BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors nonprofit publishers academic institutions research libraries and research funders in the common goal of maximizing access to critical research DETECTION OF FELINE CORONAVIRUS IN CHEETAH ACINONYX JUBATUS FECES BY REVERSE TRANSCRIPTION NESTED POLYMERASE CHAIN REACTION IN CHEETAHS WITH VARIABLE FREQUENCY OF VIRAL SHEDDING Author s Patricia M Gaffney D V M M P V M Dipl A C V P Melissa Kennedy D V M Ph D Dipl A C V M Karen Terio D V M Ph D Dipl A C V P Ian Gardner B V Sc M P V M Ph D Chad Lothamer D V M Kathleen Coleman D V M and Linda Munson D V M Ph D Dipl A C V P Source Journal of Zoo and Wildlife Medicine 43 4 776 786 2012 Published By American Association of Zoo Veterinarians DOI http dx doi org 10 1638 2011 0110R1 1 URL http www bioone org doi full 10 1638 2011 0110R1 1 BioOne www bioone org is a nonprofit online aggregation of core research in the biological ecological and environmental sciences BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies associations museums institutions and presses Your use of this PDF the BioOne Web site and all posted and associated content indicates your acceptance of BioOne s Terms of Use available at www bioone org page terms of use Usage of BioOne content is strictly limited to personal educational and non commercial use Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder Journal of Zoo and Wildlife Medicine 43 4 776 786 2012 Copyright 2012 by American Association of Zoo Veterinarians DETECTION OF FELINE CORONAVIRUS IN CHEETAH ACINONYX JUBATUS FECES BY REVERSE TRANSCRIPTION NESTED POLYMERASE CHAIN REACTION IN CHEETAHS WITH VARIABLE FREQUENCY OF VIRAL SHEDDING Patricia M Gaffney D V M M P V M Dipl A C V P Melissa Kennedy D V M Ph D Dipl A C V M Karen Terio D V M Ph D Dipl A C V P Ian Gardner B V Sc M P V M Ph D Chad Lothamer D V M Kathleen Coleman D V M and Linda Munson D V M Ph D Dipl A C V P Abstract Cheetahs Acinonyx jubatus are a highly threatened species because of habitat loss human conflict and high prevalence of disease in captivity An epidemic of feline infectious peritonitis and concern for spread of infectious disease resulted in decreased movement of cheetahs between U S zoological facilities for managed captive breeding Identifying the true feline coronavirus FCoV infection status of cheetahs is challenging because of inconsistent correlation between seropositivity and fecal viral shedding Because the pattern of fecal shedding of FCoV is unknown in cheetahs this study aimed to assess the frequency of detectable fecal viral shedding in a 30 day period and to determine the most efficient fecal sampling strategy to identify cheetahs shedding FCoV Fecal samples were collected from 16 cheetahs housed at seven zoological facilities for 30 to 46 consecutive days the samples were evaluated for the presence of FCoV by reverse transcription nested polymerase chain reaction RT nPCR Forty four percent 7 16 of cheetahs had detectable FCoV in feces and the proportion of positive samples for individual animals ranged from 13 to 93 Cheetahs shedvirus persistently intermittently or rarelyover 30 46 days Fecal RT nPCR resultswere used to calculate the probabilityof correctly identifying a cheetah known to shed virus given multiple hypothetical fecal collection schedules The most efficient hypothetical fecal sample collection schedule was evaluation of five individual consecutive fecal samples resulting in a 90 probability of identifying a known shedder Demographic and management risk factors were not significantly associated P C20 0 05 with fecal viral shedding Because some cheetahs shed virus intermittently to rarely fecal sampling schedules meant to identify all known shedders would be impractical with current tests and eradication of virus from the population unreasonable Managing the captive population as endemically infected with FCoV may be a more feasible approach Key words Acinonyx jubatus cheetah feces feline coronavirus feline infectious peritonitis virus reverse transcription nested polymerase chain reaction shedding INTRODUCTION Cheetahs Acinonyx jubatus are globally endan gered and the African subspecies is listed as vulnerable by the International Union for Con servation of Nature and as threatened by the Convention on the International Trade in Endan gered Species Appendix I 4 Estimates of the number of cheetahs remaining in the wild range from 10 000 to 12 000 in Africa with the majority in Namibia The wild population is threatened because of habitat loss poaching and conflicts with humans 23 24 To combat decline in numbers and enhance genetic diversity reproduction of the captive cheetah population in the United States is intensively managed by the American Zoo and Aquarium Association AZA Cheetah Species Survival Plan SSP Management practices in clude cohousing and movement of animals be tween zoological facilities for breeding practices that also