【病毒外文文獻(xiàn)】2001 DETECTION OF FELINE CORONAVIRUS INFECTION IN CAPTIVE CHEETAHS (ACINONYX JUBATUS) BY POLYMERASE CHAIN REACTION (1)

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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 INFECTION IN CAPTIVE CHEETAHS ACINONYX JUBATUS BY POLYMERASE CHAIN REACTION Author s Melissa KennedyD V M Ph D Scott CitinoD V M Terry DoloricoD V M Amanda Hillis McNabbB S Amy Serino MoffatB L S and Stephen KaniaPh D Source Journal of Zoo and Wildlife Medicine 32 1 25 30 Published By American Association of Zoo Veterinarians DOI http dx doi org 10 1638 1042 7260 2001 032 0025 DOFCII 2 0 CO 2 URL http www bioone org doi full 10 1638 1042 7260 282001 29032 5B0025 3ADOFCII 5D2 0 CO 3B2 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 25 Journal of Zoo and Wildlife Medicine 32 1 25 30 2001 Copyright 2001 by American Association of Zoo Veterinarians DETECTION OF FELINE CORONAVIRUS INFECTION IN CAPTIVE CHEETAHS ACINONYX JUBATUS BY POLYMERASE CHAIN REACTION Melissa Kennedy D V M Ph D Scott Citino D V M Terry Dolorico D V M Amanda Hillis McNabb B S Amy Serino Moffat B L S and Stephen Kania Ph D Abstract Feline coronavirus genetic elements were detected by polymerase chain reaction from blood fecal samples and effusive fluid collected from 33 cheetahs in the U S A Feline coronavirus specific serum antibodies were also measured by indirect immunofluorescence Ten cheetahs were positive for viral shedding by polymerase chain reaction whereas 13 were seropositive by immunofluorescence Results of serology did not consistently correlate with shedding of virus and the capture antigen used for detection of feline coronavirus specific antibodies had a significant impact on results Testing of samples from one population over a 1 yr period indicated chronic infection in some animals These relatively healthy carrier animals were a source of virus for contact animals Screening programs in cheetah populations for feline coronavirus infection may be most reliable if a combination of serologic analysis and viral detection by polymerase chain reaction is used Key words Feline coronavirus feline infectious peritonitis cheetah Acinonyx jubatus epidemiology polymerase chain reaction INTRODUCTION Feline coronavirus FCoV is an important path ogen of both domestic and nondomestic felines 1 14 Disease resulting from infection may vary in se verity from subclinical to such severe life threat ening disease as feline infectious peritonitis FIP Both host and virus related factors may influence the severity of disease but the specific factor re mains certain 9 16 Diagnosis is complicated by the existence of at least two antigenically distinct se rotypes of FCoV types I and II 9 15 Spike proteins in the two types differ with type II encoding a spike protein very similar to that of canine coro navirus 7 Within both of these serotypes virulent FIP and avirulent biotypes occur 7 15 Serious dis ease may arise from FCoV mutation in the intesti nal tract of infected cats 15 Neither serology nor ge netic analysis can distinguish the biotypes so it is not possible to screen for virulent FCoV Cheetahs Acinonyx jubatus are especially vul nerable to FCoV induced disease 1 13 so the epide miology and molecular biology of FCoV as well as optimal screening methodology must be under stood in order to manage captive cheetah popula tions From the Comparative Medicine Department College of Veterinary Medicine University of Tennessee Knox ville Tennessee 37901 1071 USA Kennedy Dolorico McNabb Soffat Kania and the White Oak Conservation Center 3823 Owens Road Yulee Florida 32097 2145 USA Citino Present address Kennedy Department of Comparative Medicine College of Veterinary Medicine The University of Tennessee Knoxville Tennessee 37901 1071 USA Polymerase chain reaction PCR technology can detect FCoV in domestic cat populations 4 8 10 Our report describes an assay for the detection of co ronavirus genomic elements in biological samples including feces and plasma from cheetahs MATERIALS AND METHODS We evaluated six serum samples 26 plasma sam ples nine whole blood samples one abdominal ef fusion sample and 82 fecal specimens from 33 cap tive cheetahs in the U S A for the presence of FCoV by PCR techniques For some animals mul tiple samples were submitted and with population F see below samples from multiple time points were submitted Feces were tested in order to detect virus shedding For six animals only blood and abdominal effusion from one animal of these was submitted The abdominal effusion was collected from a sick cheetah that later died from histopathologically confirmed FIP Eighteen of the cheetahs were in a collection in which two cheetahs may have died from FCoV see description below In this popu lation F most animals were tested every 4 mo or