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Veterinary Parasitology 153 (2008) 126–130 www.elsevier.com/locate/vetpar

Cytauxzoon felis infections are present in (Lynx rufus) in a region where is not recognized in domestic Adam J. Birkenheuer a,*, Henry S. Marr a, Camille Warren a, Anne E. Acton a, Eric M. Mucker b, Jan G. Humphreys c, Melissa D. Tucker a a Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606, b United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702-5011, United States c Indiana University of Pennsylvania, 325 Weyandt Hall, Department of Biology, Indiana, PA 15705, United States Received 3 August 2007; received in revised form 10 January 2008; accepted 11 January 2008

Abstract This study was performed to determine the prevalence of felis (C. felis) infections in bobcats (Lynx rufus) from a region where C. felis is recognized in domestic cats, North Carolina (NC), and a region where C. felis is not recognized in domestic cats, Pennsylvania (PA). Samples from NC (n = 32) were obtained post-mortem via cardiac puncture from legally trapped bobcats. Samples from PA (n = 70) were collected post-mortem onto Nobuto blood collecting strips by the PA Game Commission. Each sample was tested using a C. felis specific PCR assay as well as a PCR assay targeting host DNA to rule out the presence of PCR inhibitors. Three samples were excluded due to the presence of PCR inhibitors. Thirty-three percent (10/30) of the samples from NC and 7% (5/69) of the samples from PA tested positive for the presence of C. felis. The proportion of C. felis positive bobcats from NC was significantly different than that from PA (P < 0.005). Despite the lower prevalence of C. felis infections in bobcats from PA this finding is unique and indicates the potential for C. felis infections in domestic cats in the northeastern USA if the appropriate tick vectors are present. Veterinary practitioners in PA should be on alert for cytauxzoonosis in domestic cats. Further studies about the epidemiology and transmission of C. felis infections among both domestic cats and bobcats are needed. # 2008 Elsevier B.V. All rights reserved.

Keywords: Cytauxzoonosis; Piroplasmosis; Cytauxzoon felis; Bobcats; PCR; Tick

1. Introduction develop a rapidly progressive febrile illness and succumb to multi-organ failure secondary to schizont-laden Cytauxzoonosis is an emerging infectious disease of macrophages occluding small blood vessels. Death domestic cats in North America caused by the protozoan occurred in the majority of infected cats reported in parasite Cytauxzoon felis (C. felis)(Birkenheuer et al., the literature (Birkenheuer et al., 2006a; Ferris, 1979; 2006a). Domestic cats that become infected with C. felis Hoover et al., 1994; Wagner, 1976). Because of this, domestic cats are typically considered to be an accidental host for C. felis. They are presumed to become naturally * Corresponding author. Tel.: +1 919 513 8288; infected with C. felis via tick vectors and bobcats (Lynx fax: +1 919 513 6336. rufus) are considered to be the reservoir host. The E-mail address: [email protected] (A.J. Birkenheuer). American dog tick () has been