provide opportunity for disease trans mission Despite concerted efforts the captive cheetah population is not self sustaining 22 In the AZA Cheetah SSP population deaths outnumber births because of poor fertility and a high prevalence of diseases such as chronic gastritis associated with Helicobacter sp amyloidosis glomerulosclerosis and veno occlusive dis ease 21 27 41 Cheetahs are also susceptible to infec tion with feline coronavirus FCoV and infection From the Departments of Pathology Microbiology and Immunology Gaffney Terio Munson and Medicine and Epidemiology Gardner School of Veterinary Medicine University of California Davis California 95616 USA and the Department of Comparative Medicine College of Veterinary Medicine University of Tennessee Knoxville Tennessee 37996 USA Kennedy Lothamer Coleman Present addresses Terio Zoological Pathology Program University of Illinois Maywood Illinois 60153 USA Gardner Department of Health Management Atlantic Veterinary College University of Prince Edward Island Charlottetown Prince Edward Island C1A 4P3 Canada Correspondence should be directed to Dr Gaffney pmgaffney ucdavis edu 776 is often subclinical or causes transient mild enteritis or diarrhea 2 6 8 27 Feline infectious peri tonitis virus FIPV a mutated and deadly variant of feline enteric coronavirus FECV was the cause of an epidemic in a cheetah breeding facility with high morbidity and mortality 6 36 This epidemic combined with widespread serologic evidence of FCoV exposure in the AZA Cheetah SSP population resulted in the restriction of movement of seropositive cheetahs to prevent the spread of infection and increased risk of disease 7 Implementation of this prevention strat egy restricted movement of seropositive cheetahs between facilities thereby preventing pairing of some breeding animals used to maintain genetic diversity FCoV is a member of the family Coronaviridae and is an enveloped single stranded positive sense RNA virus with two serotypes type I and type II and two biotypes FECV and FIPV 33 FECV is transmitted mainly by the fecal oral route infects the mature apical villi of intestinal epithelium and is shed mainly in feces and rarely in saliva 1 34 In domestic cats when fecal shedding of FCoV has been monitored for as long as 7 yr there are variable patterns of shedding that have been described as absent or of low medium or high frequency 1 9 11 Initial infection may be fol lowed by complete viral clearing without shed ding If the virus is not cleared persistent infection and intermittent shedding may result Intermittent shedding of virus also may occur if a cat is reinfected Lastly infection may be followed by continuous shedding suggesting an inability to clear the virus The reasons for the different patterns of fecal shedding after infection with FECV are not completely understood but may be associated with cycles of reinfection or persis tence of virus in colonic enterocytes 13 18 20 Both intermittent and continuous shedders may be asymptomatic In cheetahs it has been suggested that shedding of FCoV in the feces may be intermittent and that shed virus is an important source of infection to other cheetahs 15 In domestic cats FECV is considered a ubiq uitous intestinal virus that is common in multicat households and catteries and it can cause mild transient enteritis and diarrhea yet infection is rarely fatal 18 26 34 35 In cheetahs unlike cats FECV may rarely be associated with chronic ulcerative colitis 15 Munson unpubl data FIPV is a mutated form of FECV and multiple mutations have been identified 3 37 39 Genetic mutations in the 7a7b open reading frame of coronavirus in cheetahs also have been identified 17 After muta tion FIPV acquires the ability to enter and replicate in macrophages and spread systemically resulting in either the effusive wet form the granulomatous dry form or a combination of the two forms of the disease Descriptions of the clinical manifestations and pathologic lesions of FIP are similar between domestic cats and cheetahs including fibrinopurulent pleuritis peri tonitis and vasculitis as well as multifocal necrosis throughout many organs 5 19 36 However cheetahs are proposed to be more susceptible to viral infections such as FCoV due to genetic monomorphism of the major histocompatibility complex associated with a genetic bottleneck Therefore it has been suggested that cheetahs may be more likely to develop fatal FIP when infected with FECV 25 30 31 Historically serologic testing for FCoV was mandated by the AZA Cheetah SSP to identify seropositive cheetahs before movement between zoologic facilities However interpretation of serologic test results is complicated because the detection of serum antibodies may only indicate previous not current infection with the virus and serology does not distinguish between FECV and FIPV In addition as in domestic cats seroposi tivity does not correlate with active infection or fecal shedding