less over a period of 1 yr during which time the second death occurred The first death occurred pri or to this investigation Two animals in this popu lation were tested monthly Single samples were obtained from the other cheetahs at the other five institutions A E in the U S A along with relevant individual health information History of population F Two female cheetahs 32 and 33 allegedly se ronegative for FCoV arrived at the institution in 26 JOURNAL OF ZOO AND WILDLIFE MEDICINE March 1995 They were quarantined for 16 mo al though three other females were housed nearby in the quarantine section These three females were not in direct contact with the imported females until January 1997 In January 1996 the first episodes of abnormal stools loose to overt diarrhea were noticed in the two imported females as well as in the three nearby resident female cheetahs In March 1996 one of these resident females died from nec rotizing colitis but tissue samples were not avail able for FCoV analysis Abnormal stools continued in the remaining resident contact females and the two imported females through 1997 1998 A male cheetah was brought into direct contact with the imported females for the first time in Jan uary 1997 with extensive fence contact from Sep tember 1997 through January 1998 This male died in January 1998 of leukoencephalomalacia Also in January 1997 two male cheetahs were moved into an enclosure immediately after the two imported females were removed from it These males subsequently had their first direct exposure to the imported females from September 1997 to March 1998 One of the males died in July 1998 from necrotizing colitis None of the remaining cheetahs in population F have been in contact with the imported females the contact females and males or any of these animals enclosures Five surviving cheetahs in population F there fore including the imported females formed the exposed group All keepers cared for the entire carnivore collection When the infection status of the exposed group was discovered quarantine pro cedures were implemented and the exposed group was not intermingled with the other cheetahs Within population F the individual s virus infec tion status over a 12 mo period was evaluated by PCR RNA extraction reverse transcription and PCR All biological samples were stored at 2708C To tal RNA was extracted from the specimens with Trizol LS Gibco BRL Baltimore Maryland 21279 USA according to the manufacturer s di rections The RNA was used for reverse transcrip tion with Moloney murine leukemia virus reverse transcriptase Gibco BRL according to the manu facturer s recommendations with the downstream external primer 10 Nested PCR was done with ExTaq polymerase Intergen Purchase New York 10577 USA 6 Primers encompassing the entire 7a7b open reading frame ORF containing ap proximately 1 000 nucleotides were used for am plification 10 11 In vitro propagated FCoV strain WSU1143 was the positive PCR control American BioResearch Sevierville Tennessee 37864 USA and water was the negative control Products were evaluated by electrophoresis on 1 agarose gels Serology FCoV specific antibody titers were measured by indirect immunofluorescence for all 28 cheetahs from which serum or plasma was provided 1 A type I UCD1 and a type II WSU 1143 FCoV Amer ican BioResearch were used as capture antigens These viruses were isolated from two separate cases of FIP Each was propagated separately in Crandell feline kidney cells The virus infected cells were applied and fixed to glass slides Twofold serial di lutions of the serum plasma were made starting at a 1 5 dilution and proceeding to a maximum of 1 5 120 Serologic testing for FCoV specific antibod ies was performed on serial dilutions of serum start ing at a 1 5 dilution in order to ensure that any antibody level was detected Though low titers are considered insignificant in terms of FIP disease di agnosis we were interested in detecting any pre vious exposure to a FCoV 9 Antibody positive and negative serologic controls VMRD Pullman Washington 99163 USA were purchased Anti body was detected with rabbit anti feline IgG con jugated to fluorescein isothiocyanate VMRD The titer was reported as the reciprocal of the highest dilution in which fluorescence was observed An tibody titers of 5 were considered negative RESULTS PCR results Results are shown in Tables 1 and 2 with a pos itive PCR result indicating that FCoV was detected by PCR in at least one sample from that animal Ten animals 30 were PCR positive Five of 15 cheetahs 33 from five U S A institutions other than population F tested positive by PCR in bio logic samples primarily feces One of the five cheetahs was positive in feces but negative in blood one tested positive in blood only no feces submitted one in effusive fluid only no feces