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Detection of a Babesia Species in a Bobcat from Georgia
Barbara C. Shock, J. Mitchell Lockhart, Adam J. Birkenheuer, and Michael J. Yabsley

Southeastern Naturalist, Volume 12, Issue 1 (2013): 243–247

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Detection of a Babesia Species in a Bobcat from Georgia Barbara C. Shock1,2,*, J. Mitchell Lockhart3, Adam J. Birkenheuer4, and Michael J. Yabsley1,2 Abstract - We describe the first detection of a Babesia sp. in a Lynx rufus (Bobcat). The Bobcat was from Georgia and was coinfected with Cytauxzoon felis and a Sarcocystis sp. The Babesia species was closely related to Babesia sp. “Coco”, a parasite previously only detected in Canis familiaris (Domestic Dog). The only other Babesia sp. in North America that infects felids is a novel Babesia species in Puma concolor coryi (Florida Puma). The low prevalence of this Babesia (less than 1%) in Bobcats suggests that they are not the normal host or reservoir and this may have been an incidental infection. Piroplasms (genera Babesia, Theileria, and Cytauxzoon) are tick-transmitted apicomplexan parasites which infect a wide range of mammals and birds worldwide (Criado- Fornelio et al. 2004). Species of all three genera infect erythrocytes, but in contrast to Theileria and Cytauxzoon, Babesia spp. do not have an extra-erythrocytic stage (Criado- Fornelio et al. 2004). Numerous species of piroplasms are important disease-causing agents for veterinary species, and Babesia are notable in that many species are zoonotic. Disease caused by Babesia is rare among wildlife, but disease can develop during stressful periods, after co-infection with immunosuppressive viruses, or when infections occur in aberrant hosts (e.g., Panthera leo L. [Lions] from Africa infected with a natural Babesia species but diseased when exposed to drought and coinfected with Canine Distemper Virus, or when exotic Rangifer tarandus L. [Reindeer] in the northeastern United States become infected with Babesia species native to the area; Bartlett et al. 2009, Munson et al. 2008). Currently, only two piroplasms have been reported from felines in North America, Cytauxzoon felis Kier in Felis catus L. (Domestic Cat), Lynx rufus Schreber (Bobcat), and Puma concolor L. (Puma) from the eastern United States, and a novel Babesia species in Puma concolor coryi Bangs (Florida Puma) from southern Florida (Glenn et al. 1983, Yabsley et al. 2006). During a surveillance study (Shock et al. 2012) on wildlife reservoirs of C. felis involving Bobcats (n = 799) and Pumas (n = 49) from thirteen states (Florida, Georgia, Kansas, Kentucky, Louisiana, Missouri, North Carolina, North Dakota, Ohio, Oklahoma, Pennsylvania, South Carolina, and West Virginia), a Babesia species was detected in a single female Bobcat from Thomas County, GA (n = 143; 0.7%). This is the first report of a Babesia sp. infection in a Bobcat and is only the second felidinfecting Babesia species reported in North America. The internal transcribed spacer (ITS)-1 region was amplified using a nested PCR that amplifies all known piroplasms (Bostrom et al. 2008). Briefly, for primary amplification, 5 ml of DNA was added to 20 ml of a master mix containing 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP (Promega, Madison, WI), 2.5 units GoTaq® Flexi DNA Polymerase (Promega), and 0.8 mM of primers ITS-15C (5’-CGATCGAGTGATCCGGTGAATTA) and ITS-13B (5’-GCTGCGTCCTTCATCGTTGTG). Cycling 1Southeastern Cooperative Wildlife Disease Study, 586 D.W. Brooks Drive, Wildlife Health Building, College of Veterinary Medicine, University of Georgia, Athens, GA, 30602. 2Warnell School of Forestry and Natural Resources, 180 E Green Street, University of Georgia, Athens, GA 30602. 3Biology Department, Valdosta State University, 1500 N Patterson, Department of Biology, Room 2035, Valdosta, GA 31698. 4NC State College of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606. *Corresponding author - barbarashock@gmail.com. Notes of the Southeastern Naturalist, Issue 12/1, 2013 243 244 Southeastern Naturalist Vol. 12, No. 1 parameters were 94 °C for 1 min followed by 35 cycles of 94 °C for 30 sec, 52 °C for 30 sec, 72 °C for 1 min, and a final extension at 72 °C for 5 min. For the nested PCR, 1 ml of primary product was used as a template in a 25-ml reaction containing the same PCR components except inclusion of primers ITS-15D (5’-AAGGAAGGAGAAGTCGTAACAAGG) and ITS-13C (5’-TTGTGTGAGCCAAGACATCCA). The cycling parameters were the same as the primary reaction except the annealing temp erature was 49 °C. To prevent and detect contamination, primary and secondary amplification, and product analysis were done in separate dedicated areas. A negative water control was included in each set of DNA extraction, and one water control was included in each set of primary and secondary PCR reactions. The amplicon from the positive Bobcat was purified with a Qiagen gel extraction kit (Germantown, MD) and bi-directionally sequenced at the University of Georgia Integrated Biotechnology Laboratory (Athens, GA). Sequence analysis of the ITS-1 region (601 bp) indicated that the greatest similarity (92%) was with a novel large Babesia sp. “Coco’” that was first identified in a Canis familiaris L. (Domestic Dog) from North Carolina in 2002 (GenBank accession number: AY618928; Fig. 1; Birkenheuer et al. 2004). The phylogenetic relationship based on ITS1 between this Babesia and other Babesia spp. is similar to the relationship between Babesia sp. “Coco” and other Babesia spp. based on analysis of the 18S rRNA gene (Birkenheuer et al. 2004). The only difference between the Bobcat Babesia sequence and Babesia sp. “Coco” was the presence of a 45-bp insert in the Bobcat Babesia sp. at nucleotide site 434. Outside the insert region, bases 1–434 and 435 to 557 of Babesia sp. “Coco” (EU109720) were 100% similar to our Bobcat Babesia. Thus, we believe that this Figure 1. Phylogenetic relationships between Babesia spp. inferred from internal transcribed spacer (ITS)-1 rRNA region sequences. 