of the virus in cheetahs 11 14 16 To use fecal detection of FCoV in cheetah feces as a complementary screening tool to identify actively shedding animals the frequency of fecal shedding of virus needs to be more closely evaluated This study had three objectives 1 to determine the optimal handling and storage procedures for detecting FCoV by reverse transcription nested polymerase chain reaction RT nPCR in fecal samples 2 to assess the frequency of FCoV shedding in cheetahs naturally exposed to FCoV so that a reasonable fecal sampling schedule with a high probability of identifying a cheetah shed ding FCoV can be recommended and 3 to assess potential demographic and management risk factors for association with fecal shedding of virus This information will be used to assess whether fecal RT nPCR for FCoV is a useful test for identifying actively shedding animals and to recommend appropriate testing protocols MATERIALS AND METHODS Study population Twenty five AZA Cheetah SSP cheetahs 8 males and 17 females ranging in age from 1 to 15 yr with a median age of 10 yr from seven geographically distinct zoological facilities in the GAFFNEY ET AL DETECTING FCOV IN SHEDDING CHEETAHS 777 United States Facilities A G were included in the study Table 1 Two cheetahs were wild caught and 23 were captive born Cheetahs selected for the study met one or more of the following criteria prior positive FCoV serology antibody titer 1 40 prior positive FCoV fecal RT PCR housed in or originating from an institution with endemic FCoV or scheduled to move to a facility free of FCoV Positive serologic results were based on previously published crite ria 14 15 In brief FCoV specific antibody titers were measured by indirect immunofluorescence using type I UCD1 and type II WSU 1143 FCoV as capture antigens Antibody titers were defined as the highest dilution that resulted in fluorescence and a titer of C201 40 was considered negative Additional variables including cohous ing number of cage mates sharing of exhibit space and institutional information were ob tained from questionnaires Table 1 Sample collection and storage For each of the 25 cheetahs fecal samples were collected daily for a minimum of 30 and a maximum of 46 days stored at C0208CorC0808C at the participating zoological facility and Table 1 Zoological facility and individual animal information for 25 cheetahs including prior FCoV serology prior FCoV fecal RT nPCR this study s FCoV fecal RT nPCR results and percentage of positive FCoV fecals by RT nPCR for this study s collection period ID no Facility Facility size Facility FCoV Group Age yr Sex Origin Cohoused Diarrhea Prior cheetah FCoV serology Prior cheetah FCoV fecal shedding Current cheetah FCoV fecal shedding C5 C S a N b 2 c 10 F d C e N N Neg f Neg Neg C6 C S N 2 12 M g C N N Neg Neg Neg C24 G S N 2 2 M C Y N Neg Neg Neg C25 G S N 2 2 M C Y N Neg Neg Neg C4 B S Y h 1 i 8F C Y N NS j Neg Neg C3 B S Y 1 14 M C Y N NS Neg Neg C12 D L k Y 1 11 F C N N Neg Neg Neg C13 D L Y 1 4 F W l N N Neg Neg Neg C1 A L Y 2 4 F C N N Neg Neg Neg C2 A L Y 2 14 F C N N Neg Neg Neg C14 E S Y 1 12 F C Y Y Neg Pos Neg C17 D L Y 1 4 F C Y N Neg Pos Neg C18 D L Y 1 10 F C N N Neg Pos Neg C20 F S Y 1 10 F C Y N Neg Pos Neg C21 F S Y 2 10 F C Y Y Neg Pos Neg C22 D L Y 2 1 M C Y N Neg Pos Neg C23 D L Y 2 11 M C Y N Neg Pos Neg C19 F S Y 1 10 M C Y N Pos m Pos Neg C7 D L Y 1 2 F C Y N Neg Pos Pos 43 C8 D L Y 1 2 F C Y N Neg Pos Pos 33 C9 D L Y 1 6 F C N N Neg Pos Pos 13 C10 D L Y 1 2 F W Y N Neg Pos Pos 24 C11 D L Y 1 6 F C N N Neg Pos Pos 35 C15 E S Y 1 13 M C Y Y Neg Pos Pos 23 C16 E S Y 1 15 F C Y Y Pos Pos Pos 93 a S small institution with C2015 cheetahs b N no c 2 five fecal samples assessed d F female e C captive born f Neg negative g M male h Y yes i 1 C2130 days of fecal samples assessed j NS not sampled k L large institution with 15 cheetahs l W wild caught m Pos positive 778 JOURNAL OF ZOO AND WILDLIFE MEDICINE shipped on dry ice to the University of California Davis Before collection of fecal samples avariety of storage scenarios were assessed to determine what storage conditions would allow for optimal detection of FCoV in feces in the event C0808C storage was not available Feces from two chee tahs known to shed FCoV persistently were collected fresh and shipped immediately on dry ice to the laboratory Each sample was thoroughly mixed and separated into different aliquots that were then subjected to variable temperatures and times in storage with and without RNA stabili zation solution RNAlatert Invitrogen Carlsbad California 92008 USA RT nPCR was done in triplicate as described for the study samples see next section Results indicated that FCoV was detectable in these fecal samples when processed immediately and when processed after storage at 48C C0208C andC0808C for up to 7 days Table 2 In addition positive control feces stored at both C0208CandC0808Cfor4yrconsistentlyhad detectable virus