Introduction

Babesiosis is a hemoprotozoal tick-borne disease that can infect humans and a variety of domestic species worldwide.¹ The most common Babesia species in North America that infect dogs are B. gibsoni, B. canis vogeli, B. conradae, and an unnamed large species referred to as B. coco.^2–5 In addition to tick bites, transmission of Babesia sp. occurs via bite wounds or blood transfusions.^6,7 Typical disease manifestations include thrombocytopenia, anemia, and splenomegaly. Severe infections can lead to hypotension, hypoxia, cerebral ischemia, or renal disease.^8,9

Clinical and histopathologic renal involvement of canine babesiosis caused by B. canis rossi, B. microti-like sp., B. canis canis, and B. conradae have been documented in a number of dogs.^10–17 In one report, renal disease was present in 3 of 134 (2.2%) Babesia-infected dogs.⁸ In retrospective studies performed in Spain, Hungary, and South Africa, azotemia was identified in 30–60% of Babesia-infected dogs.^10,12–14 A retrospective study of dogs infected with Babesia canis rossi concluded that renal injury resulted in a fivefold increased risk of mortality.¹¹ None of the studies evaluated the outcome of renal disease in response to treatment of babesiosis. A search performed on Pubmed on January 5, 2017, using the search terms Babesia and dogs retrieved 832 results. Of these, 50 described natural infection of dogs in North America or experimental infection of dogs with an organism isolated from a North American dog. Eight of these studies explicitly state that serum biochemical analyses were performed. Hypoalbuminemia or azotemia are occasionally reported, but only two, one case report of a protein-losing nephropathy in a B. gibsoni-infected dog and one histopathologic study of six dogs experimentally infected with B. conradae, explicitly address the potential for glomerular injury.^4,17,18 Therefore, renal disease, or specifically, glomerular injury, caused by Babesia spp. in North America, is a valuable area of study.

The goals of this retrospective study were twofold: (1) to document azotemia and/or proteinuria in dogs infected with Babesia, and (2) to describe improvement in azotemia and/or proteinuria in response to treatment of babesiosis.

Materials and Methods

A retrospective search was performed of the electronic database (January 1, 2006, to August 20, 2015) from the North Carolina State University Vector Borne Disease Laboratory to identify dogs who were ≥1:64 seroreactive, polymerase chain reaction (PCR)-positive, or both, for Babesia sp. The medical record of each dog identified was examined further. Dogs were excluded based on the following criteria: absence of azotemia, absence of proteinuria, coinfections with other vector-borne disease (Ehrlichia sp., Rickettsia sp., Borrelia sp., Mycoplasma sp. Anaplasma sp., Bartonella, and Dirofilaria immitis), or presence of comorbidities known to cause proteinuria (e.g., neoplasia, endocarditis, urinary tract infections).^19,20 Screening for exposure or infection to vector-borne infections were performed in a diagnostic laboratory and included serology for Babesia spp. (immunofluorescence antibody assay contains both B. canis and B. vogeli organisms), B. gibsoni, E. canis, R. rickettsia, B. henselae, B. vinsonii, B. khoelerae by immunofluorescence assay, enzyme-linked immunosorbent assay for antibodies against Borrelia burgdorferi, Anaplasma spp., Ehrlichia spp., and antigens from Dirofilaria immitis using a commercially available assay according to the manufacturer’s instructions. PCR testing for the presence of Babesia spp., Bartonella spp., Anaplasma spp., Ehrlichia spp., Rickettsia spp., and hemotropic Mycoplasma spp. was performed. Testing for infectious agents was performed at the discretion of the managing clinician; not all dogs were tested for all pathogens. Dogs were included if they showed evidence of exposure to or infection with Babesia sp. and had proteinuria and/or azotemia.

Signalment (age at diagnosis, sex, breed), presenting complaints, and pertinent physical exam findings at presentation (i.e., pale mucous membranes, pyrexia, petechiae, splenomegaly) were recorded for each dog. Results of complete blood count, biochemical panel, and urinalyses were recorded from two time-points: pretreatment and posttreatment. Urine protein/creatinine (UPC) ratios and urine microbial culture results were also recorded from these time points when available. Pretreatment data were included only if they had been performed within 5 days of the time of diagnosis. Urine protein/creatinine ratios were included only if they had been submitted within 24 hr of a complete blood count, biochemical panel, and urinalysis, and if the urinalysis was free of pyuria (<5 white blood cells per high power field) and hematuria (<10 red blood cells per high power field) or a urine sample submitted for microbial culture was negative for bacterial growth within 72 hr of sample collection. Posttreatment data were obtained at any time-point after a 10-day course of azithromycin (10 mg/kg per os q 24 hr) and atovaquone (13.3 mg/kg per os q 8 hr administered with a fatty meal).

