Fecal Volatile Organic Ccompound Profiles from White-Tailed Deer (Odocoileus virginianus) as Indicators of Mycobacterium bovis Exposure or Mycobacterium bovis Bacille Calmette-Guerin (BCG) Vaccination

RESEARCH ARTICLE Fecal Volatile Organic Ccompound Profiles from White-Tailed Deer (Odocoileus virginianus) as Indicators of Mycobacterium bovis Exposure or Mycobacterium bovis Bacille Calmette-Guerin (BCG) Vaccination Randal S. Stahl1*, Christine K. Ellis1, Pauline Nol2, W. Ray Waters3, Mitchell Palmer3, Kurt C. VerCauteren1 1 United States Department of Agriculture (USDA)-Animal and Plant Health Inspection Service (APHIS)Wildlife Services, National Wildlife Research Center, Fort Collins, Colorado, United States of America, 2 Wildlife Livestock Disease Investigations Team, USDA-APHIS-Veterinary Services-Science, Technology, and Analysis Services, National Veterinary Services Laboratory, Fort Collins, Colorado, United States of America, 3 Infectious Bacterial Diseases Research Unit, USDA-Agricultural Research Service, National Animal Disease Center, Ames, Iowa, United States of America * OPEN ACCESS Citation: Stahl RS, Ellis CK, Nol P, Waters WR, Palmer M, VerCauteren KC (2015) Fecal Volatile Organic Ccompound Profiles from White-Tailed Deer (Odocoileus virginianus) as Indicators of Mycobacterium bovis Exposure or Mycobacterium bovis Bacille Calmette-Guerin (BCG) Vaccination. PLoS ONE 10(6): e0129740. doi:10.1371/journal. pone.0129740 Academic Editor: Srinand Sreevatsan, University of Minnesota, UNITED STATES Received: November 14, 2014 Accepted: May 12, 2015 Published: June 10, 2015 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Abstract White-tailed deer (Odocoileus virginianus) serve as a reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and can be a source of infection in cattle. Vaccination with M. bovis Bacille Calmette Guerin (BCG) is being considered for management of bovine tuberculosis in deer. Presently, no method exists to non-invasively monitor the presence of bovine tuberculosis in deer. In this study, volatile organic compound profiles of BCG-vaccinated and non-vaccinated deer, before and after experimental challenge with M. bovis strain 95–1315, were generated using solid phase microextraction fiber head-space sampling over suspended fecal pellets with analysis by gas chromatography/mass spectrometry. Chromatograms were processed using XCMS Online to characterize ion variation among treatment groups. The principal component scores resulting from significant (α = 0.05) ion responses were used to build linear discriminant analysis models. The sensitivity and specificity of these models were used to evaluate the feasibility of using this analytical approach to distinguish within group comparisons between pre- and post-M. bovis challenge: non-vaccinated male or female deer, BCG-vaccinated male deer, and the mixed gender non-vaccinated deer data. Seventeen compounds were identified in this analysis. The peak areas for these compounds were used to build a linear discriminant classification model based on principal component analysis scores to evaluate the feasibility of discriminating between fecal samples from M. bovis challenged deer, irrespective of vaccination status. The model best representing the data had a sensitivity of 78.6% and a specificity of 91.4%. The fecal head-space sampling approach presented in this pilot study provides a non-invasive method to discriminate between M. bovis challenged deer and BCG-vaccinated deer. Additionally, the technique may prove invaluable for BCG efficacy studies with PLOS ONE | DOI:10.1371/journal.pone.0129740 June 10, 2015 1 / 20 Use of Fecal VOC as Indicators of bTB Exposure in WTD free-ranging deer as well as for use as a non-invasive monitoring system for the detection of tuberculosis in captive deer and other livestock. Introduction Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a disease of importance to public health, domestic agriculture, and international trade [1, 2]. Implementation of disease surveillance and eradication programs in the United States (US) has dramatically reduced the prevalence of bTB in domestic livestock herds [3]; however, import of infected animals from Mexico, infrequent inter-herd transmission (including transmission from captive cervids to cattle), and the endemic presence of bTB in free-ranging populations of white-tailed deer (Odocoileus virginianus; WTD) in Michigan, USA and feral swine (Sus scrofa) on Molokai Island, Hawaii, USA have been major obstacles to achieving disease-free status [4–7]. According to the World Health Organization (WHO), approximately 8.8 million incident cases of human tuberculosis occurred globally in 2010 [8]. Mycobacterium tuberculosis was responsible for the majority of those cases; however, an unknown proportion of cases were likely attributable to M. bovis [9, 10]. Eradication programs [11] and milk pasteurization have decreased the incidence of bTB in developed countries; however, in some developing countries, disease prevalence in cattle may exceed 10% [12, 13]. Bovine tuberculosis is endemic at low prevalence in the WTD population in northeastern Michigan, which serves as a reservoir for transmission to cattle [14, 15]. Surveillance data identified core outbreak areas with a prevalence rate of approximately 2%, with focal areas within the core area having higher prevalence (> 3.5%)[16]. Primary surveillance and control strategies for WTD have historically relied on reducing WTD densities through hunting and by restricting baiting and supplemental feeding. Oral vaccination with M. bovis Bacille CalmetteGuerin (BCG) has been shown to be effective in protecting WTD from disease and is being considered as a management tool in addition to the existing tools already in place [17–19]. Standard procedures for monitoring captive cervids for bTB are based on the administration of a single cervical tuberculin test (SCT) followed by a comparative cervical tuberculin test (CCT) [20] and more recently, the Dual Path Platform VetTB Assay (DPP; Chembio Diagnostic Systems, Inc., Medford, NY, USA) which has been approved for use as both primary and secondary tests [21]. These testing strategies require one or more animal handling events and DPP-based approaches may falsely identify BCG-vaccinated animals as M. bovis-infected [18, 22]. The United States Department of Agriculture (USDA) performed a comprehensive evaluation in cervids of the SCT and CCT used in series and reported that the sensitivity and specificity were 87.1 and 90.4% respectively [23]. Palmer et al. [24] demonstrated the sensitivity and specificity of the CCT alone to be 97% and 81% respectively in 169 known infected and non (...truncated)