by RT nPCR The decision was made that for the study samples normally voided feces could be stored at C0208CorC0808C at the zoological facility depending on the facility s capabilities and then shipped overnight on dry ice and stored at C0808C until samples were processed Sample preparation RNA extraction RT nPCR and sequence analysis Fecal samples were defrosted and then thor oughly mixed A 1 0 ml aliquot was suspended in a 1 2 vol vol of Dulbecco s modified Eagle s medium with 5 fetal bovine serum Invitrogen and the sample was then homogenized by vortex ing and insoluble particles were allowed to settle Total RNA was extracted using TRIzolt LS Invitrogen according to the manufacturer s in structions for biological fluids Reverse transcrip tion was done using the Moloney murine leukemia virus reverse transcriptase kit Invitro gen replacing the oligo dT with a previously published primer primer 211 and nPCR was done as described previously with primers of high sensitivity and specificity for FCoV encompass ing a highly conserved 177 base pair bp region of the 39 untranslated region of the FCoV genome 12 14 Amplification was performed using AmpliTaq polymerase Applied Biosystems Fos ter City California 94404 USA in a Gene Amp PCR System 9700 thermocycler Applied Biosys tems with the following cycling conditions for each of the two amplification reactions one cycle of 1 5 min at 908C followed by 30 cycles of 0 5 min at 508C 1 min at 728C and 1 min at 948C and completed with 2 min at 508C and 5 min at 728C Positive controls for RT nPCR were feces from a persistently shedding cheetah and in vitro prop agated FCoV strain WSU1143 American BioRe search Seviervile Tennessee37864 USA Water Invitrogen was the negative control A 10 ll sample of amplified product was separated by electrophoresis on a 1 5 agarose gel and visualized with UV light after staining with ethidium bromide Bio Rad Laboratories Hercu les California 94547 USA PCR product identi ty was confirmed by direct sequencing of a subgroup n 10 of 177 bp amplification prod ucts PCR products were purified using a Cen tricon 100 column Millipore Bedford Maryland 01730 USA and nucleotide sequencing was done using an ABI 3730 capillary electrophoresis Table 2 Results of RT nPCR for FCoV on cheetah feces stored for increasing lengths of time at different temperatures with and without RNA stabilization solution for fecal samples run in triplicate Condition Temp 8C Days to extraction RNAlater RT nPCR result chronic shedder RT nPCR result intermittent shedder A 20 25 0 N a b C0 c B43 C0 C45 D47N C0 E C020 7 N C0 F C080 7 N C0 G 20 25 7 Y d C0 H4 C0 I C020 7 Y C0 a N no b all three replicates positive c C0 one or two replicates positive d Y yes GAFFNEY ET AL DETECTING FCOV IN SHEDDING CHEETAHS 779 genetic analyzer and BigDye terminator v 31 cycle sequencing University of California Davis Cal ifornia 95616 USA Subsequent amplification products visualized at 177 bp were interpreted as positive One subset of cheetahs n 16 referred to hereafter as group 1 had 30 to 46 days of feces analyzed A second subset of cheetahs n 9 referred to hereafter as group 2 had five consec utive fecal samples analyzed to assess the use of five consecutive individual samples for detection of a shedding animal Statistical analysis The presence or absence of detectable FCoV in consecutive fecal samples as detected by RT nPCRwas the outcome variable To determine the probability of correctly identifying a cheetah shedding FCoV by analyzing fewer than 30 samples cheetah 12 hypothetical schedules of fecal sample collection and analysis by RT nPCR were applied to the known daily fecal RT nPCR results of the seven cheetahs with detectable FCoV in their feces The proportion of the seven cheetahs that would be correctly identified as fecal shedders with each collection schedule was calculated and compared with the number of samples needed for that schedule The schedule correctly identifying the highest proportion of shedding cheetahs using the smallest number of samples over the fewest sampling days was considered the most efficient The first day a fecal result was to be assessed was assigned using a random number generator Microsoft Office 2007 Excel Microsoft Redmond Washington 98052 USA Scenarios were run in triplicate and the results averaged Table 3 If the day s to be assessed had no feces collected the result from the next consecutive sample was used Fisher s exact test EpiInfo v 3 5 1 Centers for Disease Control and Prevention Atlanta Georgia 30333 USA was used to assess the association between potential risk factors and the presence or absence of FCoV in feces in group 1 Risk factors evaluated included age dichotomized into juve nile C202 yr or adult 2 yr sex facility size dichotomized into small C2015 cheetahs or large 15 cheetahs wild caught or captive born status termed origin presence or absence of diarrhea at the time of fecal collection cheetah s