Babesia gibsoni (Asian genotype) is the cause of an

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1 J Vet Intern Med 2;1: Efficacy of Combined Atovaquone and Azithromycin for Therapy of Chronic Babesia gibsoni (Asian Genotype) Infections in Dogs Adam J. Birkenheuer, Michael G. Levy, and Edward B. Breitschwerdt Babesiosis caused by Babesia gibsoni (Asian genotype) is an emerging disease in dogs in the United States. To date, no drugs have been shown to eliminate B gibsoni (Asian genotype) infections from dogs. Twenty-two dogs that remained persistently infected with B gibsoni (Asian genotype) after either imidocarb diproprionate and or diminazine aceturate therapy were identified and randomly and evenly distributed into 2 groups. One group was treated with atovaquone and azithromycin combination therapy, and the other group received a placebo. Eight of 1 dogs in the treatment group had no detectable B gibsoni (Asian genotype) DNA, as determined by a sensitive and specific polymerase chain reaction (PCR) assay, in any of their posttreatment samples. In contrast, B gibsoni (Asian genotype) DNA was detectable by PCR in the posttreatment samples from 11 of 11 of the placebotreated dogs. One dog in the treatment group was excluded from the treatment outcome analysis. This dog had 2 consecutive negative PCR assay results and was euthanized because of ongoing degenerative joint disease prior to completion of the study. No adverse effects of treatment were reported in any dog during the study period. A combination of atovaquone and azithromycin is the 1st described treatment that will either eliminate B gibsoni (Asian genotype) infections or suppress the parasitemia below the limit of detection in the majority of treated dogs. Key words: Piroplasm; Polymerase chain reaction; Protozoa;. Babesia gibsoni (Asian genotype) is the cause of an emerging infectious disease with an expanding region of endemicity. 1 Infection with B gibsoni (Asian genotype) can result in severe clinical manifestations in some dogs, while other dogs exhibit only mild clinical signs and minimal hematologic or biochemical abnormalities. 1,2, There is no vaccine available to prevent B gibsoni (Asian genotype) infections, and because the parasites are not eliminated by any of the antibabesial therapies tested to date, pets that survive acute infections are at risk for recurring clinical disease and serve as reservoir hosts. 11 Canine babesiosis caused by B gibsoni (Asian genotype) is very common in areas where tick transmission occurs. For example, in Japan 3% of the veterinarians surveyed had treated dogs for B gibsoni (Asian genotype) infections between 11 and Seroprevalence studies in Malaysia, Taiwan, Korea, Japan, and Okinawa reported seroprevalences of 1., 1, 1., 1., and.%, respectively Recent molecular investigations have revealed at least 3 genetically distinct small piroplasms that are capable of infecting dogs 1 ; the 1S ribosomal RNA sequences for 2 of these piroplasms have been submitted to GenBank 1 as B gibsoni. One of these genotypes has been identified in dogs from Asia and North America and is presumed to be the same as the B gibsoni originally described by Patton in The other genotype has been reported only in dogs in California. The genotype of the majority of small Ba- From the Departments of Clinical Sciences (Birkenheuer, Breitschwerdt) and Farm Animal Health and Resource Management (Levy), College of Veterinary Medicine, North Carolina State University, Raleigh, NC. Previously presented in part at the 23 ACVIM forum, Charlotte, NC. Reprint requests: Edward Breitschwerdt, DVM, College of Veterinary Medicine, North Carolina State University, Hillsborough Street, Raleigh, NC 2; edbreitschwerdt@ncsu.edu. Submitted October, 23; Revised December 22, 23; Accepted January 2, 2. Copyright 2 by the American College of Veterinary Internal Medicine 1-//1-/$3./ besia organisms reported in the United States have been of the Asian genotype, 2,1,21 not the California genotype. 1,22,23 The number of B gibsoni (Asian genotype) infections reported in the United States is increasing. Prior to 1 there were only 2 cases of presumed B gibsoni (Asian genotype) infections in the United States, and 1 of those was in a dog that had been imported from a region where the disease is endemic. 2,2 Since 1, the number of reports of B gibsoni (Asian genotype) infections in the United States has increased considerably. 