sub mitted and two tested positive in both feces and blood Five of the cheetahs in population F 28 all either imported or exposed to the imported chee tahs tested positive for FCoV in feces Table 2 Three of these 17 were positive on more than one occasion Four of them 22 tested positive in samples collected in July 1998 which thus ap pears to have been a peak shedding time 27KENNEDY ET AL DETECTION OF CHEETAH CORONAVIRUS INFECTION Table 1 Health status and feline coronavirus FCoV polymerase chain reaction PCR and serologic results for cheetahs from institutions A E Cheetah number Facility Health status PCR results Serology FCoV I FCoV II 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 A A A B C C C C D D D D E E E Feline infectious peritonitis Healthy Healthy Healthy Healthy Healthy Healthy Healthy Gastritis Loose feces Chronic diarrhea Healthy Healthy Weight loss poor appetite Healthy 1 2 2 1 2 2 1 2 1 2 2 2 1 2 2 640 5 320 5 5 5 5 640 5 5 20 5 160 5 ND a 5 5 40 5 5 5 5 5 5 5 5 5 5 5 ND a ND 5 not done Table 2 Health status and feline coronavirus FCoV polymerase chain reaction PCR and serologic results for captive cheetahs from institution F Cheetah number Facility Health status PCR results Serology FCoV I FCoV II 16 17 18 19 20 21 22 23 24 25 26 27 b 28 b 29 b 30 b 31 b 32 d 33 d F F F F F F F F F F F F F F F F F F Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Intermittent abnormal feces Intermittent abnormal feces Intermittent abnormal feces c Intermittent abnormal feces Intermittent abnormal feces Intermittent abnormal feces 2 2 2 2 2 2 2 2 2 2 2 2 1 1 2 1 1 1 5 5 5 5 ND a 40 10 5 5 ND ND 20 320 10 ND 20 640 5 120 5 5 5 5 ND 5 5 5 5 ND ND 5 5 5 ND 5 5 640 a ND 5 not done b Exposed to imported females c Death due to necrotizing colitis July 1998 d Imported females Samples were available from only one of the dead animals the male that died of necrotizing co litis in January 1998 PCR tests on feces collected monthly for the 3 mo preceding its death were neg ative However the cheetah s FCoV specific anti body level rose from negative in 1996 to 625 in March 1997 2 mo postexposure to imported fe males to 3 125 in June 1998 1 mo prior to death 28 JOURNAL OF ZOO AND WILDLIFE MEDICINE This testing done at another reference laboratory as part of the routine monitoring of the cheetah population is not included in our other data Fecal samples from the two imported females collected monthly from May to August 1998 in December 1998 and in March 1999 were positive by PCR for FCoV showing constant shedding from these animals The individuals may be chronically FCoV infected FCoV serology Serology with type I FCoV as the capture antigen identified 13 cheetahs 39 with detectable anti body levels Tables 1 2 However when type II FCoV was used only two cheetahs 6 were se ropositive Five cheetahs 15 had antibody titers 80 to type I FCoV but were seronegative for type II FCoV Three of the 10 PCR positive cheetahs 33 were seronegative to both types I and II FCoV Ta ble 1 Six of the 13 seropositive cheetahs 46 were negative by PCR Tables 1 2 Serologic screening did not identify all cheetahs shedding vi rus Conversely PCR did not detect all animals pre viously exposed to FCoV All five PCR positive cheetahs from population F were seropositive to type I FCoV Table 2 Two of these resident females had low titers to type I whereas a male had a titer of 320 to type I The two imported females had titers 640 Four of the five were seronegative to type II One of the im ported females with a titer of 640 to type I was seronegative to type II The other imported female had a titer of 5 120 to type I and 640 to type II The contact male that died in July 1998 had a rising antibody level in March 1997 and June 1998 test ing done at another laboratory Cheetah health status Three deaths occurred within population F since the importation of two females in 1995 one from leukoencephalomalacia and two from necrotizing colitis that may have involved FCoV although only one of the cheetahs with colitis was tested This animal was negative by PCR with the 7a7b primer set three times between May and July 1998 when it died The remaining cheetahs from population F were healthy though abnormal stools were noted on repeated occasions in five animals Each of these was PCR positive in at least one sample Of the remaining cheetahs not members of pop ulation F one was verified by histopathology to have died of FIP This animal s abdominal effusion was PCR positive One cheetah at another institu tion was suffering from chronic diarrhea and al though this animal was PCR negative a contact cheetah was PCR positive A third PCR positive cheetah was from a population in which a contact animal had experienced weight loss and decreased appetite and another contact had suffered diarrhea The