,68 Although common, muscle damage has not been shown to affect outcome.⁷¹

Acute respiratory distress syndrome (ARDS) is a common and important complication of the more pathogenic strains of Babesia spp. Clinical features include tachypnea, dyspnea, a moist cough, serosanguinous frothy respiratory secretions, and hypoxemia. Radiographs reveal either a diffuse or caudodorsal patchy alveolar infiltrate with normal cardiac silhouette and vessel size. Pulmonary capillary wedge pressure, when measured, is normal. This syndrome may arise at any point in the disease process and tends to be rapidly progressive. A systemic inflammatory response syndrome, secondary to the production of inflammatory cytokines and reactive oxygen species, likely plays an important role in the pathogenesis of this complication.⁴¹ Inflamed pulmonary arteries with multifocal deposits of immunoglobulin M antibodies within the walls have been demonstrated in dogs with B. gibsoni.⁸⁴ The development of ARDS is a catastrophic complication and is associated with a marked elevation in mortality.^71,73,74

Both cerebral and cerebellar signs are reported uncommonly in dogs infected with B. canis subsp. rossi, and include loss of consciousness, stupor, coma, tremors, seizures, paddling, nystagmus, anisocoria, central blindness, ataxia, tetra- and paraparesis, aggression, and vocalization.^41,70,88 To date, no reports exist documenting the presence of cerebral or cerebellar signs in dogs infected with Babesia spp. present in North America. Peracute to acute onset is common. Postulated pathogenesis includes endothelial cell damage and necrosis followed by segmental microvascular necrosis with perivascular edema and hemorrhage.⁷³ Although clinical signs resolve in some patients following anti-babesial therapy, the development of neurologic signs is associated with a high mortality rate and a 57-fold increased risk of death.^71,73,74

Acute pancreatitis is a newly described but common occurrence in canine babesiosis.^73,74,76 The onset of diagnosis tends to be acute with the median time to diagnosis reported as 2.5 days. Young animals (median age of 3 y) and sexually intact dogs appear to be at increased risk. Clinical signs include inappetance, vomiting, diarrhea, and abdominal pain. Proposed mechanisms of injury include ischemia-reperfusion, altered blood flow and oxygen delivery due to hypotensive shock, anemia, and hemoconcentration, altered lipid metabolism and pro-inflammatory cytokine production.⁷⁴ Pancreatitis more commonly occurs in patients with MODS (80% suffer concurrent complications) and is associated with an 18% increase in mortality.^73,74

Hypotensive shock commonly accompanies canine babesiosis, and can occur at any point in the disease process.^34,86,89 Proposed mechanisms include parasite-induced kallikrien activation with resultant vasodilation (relative hypovolemia), production of vasoactive proteins, increased capillary permeability, decreased intravascular volume, and myocardial depression.^3,34 Hypotension enhances parasite sequestration and hypoxia, leading to a vicious cycle of increased disease severity with perpetuated inflammation and hypotension.³ The incidence of hypotension increases with disease severity but does not differentiate survivors from nonsurvivors.⁸⁹

Cardiac dysfunction is a rare occurrence in canine babesiosis. Cardiac troponin I levels are increased in infected dogs, and correlate with histologic cardiac changes, clinical severity, and survival. Reported ECG abnormalities include heart block, ventricular premature contractions, and prolonged QRS and ST segment changes. These ECG findings are not associated with disease severity, histopathologic changes, cardiac troponin levels, or outcome with the exception of ventricular premature contractions which are significantly associated with cardiac troponin I concentration.^90,91 Left ventricular failure with volume overload and hypotension has been reported, and associated histopathologic changes include hemorrhage, necrosis, inflammation, and the presence of fibrin microthrombi in the myocardium.⁹¹

Hypoglycemia is a relatively common complication of the more pathogenic strains of Babesia spp., with a reported incidence of 20%.⁷⁵ Hypoglycemia has been significantly correlated with mortality.^75,92–94 Reported risk factors include a collapsed state, severe anemia, icterus, vomiting, and immaturity (less than 6 mo of age). Collapsed state and young age are the most strongly correlated variables with an increased occurrence of hypoglycemia of 17.8 and 2.8 times, respectively.⁷⁵ Hypoglycemia must be differentiated from cerebral babesiosis as cerebral disease carries a far poorer prognosis. Speculated causes of hypoglycemia include increased glucose consumption secondary to anaerobic glycolysis, hypermetabolism and enhanced cellular uptake of glucose, depletion of hepatic glycogen stores, and hepatic dysfunction with impaired gluconeogenesis. Hyperinsulinemia was once a speculated cause; a recent study, however, failed to demonstrate any significant alterations in insulin concentrations.^75,92

