The detection of Cytauxzoon felis in apparently healthy free-roaming cats in the USA

Veterinary Parasitology 146 (2007) 316 320 www.elsevier.com/locate/vetpar The detection of Cytauxzoon felis in apparently healthy free-roaming cats in the USA Marion D. Haber a, Melissa D. Tucker a, Henry S. Marr a, Julie K. Levy b, Jill Burgess c, Michael R. Lappin d, Adam J. Birkenheuer a, * a Department of Clinical Sciences, North Carolina State University, College of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606, United States b Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, 2015 SW 16th Avenue, Gainesville, FL 32610, United States c 222 Chamberlain Blvd, Knoxville, TN 37920, United States d Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1678, United States Received 1 January 2007; received in revised form 22 February 2007; accepted 22 February 2007 Abstract Cytauxzoon felis typically causes fatal disease in domestic cats. Survival after infection and persistent parasitemia without clinical illness has been documented in a few cases. To our knowledge there are no prevalence studies of C. felis in domestic cats. The purpose of this study was to estimate the prevalence of C. felis infected cats that were presented to trap-neuter-return programs in Florida, North Carolina and Tennessee. Cats that were presented to trap-neuter-return programs were tested using a C. felisspecific PCR assay. A total of 961 domestic cats were tested (494 from Florida; 392 from North Carolina; 75 from Tennessee). Prevalence of C. felis infection in this population was 0.3%. Two cats from Florida and one cat from Tennessee tested positive for the presence of C. felis DNA. These amplicons were sequenced and confirmed to be C. felis. The cat from Tennessee was alive without evidence of illness 2 months post-surgery. The other two cats were alive 24 h post-surgery, but were then lost to follow-up. This is the first report documenting C. felis infections in free-roaming cats. Despite the low prevalence rate, the presence of apparently healthy infected free-roaming cats suggests that they may have the capacity to serve as an additional reservoir host for C. felis. Further investigations should evaluate the potential vector competence of domestic cats as well as the role of chronically infected cats in areas in which cytauxzoonosis appears hyperendemic. # 2007 Published by Elsevier B.V. Keywords: Cytauxzoonosis; PCR; Free-roaming; Survival; Hyperendemic 1. Introduction Cytauxzoon felis is a tick-transmitted, intracellular protozoan that is known to cause rapidly progressive * Corresponding author. Tel.: +1 919 513 8288; fax: +1 919 513 6336. E-mail addresses: adam_birkenheuer@ncsu.edu, ajbirken@ncsu.edu (A.J. Birkenheuer). fatal disease in domestic cats (Kier et al., 1982; Walker and Cowell, 1995; Meinkoth et al., 2000; Birkenheuer et al., 2006a,b). Domestic cats are typically affected in the summer months and present with fever and a variety of signs ranging from lethargy and decreased appetite, to severe dyspnea and a moribund state. The pathophysiology and clinical progression of this infection has been documented in previous reports (Wagner et al., 1980; Kier et al., 1982, 1987; Blouin et al., 1987). This disease has been recently recognized 0304-4017/$ see front matter # 2007 Published by Elsevier B.V. doi:10.1016/j.vetpar.2007.02.029

M.D. Haber et al. / Veterinary Parasitology 146 (2007) 316 320 317 in regions where it was not believed to be endemic (Birkenheuer et al., 2006a,b). The causes for this apparent expansion of the parasites geographic range are unknown. Due to the devastating nature of the illness and the relative ease with which the disease is identified, poor clinician recognition seems unlikely. Bobcats (Felis rufus) are the presumed reservoir host due to the high prevalence of C. felis in this species and their ability to survive the schizogenous phase of infection followed by persistent red blood cell parasitemia without the consequence of illness (Glenn et al., 1982, 1983; Blouin et al., 1984, 1987; Kocan and Blouin, 1985). C. felis has been experimentally transmitted to domestic cats from infected bobcats via the tick vector Dermacentor variabilis (Blouin et al., 1984). Historically, it was believed that the disease was 100% fatal in domestic cats and therefore domestic cats were presumed to be a dead-end host. However, there are several reports of cats surviving C. felis infections (Kier et al., 1982; Walker and Cowell, 1995; Greene et al., 1999; Meinkoth et al., 2000; Birkenheuer et al., 2006a). Some of these cats remained parasitemic without clinical illness for up to 154 days (Meinkoth et al., 2000). In addition, there are cats infected with C. felis that have never exhibited clinical signs (Meinkoth et al., 2000; Birkenheuer, unpublished data, 2006). This evidence, along with the social dynamics of freeroaming cats and their substantial exposure to ticks, led us to speculate that free-roaming cats may serve as an additional reservoir host for C. felis, which could contribute to the apparent spread of the disease s geographic range. To the authors knowledge, no large epidemiological surveys for C. felis in domestic cats have been performed. The purpose of this study was to screen for the presence of C. felis infected free-roaming cats that were presented to trap-neuter-return (TNR) programs in Northern Florida, Central North Carolina, and Tennessee. In trap-neuter-return programs, freeroaming cats are trapped, neutered, and returned to the site of where they were captured. These programs are intended to limit the reproduction of free-roaming cats. Although these cats are not owned, they frequently have caretakers that feed the cats on a regular or irregular basis and these are often the individuals that trap the cats and bring them to the TNR programs. 