【病毒外文文献】2012 No Serologic Evidence for Zoonotic Canine Respiratory Coronavirus Infections among Immunocompetent Adults_span___sp

ORIGINAL ARTICLE No Serologic Evidence for Zoonotic Canine Respiratory Coronavirus Infections among Immunocompetent Adults W S Krueger G L Heil and G C Gray Emerging Pathogens Institute and College of Public Health and Health Professions University of Florida Gainesville Florida USA Impacts An emerging infectious disease first seen in persons with intense canine exposures could indicate that a canine pathogen has gained the ability to spread across species While seroepidemiological studies have their limitations they are often employed as a valid first step in examining the potential for zoonotic spread of animal pathogens This study supports the premise that immunocompetent adults are not at risk for canine respiratory coronavirus CRCoV infections however infrequent cross species transmission of CRCoV cannot be ruled out Introduction It is important to monitor the human animal nexus for emerging zoonotic pathogens that gain the ability to cross the species barrier This is especially true among high risk occupational settings where animal workers have close and prolonged contact with many animals Recently agri cultural professionals in routine contact with production animals have been shown to have an increased risk of zoonotic infections and often experience symptomatic ill nesses Meng et al 2002 Olsen et al 2002 Koopmans et al 2004 Myers et al 2006 Gray and Baker 2007 Gray et al 2007a b 2008 Kayali et al 2010 Dogs are a popular companion animal but the canine industry is a less studied occupational setting Overcrowded shelters and breeding kennels create the perfect environment for Keywords Coronavirus zoonoses occupational exposure communicable diseases emerging seroepidemiological studies Correspondence Whitney Krueger Global Pathogens Laboratory Emerging Pathogens Institute and Department of Environmental and Global Health College of Public Health and Health Professions University of Florida 2055 Mowry Road PO Box 100009 Gainesville FL 32611 USA Tel 352 273 9569 Fax 352 273 9420 E mail wsbaker phhp ufl edu Received for publication March 2 2012 doi 10 1111 zph 12005 Summary Zoonotic diseases continue to emerge and threaten both human and animal health Overcrowded shelters and breeding kennels create the perfect environ ment for amplified infectious disease transmission among dogs and present a critical opportunity for zoonotic pathogens to emerge and infect people who work in close contact with dogs Coronaviruses widespread prevalence exten sive host range various disease manifestations and increased frequency of recombination events all underline their potential for interspecies transmission Methods Mol Biol 2008 454 43 The objectives of this study were to deter mine whether people with occupational contact with dogs were more likely to have antibodies against canine respiratory coronavirus CRCoV compared to persons with no dog exposure A seroepidemiological cohort study was com pleted for which 302 canine exposed and 99 non canine exposed study sub jects enrolled in the study by providing a serum sample and completing a self administered questionnaire A competitive enzyme linked immunosorbent assay ELISA was developed to detect human antibodies against CRCoV while con trolling for cross reacting antibodies against the human coronavirus OC43 All study subjects were negative for antibodies against CRCoV by this competitive ELISA This study supports the premise that humans are not at risk for CRCoV infections however infrequent cross species transmission of CRCoV cannot be ruled out Zoonoses and Public Health 2012 Blackwell Verlag GmbH Zoonoses and Public Health 1 amplified infectious disease transmission among dogs and a critical opportunity for zoonotic pathogens to emerge and infect people who work in close contact with dogs First identified in 2003 in the United Kingdom canine respiratory coronavirus CRCoV is a newly emerged host variant of the enteric canine coronavirus CCoV Similar to how other coronaviruses undergo genetic evolution CRCoV evolved through accumulations of point muta tions insertions and deletions within the coronavirus genome Decaro and Buonavoglia 2008 Following its discovery evidence of CRCoV infection or seropositivity has been documented in dogs with various clinical histories in Japan Italy New Zealand Korea Canada and the United States CRCoV plays a role in the canine infectious respiratory disease complex CIRD or kennel cough and is now considered enzootic among dog pop ulations Priestnall et al 2006 Erles and Brownlie 2008 Human infections with CRCoV have never been reported or studied To investigate evidence of CRCoV infections in humans a seroepidemiological cohort study was con ducted in the United States We sought to test the hypothesis that dog workers would have a higher prevalence of antibodies against CRCoV compared to non dog exposed controls Materials and Methods