2013 Southeastern Naturalist Notes 245 Bobcat Babesia sp. represents a variant of Babesia sp. “Coco” and not a novel Babesia sp., although additional studies are needed to definitively determine the con-specificity of these two Babesia spp. Insertions and deletions are common in the ITS regions of other piroplasms (Aktas et al. 2007, Brown et al. 2009, Shock et al. 2012). Attempts to amplify other gene targets failed as the Bobcat was also co-infected with C. felis, and other targets (e.g., 18S rRNA gene and ITS-2) were positive but when sequenced were confirmed to be C .felis. These data highlight the need to utilize multiple gene targets when conducting pathogen surveillance. Unfortunately, a blood smear was not available from the Babesia sp.-infected Bobcat so no morphologic data is available. Babesia sp. “Coco” has only previously been reported from immunosuppressed Domestic Dogs (Sikorski et al. 2010), so in an effort to better understand why this Bobcat was infected, we conducted additional pathogen screening using the limited samples available from this trapper-harvested animal. Serum from the Bobcat was negative for Feline Immunodeficiency Virus (FIV) antibodies and Feline Leukemia Virus (FeLV) antigens (IDEXX, Westbrook, ME). A low antibody titer (1:10) for Feline Panleukopenia Virus was detected (Animal Health Diagnostic Center, Cornell University, Ithaca, NY), which was not interpreted as an infection. PCR testing for other pathogens revealed that the Bobcat was positive for “Candidatus Bartonella volans” and was negative for hemoplasmas (Cadenas et al. 2008, Jensen et al. 2001). Histological examination of available tissues was unrewarding due to advanced autolysis, but Sarcocystis sp. cysts were observed in muscle tissue. All of these findings were considered incidental. Currently, little is known about the natural history of Babesia sp. “Coco” and the Babesia sp. detected in the Bobcat from Georgia. Babesia sp. “Coco” was first reported from an immunosuppressed dog undergoing chemotherapy for lymphoma (Birkenheuer et al., 2004). Since the initial detection, eight additional canine infections have been reported from dogs, all with a travel history to Mid-Atlantic states. Six of these dogs were splenectomized, and two were immunosuppressed due to oncolytic drugs (Birkenheuer et al. 2004, Holman et al. 2009, Sikorski et al. 2010). At least 5 of the 9 dogs infected with Babesia sp. “Coco” had a history of tick exposure, and at least one sustained bites to the face, a risk factor for other Babesia sp., such as B. gibsoni Patton (Holman et al. 2009, Sikorski et al. 2010, Yeagley et al. 2009). Worldwide, several Babesia spp. have been reported from felids including B. herpailuri Dennig and B. pantherae Dennig and Brocklesby from wild felids in Africa, B. felis Davis and B. leo Penzhorn from domestic cats and wild felids in Africa, B. cati Mudaliar in Domestic Cats from India, B. canis canis Uilenberg from domestic cats in Spain, B. canis presentii Baneth from Domestic Cats in Israel; a Babesia sp. from Domestic Cats in Portugal, (Baneth et al. 2004, Criado-Fornelio et al. 2004, Penzhorn et al. 2004). In the United States, the only previous report of a Babesia species in a felid is a novel Babesia sp. from Florida Pumas (Yabsley et al. 2006). The Puma Babesia sp. appears to be restricted to Florida Pumas because, in the current study, infections were not detected in 49 Pumas from Texas, Louisiana, Georgia, or North Dakota. Interestingly, the Babesia sp. from the Florida Puma is a small piroplasm morphologically and is indistinguishable from C. felis, whereas Babesia sp. “Coco” is a large Babesia. In addition, the two feline-infecting Babesia species from North America are easily distinguished based on sequence analysis of the ITS-1 region (B.C. Shock et al., unpublished data). In summary, a Babesia sp. closely related to Babesia sp. “Coco” was detected in a single Bobcat from Georgia, which is the first report of Babesia infection of a Bobcat and the second report of Babesia in felids from North America. Bobcats likely do not 246 Southeastern Naturalist Vol. 12, No. 1 represent a natural host of this Babesia sp.; thus, additional surveillance studies are needed to understand the natural host of this parasite. Acknowledgments. The authors thank numerous personnel from state agencies who assisted with the collection of felid samples. This study was primarily funded by the Morris Animal Foundation (DO8FE-003), and additional support was provided by the Federal Aid to Wildlife Restoration Act (50 Stat. 917) and through sponsorship from fish and wildlife agencies in Alabama, Arkansas, Florida, Georgia, Kansas, Kentucky, Louisiana, Maryland, Mississippi, Missouri, North Carolina, Oklahoma, Pennsylvania Puerto Rico, South Carolina, Tennessee, Virginia, and West Virginia. Literature Cited Aktas, M., K.G. Bendele, K. Altay, N. Dumanli, M. Tsuji, and P.J. Holman. 2007. Sequence polymorphism in the ribosomal DNA internal transcribed spaces differs among Theileria species. Veterinary Parasitology 147:221–230. Baneth, G., M.J. Kenny, S. Tasker, Y. Anug, V. Shkap, A. Levy, and S.E. Shaw. 2004. Infection with a proposed new subspecies of Babesia canis, Babesia canis subsp. presentii, in domestic cats. 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