2. Materials and methods This was a cross-sectional study utilizing a convenience sample from free-roaming cats that were presented to trap-neuter-release programs. This study was performed in accordance with a protocol approved by the North Carolina State University Institutional Animal Care and Use Committee and in accordance with UF IACUC protocol B345. 2.1. Animals and sample collection Ethylene diamine tetraacetic acid (EDTA) anticoagulated whole blood was collected from domestic cats that were presented to TNR programs in Alachua County Florida, Wake County North Carolina, and Wilson County Tennessee. Samples were collected from Florida from June 1999 through February 2000, and had been used for a previous study and were then sent to North Carolina State University for analysis (Luria et al., 2004). These samples were stored at 80 8C until the initial testing was performed. Then the total DNA was stored at 80 8C until the assays described in this study were performed. Samples were collected from North Carolina from April 2005 through August 2005. Samples were collected from Tennessee in June 2006. When available, the caretakers of infected cats were contacted via telephone for follow-up information. 2.2. DNA extraction and PCR amplification and DNA sequencing Total DNA was isolated from 200 ml of whole blood using a commercial kit and an automated workstation according to manufactures instructions (QIAamp DNA Blood Mini Kit or Magattract DNA Blood Mini M48 Kit, Qiagen Inc., Valencia, CA). A C. felis-specific PCR assay was used for the amplification of 18S rrna gene sequence that is able to detect 10 copies of target DNA/ml of sample with 100% sensitivity as previously described (Birkenheuer et al., 2006b). Primer sequences were selected to specifically amplify a 284 bp fragment of the C. felis 18S rrna gene. The selected primer sequences were: 5 0 -GCGAATCGCATTGCTTTATGCT-3 0 and 5 0 - CCAAATGATACTCCGGAAAGAG-3 0. Each reaction contained a 1 concentration of PCR Buffer II (Applied Biosystems, Foster City, CA), 1.25 U of Taq polymerase, 5 ml of DNA template, 1.5 mm MgCl 2,25pmolofeach primer, and 200 mm of each dntp. The thermal cycling conditions consisted of an initial denaturation at 95 8C for 5 min, followed by 40 amplification cycles (95 8C for 45 s, 59 8C for 45 s, and 72 8C for 60 s), and a final extension step at 72 8C for 5 min (PCR Express; Thermo Hybaid, Middlesex, UK). Positive controls consisted of C. felis infected feline whole blood and negative controls consisted of water (no DNA). C. felis-pcr negative samples were screened for the presence of PCR inhibitors

318 M.D. Haber et al. / Veterinary Parasitology 146 (2007) 316 320 via amplification of a GAPDH pseudogene as previously described (Birkenheuer et al., 2003). In order to prevent amplicon contamination, precautions were taken such as the use of disposable gloves and a clean to dirty flow of procedures. Sample processing, DNA extraction, reaction setup, PCR amplification and post-amplification processing were performed in separate rooms to avoid amplicon contamination. Positive samples were sequenced bi-directionally either directly or after cloning into a plasmid vector (pcr2.1-topo, Invitrogen Life Technologies, Carlsbad, CA; Davis Sequencing Inc., Davis, CA). Comparisons to the sequences deposited in GenBank were made using the basic local alignment search tool (Altschul et al., 1990). 2.3. Statistical analysis Differences between the proportions of infected cats from each group were evaluated using a Fisher exact test. Statistical calculations were completed using a computer program (Epi Info TM, Centers for Disease Control and Prevention, Atlanta, GA). 3. Results A total of 494 samples from Northern Florida (FL), 392 samples from Central North Carolina, and 75 samples from Tennessee (TN) were collected. Two samples from Florida and one from Tennessee tested positive for the presence of C. felis DNA. The DNA sequences from all three samples were 100% identical to the C. felis sequence recorded in GenBank (accession number AF399930). Amplicons were never detected in negative control samples. Six samples were excluded due to the presence of PCR inhibitors. Fig. 1 demonstrates the results of representative PCR assays; cats from which C. felis DNA was detected, cats from which C. felis DNA was not detected, positive and negative controls. There were no statistical differences between the proportions of infected cats from sampling locations. The overall prevalence in this population was 0.3%. If our samples are representative of the freeroaming cat populations, the true prevalence is 0 0.8% (99% CI). The infected cat from TN was an adult male domestic short haired cat, approximately 4 years of age, from a colony in Lincoln County, TN. According to the colony caretaker this cat had been born into that colony and they were not aware of any illness in