1,2 No drugs, including diminazine aceturate and imidocarb diproprionate, have been demonstrated to clear B gibsoni (Asian genotype) infections from dogs. 1,,,1,11,2 3 The antiprotozoal drug atovaquone has produced excellent antibabesial activity against 2 Babesia species, B microti and B divergens Atovaquone combined with the azalide antibiotic azithromycin has eliminated B microti parasitemia in both humans and hamsters. 3,3 These 2 drugs appear to have an additive effect because neither drug alone will eliminate Babesia parasitemia. 32,3,3 The mechanism of action of either atovaquone or azithromycin against Babesia spp. is unknown. The mechanism of action of atovaquone against other protozoa is believed to involve the inhibition of cytochrome b and electron transport. 3 We have performed a controlled pilot study evaluating an atovaquoneazithromycin drug combination in B gibsoni infected dogs. a Both atovaquone and azithromycin have been shown to be safe in a large number of vertebrate species, and throughout the pilot study or in follow-up phone calls up to months after the end of the study period no adverse effects were reported in any of the treated dogs. To our knowledge, this is the 1st report on the use of atovaquone in dogs, and there is no information about its pharmacokinetics or safety in dogs. In humans, atovaquone is excreted primarily in the feces after extensive enterohepatic recirculation, resulting in a long half-life. The pharmacokinetics of azithromycin in dogs has been studied, and this drug has a very large safety margin and a long tissue halflife. 1 The most commonly reported adverse effects in humans treated with an atovaquone-azithromycin combination was diarrhea and rash in % of the recipients. 3 Here, we

2 of Canine Babesiosis report the results of a double-blind placebo-controlled trial to evaluate the safety and efficacy of an atovaquone-azithromycin combination for the treatment of B gibsoni (Asian genotype) infections in dogs. Materials and Methods Case Enrollment Criterion and Study Population Dogs were selected for inclusion in the study if they had persistent B gibsoni (Asian genotype) infection documented by polymerase chain reaction (PCR) assay and light microscopy after failure of conventional antibabesial therapy (imidocarb diproprionate, diminazine aceturate, or both). B gibsoni (Asian genotype) infection had to be documented 3 days after the last antibabesial treatment. Dogs were excluded if the attending clinician determined that they were ill enough to require hospitalization. Seventeen dogs had been treated with imidocarb diproprionate alone (dose:. mg/kg IM) for 2 doses 1 2 weeks apart. Four dogs had been treated with diminazene aceturate (3. mg/kg IM) for 2 doses 2 weeks apart. One dog was treated with imidocarb diproprionate (. mg/kg IM) for 2 doses 2 weeks apart followed by dimiazene aceturate (3. mg/kg IM) for 2 doses 2 weeks apart. After randomization (data collected after study completion), there were dogs in the treatment group that had been treated with imidocarb diproprionate alone and 3 dogs that had been treated with diminazene aceturate alone. The placebo group consisted of dogs that had been treated with imidocarb diproprionate alone, 1 dog that had been treated with diminazene aceturate alone, and 1 dog that had been treated with both drugs. No concurrent anti-infective therapy other than heartworm prophylaxis was administered during the study period. Twenty-two dogs satisfied the enrollment criteria and were randomly and evenly divided into 2 groups (treatment and placebo). Eleven dogs were male and 11 were female. Ages ranged from 1 to 12 years. Breeds included 1 American Pit Bull Terriers and 1 each of the following: American Staffordshire Terrier, German Shepherd Dog, Rottweiler, Boxer, and mixed-breed dog. Owners of the dogs were required to sign an informed consent form including information regarding the use of placebo and that possible adverse effects of the atovaquone-azithromycin drug combination were unknown. Study Protocol The study was conducted as a randomized, double-blind, placebocontrolled therapeutic trial designed to detect at least a % difference between the 2 groups, with a power (1 ) of. and an level of.. 2 Owners, clinicians, and laboratory technicians were blinded throughout the study until all of the posttreatment PCR results had been analyzed. The treatment group received atovaquone b and azithromycin. c The atovaquone dosage was 13.3 mg/kg PO