Participant recruitment and enrolment This study was approved by the University of Iowa and the University of Florida s institutional review boards The target population included breeders kennel employ ees veterinary personnel animal shelter workers grey hound racetrack employees and dog show handlers whose work or hobby involved exposure to multiple dogs A non exposed non matched control group consisted of individuals who had neither been exposed to multiple dogs as part of their work or hobby nor had pet dogs in their household in the last 5 years All participants had to be at least 18 years of age and self report no current immunocompromising conditions Recruitments were based on a convenience sample of the target population primarily from Iowa and Florida Breeders shelters and veterinary clinics were identified through state databases of licensed breeders and practic ing veterinarians as well as through internet searches Organizations and staff were invited to participate in the study via a mailed letter with a telephone call follow up Enrolments typically occurred at the participants place of employment Recruitments also occurred at large public venues including dog shows agility trials and trade shows Non exposed controls were faculty staff and stu dents from the University of Iowa and the University of Florida After informed consent was obtained participants completed a self administered questionnaire and permit ted collection of a blood specimen via venipuncture at a single encounter The questionnaire collected demo graphic data specific dog exposure data and behavioural data including personal hygiene practices when caring for dogs Dog years of exposure for a specific occupation hobby was calculated by multiplying the average number of dogs with which the subject came in close contact on a given day for the occupation hobby by the total years worked in the occupation hobby Whole blood specimens were transported on ice to the laboratory within a few hours of collection Blood tubes were centrifuged at 3000 g for 15 min at room tempera ture to separate serum All collected serum was aliquoted and frozen at 80C176C Laboratory methods Culturing of CRCoV and HCoV for use as a capture antigen and antagonist in a competitive ELISA The human colorectal adenocarcinoma cell line HCT 8 ATCC catalog CCL 224 was propagated in modified Roswell Park Memorial Institute RPMI media RPMI 1640 Gibco C210 Invitrogen Carlsbad CA USA 10 mm HEPES buffer Fisher Scientific Pittsburgh PA USA 5 foetal bovine serum FBS 10 glucose 100 mm sodium pyruvate 100 mg ml streptomycin Fisher Scien tific and 100 000 IU penicillin Fisher Scientific as pre viously described Erles et al 2007 A CRCoV polymerase chain reaction PCR positive canine respira tory swab provided by Dr Edward Dubovi at Cornell University was diluted 1 4 in RPMI infection media FBS dropped to 2 and used to inoculate a suspension of freshly trypsinized HCT 8 cells The suspension was then incubated for 1 h at 35C176C with 5 CO 2 on a rock ing platform The infected suspension was then seeded onto a 150 cm 2 cell culture flask Corning Corning NY USA 15 ml of infection media was added and the cells were allowed to adhere A mock infected flask of HCT 8 cells was included as a negative control After 5 days the cells had reached 80 90 confluency and were harvested as previously described Priestnall et al 2006 Propagation of CRCoV was also attempted on two canine respiratory tract cell lines however viral titre never surpassed the threshold of that provided by cultur ing on HCT 8 cells In attempts to obtain higher titres of CRCoV by serial passage on HCT 8 cells it was observed that after 5 7 viral passages the titre would dramatically decrease therefore for development of the competitive ELISA virus at passage 1 on HCT 8 cells was used For use as the antagonist for a competitive ELISA human coronavirus HCoV OC43 was also propagated in HCT 8 cells Briefly cells were seeded in 150 cm 2 cell Canine Respiratory Coronavirus and Man W S Krueger et al 2 2012 Blackwell Verlag GmbH Zoonoses and Public Health culture flasks Corning and upon reaching 90 conflu ency monolayers were washed three times with plain RPMI 1640 media GibcoC210 Invitrogen and inoculated with 1 ml of HCoV OC43 passage 7 ATTC VR 1558 at 9 10 7 TCID 50 ml diluted 1 4 in RPMI infection media An additional 15 ml of RPMI infection media was then added and cells were incubated at 37C176C with 5 CO 2 until 70 90 cytopathic effect CPE was observed microscopically 24 h This virus stock was saved as pas sage 8 and later blind passaged as described above with out re calculating its TCID 50 ml to ensure fresh viral culture supernatant was used during the competitive ELISA A mock infected flask was included as a negative control Real time RT PCR for the detection of CRCoV in cell culture Real time reverse transcription polymerase chain reaction qRT PCR was performed with proprietary primers and probe obtained from the University of Wisconsin to detect the presence