Canine babesiosis is an emerging hemoprotozoan

J Vet Intern Med 2010;24:127 131 Babesiosis Caused by a Large Babesia Species in 7 Immunocompromised Dogs L.E. Sikorski, A.J. Birkenheuer, M.K. Holowaychuk, A.L. McCleary-Wheeler, J.M. Davis, and M.P. Littman Background: A large unnamed Babesia species was detected in a dog with lymphoma. It was unknown if this was an underrecognized pathogen. Objective: Report the historical and clinicopathologic findings in 7 dogs with babesiosis caused by a large unnamed Babesia species characterize the 18S ribosomal ribonucleic acid (rrna) genes. Animals: Seven immunocompromised dogs from which the Babesia was isolated. Methods: Retrospective case review. Cases were identified by a diagnostic laboratory, the attending clinicians were contacted and the medical records were reviewed. The Babesia sp. 18S rrna genes were amplified and sequenced. Results: Six of 7 dogs had been splenectomized; the remaining dog was receiving oncolytic drugs. Lethargy, anorexia, fever, and pigmenturia were reported in 6/7, 6/7, 4/7, and 3/7 dogs. Laboratory findings included mild anemia (7/7) and severe thrombocytopenia (6/7). Polymerase chain reaction (PCR) assays used to detect Babesia sensu stricto species were all positive, but specific PCR assays for Babesia canis and Babesia gibsoni were negative in all dogs. The 18S rrna gene sequences were determined to be identical to a large unnamed Babesia sp. previously isolated. Cross-reactive antibodies against other Babesia spp. were not always detectable. Five dogs were treated with imidocarb dipropionate and 1 dog with atovaquone/azithromycin; some favorable responses were noted. The remaining dog was untreated and remained a clinically stable carrier. Conclusions and Clinical Importance: Dogs with pigmenturia, anemia, and thrombocytopenia should be tested for Babesia sp. by PCR. Serology is not sufficient for diagnosis of this Babesia sp. Asplenia, chemotherapy, or both might represent risk factors for persistent infection, illness, or both. Key words: Anemia; Chemotherapy; Splenectomy; Thrombocytopenia. Canine babesiosis is an emerging hemoprotozoan parasitic disease that affects dogs worldwide. Classically, canine piroplasmosis is characterized by anemia, pigmenturia, thrombocytopenia, hyperglobulinemia, waxing and waning fever, and splenomegaly. 1,2 These findings are fairly consistent in the majority of dogs regardless of geographic region, genotype of the piroplasm, or species of the host. Historically, there have been 3 large Babesia spp. or subspecies recognized to cause clinical disease in dogs: Babesia canis vogeli, Babesia canis canis, and Babesia canis rossi. 1,2 Recently, with the use of phylogenetic analyses of the 18S ribosomal ribonucleic acid (rrna) genes, 2 additional unique large unnamed Babesia sp. have been detected in clinically ill dogs. 3 One genetically distinct organism was identified in a dog from Britain. 4 The other organism, a large unnamed Babesia sp. (GenBank accession number AY618928) was first reported in a Labrador Retriever from North Carolina From the Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA (Sikorski, Littman); the Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC (Birkenheuer, Holowaychuk, McCleary-Wheeler, Davis); and the Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada (Holowaychuk). These results were reported in part at the 2008 American College of Veterinary Internal Medicine Forum, San Antonio, TX. Corresponding author: Adam J. Birkenheuer, DVM, PhD, DAC- VIM, Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606; e-mail: ajbirken@ncsu.edu. Submitted July 16, 2009; Revised August 28, 2009; Accepted October 7, 2009. Copyright r 2009 by the American College of Veterinary Internal Medicine 10.1111/j.1939-1676.2009.0440.x Abbreviations: BLAST IFA L-CHOP PCR rrna basic local alignment search tool immunofluoresencent antibody l-asparaginase, cyclophosphamide, doxorubicin, vincristine, prednisone polymerase chain reaction ribosomal ribonucleic acid undergoing chemotherapy for lymphoma. 3 On the basis of light microscopic identification, this piroplasm is difficult to distinguish from the 3 previously defined Babesia canis species/subspecies. Based on 18S rrna gene sequence analyses, this organism appeared to share the highest sequence identity to Babesia bigemina (93.9%), which is an etiological agent for bovine babesiosis. 3 Cross-reactive antibodies against B. canis, Babesia gibsoni, or Babesia conradae antigens were not detected by immunofluoresencent antibody (IFA) testing in acute or convalescent serum samples in the original case. 3 Because the description of the original case in North Carolina, we have identified 7 additional dogs from across the eastern United States that were infected with this large unnamed Babesia sp. The purpose of this report is to summarize the historical and clinicopathologic findings in these 7 dogs and to characterize the Babesia 18S rrna genes from these dogs. Materials and Methods Medical Records The medical records of all 7 dogs were reviewed. Dogs were identified by a university diagnostic laboratory (North Carolina State