Mixed metabolic and respiratory acid-base imbalances are commonly seen. One study documented concurrent metabolic acidosis with respiratory alkalosis in 91% of dogs infected with B. canis subsp. rossi.⁷² Eighty-two percent of patients in this study were hyperlactatemic, suggestive of lactic acid production secondary to tissue hypoxia as the primary mechanism for the observed metabolic acidosis. Concentrations of lactate and pyruvate are significantly higher in nonsurvivors and can be used as an indicator of disease severity.^73,93,94 The respiratory alkalosis is likely due to hypoxemia-induced hyperventilation. In addition, a hypoalbuminemic alkalosis was present in all dogs.⁷²

Clinical Presentation

The clinical presentation of Babesia spp. infection ranges from peracute to subclinical. Peracute infection is rare and is characterized by severe, extensive tissue damage and a high mortality rate.^16,34 Dogs with acute babesiosis typically present with hemolytic anemia, fever, lethargy, anorexia, splenomegaly, lymphadenomegaly, thrombocytopenia, and vomiting.^41,95,96 Although fatalities are common in puppies with acute disease, adult dogs with acute babesiosis typically survive with appropriate therapy.³ Chronic infection is recognized but its manifestation is poorly characterized.⁴¹ Low-grade or subclinical infections are seen with many babesial strains, most notably B. canis subsp. vogeli in Greyhounds and B. gibsoni in APBTs.^12,18 Although most subclinically infected dogs never manifest clinical signs, disease can be precipitated by stress or glucocorticoid therapy.^3,97

Canine babesiosis is classified as uncomplicated or complicated. An uncomplicated presentation refers to hemolytic anemia and its accompanying clinical signs (fever, pallor, anorexia, depression, splenomegaly, and tachycardia with hyperdynamic pulse pressures). These signs range from mild to severe and life threatening. Complicated babesiosis refers to clinical manifestations not directly associated with hemolytic anemia, including cardiovascular, respiratory, hepatic, renal, gastrointestinal, neurologic, and coagulopathic dysfunction.

B. canis infection usually results in mild disease in the American strains; the African, Australian, and European forms are more pathogenic.^3,16 Adult dogs infected with B. canis subsp. vogeli typically lack overt clinical signs.³B. canis subsp. canis infection is characterized by a low-level parasitemia with congestion of organs of the mononuclear phagocytic system.⁹⁸ The acute, uncomplicated form of the disease characterizes B. gibsoni infection.^3,16 The most common clinical signs include intermittent fever, pallor with a low-grade or compensated hemolytic anemia, splenomegaly, inappetance, poor body condition, and a variable thrombocytopenia.^3,99 Additionally, a chronic form of the disease occurs characterized by mild fever, pallor, splenomegaly, hepatomegaly, lymphadenopathy, and lethargy.¹⁷ An asymptomatic carrier state of B. gibsoni is also reported, primarily in APBT dogs.^5,11,12

B. canis subsp. rossi is the most virulent strain of the organism, resulting in profound intravascular hemolysis, splenomegaly, and pigmenturia. Complicated babesiosis is more likely to occur with this than with other species. Complications correlate with the degree of parasitemia, and may include severe thrombocytopenia (with a reported prevalence rate of 99%), metabolic acidosis, hyperlactatemia, hepatopathy, ARF, rhabdomyolysis, hypoglycemia, pancreatitis, DIC, MODS, cerebral dysfunction, ARDS, and cardiovascular disturbances.^{68,70–75,86,100}

A moderate to severe disease process characterizes the small California Babesia species. Clinical signs associated with infection include lethargy, vomiting, anorexia, fever, and pallor secondary to hemolytic anemia. Severe acute hemolytic anemia with concurrent thrombocytopenia is present in the majority of cases.¹⁶