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.01.020 A.J. Birkenheuer et al. / Veterinary Parasitology 153 (2008) 126–130 127 shown to transmit C. felis to domestic cats from bobcats the carcasses were either fresh or had been frozen at under laboratory conditions and is assumed to be the À20 8C and thawed immediately prior to sample primary tick vector in nature (Blouin et al., 1984). Unlike collection. Blood samples from NC (n = 32) were in domestic cats, C. felis appears to undergo a limited obtained via cardiac puncture. All samples were frozen schizogenous phase in bobcats and therefore bobcats at À20 8C until processing. Total deoxyribonucleic acid exhibit minimal morbidity and mortality as a result of the (DNA) was extracted from 100 mg of clotted whole infection (Blouin et al., 1987; Glenn et al., 1983). blood samples using a commercially available kit Cytauxzoonosis was first reported in Missouri in (QIAamp DNA blood mini kit, Qiagen, Valencia, CA). 1979 and the disease was only recognized in the south Blood samples from bobcats from PA (n = 70) were central and southeastern United States for the past 20 collected directly onto Nobuto blood collecting strips years. The geographic distribution of cytauxzoonosis in (Advantec, Toyo Roshi Kaisha, Japan) as previously domestic cats has been recently documented to extend described (Mucker et al., 2006). Samples from PA were east and northeast of its previously recognized range collected between October and February of 2002. (Birkenheuer et al., 2006a; Jackson and Fisher, 2006). Samples from NC were collected in January and Since cytauxzoonosis is not a difficult disease to February during 2004, 2005 and 2006. Total DNA was diagnose, it seems more likely that these infections extracted from dried blood strips using a commercially represent a true expansion of the disease distribution available kit (DNeasy tissue kit, Qiagen, Valencia, CA). rather than a new recognition of a disease that has been Each sample was tested using a C. felis specific historically present in these regions at low levels. polymerase chain reaction assay designed to amplify a Even with this expanded distribution, cytauxzoono- 284 base pair fragment of the 18S rRNA gene that is sis in domestic cats does not appear to correlate with the able to detect as few as ten copies of the target DNA per natural distribution of the presumed reservoir host, L. microliter of sample with 100% sensitivity (Birken- rufus, which includes most of North America and heuer et al., 2006b). The assay did not produce extends as far south as southern Mexico (Long, 2003). amplicons when feline DNA, canis vogeli, The cause or causes for this discrepancy are unknown. B. c. canis, B. c. rossi, B. gibsoni (Asian genotype), B. Some possibilities include a lack of C. felis in bobcats gibsoni (CA genotype), B. annae, Ehrlichia canis, E. throughout their entire range or the absence of ewingii, E. chafeensis, Neorickettsia risticii, Anaplasma competent vectors that can transmit C. felis from phagocytophilum, A. platys, , bobcats to domestic cats in areas where cytauxzoonosis M. haemominutm, Rickettsia rickettsii, Leishmania is not recognized. Studies investigating the prevalence infantum, Bartonella henselae or Bartonella vinsonii of C. felis in bobcats are limited to the southcentral ssp. berkhoffii were used as templates (Birkenheuer region of the US. These studies found that C. felis could et al., 2006b). Positive controls consisted of DNA be detected by examination of stained blood smears in extracted from the blood of C. felis infected domestic 30–50% of wild-trapped bobcats (Glenn et al., 1982; cats. The C. felis 18S rRNA genes from these positive Kocan et al., 1985). To the authors’ knowledge, studies control cats have been characterized previously examining the prevalence of C. felis infections in (Birkenheuer et al., 2006b). Negative (i.e. no DNA) bobcats from areas of the US where cytauxzoonosis is water controls were included with each amplification not recognized in domestic cats have not been run. To demonstrate that the extracted DNA was performed. The purpose of this study was to determine amenable to PCR testing, each sample was tested using and compare the proportion of bobcats that were an assay designed to amplify a fragment of a infected with C. felis from a region where cytauxzoo- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nosis is recognized in domestic cats to that where it is pseudogene from most mammals as previously not recognized. described (Birkenheuer et al., 2003). Amplicons were visualized by ethidium bromide staining and ultraviolet 2. Materials and methods trans-illumination after electrophoresis in a 2% agarose gel. Randomly selected amplicons (n = 5) were purified Bobcats were sampled from a state where cytaux- using a commercially available kit (Qiaquick PCR is recognized in domestic cats, North Carolina purification kit, Qiagen, Valencia, CA) and sequenced (NC) and another state where cytauxzoonosis in directly (Davis Sequencing, Davis, CA). These domestic cats has never been reported, Pennsylvania sequences were aligned with C. felis 18S rRNA (PA). All bobcats were legally trapped by licensed sequences obtained from domestic cats with cytaux- personnel. All samples were obtained post-mortem and zoonosis (ClustalW, http://www.mbio.ncsu.edu/BioE- 128 A.J. Birkenheuer et al. / Veterinary Parasitology 153 (2008) 126–130 dit/bioedit.html). The proportions of C. felis infected specific amplicons were detected in 33% (10/30) of the bobcats from each region were compared using a samples from NC, the region where cytauxzoo- Fisher’s exact test. nosis is recognized in domestic cats. In contrast, C. felis DNA was detected by PCR from only 7% (5/69) of the 3. Results bobcat samples from PA, the region where domestic cytauxzoonosis is not recognized. Amplicons were not Three samples, two from NC and one from PA, had detected in the negative controls (i.e. no DNA) to be excluded from the analyses because the GAPDH throughout the study. The sequences (n =5)ofthe pseudogene fragment could not be amplified. C. felis randomly selected amplicons, three from PA and two

