cytauxzoon felis is a tick - transmitted protozoan parasite that can cause cytauxzoonosis in wild and domestic felids . cytauxzoonosis in domestic cats has been reported throughout central , south - eastern and south - central usa . oklahoma is considered enzootic for c felis and iowa ( ia ) is a non - enzootic state but borders other enzootic states . domestic cats infected with c felis often show severe , acute clinical signs characterized by fever , inappetence , anorexia , dyspnea and icterus . onset of disease typically occurs 1014 days after c felis - infected ticks feed on naive cats and progresses quickly , with fatalities reported 17 days after onset of clinical signs . a recent study demonstrated 60% survival in c felis - infected cats that received a combination therapy of azithromycin and atovaquone with supportive care . a free - roaming cat is a domestic cat that has been born and raised without or limited contact to humans and is unsocialized . as free - roaming cats live outdoors and are exposed to ticks , they are favorable populations in which to examine and monitor the distribution and range of cytauxzoonosis . our objective was to determine the prevalence of c felis infections in free - roaming cats in enzootic north - central oklahoma ( ok ) and non - enzootic central ia . blood samples were collected from free - roaming cats in stillwater , ok , and ames , ia , as part of community trap cats were trapped mainly in north - central ok and in central ia . in ok , all cats were sedated with a mixture of tiletamine hydrochloride and zolazepam hydrochloride ( telazol ; zoetis ) , ketamine hydrochloride ( ketamine ; putney ) and xylazine ( anased ; akorn ) . in ia , a mixture of ketamine hydrochloride ( ketamine ; putney ) , dexmedetomidine hydrochloride ( precedex ; orion pharma ) , buprenorphine ( simbadol ; zoetis ) and butorphanol tartrate ( torbugesic - sa ; zoetis ) was used for sedation in 20122013 . in 2014 , butorphanol tartrate ( torbugesic - sa ; zoetis ) was added to the mixture but only for fractious cats . the approximate ages of cats were determined based on dentition , and cats that were 46 months or older were selected for blood collection to increase the chance of finding c felis - infected cats . cats were placed in dorsal recumbency , fur around the neck was clipped and 70% isopropyl was sprayed in the area . approximately 1 ml blood was collected from the jugular vein and was immediately placed in an edta collection tube . genomic dna was extracted from peripheral whole blood samples using genejet whole blood dna purification mini kit ( thermo scientific ) . briefly , 200 l whole blood was mixed with 20 l proteinase k solution and 400 l lysis solution . after incubating the sample at 56c for 10 mins , the prepared mixture was transferred to the spin column and centrifuged for 1 min at 6000  g ( ~8000 rpm ) . the column was then washed with 500 l wash buffer i and centrifuged again for 1 min at 8000  g ( ~10,000 rpm ) . the column was then washed with wash buffer ii and centrifuged for 3 mins at 20,000  g ( 14,000 rpm ) . dna was extracted with 200 l preheated ( approximately 56c ) pcr - quality water added to the center of the column membrane to elute genomic dna . the sample was incubated for 2 mins at room temperature and centrifuged for 1 min at 8000  g ( ~10,000 rpm ) . a conventional pcr was performed to amplify the c felis small subunit rrna ( ssu - rrna ) . cycling conditions of nested pcr were as follows : denaturation at 95c for 5 mins , annealing at 54c for 1 min and extension at 72c for 1 min . pcr products were separated on 1.75% agarose gel and viewed with ultraviolet light . positive control templates consisted of dna extracted from whole blood of a cat that died from an infection of c felis , whereas negative control reactions used dna isolated from purified water . pcr products were purified using a qiaquick pcr purification kit ( qiagen ) and amplicons were sequenced by eurofin genomics ( huntsville , al ) . ninety - five percent confidence intervals ( cis ) were calculated according to sterne s exact method , using quantitative parasitology 3.0 . a total of 380 blood samples were collected from january to may 2014 in ok . three of 380 ( 0.8% ; 95% ci 0.222.3% ) samples showed approximately 250 bp bands , which were targeted product size for c felis . blast comparison of these three sequences showed 100% identity to c felis ( eg , l19080 , ay531524 , ay679105 , af399930 , gu903911 ) . all c felis - infected cats were male and older than 12 months of age . two infected cats were from stillwater , ok , and caught separately in march and april respectively . one c felis - infected cat originated from drumright , ok , in march ( figure 1 ) . a total of 292 blood samples were collected from august 2012 to april 2014 in ia . neuter  return ( tnr ) programs were conducted in stillwater , ok , and ames , ia . the prevalence of c felis ( 0.8% ) in north - central ok found in the current study was lower than the previously reported 3.4% in domestic cats in ok . rizzi et al reported a difference in prevalence of c felis infection depending on geographic locations within ok ; 13/77 ( 16.9% ) cats were infected with c felis in eastern ok , while 10/602 ( 1.7% ) cats were infected with c felis in north - central ok . rizzi et al postulated differences in the prevalence of c felis within ok could include strain variation in virulence of c felis , differences in immunologic responses of cats to infection with c felis and differences in c felis inoculation from ticks . three different genotypes of c felis , ribosomal internal transcribed spacer regions ( its ) a , b and c , have been demonstrated , and itsb and itsc seem to be more pathogenic with a higher mortality rate than itsa . although it has not been established which genotype is more prevalent or pathogenic than others in ok , genotypic variations could influence prevalence because cats infected with the more pathogenic genotype are likely to succumb to infection . as cats become infected with c felis through tick bites , prevalence of c felis infection in domestic cats is likely affected by geographic variation of the abundance and activity of ticks and reservoir hosts . our study supports the report of rizzi et al , which demonstrated that the prevalence of c felis in a given enzootic area can vary from location to location and from the population of cats sampled . dna of c felis was not detected in 291 blood samples collected from free - roaming cats in central ia , a non - enzootic state that borders enzootic states . one case of c felis infection in a domestic cat has been reported in south - western ia along the missouri river . unfortunately , this report did not provide details regarding travel history , age , or sex of the infected cat . in our study , blood samples were collected from relatively young cats , and that might have influenced the results as those cats had experienced less time being exposed to tick vectors . as the geographic distribution and range of a americanum expands northwards , it is important to keep monitoring free - roaming cat populations where cytauxzoonosis has not been considered enzootic . historically , only bobcats were thought to be reservoirs for c felis ; however , reports have indicated domestic cats that survive acute cytauxzoonosis become chronically infected with c felis and can also serve as a source of c felis infection . as free - roaming outdoor cats encounter ticks , the source of c felis infections , they are an excellent population to monitor the expansion of c felis distribution in the usa .
case summary cytauxzoonosis is a tick - borne disease of cats , and oklahoma ( ok ) , usa , is considered an enzootic state . to determine the prevalence of cytauxzoon felis , 
 blood was collected from free - roaming cats , as they are frequently exposed to tick vectors . 
 our objective was to determine the prevalence of c felis infection in free - roaming cats in north - central oklahoma and central iowa ( ia ) . 
 infection with c felis was determined using dna extracted from blood and pcr amplification . 
 blood was collected from 380 free - roaming cats between january and april in 2014 in ok . 
 dna from c felis was detected in 3/380 ( 0.8% ; 95% confidential interval [ ci ] 0.222.3% ) . in ia 
 , 292 blood samples were collected between 2012 and 2014 . 
 no c felis - infected cats were detected ( 0 ; 95% ci 00%).relevance and novel information the prevalence of c felis ( 0.8% ) in north - central ok reported herein was lower than the previously reported 3.4% in domestic cats in ok . 
 our study supports that the prevalence in a given enzootic area can vary by location and from the pool of cats sampled . 
 none of 291 ( 0% ) cats were infected with c felis in central ia . to date , only one case of cytauxzoonosis in a domestic cat has been reported in ia . 
 it is important to monitor cats for c felis infections in northern us states , as geographic distribution of amblyomma americanum expands northward . as free - roaming cats have more contact with the tick vectors of c felis , this population allows us to monitor the expansion of c felis distribution .