Diagnosis

Definitive diagnosis of Babesia infections requires demonstration of organisms within RBCs. Light microscopy evaluation of blood smears is an excellent diagnostic tool for acute infections with moderate or high parasitemia. It is often unrewarding, however, in peracute or chronic infections, asymptomatic carriers and patients with circulatory compromise.³ Evaluation of smears prepared from capillary blood, such as the ear tip or nail bed enhances the likelihood of organism detection, because parasitized erythrocytes tend to sludge in the capillaries.^96,101 Similarly, evaluation of smears prepared from a concentrated and stained Buffy coat (Percoll gradient separation) may facilitate diagnosis, as Babesia organisms preferentially parasitize reticulocytes over mature RBCs.^102–107 Platelet count is routinely used as a screening test for B. canis subsp. rossi infection, possessing a negative predictive value of 99% when platelet count is ≥110 × 10⁹/L.¹⁰⁰ This has yet to be evaluated for other Babesia infections. Electron microscopy improves visualization of Babesia piroplasms in RBCs.¹⁰⁸

The sensitivity of flow cytometry for documentation of acute babesial infections parallels that of conventional light microscopy, with a correlation coefficient as high as 0.97 when used in combination with fluorescent nucleic acid staining.^107,109,110 Flow cytometry has also successfully documented chronic infection, with a higher sensitivity than conventional light microscopy, but lower sensitivity than indirect fluorescent antibody (IFA) testing.¹¹¹

IFA evaluation detects anti-babesial antibodies in the blood of infected or exposed animals.^112–114 It is the most specific indirect methodology for documentation of infection, and a superior diagnostic test for occult and patent babesiosis.¹¹¹ Some authors suggest that single sample titers≥1:80 for B. canis and≥1:320–1:1280 for B. gibsoni infections or a 4-fold increase in convalescent titers are consistent with active infection; however due to variability in assay results titers should be viewed as circumstantial rather than definitive evidence of active infection.^3,115 A false-negative rate of 36% is reported with IFA testing, occurring most commonly in the very young, immunocompromised patient, or in early acute infection before an immune response is mounted.⁴⁰ In these cases, the evaluation of convalescent titers is recommended. Additionally, false-positive results may be seen in areas endemic to B. canis where the production of large numbers of antibodies occurs without clinical disease.¹¹⁶

ELISA represents another form of indirect diagnosis. Traditional ELISA and dot-ELISA tests have a superior sensitivity but significantly lower specificity when compared with IFA testing.^3,117 The recent development of recombinant ELISA assays has improved test specificity.^118–125 Caution must be exercised when interpreting results (especially with whole cell antigen tests), as cross-reactivity occurs between B. canis, B. gibsoni, Toxoplasma spp., and Neospora spp. infections.¹¹⁵ While ELISA is used commonly in seroepidemeologic studies, IFA testing is utilized more frequently as supportive evidence of clinical infection.^40,112,118

The most sensitive and specific tests available for diagnosis of babesial infections are genetic-based methods, including PCR testing.³ This methodology targets parasitic DNA rather than anti-babesial antibodies, and is therefore a reliable diagnostic tool in peracute, acute, and chronic infections, very young dogs, and immunocompromised patients.¹²⁶ Sensitivities and specificities approaching 100% have been reported.^127,128 PCR is capable of diagnosing babesial infections from small-volume blood samples with extremely low parasitiemia^126,129,130 Nested PCR tests display superior sensitivity than do semi-nested PCR tests, and newer single-step tests are capable of subspecies differentiation.^{119,126,129,131} PCR is commonly used in clinical diagnosis, identification of new strains, and to differentiate alike and genetically distinct Babesia spp. in epidemiologic studies internationally.^{12,20,119,126,128,129,131,132}

Treatment and Prevention

Antiprotozoal drugs, antimicrobials, and supportive care encompass the mainstays of anti-babesial therapy. Although mild to moderate infections may resolve without specific treatment, anti-babesial drugs hasten the resolution of clinical symptoms often within the first 24 hours. Complete disease eradication may not be possible, however, and there is a high incidence of relapse.³