Anemia and thrombocytopenia were defined as packed cell volume <39% and platelets <190,000/uL, respectively, based on laboratory reference ranges. Regenerative anemia was defined as >60,000 reticulocytes/uL. Hypoalbuminemia was also defined by laboratory reference range (albumin <3.0 g/dL). Azotemia was determined based on the presence of increased serum creatinine (>1.6 mg/dL). Proteinuria was defined as an increased UPC (>0.5) in the absence of hematuria, pyuria, or positive urine microbial culture.²¹ If a UPC had not been performed, proteinuria was defined as a ≥1+ positive urine protein sulfosalicylic acid precipitation test result.²¹

Results

Of 35 dogs identified during the initial search to be Babesia PCR-positive or seropositive, 12 (34%) had azotemia, proteinuria, or both. Four of the twenty-three dogs were excluded because of unavailable initial bloodwork or urinalysis data. The remaining 21 dogs were excluded based on absence of azotemia and/or absence of proteinuria. Of the 12 dogs with azotemia, proteinuria, or both, 7 were excluded and 5 were included. Four dogs were excluded based on evidence of coinfections with other vector-borne disease agents (Borrelia burgdorferri, one; Dirofiliaria immitis and Borrelia burgdorferri, one; Ehrlichia sp., two) based on positive antibody testing. The remaining three dogs were excluded because of comorbidities (lymphoma, one; suspected endocarditis, one) or limited available data (one). One of the five dogs included in the study died before posttreatment data were obtained.

All five dogs were American pit bull terriers or American pit bull terrier-mixed breed dogs (Table 1). Two were female spayed, one was female intact, and the remaining two were male castrated. Dogs were referred for further evaluation of azotemia and proteinuria (three) or anemia (one) or for treatment of babesiosis (one). At presentation, these dogs all had a pattern consistent with glomerular injury as defined by hypoalbuminemia and proteinuria (3-4+ sulfosalicylic acid precipitation with concomitant increase in UPC when available), with or without azotemia.

TABLE 1 Signalment, Presenting Complaint, and Babesia sp. Test Results of Five American Pit Bull Terriers or American Pit Bull Terrier-Mixed Dogs with Renal Disease Secondary to Babesia gibsoni

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All five dogs had Babesia gibsoni as determined by PCR. Two of three dogs were seroreactive for B. gibsoni and B. spp.; the remaining two did not have serologic testing (Table 1). Babesia gibsoni infection was treated with a combination of azithromycin (10 mg/kg q 24 hr) and atovaquone (13.3 mg/kg q 8 hr) in all five dogs. Additional antiprotozoal medications used in individual dogs included imidocarb (one) or a doxycycline/clindamycin/metronidazole combination therapy (one).

Three of five dogs were anemic pretreatment; two had regenerative anemia and results of a reticulocyte count were not available for one (Table 2). Two of the three anemic dogs had concurrent thrombocytopenia. Anemia improved posttreatment in two dogs; one of these also had resolution of thrombocytopenia. One dog died before posttreatment blood or urine tests could be performed.

TABLE 2 Pre- and Posttreatment Clinicopathologic Test Results from Five American Pit Bull Terriers or American Pit Bull Terrier-Mixed Dogs with Renal Disease Secondary to Babesia gibsoni

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All five dogs had hypoalbuminemia pre-treatment. Four dogs had serum globulin concentrations within the reference range; globulin concentration was not available for the remaining dog. Three of the four dogs had normal serum albumin concentrations posttreatment, although there was a further reduction in serum albumin in the remaining dog.

Three of five dogs had azotemia pre-treatment. Of the two remaining dogs, one did not have serum creatinine available pre-treatment and one had a normal serum creatinine concentration. Azotemia resolved after treatment in one dog and improved in another.

Urine specific gravity ranged from 1.013 to 1.022 pre-treatment. Complete urinalyses were only available from three dogs posttreatment. In one dog, a urinalysis was not performed posttreatment, but a UPC was evaluated. All dogs had proteinuria pre-treatment. The UPC decreased posttreatment in the two dogs for whom pre- and posttreatment results were available.

Abdominal ultrasonography was performed in all dogs. Four dogs had splenomegaly; the remaining dog had a prior splenectomy. Hyperechoic renal medullary sign was present bilaterally in one dog and interpreted as being nonspecific. Bilateral renal pyelectasia was present in one dog. The remaining three dogs had ultrasonographically normal-appearing kidneys.

Discussion

This study documented azotemia and proteinuria in dogs infected with Babesia gibsoni. Although babesiosis is classically characterized by anemia and thrombocytopenia, only three of five dogs were anemic and two of five dogs were thrombocytopenic. This finding emphasizes that although hemolytic anemia is the classically recognized manifestation of babesiosis, testing for Babesia sp. in an azotemic and/or proteinuric patient should be considered regardless of the presence of anemia or thrombocytopenia. This is particularly true in American pit bull terriers and American pit bull terrier-mixed breed dogs.