qh, and the dose was administered with a fatty meal. The azithromycin dosage was 1 mg/kg (1 11. mg/kg) PO q2h. The dosages and course of therapy were extrapolated from published veterinary and human dosages and based on the pilot study. a,3,1,3 For ease of administration, the azithromycin was reformulated by the North Carolina State University College of Veterinary Medicine Pharmacy as -mg gelatin capsules. Both drugs were administered simultaneously for 1 days. The placebo group received color- and consistency-matched encapsulated and liquid placebos consisting of dextrose in gelatin capsules and white liquid food color d mixed with yellow food color. e Both the treatment and placebo groups were treated with fipronil f throughout the course of the study. To avoid transmission of B gibsoni (Asian genotype) via bite wounds and/or blood transfer, physical contact with other dogs was prohibited. g Each dog was scheduled to be evaluated a total of times: pretreatment (day ), end of treatment (day 1), and 3 posttreatment evaluations at approximately,, and 12 days. A physical examination, CBC, and Babesia-specific PCR assay were performed at each sample collection time. A serum biochemical profile was performed at days, 1, and 12. Based on pharmacokinetic studies in dogs and other mammals, there should be no detectable amounts of either drug by the -day posttreatment evaluation. 1, This study was performed in compliance with the North Carolina State University Animal Care and Use protocol 1-2. DNA Extraction and PCR DNA was isolated from anticoagulated (ethylenediaminetetraacetic acid) whole blood samples using a comercial kit h according to the manufacturer s instructions. B gibsoni (Asian genotype) DNA was amplified as previously described. 21 The presence of PCR inhibitors in DNA samples that tested negative in our PCR assay was excluded by the amplification of a fragment of the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene as previously described. 21 To prevent PCR amplicon contamination, sample preparation, reaction setup, PCR amplification, and amplicon detection were all performed in separate areas. Positive and negative controls were used in all processing steps, including the DNA extraction. Statistical Analyses A dog was considered to have a positive treatment outcome if no B gibsoni (Asian genotype) DNA was detected by PCR in any posttreatment sample. A dog was considered to have a negative treatment outcome if B gibsoni (Asian genotype) DNA was detected by PCR in any posttreatment sample. Difference in treatment outcome between the 2 groups was assessed using a Fisher s exact test. Statistical analyses were carried out using a statistical program. i Results B gibsoni (Asian genotype) DNA was detectable by PCR in posttreatment samples from 2/1 dogs in the treatment group and 11/11 dogs in the placebo group. There was a significant difference in outcome (P.1) between the treatment and placebo groups. One of the treatment group dogs was positive and days after treatment, and another was positive days after treatment. Nine of 11, of 1, and of dogs tested in the control group were positive,, and 12 days after treatment, respectively (Table 1). The G3PDH gene fragment was amplified from all DNA samples that tested negative by PCR for the presence of Babesia DNA, which ruled out the presence of PCR inhibitors in these samples. Five dogs did not complete all posttreatment visits: 3 dogs in the treatment group and 2 dogs in the placebo group. Of the 3 dogs in the treatment group, 1 dog was euthanized because of progressive degenerative joint disease, 1 dog died from a heat stroke after being left in a car, and 1 dog was lost to follow-up because of lack of owner compliance. In the placebo group, 2 dogs were lost to follow-up because of lack of owner compliance. One dog was excluded from the outcome analyses. This dog was in the treatment group and had 2 consecutive negative PCR results prior to being euthanized for progressive degenerative joint disease. The remaining dogs were included in the outcome analyses because they were considered to have a negative treatment outcome based on at least 1 positive PCR result in a posttreatment sample. The hematologic and biochemical values for both the treatment and placebo groups for which the mean values were outside of the reference range are presented in Table 2. No clinically apparent adverse effects were reported in either the treatment or placebo group.