Diminazine aceturate, an aromatic diaminidine derivative, which traditionally is viewed as the most effective treatment for large babesial species, is not approved for use in the United States.³B. canis displays a higher susceptibility to diminazine than does B. gibsoni. A single dose of 3–5 mg/kg IM has proven efficacy. Diminazine has a very narrow therapeutic range; the drug is inconsistently cleared and results in a high rate of toxicity.^133–135 Adverse effects include pain and swelling at the injection site, gastrointestinal signs, and neurologic dysfunction.^3,133 The latter effect can be severe and fatal. Moreover, it is cumulative over an extended period. The dose cannot be repeated with this or another diaminidine derivative within a 6-week period. A relapse rate as high as 50% has been reported following diminazine treatment. Complete sterilization of the infection is a questionable goal, as rechallenge studies in diminazine-treated B. canis-infected dogs suggest a decreased rate of relapse in animals who remain chronic carriers.^134,136 Phenamidine isethionate, the second most effective anti-babesial drug, is also unavailable in the United States.³ As is seen with diminazine, the large babesial (specifically B. canis) species show a high susceptibility to phenamidine therapy. A dose of 15–20 mg/kg administered SC once daily for 2 consecutive days results in a rapid resolution of clinical signs.³

Approved for use in the United States, the antiprotozoal, pentamide, has documented efficacy against both B. canis and gibsoni. B. gibsoni shows a decreased susceptibility compared with B. canis. The recommended dose is 15–20 mg/kg, SC, every 12 hours for 2 consecutive days. Adverse effects include injection site pain, hypotension, tachycardia, and vomiting.^3,137

Imidocarb diproprionate, a carbanilide member of the diaminidine family, is the most commonly used drug in the United States for treatment of babesial infections. It has shown high efficacy against B. canis. A single dose of 7.5 mg/kg, IM or co-administration with diminazine have both proven efficacious in resolution of clinical signs in B. canis infected dogs. A dose of 5–6.6 mg/kg, IM on days 1 and 14 eliminates clinical signs and decreases the infectivity of tick vectors who feed on treated blood for up to 4 weeks post-therapy.^{134,137–139} In addition, a prophylactic protective post-therapy effect has been documented for up to 6 weeks.¹⁴⁰ Anecdotally, the use of imidocarb in B. gibsoni infection results in a rapid resolution of clinical signs with a reduction in morbidity and mortality. Although uncommon, adverse effects are severe and are anticholinesterase-related in nature; they include salivation, lacrimation, vomiting, diarrhea, muscle tremors, restlessness, tachycardia, and dyspnea. One report of overdose in a dog resulted in hepatic necrosis and subsequent death.^141,142

Atovaqoune, an antiprotozoal drug used to treat Pneumocytis infection in humans, is also effective in babesial infection. This drug has documented efficacy against B. gibsoni and may be the treatment of choice for this species. However, it displays little to no efficacy against B. canis.³ Disadvantages include expense and relative unavailability. Additionally, drug resistance has been documented, with relapse infection developing 1 month or more post-therapy. Combination therapy with azithromycin may reduce the incidence of resistance and relapse.^132,143,144 The combination of atovaquone (13.3 mg/kg, PO, q 8 h, for 10 d) and azithromycin (10 mg/kg, PO, q 24 h, for 10 d) is the first therapy described that is capable of producing an undetectable parasitemia in both B. gibsoni and B. microti infection.^{107,144–147}

Clindamycin alone or in combination with quinine is the treatment of choice for B. microti infections in humans.³ A dose of 25–50 mg/kg/d divided every 12 hours for 10 days has been reported to resolve clinical signs in canine B. gibsoni infection. However, clindamycin has not been shown to affect level of parasitemia or antibody titer.^148,149 A decreased B. canis parasitemia level has been shown with doxycycline therapy when administered at 10 mg/kg, PO, every 12 hours, for 7–10 days.^3,150 One clinical study suggested successful therapy for resistant or relapsed cases of B. canis infection using of a combination of clindamycin (25 mg/kg, PO, q 12 h), metronidazole (15 mg/kg, PO, q 12 h), and doxycycline (5 mg/kg, PO, q 12 h).¹⁵¹ Resolution of clinical signs and normalization of PCV was noted in 3 of 4 dogs; however, clinical efficacy was not reached until an average of 50 days after initiation of therapy. Results of this study are difficult to interpret due to its small sample size (4 dogs) and lack of controls. A 1% trypan blue solution administered at 10 mg/kg, IV, every 24 hours has also proven variably effective in B. canis infection.¹³⁵ The main advantage of this agent is lack of the anticholinesterase-related and CNS toxicities observed with drugs in the diamindine family. Disadvantages includes its variable efficacy and a blue discoloration to the plasma and tissues.¹³⁷ Additional therapies with some documented efficacy include quinuronium sulfate at a dosage of 0.25 mg/kg, SQ, every 48 hours for 2 dosages (effective against B. canis); and homeopathic treatment with Crotalus horridus 200C, 4 pills, PO, every 6 hours, for 14 days (appearing as effective as diminazine therapy in one reported study).^3,152