remaining two PCR positive cheetahs and their contacts are healthy to date DISCUSSION The 7a7b genes the 39 most ORFs of the FCoV genome were targeted for amplification These genes were used for several reasons including the consistent success our laboratory has had in ampli fying FCoV genetic material regardless of virus strain the proposed association of this region with virulence and the possibility that the avirulent form of FCoV does not express the 7b protein 15 16 Al though the functions of the 7a7b gene products are unknown this region is associated with virulence because deletions that occur in this region in some virus isolates have been shown to lead to decreased virulence of the virus 8 15 16 PCR proved to be a sensitive assay for detection of virus shedding FCoV is prevalent among cap tive cheetahs in the U S A because nearly one third of the animals we tested were shedding FCoV in their feces or had detectable virus in plasma Al though not all infected animals exhibited charac teristic FIP disease consistent with FCoV infection had occurred in four of the six infected cheetah populations FCoV may be a factor in cheetah gas trointestinal diseases particularly those exhibiting such vague signs as abnormal stools decreased ap petite and or weight loss Our results indicate that a combination of sero logic analysis and PCR detection of virus shedding may be needed to detect FCoV infection Serology results do not consistently correlate with PCR re sults for detection of virus shedding as in FCoV infected domestic cat populations 5 10 Seronegative animals were occasionally virus positive whereas the converse was also true Thus although serology can detect previous exposure to the virus PCR is more sensitive for detecting viral shedding Addi tionally the capture antigen used for detection of FCoV specific antibodies had a significant impact on the results with type II FCoV frequently leading to false negative results Type II FCoV biotypes are more closely related to canine coronavirus antigen ically than to type I FCoV 7 Although types I and II FCoV cross react their antigenicities are suffi ciently different that low titers to one type may be missed when using the other to measure virus spe cific antibody Thus serologic screening that uses only one serotype is inherently flawed In addition 29KENNEDY ET AL DETECTION OF CHEETAH CORONAVIRUS INFECTION one isolate of cheetah FCoV is antigenically dis tinct from FCoV of domestic cats 2 6 This antigenic diversity impacts serology results and may explain our findings The antigenic disparity between the infection and assay viruses may result in failure of antibody detection The epizootiology of captive population F was examined From the history and PCR data the two imported females were probably chronically infect ed with FCoV when they arrived at this institution Chronic carrier states lasting a period of months occur in domestic cats 12 15 Infection of additional cheetahs probably occurred by direct and or indi rect exposure primarily through shared enclosures and through the fence contact As noted peak shedding in the population occurred in July 1998 Male stimulation trials involving across the fence access of these males to females for the purpose of stimulating reproductive activity in the female and selection of male mates began in May 1998 Re sulting stress may have induced viral shedding or predisposed the cheetahs to infection Similar pat terns of shedding have been noted in closed popu lations of domestic cats with waxing and waning of both infection and viral shedding 3 Recurrent ab normal feces occasionally severe were noted in this group Two cheetahs died of necrotizing colitis possibly related to FCoV infection Samples were available from only one of these and were PCR negative possibly due to viral nucleotide sequence variation including deletions in the genomic region amplified that we have documented in population F leading to failure of amplification due to loss of primer binding sites unpubl results FCoV is clearly an important contagious patho gen of captive cheetahs Carrier animals may be an important source of infection through direct and in direct contact with susceptible animals and serious disease may occur in some infected cheetahs Screening for infection only with serology espe cially if only one serotype is used is not ideal The optimal screening methodology uses both serologic analysis and PCR fecal virus detection Total RNA extraction reverse transcription and nested PCR were used to amplify the 7a7b ORFs of the FCoV genome This region amplifies consis tently in samples from domestic cats 9 We also eval uated the level of FCoV specific antibodies by in direct immunofluorescence in the majority of ani mals tested by PCR Comparison of virus infection by PCR for viru