128 Sikorski et al University Vector Borne Disease Diagnostic Laboratory, Raleigh, NC). The attending clinicians were contacted by telephone or electronic mail to retrieve the medical records. Data abstracted from the medical records included history, physical examination, clinicopathologic data, and diagnostic imaging at the time of diagnosis and after treatment. Additional data collected from the medical record or from the owner during a telephone interview included exposure to ticks, use of tick preventative, history of dog bites, history of blood transfusion, history of splenectomy, splenic histopathology, and concurrent illnesses. Nucleic Acid Isolation Total DNA was extracted from 200 ml of EDTA anticoagulated whole blood WITH the QIAamp DNA Blood Mini Kit according to the manufacturer s instructions. a Samples were used as template to amplify a fragment of the glyceraldehyde-3-phosphodehydrogenase pseudogene as described previously to demonstrate integrity of the DNA and the absence of polymerase chain reaction (PCR) inhibitors. 5 PCR and Sequencing of 18S rrna Genes Partial 18S rrna genes were amplified by PCR in a 50 ml reaction volume using the following primers: 5 0 -GTTGATCCTGCCAGT AGT-3 0 and 5 0 -TGCTTTCGCAGTAGTTCGTC-3 0. 3 Each reaction contained a 1 concentration of PCR buffer II, b 1.25 U of Taq polymerase, 5 ml of DNA template, 1.5 mm MgCl 2,50pmolofeach primer, and 200 mm of each dntp. The thermal cycling conditions were as follows: initial 951C for 5 minutes, followed by 55 cycles (951C for 45 seconds, 581C for 45 seconds, and 721C for 1.0 minutes) with a final extension at 721C for 5 minutes. c Amplicons were visualized by ethidium bromide staining and ultraviolet transillumination after electrophoresis in a 2% agarose gel. Positive controls (previously characterized Babesia spp.) and negative controls (no DNA) were used with each reaction. Amplicons were either cloned into a plasmid vector d according to the manufacturer s instructions before sequencing or the amplicons were sequenced directly in both directions. The resulting sequences were compared with the existing sequences in GenBank by the basic local alignment search tool (BLAST). Results DNA Sequencing The resulting sequences were 845 base pairs in length and included the V4 hypervariable region of the 18S rrna gene. A BLAST search revealed that the sequences were 100% similar to each other as well as the recently diagnosed large unnamed Babesia sp. (nucleotide positions 1 845 of GenBank accession number AY618928). 3 Case Descriptions Seven dogs infected with the large unnamed Babesia species were initially identified via microscopic examination of thin stained blood smears. The average age was 11.5 years (range 10 13 years). Three dogs were castrated males and 4 were spayed females. Two dogs were Labrador Retrievers and there was 1 of each of the following breeds: German Shepherd dog, Great Pyrenees, Miniature Schnauzer, Miniature Dachshund, and mixed. Five dogs were residing in North Carolina and 1 dog each were residing in New Jersey and New York at the time of diagnosis. The dog from New Jersey had a history of travel to Pennsylvania and Wisconsin. The dog from New York had a history of travel to Massachusetts. One dog from North Carolina had a history of travel to Kentucky and Ohio, 1 had a history of travel to South Carolina, and the remaining 3 dogs from North Carolina had no history of travel. Dogs were presented by their owners for nonspecific complaints including lethargy and anorexia. Pigmenturia was reported by the owners in 3 dogs. Six dogs had a history of a splenectomy before the diagnosis of babesiosis (range: 1 5 years before the diagnosis of babesiosis). Splenic histopathology revealed splenic hematoma in 2 dogs, benign nodular hyperplasia in 2 dogs, and splenic torsion and fibrohistiocytic proliferation in 1 dog each. Six of 7 dogs were reported to be routinely treated with fipronil e at monthly intervals at the time of the initial diagnosis. Despite this, 4/7 dogs were reported to have been infested with ticks at some time during their lives. One dog was reported to have