Aggressive supportive care is required for canine babesiosis, especially with complicated forms. Fluid therapy is paramount for maintenance of blood volume and adequate end-organ perfusion, correction of acid-base and electrolyte abnormalities, diuresis, and prevention of RBC sludging in capillaries. Blood transfusion is often necessary, the transfusion trigger being clinical signs referable to anemia (tachycardia, tachypnea, weakness, collapse, bounding pulses, and increased lactate levels). The degree of parasitemia in affected dogs does not correlate with anemia and should not be used as a decision to transfuse.³ Packed RBCs are the blood component of choice, as the anemia results from RBC lysis rather than whole blood loss. The development of DIC and coagulopathy may warrant treatment with platelet, or plasma products. Additional management that may be indicated in the complicated forms of babesiosis includes vasopressors for refractory hypotension, nutritional support, glucocorticoids for secondary IMHA development, heparin for DIC, and oxygen and mechanical ventilation for ARDS.¹⁵³

Prevention

The most effective preventative strategy is control of the tick vector. Frequent visual inspection of the skin and haircoat is an effective means of tick control as a minimum of 2–3 days of tick engorgement is necessary for parasite transmission.²⁹ Visual inspection should be combined with topical acaricide therapy to prevent tick infestation. Topical therapies of proven benefit include amitraz-impregnated collars, fipronil and imidacloprid-permethrin applied once monthly, all resulting in prevention of tick attachment or tick death within 24–48 hours.^154–161

While imidocarb may have a posttreatment prophylactic effect for up to 6 weeks, results are variable, and this drug is not currently recommended for prophylaxis.^139,140 As previously discussed, complete sterilization may not be advised in endemic areas by some clinicians, as premunition confers some immunity. Frequent blood smear evaluation and PCR testing of blood donors is recommended to prevent the transmission of infected blood.

Two anti-babesial vaccines are licensed for use in Europe. One vaccine contains culture-derived soluble parasite antigens from European homologous B. canis in combination with adjuvant.^{38,81,162–164} The production of antibodies that react with parasitized erythrocytes results in a less severe anemia, decreased clinical signs, and decreased morbidity within 6 days of infection.^38,81,162 However, the vaccine has been met with contradictory results as the antibodies produced are strain specific; thus, no cross-protection is conferred.^38,164

The other vaccine contains culture-derived soluble parasite antigens of heterologous European B. canis and South African B. canis subsp. rossi in combination with an adjuvant (saponin).¹⁶² Induction of immunity against heterologous strains of B. canis was seen with a decrease in the duration and severity of clinical signs.^165–168 Additionally, immunologic protection against B. canis subsp. rossi is seen manifested as a decrease in clinical signs as well as a decrease in the parasitemia level.¹⁶⁷ Antibody production has been documented within 3 weeks of vaccination with the former vaccine and lasts at least 6 months.¹⁶⁶ For optimal results the manufacturer recommends administering an initial primer vaccine with a booster vaccine 3 weeks later, then follow-up with boosters every 6 months. No similar data are available regarding the latter vaccine.

Public Health and Zoonosis

Human babesiosis is a significant and emerging tick-borne zoonosis. Documented infections of B. microti, B. equi, B. divergens-like and B. gibsoni-like organisms have been reported in humans in the United States.^169–175 The Ixodes and Dermacentor family of ticks are suspected vectors. No human-specific species of Babesia exists, rather humans appear to serve as accidental hosts in a Sylvan cycle with wildlife reservoirs.³ Documented cases of transfusion-related transmission also exist.¹⁷⁵ The majority of human infections are symptomatic or may present as mild self-limiting flu-like symptoms. However, immunocompromised, geriatric, and splenectomized people are at risk for a complicated form of the disease that presents as a severe hemolytic anemia with multiple-organ involvement and confers a high risk of death. The treatment of choice is a combination of clindamycin and quinine treatment. Combined therapy with atovaquone and azithromycin is also efficacious.¹⁷⁶