the cat s life. The cat recovered from surgery without incident and continues to behave normally up to 2 months after the procedure. There are approximately 20 Fig. 1. Representative results of PCR assays from free-roaming cats with and without Cytauxzoon felis infections. (A) PCR products from the amplification of a 284 bp fragment of the C. felis 18S rrna gene visualized by ethidium bromide staining and trans-illumination with ultra-violet light after electrophoresis in a 2% agarose gel. Lane 1: molecular weight marker. Lanes 2 and 3: free-roaming cats that tested positive for C. felis. Lanes 4 and 5: free-roaming cats that tested negative for C. felis. Lane 6: positive control (C. felis infected feline whole blood). Lane 7: negative control (no DNA). (B) PCR products from the amplification a 400 bp fragment of a GAPDH pseudogene visualized by ethidium bromide staining and trans-illumination with ultra-violet light after electrophoresis in a 2% agarose gel. Lane 1: molecular weight marker. Lanes 2 and 3: free-roaming cats that tested positive for C. felis. Lanes 4 and 5: free-roaming cats that tested negative for C. felis. Lane 6: positive control (C. felis infected feline whole blood). Lane 7: negative control (no DNA). other cats in the colony and none have displayed outward signs of illness and none have died unexpectedly. The caretakers have had two owned cats living on the same property die of cytauxzoonosis in the past 4 years. The only information available for the infected cats from FL was that they did not demonstrate obvious clinical signs of C. felis infection before capture, and they were released 24 h following surgery without complications. The individuals that presented them to the TNR program could not be contacted regarding long-term survival.

M.D. Haber et al. / Veterinary Parasitology 146 (2007) 316 320 319 4. Discussion This is the first study to survey free-roaming cats for the presence of C. felis infections. We were able to document the presence of C. felis in a small percentage (0.3%) of free-roaming cats. This is a low prevalence when compared to that of bobcats, which has been reported between 30 and 50% (Glenn et al., 1982; Kocan and Blouin, 1985). The prevalence in the Wilson County, TN group was the highest detected, at 1.3%, compared to 0% for Wake County, NC, but we were unable to detect a significant difference between the populations. Of the two cats from FL from which C. felis DNA was detected by PCR, one also tested positive for the presence of IgM antibodies against Toxoplasma gondii by microtiter plate ELISA with a titer of 1:64 (Luria et al., 2004). Both of these cats tested positive, for the presence of antibodies against Dirofilaria immitis using microtiter plate ELISA test kits with titers of 1:80 (Luria et al., 2004). These cats were from different colonies and presented 1 month apart from each other. There have been several reports of domestic cats surviving C. felis infection, as well as carrying infection without demonstrating clinical signs of disease (Kier et al., 1982; Walker and Cowell, 1995; Greene et al., 1999; Meinkoth et al., 2000; Birkenheuer et al., 2006a). Several of these reported cats that survived infection remained parasitemic for months to years without clinical illness. In one of the original publications describing cats that survived cytauxzoonosis, it was considered that survival may be a geographically limited phenomenon (Meinkoth et al., 2000). The cat in this study from TN from which C. felis DNA was detected by PCR was born in TN and is presumed to have lived its entire life there, based on frequent sightings in that location over the 4 years prior to the cat s enrollment into this study. This cat lived over 500 miles from the nearest cat in the report by Meinkoth et al. We have also previously reported a cat that survived C. felis infection from the North Carolina coast nearly 1000 miles away (Birkenheuer et al., 2006a). Together these findings indicate that survival of cytauxzoonosis in domestic cats is not geographically limited. Similar to the infected cat from TN, there are documentations of C. felis infections in multiple cats living on the same property or from individual cats from multiple households from the same neighborhood (Wagner, 1976; Greene et al., 1999; Meinkoth et al., 2000; Birkenheuer et al., 2006a). Likewise, we have documented several households in which we detected asymptomatic C. felis infected cats after one or more cats in that household have succumbed to cytauxzoonosis (Birkenheuer, unpublished data). This type of concentrated prevalence may represent hyperendemic foci or hot spots of C. felis infection. Together these findings support the possibility that domestic cats may be able to serve as an additional reservoir host for C. felis. Future studies should evaluate the potential vector competence and capacity of domestic cats for the transmission of C. felis as well as prevalence studies of domestic cats in geographic areas that may constitute hot spots of C. felis. Acknowledgments Supported in part by a grant from the Merck-Merial Scholars Program. We would like to thank the following organizations for allowing us to obtain samples from their free-roaming cats: Operation Catnip, Raleigh, NC and Gainesville, FL Snip and Tip, Knoxville, TN. 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