received a blood transfusion 1 year before the diagnosis of babesiosis at the time that the splenectomy was performed; the donor was reported to be a mixed-breed dog. None of the dogs were reported to have been in fights with other dogs, but 1 dog did have a wound on its tail at the time of presentation. Concurrent noninfectious conditions that were identified or already undergoing therapy at the time babesiosis was diagnosed included 1 dog each with a tail wound of indeterminate origin, a cranial abdominal mass of indeterminate origin, hyperadrenocorticism, degenerative joint disease, B-cell lymphoblastic lymphoma, and a hepatic mass that was not characterized by histopathology. The dog with B-cell lymphoblastic lymphoma was diagnosed with lymphoma in September 2005 and had been treated with a standard L-CHOP weekly sequential chemotherapy and had received 2 courses of half-body radiation. A strong partial remission was maintained on a multiagent maintenance chemotherapy protocol until July 2008 when enlarging lymph nodes were noted. Babesiosis was diagnosed in this dog August 2008. Screening for other infectious diseases was not standardized between cases. Three dogs had in-house ELISA testing for antibodies against Ehrlichia canis, Borrelia burgdorferi as well as the presence of heartworm antigens and all dogs tested negative. f Three dogs were tested for the presence of antibodies against 1 or more Babesia spp. One dog was tested for antibodies against B. canis, B. gibsoni, and B. conradae, 1 dog was tested for antibodies against B. canis and B. gibsoni and the final dog was only tested for antibodies against B. canis. The dog that was only tested for antibodies against B. canis was found to have an antibody titer against that organism of 1 : 128, the remaining dogs tested negative against all antigens. Two dogs were tested by IFA for the presence of antibodies against Bartonella vinsonii, Rickettsia rickettsii, and E. canis. The same dog that tested positive for the presence of antibodies against B. canis also tested positive for antibodies against E. canis with a titer of 1 : 128. The remaining dogs tested negative. One dog each was tested for Bartonella spp. or Ehrlichia spp. by PCR. The dog that was tested for Bartonella spp. DNA tested positive, but the species of Bartonella was not specifically identified. The remaining dog tested negative for the presence of Ehrlichia spp. DNA.

Babesiosis in Immunocompromised Dogs 129 Table 1. CBC and plasma protein results on presentation for 7 dogs diagnosed with a large unnamed Babesia sp. Parameter Mean (Range) below RI above RI Red blood cells ( 10 6 /ml) 4.78 (3.9 5.43) 7 0 Hematocrit (%) 32.7 (28.2 36.5) 7 0 Hemoglobin (g/dl) 11.6 (10.3 13.2) 7 0 MCV (fl) 70.5 (62.8 74) 1 0 MCHC (g/dl) 35.4 (32.7 37.5) 0 3 Platelets (/ml) 98.6 (o 5 478) 6 1 Plasma protein (g/dl) a 7.2 (6.6 7.8) 2 2 WBC ( 10 3 /ml) 8.77 (1.78 23.14) 2 1 Neutrophils ( 10 3 /ml) 6.59 (0.55 18.97) 2 1 Bands ( 10 3 /ml) 0.23 (0 1.62) 0 1 Lymphocytes ( 10 3 /ml) 0.76 (0.24 1.74) 4 0 Eosinophils ( 10 3 /ml) 0.07 (0.0 0.96) 0 2 Monocytes ( 10 3 /ml) 0.5 (0.096 1.15) 1 0 NRBC (/100 WBC) 0.6 (0 2) 0 2 Reference intervals not reported because of laboratory variations. a Values only reported for 5 dogs. RI, reference interval; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; RDW, red blood cell distribution width; WBC, white blood cell; NRBC, nucleated red blood cell. Fever, defined as a rectal temperature 102.51F, was noted in 4/6 cases in which a temperature was recorded. One dog had a palpable mass in the cranial abdomen. One dog was reported to have mildly icteric pinnae and sclera. The dog that was undergoing treatment for lymphoma was reported to have mild generalized lymphadenomegaly. The lymph nodes were not aspirated or biopsied at the time of presentation so it was unclear whether or not they were an indication that the dog s lymphoma had relapsed or that the lymph nodes were reactive as a consequence of the Babesia infection. Asplenia was the only other abnormality recorded during physical examinations. Hematological and biochemical abnormalities detected at the time of diagnosis are summarized in Tables 1 and 2. Four dogs had urinalyses performed at the time of initial diagnosis. Urine color was reported as red for 1 dog and yellow for the remaining 3. Urine specific gravities ranged from 1.005 to 1.027. All dogs had blood reported on the dipstick (2 31). Three dogs had no erythrocytes seen in their urine sediment and 1 dog had 3 10 erythrocytes per high power field. No white blood cells were seen in any sediment. Three of 4 dogs had bilirubinuria reported