of CRCoV in the cell culture RNA was extracted from the HCT 8 cell culture supernatant with the QIAamp Viral RNA Mini Kit Qiagen Valen cia CA USA and from infected HCT 8 cells with the RNeasy Mini Kit Qiagen according to the manu facturer s instructions One step qRT PCR was run using Superscript III Reverse Transcriptase with Platinum Taq Invitrogen at the following conditions 42C176C for 15 min 95C176C for 2 min 40 cycles of 95C176C for 15 s and 53C176C for 30 s Cycle threshold C T values were exam ined to determine the number of cycles required for the fluorescent signal to cross a threshold background level As per a previous report Priestnall et al 2006 the protein concentration of the CRCoV HCoV OC43 and negative control cell lysates was determined with the Pierce Coomassie Bradford UK colorimetric protein assay kit Thermo Fisher Scientific Pierce Biotechnology Rockford IL USA according to the manufacturer s instructions Optical densities of the samples were determined using the Powerwave 340 automated microplate spectrophotometer Biotek Winooski VT USA Referencing a standard curve with bovine serum albumin protein concentrations reported as lg ml were extrapolated Development of a competitive ELISA for CRCoV To detect CRCoV antibodies in human sera while also controlling for cross reacting antibodies against HCoV OC43 an ELISA designed to detect CRCoV antibodies in canine serum Erles et al 2003 Priestnall et al 2006 was adapted Sera from CRCoV positive and CRCoV neg ative dogs were used as positive and negative assay con trols CRCoV infected and uninfected control cell culture lysates were diluted to approximately 20 lg ml protein in carbonate bicarbonate buffer pH 9 6 The diluted antigen suspensions were added to alternating duplicate columns of clear 96 well flat bottom high binding Immulon C210 2HB polystyrene microtiter plates Thermo Scientific Rochester NY USA Plates were sealed to prevent evap oration or contamination and incubated overnight at 4C176C for optimal protein binding Next the plates were washed three times with phosphate buffered saline PBS and blocked by the addition of 300 ll of a solution of 5 non fat milk Nestle Carnation Wilkes Barre PA USA in PBS for 1 h at room temperature Plates were washed once with PBS Undiluted human sera were mixed 1 1 with HCoV OC43 culture supernatant and incubated at 37C176C for 1 h Then 50 ll of the mixture diluted 1 50 in dilution buffer 5 non fat milk and 0 05 Tween C210 20 Fisher Scientific in PBS resulting in a final 1 100 dilu tion of the sera was added to the plates in duplicate to wells coated with CRCoV infected and uninfected cell culture lysates ELISA plates were incubated at 37C176C for 1 h and then washed three times in wash buffer 0 05 Tween C210 20 in PBS Detection of human IgG bound to the plates was accomplished by the addition of 50 llof goat anti human IgG conjugated to horseradish peroxi dase HRP KPL diluted 1 6000 in dilution buffer was added to wells where blocked human sera had been added To detect dog IgG in the positive and negative control wells 50 ll of rabbit anti dog IgG conjugate con jugated to HRP Sigma Aldrich St Louis MO USA diluted 1 5000 was added to the control wells Following a 1 h incubation at room temperature plates were washed three times in wash buffer Detection of the HRP conju gated antibodies bound to the plates was accomplished by the addition of 100 ll of tetramethylbenzidine peroxidase substrate 2 TMB KPL After a 10 min incubation in the dark at room temperature the TMB reaction was stopped by the addition of 100 ll 1 N sulphuric acid Fisher Scientific Within 30 min of stopping the reaction absorbance was read at 450 nm wavelength using the Pow erwave 340 automated microplate spectrophotometer Biotek Values from the duplicate wells of CRCoV coated and uninfected cell control wells were averaged for each serum sample A serum sample was considered positive for antibodies against CRCoV when the average absorbance of the CRCoV well exceeded three standard deviations above the average absorbance of the control wells Student s t test was used to compare continuous vari ables and Wald chi square test was used to compare cat egorical variables Logistic regression was used to compare optical density OD levels between the exposure groups and ascertain odds ratios and associated confi dence intervals Analysis was performed using sas v9 2 SAS Institute Cary NC USA W S Krueger et al Canine Respiratory Coronavirus and Man 2012 Blackwell Verlag GmbH Zoonoses and Public Health 3 Results Between 2007 and 2010 a total of 302 canine exposed subjects and 99 non canine exposed controls granted informed consent completed the enrolment questionnaire and submitted a serum sample Demographically the gender distribution was identical between exposure groups but the controls tended to be younger than the exposed group means of 33 and 43 years old respec tively Overall the participants were more likely to be women 68 and 79 resided in Iowa or Florida where the