Fig. 1. (A) The approximate distribution of bobcats (Lynx rufus) in the United States (shaded area) (Long, 2003). (B) The states in which cytauxzoonosis has been documented in domestic cats (shaded area) (Birkenheuer et al., 2006a; Greene, 2006; Jackson and Fisher, 2006). (C) The approximate distribution of Dermacentor variabilis in the United States (shaded area) (redrawn based on the distributions reported by the centers for disease control: http://www.cdc.gov). (D) The approximate distribution of in the United States (shaded area) (redrawn based on the distributions reported by the centers for disease control: http://www.cdc.gov). (E) The approximate distribution of Ixodes scapularis in the United States (shaded area) (redrawn based on the distributions reported by the centers for disease control: http://www.cdc.gov). A.J. Birkenheuer et al. / Veterinary Parasitology 153 (2008) 126–130 129 from NC, were 100% identical to the C. felis 18S rRNA from A. americanum ticks (Bondy et al., 2005). Based sequences from North America deposited in GenBank on these findings, further studies are indicated (Accession numbers L19080, AY679105, AY531524, evaluating the vector competence and vectorial and AF399930). The proportion of C. felis infected capacity of A. americanum and perhaps I. scapularis bobcats from PA was significantly lower (P < 0.005) for the transmission of C. felis. than that of bobcats from NC. This study represents the first to screen large numbers of bobcats for C. felis infections by PCR, 4. Discussion and the first to screen bobcats from an area where cytauxzoonosis is not recognized in domestic cats. Very The geographic distribution of bobcats (Fig. 1A) little is known about the epidemiology of C. felis does not correlate well with recognized distribution of infections and further studies are clearly indicated. cytauxzoonosis in domestic cats (Fig. 1B). The Studies evaluating the reservoir competence and findings of this study suggest that the lack of capacity of bobcats as well as the vector competence recognition of cytauxzoonosis in domestic cats in and capacity of several ixodid tick species for the PA is not due to the absence of C. felis infections in maintenance and transmission of C. felis infections are bobcats in that state. Assuming that the prevalence of needed. C. felis infections in the PA bobcats tested in this study is representative of the prevalence of C. felis in References all PA bobcats, this could contribute to a decreased reservoir capacity with a subsequent lower preva- Birkenheuer, A.J., Le, J.A., Valenzisi, A.M., Tucker, M.D., Levy, lence, or absence, of cytauxzoonosis in domestic cats. M.G., Breitschwerdt, E.B., 2006a. Cytauxzoon felis infection in The possibility that C. felis infections in PA bobcats cats in the mid-Atlantic states: 34 cases (1998–2004). J. Am. Vet. are a recent occurrence and that cytauxzoonosis in Med. Assoc. 228, 568–571. domestic cats may occur there in the future must also Birkenheuer, A.J., Levy, M.G., Breitschwerdt, E.B., 2003. Develop- ment and evaluation of a seminested PCR for detection and be considered. Ultimately, the presence of C. felis differentiation of Babesia gibsoni (Asian genotype) and B. canis infections in PA bobcats mandates that veterinary DNA in canine blood samples. J. Clin. Microbiol. 41, 4172–4177. clinicians in PA should be vigilant for cytauxzoonosis Birkenheuer, A.J., Marr, H., Alleman, A.R., Levy, M.G., Breitsch- in domestic cats. werdt, E.B., 2006b. Development and evaluation of a PCR assay The presence of C. felis in PA bobcats along with the for the detection of Cytauxzoon felis DNA in feline blood samples. Vet. Parasitol. 137 (1/2), 144–149. apparent absence of cytauxzoonosis in PA domestic Blouin, E.F., Kocan, A.A., Glenn, B.L., Kocan, K.M., Hair, J.A., 1984. cats may suggest that the routes of C. felis infection Transmission of Cytauxzoon felis Kier, 1979 from bobcats, Felis among bobcats and domestic cats differ. Perhaps rufus (Schreber), to domestic cats by Dermacentor variabilis similar to other piroplasma, C. felis infections can be (Say). J. Wildl. Dis. 20, 241–242. transmitted transplacentally and can be perpetuated in Blouin, E.F., Kocan, A.A., Kocan, K.M., Hair, J., 1987. Evidence of a limited schizogonous cycle for Cytauxzoon felis in bobcats fol- bobcats in the absence of a tick vector. Alternatively lowing exposure to infected ticks. J. Wildl. Dis. 23, 499–501. intra-species and inter-species transmission of C. felis Bondy Jr., P.J., Cohn, L.A., Tyler, J.W., Marsh, A.E., 2005. Poly- may occur via different tick species. While D. merase chain reaction detection of Cytauxzoon felis from field- variabilis has been demonstrated to have vector collected ticks and sequence analysis of the small subunit and competence for both inter- and intra-species transmis- internal transcribed spacer 1 region of the ribosomal RNA gene. J. Parasitol. 91, 458–461. sion of C. felis from bobcats (Blouin et al., 1984, 1987), Ferris, D.H., 1979. A progress report on the status of a new disease of the geographic distribution of D. variabilis (Fig. 1C) American cats: cytauxzoonosis. Comp. Immunol. Microbiol. does not appear to correlate well with the recognized Infect. Dis. 1, 269–276. distribution of cytauxzoonosis in domestic cats. In fact Glenn, B.L., Kocan, A.A., Blouin, E.F., 1983. Cytauxzoonosis in the distribution of cytauxzoonosis in domestic cats bobcats. J. Am. Vet. Med. Assoc. 183, 1155–1158. Glenn, B.L., Rolley, R.E., Kocan, A.A., 1982. Cytauxzoon-like pir- appears to correspond best with the distribution of the oplasms in erythrocytes of wild-trapped bobcats in Oklahoma. J. lonestar tick (Amblyomma americanum)(Fig. 1D) and Am. Vet. Med. Assoc. 181, 1251–1253. the black-legged tick (Ixodes scapularis)(Fig. 1E). In Greene, C., 2006. Infectious Diseases of the Dog and Cat, third ed. North Carolina, A. americanum is frequently detected WB Saunders, Philadelphia. feeding on domestic cats that have access to outdoors Hoover, J.P., Walker, D.B., Hedges, J.D., 1994. Cytauxzoonosis in cats: eight cases (1985–1992). J. Am. Vet. Med. Assoc. 205, (Birkenheuer, unpublished data). 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