on the dipstick (1 4 1). Two of 4 dogs had urine cultures completed at the time of urinalysis, which were negative for growth of bacteria. Four dogs required hospitalization at the time of initial diagnosis with a mean hospitalization time of 3.5 days (range 3 4 days). The remaining 3 dogs were treated on an outpatient basis. All hospitalized dogs received crystalloid fluid therapy IV. Six dogs were treated with antiprotozoal medications. Five dogs were treated initially with an injection of imidocarb dipropionate Table 2. Serum biochemical profile results at the time of initial presentation for 6 a dogs diagnosed with a large unnamed Babesia sp. Analyte Mean (Range) below RI above RI Glucose (mg/dl) 90 (56 108) 1 0 BUN (mg/dl) 31 (16 53) 0 3 Creatinine (mg/dl) 1.0 (1 1.4) 0 0 Phosphorus (mg/dl) 4.6 (4 5.6) 0 0 Calcium (mg/dl) 9.9 (8.8 10) 1 0 Sodium (mmol/l) 148 (145 150) 0 0 Potassium (mmol/l) 4.1 (3.2 4.5) 1 0 Chloride (mmol/l) 117 (114 120) 0 0 Carbon dioxide (mmol/l) b 23 (19 25) 0 0 Total protein (g/dl) 5.6 (4.2 6.9) 3 0 Albumin (g/dl) 2.7 (2.1 3.1) 3 0 Globulin (g/dl) 2.9 (2.1 4.1) 2 1 A/G Ratio 0.9 (0.7 1.0) 1 0 ALT (U/L) 74 (47 144) 0 2 AST (U/L) b 67 (30 250) 0 2 ALP (U/L) 38 (71 1,273) 0 3 GGT (U/L) c 10 (1.0 21) 1 0 Total bilirubin (mg/dl) 0.3 (0.2 0.5) 0 0 Cholesterol (mg/dl) 242 (164 329) 0 1 Reference intervals not reported because of laboratory variations. a Complete chemistry panel results were not available from 1 dog, but were noted unremarkable in the record. b Values only reported for 5 dogs. c Values only reported for 4 dogs. RI, reference interval. followed by a 2nd injection at the same dose 14 17 days later. Four dogs were treated with the recommended dose (6.6 mg/kg IM) and 1 dog was treated erroneously with a lower dose (3 mg/kg IM). Because of owner concerns about the potential adverse effects of imidocarb dipropionate, g 1 dog was treated with azithromycin h (10 mg/kg PO q24h) and atovaquone i (13.4 mg/kg PO q8h) for 10 days. The remaining dog received no specific antiprotozoal treatment. The owner elected not to treat this dog because of concern over the potential adverse effects of treatment and the perception of an overall poor prognosis for long-term survival given presence of a palpable abdominal mass. Three dogs that were treated with imidocarb were also treated with antibiotics that have some antiprotozoal activity. Two were administered doxycycline hyclate (average dose 6.65 mg/kg q12h; dose range 5 8.3 mg/kg). Another was treated with azithromycin (250 mg q24h 1 week, then q48h 5 weeks) for a concurrent Bartonella sp. infection. Five dogs had repeat PCR testing for Babesia spp. after treatment. The dog that received no specific antiprotozoal therapy remained PCR positive at days 30 and 670. The dog that received imidocarb dipropionate g below the recommended dosage remained PCR positive on days 30 and 60. Of the dogs treated with imidocarb dipropionate at the standard dose, 2 dogs were only tested once posttreatment and both tested negative on days 45 and 77, respectively. The remaining dog tested negative 42 days posttreatment. However, that same dog presented 340 days after treat-

130 Sikorski et al ment for pigmenturia and was once again diagnosed with babesiosis by PCR and microscopy. Thrombocytopenia resolved in all dogs after treatment with antiprotozoal therapy. The dog that received no specific antiprotozoal therapy had a thrombocytosis at the time of diagnosis that was also detected over 1 year later. Thrombocytosis was detected in 4 other dogs after treatment for babesiosis. Anemia resolved in 3/6 dogs after antiprotozoal treatment. One of the dogs in which the anemia resolved initially had reinfection or recrudescence of babesiosis, including anemia and thrombocytopenia, again 340 days after the initial episode. Three dogs that were treated remained mildly anemic (PCV ranging from 33% to 37%). One of these dogs was treated with the lower dose of imidocarb and remained PCR positive. The other 2 dogs that remained anemic were not retested for Babesia, and 1 was euthanized 1 month after the diagnosis of Babesia because of the identification of a hepatic mass. The dog that received no antiprotozoal therapy was persistently anemic (PCV 27%) for two and a half years at which time the dog was euthanized due the presence of an intra-abdominal mass. Discussion This report further supports that domestic dogs are susceptible to infection with a large unnamed Babesia sp., 3 and these infections can be associated with clinical babesiosis. Immunocompromised dogs appear to be at risk for infection. Further studies are warranted to determine