3 Birkenheuer, Levy, and Breitschwerdt Table 1. Results of the PCR assays for the treatment and placebo groups. Dog no. Group Day Day Day Day , positive PCR result;, negative PCR result;, not determined. Discussion Based on the results of the present study, atovaquoneazithromycin combination therapy is an effective treatment for B gibsoni (Asian genotype) infections in dogs. Because the dogs in this study did not undergo splenectomy and drug-induced immune suppression, we cannot definitively conclude that the B gibsoni (Asian genotype) infections were sterilized by the combination therapy. However, even if complete parasite elimination did not occur and B gibsoni (Asian genotype) infections were only suppressed below the limits of PCR detection, reduction of the levels of parasitemia has been shown to significantly reduce the transmission of tick-transmitted protozoan pathogens such as Babesia and Theileria. Atovaquone is commercially available in the United States in 2 formulations: a single drug formulation b and a 2.:1 combination with proguanil hydrochloride. j The single drug formulation was used in the current study. The use of the 2.:1 combination with proguanil hydrochloride j in dogs has resulted in vomiting and diarrhea (Birkenheuer, unpublished data). The efficacy of an atovaquone-azithromycin combination therapy in acutely or critically ill dogs has not been evaluated. The lack of adverse clinical, hemtologic, and biochemical abnormalities reported in the treatment group is promising, but larger studies must be performed to fully assess the safety of the atovaquone-azithromycin drug combination in dogs. It is not known whether the 2 dogs in the treatment group that tested positive in the posttreatment samples represented treatment failures, reinfection, or lack of dosing compliance. Plasma concentrations of atovaquone and azithromycin were not evaluated to assess treatment compliance or drug absorption. Resistance to atovaquone by other protozoa has been reported and is associated with mutations in the cytochrome b gene. 3,3,, Some B gibsoni (Asian genotype) isolates may be resistant to or could acquire resistance to atovaquone-azithromycin combination therapy. It is not clear what effect if any was introduced by the overrepresentation of American Pit Bull Terriers in the study population. This is the most commonly reported affected breed in the United States 1 3, and % of the B gibsoni (Asian genotype) infections identified in the North Table 2. Hematologic and biochemical variables (mean and SD) for dogs for which the mean values were outside of the reference range. Parameter Reference range 1 12 n Plasma protein (g/dl) Mean cell volume (Fl) Mean corpuscular hemoglobin (g/dl) Red cell distribution width (%) Total Protein (g/dl) Globulin (g/dl) (1.). (.) 2. (3.) 1. (2.). (1.2). (1.2). (.) 3. (3.) 1.1 (3.). (1.) 1. (.) 3.3 (3.3) 21. (2.). (.2) 1. (3.) n Plasma protein (g/dl) Mean cell volume (Fl) Red cell distribution width (%) Total protein (g/dl) Globulin (g/dl) (.) 1. (3.) 1. (3.). (1.1). (1.3) (.) 3. (.) 1. (3.1).3 (.).1 (1.).1 (.) 2. (2.) 1. (3.). (.) 1. (3.), within reference range;, not determined; dog number of dogs that had values outside of the reference range.. (.).2 (2.) 2. (3.). (.) 3. (3.) 1.2 (3.). (1.). (1.2)

4 of Canine Babesiosis Carolina State University College of Veterinary Medicine Vector-Borne Disease Diagnostic Laboratory Database have been in this breed (Breitschwerdt and Levy, unpublished data). In the present study, we demonstrated that an atovaquone-azithromycin drug combination is an effective treatment for dogs that are chronically infected with B gibsoni. It is the only treatment for B gibsoni (Asian genotype) infections that has resulted in either elimination of infection or the suppression of parasitemia below the limit of detection. An effective treatment for B gibsoni (Asian genotype) will have important implications for dogs around the world. Footnotes a Birkenheuer AJ, Green CE, Silver S, et al. Babesia gibsoni infections in non-american Pit Bull Terriers. ACVIM Forum, Charlotte, NC, 23. Research Abstract. b Mepron, GlaxoSmithKline, Research Triangle Park, NC c Zithromax, Pfizer, New York, NY d Professional Compounding Centers of America, Houston, TX e McCormick and Company Inc, Sparks, MD f Frontline, Merial Ltd, Duluth, GA g Birkenheuer AJ, Levy MG, Stebbins M, Breitschwerdt EB, et al. Atovaquone and azithromycin combination therapy for the treatment of Babesia gibsoni infections in dogs. ACVIM Forum, Charlotte, NC, 23. Research Abstract. h Qiamp DNA blood mini kit, Qiagen, Valencia, CA i SigmaStat 2.3, SSPS Inc, Chicago, IL j Malarone, GlaxoSmithKline, Reseach Triangle Park, NC Acknowledgments The authors thank Drs Martha Stebbins and Maria Correa for their thoughtful discussions and the NCSU Clinical Pathology Laboratory and Vector-Borne Disease Diagnostic Laboratory for their expert technical assistance. All laboratory work was performed at the North Carolina State University College of Veterinary Medicine. 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