majority of enrolments took place Table 1 illustrates the work hobbies involving close contact approximately 3 ft with dogs as reported by participants respondents were allowed to indicate more than one occupation hobby A single occupation hobby involved a median of 80 dog years of exposure Breeders tended to a median of three breeding females in their kennels There was no serological evidence of previous exposure to CRCoV among the study population based upon results of the competitive ELISA The frequency of mean OD levels indicated no apparent outliers In addition there was no significant difference in the mean OD levels average of the test wells average of the negative control wells 3 standard deviations between the two groups The mean OD was 0 03 for both canine exposed subjects and non exposed controls with no significant difference between the groups examining both continu ous OD data and OD levels categorized into quartiles Table 2 The canine positive control serum had a mean OD of 0 013 which was 3 standard deviations above its negative control well The canine negative control serum had a mean OD of 0 066 Discussion Variations in CoV host range specificity and pathogenesis are attributed to the spike glycoprotein Gallagher and Buchmeier 2001 After entry into the body CoVs attach to specific cellular receptors via the spike protein Weiss and Navas Martin 2005 CRCoV is transmitted through inhalation of infected aerosolized droplets however its pathogenesis in dogs is still unknown CRCoV likely elic its only a subclinical or asymptomatic disease in dogs but damage to the respiratory epithelium during viral replication may lead to clinical secondary infections by other respiratory pathogens Buonavoglia and Martella 2007 CRCoV may also function as a primary pathogen for infection Priestnall et al 2006 If CRCoV s spike protein was to gain affinity for the human respiratory epithelial cell receptor that HCoV OC43 employs Weiss and Navas Martin 2005 CRCoV could potentially replicate in human cells and cause human infections Based on the current published litera ture this is the first study to examine the possibility of zoonotic infections with CRCoV among humans however results show no evidence of previous exposure to CRCoV among immunocompetent adults as no antibodies against CRCoV were detected There was no difference in ELISA OD between dog workers and unexposed controls Cross reactivity was a substantial obstacle to overcome when designing a serological assay A competitive ELISA was developed to control for cross reacting antibodies and detect specific antibodies against CRCoV The Group 2a HCoV OC43 was chosen as the CRCoV ELISA com petitor owing to the high amino acid identities between various viral proteins up to 98 homologous Erles et al 2007 Lorusso et al 2009 Strain OC43 is the HCoV most closely related to CRCoV by phylogenetic analyses Kaneshima et al 2006 and therefore consi dered most likely to cross react with CRCoV antibodies Table 1 Occupations hobbies and associated levels of dog exposure as cited by subjects Occupation a N Median dog years of exposure IQR b c Breeder 101 60 25 250 Veterinary staff 90 79 24 200 Kennel staff 72 60 30 300 Veterinarian 63 140 80 264 Shelter staff 47 54 16 160 Trainer 38 50 12 160 Kennel owner 30 225 117 520 Groomer 23 50 14 210 Racetrack staff 16 540 200 1560 Dog show handler 12 60 26 286 Owner Hobbyist 7 50 18 90 Researcher 2 19 5 32 Pet store staff 1 180 180 180 a Subjects allowed to cite multiple occupations b Calculated as the reported number of years multiplied by the average number of dogs per day c Interquartile range Table 2 Serologic results for human antibodies against canine respi ratory coronavirus based on optical density OD readings between exposed and non exposed study groups Variable Exposed n 302 Controls n 99 P value OR 95 CI Mean OD SD 0 03 0 11 0 03 0 12 0 97 1 02 0 7 1 5 OD Quartiles First 75 24 8 24 24 2 0 80 1 1 0 7 1 6 Second 76 25 2 25 25 3 Third 76 25 2 25 25 3 Fourth 75 24 8 25 25 3 Canine Respiratory Coronavirus and Man W S Krueger et al 4 2012 Blackwell Verlag GmbH Zoonoses and Public Health This study had a number of limitations A key limit ing factor was the inherently imperfect nature of sero logical assays By design antibodies are not rigidly specific Infection with one virus or bacterium can render a person immune to attack by a closely related pathogen thus reducing the incidence of infections Although fortuitous in nature this can present a difficult obstacle in serological diagnoses Because com pletely controlling for cross reacting antibodies is often unachievable epidemiological studies frequently employ comparison groups and statistical adjustments to control for this limitation In the case of this study both of these approaches did not overcome the lack of antibody specificity seen for CRCoV The negative results of this study suggest several possi ble scenarios i no one in the study population has been previously exposed to CRCoV which could be extrapo l