All five dogs in the study were found to have hypoalbuminemia and proteinuria. This paired finding suggests that Babesia sp. preferentially causes glomerular injury in the canine kidney. Inflammation, decreased hepatic production, or gastrointestinal losses could contribute to the decreased serum albumin concentrations; however, the concurrent finding of proteinuria makes glomerular injury the most likely cause of hypoalbuminemia.²¹ Reported histopathologic analyses corroborate these findings. Multifocal glomerular deposits of immunoglobulin M and a membranoproliferative pattern of renal injury were found in Babesia conradae-infected dogs.¹⁷ Three individual case reports have documented protein-losing nephropathy attributed to Babesia sp. infections.^18,22,23 In one of the cases, membranoproliferative glomerulonephritis was diagnosed.¹⁸ In the same case report, treatment of babesiosis with azithromycin/atovoquone and a single dose of a corticosteroid and cyclophosphamide led to complete resolution of the protein-losing nephropathy.¹⁸ Similarly, in the dogs of this report, UPC ratios decreased following treatment of babesiosis in both dogs who had both pre- and posttreatment data. One of these dogs had complete resolution of the proteinuria and the other dog had a marked reduction. A previous study demonstrated that treatment of babesiosis resulted in increased activity of antithrombin, suggesting that effective treatment of the infection resulted in glomerular recovery and improved protein retention.²⁴

Given that all five dogs included in the study were infected with Babesia gibsoni, one can hypothesize that B. gibsoni may directly or indirectly (type III hypersensitivity) induce glomerular injury. No B. canis vogeli-infected dogs were definitively identified by PCR, so nothing can be learned about potential relationships between this infection and glomerular disease. During initial screening, six dogs were identified to be infected with an unnamed large Babesia sp. (Babesia “Coco”), but all of these dogs were excluded from the final analysis because of lack of available laboratory data, lack of azotemia and/or proteinuria, or presence of comorbidities such as neoplasia. This is not surprising because the majority of dogs reported in the literature to be infected with this large Babesia sp. (Babesia “Coco”) have either been splenectomized or received chemotherapy for a diagnosis of neoplasia. A histopathologic analysis of dogs with Babesia canis did not reveal a pattern of glomerular injury.¹⁶ Of the three case reports of glomerular disease in dogs with babesiosis, two were infected with B. gibsoni.^18,22,23 B. canis was suspected from the dog of the third case report but the subspecies was not identified.²³

Babesiosis can also be associated with renal tubular injury. Renal disease in dogs with babesiosis was first attributed to hemoglobinuric tubular nephropathy.⁸ However, recent studies were unable to demonstrate severe renal pathology when hemoglobinemia was experimentally induced by infusion of canine hemoglobin in healthy dogs.²⁵ Recent theories have postulated that renal injury and subsequent azotemia and proteinuria are due to a combination of hypovolemia, renal vasoconstriction, and anemic hypoxia as well as immunologically mediated glomerular injury.^25–28 Because not all dogs in the present study were anemic, mechanisms other than anemic hypoxia or hemoglobinemia likely contributed to the pathogenesis of renal disease.^29,30

Specific challenges and limitations of this study include the retrospective nature of data collection and analysis. The sample size obtained was small and statistical comparisons could not be performed. Furthermore, data sets were incomplete. The real or perceived poor prognosis associated with the renal disease may have led to euthanasia, limiting the number of dogs for whom posttreatment data were available. Another challenge of this study was the need to eliminate confounding effects of coinfections or comorbidities that were also known to cause glomerular injury. This study attempted to effectively exclude all such diseases and demonstrate the singular relationship between glomerular disease and babesiosis, although not every dog who was ultimately included had comprehensive screening for all known comorbidities that can cause glomerular disease. It is possible that dogs who had alternate causes of renal damage were included in the final data set. However, exclusion of dogs with potential comorbidities did dramatically reduce sample size to five dogs who were all the same breed and all infected with B. gibsoni. A larger sample size or prospective study would benefit in establishing a relationship between renal disease and babesiosis across a variety of dog breeds. This might also help determine if the various Babesia species have differing predilections for causing renal injury. Combined histopathologic analysis would help to confirm patterns of tubulointerstitial versus glomerular injury. Patients with histopathologic evidence of glomerular and renal disease may have been excluded on the basis of normal renal values and lack of proteinuria. It remains to be determined whether similar renal complications occur with other Babesia species endemic to North America.

Conclusion

Despite limitations of this retrospective evaluation, this study confirmed that Babesia gibsoni is associated with renal disease, specifically glomerular disease, and that treatment of the Babesia can lead to improvement in the clinical manifestations of renal disease.