whetherornotimmunocompetentdogsaresusceptibleto infection with this Babesia sp. Additionally this report documents that infection with this agent is not geographically limitedtonorthcarolinaandataminimumthegeographic range should be considered to expand through the Mid-Atlantic region of the United States. Dogs infected with the large unnamed Babesia sp. were presented for nonspecific complaints such as lethargy and anorexia. Anemia and thrombocytopenia appear to be the most common laboratory abnormalities associated with this infection. A mild normocytic normochromic anemia was found in all dogs. Anemia is a common finding in canine babesiosis and is because of secondary immune-mediated destruction and direct parasitic damage. 1,2 Asevere thrombocytopenia (o35,000 platelets/ml) was found in 6/7 dogs, but no dogs displayed overt bleeding tendencies at the time of diagnosis. Pigmenturia was reported by the owners in 3 dogs, but the exact cause of the pigmenturia was not easily identifiable because of the retrospective nature of the study. Pigmenturia (hemoglobinuria, bilirubinuria, or both) associated with intravascular, extravascular hemolysis, or both likely is the most plausible explanation for the pigmenturia reported in these cases. The serum biochemical abnormalities of hypoproteinemia, increased ALT, AST, ALP, and BUN identified in dogs infected with the large unnamed Babesia sp. are similar to those reported with infection with other Babesia spp. They do not, however, appear to be common or specific enough to warrant investigation for babesiosis in the absence of hematologic abnormalities. Co-infections could not be completely excluded as contributors to clinical illness. Clinical signs of babesiosis and hematological abnormalities of anemia and thrombocytopenia improved or completely resolved in all dogs that were treated with specific antiprotozoal therapy. Two of 4 dogs that were tested for Babesia by PCR posttreatment, tested negative. One dog that tested positive by PCR after treatment was treated erroneously with a dosage of imidocarb dipropionate g below the standard recommendation. Another dog had 1 posttreatment PCR test that was negative, but was once again PCR positive during a 2nd course of clinical disease. It is unknown whether or not the 2nd bout of babesiosis represented a reinfection or a relapse with a false-negative PCR test because of low numbers of parasites when the dog was doing well posttreatment. Three dogs were treated concurrently with corticosteroids in addition to imidocarb dipropionate. g In 1 dog, clinical signs resolved with corticosteroid treatment alone but persistent Babesia infection of approximately 2 years duration was identified. This dog appeared to be in a state of premunition or have immunity of infection similar to cattle that have been vaccinated using a less pathogenic strain of Babesia. Whereas in a state of premunition, animals have mild or no disease and are resistant to developing disease when challenged with virulent strains of the same parasite. We cannot determine whether or not this dog was challenged and resistant to reinfection. In this report, 6/7 dogs had been splenectomized before diagnosis, and similar to the original report, the remaining spleen-intact dog was undergoing chemotherapy for lymphoma. 1 Accordingly, we suspect that asplenic and or immunosuppressed dogs are at risk of either infection or clinical disease from this parasite. It remains unknown if domestic dogs are a primary reservoir host for this Babesia sp. and disease only develops when the immune system is no longer able to control the level of parasitemias; or that a larger number of dogs are exposed to this parasite and only those with immune suppression or asplenia develop persistent infections and disease. The best diagnostic assay for the detection of this Babesia sp. in dogs remains unknown. Antibodies that cross-react with B. canis or B. gibsoni antigens are not consistently found. Piroplasms were identified via light microscopy in all dogs on at least 1 occasion. In 2 dogs that underwent repeated testing, piroplasms were not detected by light microscopy at the same time Babesia sp. DNA was detected by PCR assay and B. canis infection was suspected by the clinical pathologists based on parasite morphology in nearly every case. These findings emphasize the utility of PCR as a diagnostic tool for the detection and accurate identification of this large unnamed Babesia sp. Footnotes a Qiagen Inc, Valencia, CA b Applied Biosystems, Foster City, CA c PCR Express; Thermo Hybaid, Middlesex, UK

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