Regular issues
Monographs
Special Issues



Southeastern Naturalist
    SENA Home
    Range and Scope
    Board of Editors
    Staff
    Editorial Workflow
    Publication Charges
    Subscriptions

Other EH Journals
    Northeastern Naturalist
    Caribbean Naturalist
    Urban Naturalist
    Eastern Paleontologist
    Eastern Biologist
    Journal of the North Atlantic

EH Natural History Home

Bacterial Fauna of the Forehead, Tongue, and Nasal Mucosa of Odocoileus virginianus (White-tailed Deer) in Georgia
Emily H. Belser, Bradley S. Cohen, David A. Osborn, Shamus P. Keeler, Scott M. Russell, and Karl V. Miller

Southeastern Naturalist, Volume 15, Issue 3 (2016): 488–495

Full-text pdf (Accessible only to subscribers.To subscribe click here.)

 

Site by Bennett Web & Design Co.
Southeastern Naturalist E.H. Belser, B.S. Cohen, D.A. Osborn, S.P. Keeler, S.M. Russell, and K.V. Miller 2016 Vol. 15, No. 3 488 2016 SOUTHEASTERN NATURALIST 15(3):488–495 Bacterial Fauna of the Forehead, Tongue, and Nasal Mucosa of Odocoileus virginianus (White-tailed Deer) in Georgia Emily H. Belser1, Bradley S. Cohen2,*, David A. Osborn2, Shamus P. Keeler3, Scott M. Russell4, and Karl V. Miller2 Abstract - Identification of the bacterial fauna associated with the skin, nasal mucosa, and tongue of Odocoileus virginianus (White-tailed Deer) may provide some insight into deer health and potential risks for humans from contact with deer. Few bacterial surveys have investigated White-tailed Deer, despite the commonality of human–deer interactions. From October to December 2011, we collected swab samples from the forehead, nose, and tongue of 39 hunter-harvested White-tailed Deer in Georgia. We inoculated and incubated agar plates for 48 h at 35 °C and identified the isolated bacterial colonies to genus using the bioMerieux Vitek 2 system. We amplified and sequenced portions of the 16s ribosomal-RNA gene, and used the products to identify 60 species of bacteria, including 342 Gram-positive isolates and 93 Gram-negative isolates. Although most species isolated from White-tailed Deer were nonpathogenic environmental bacteria, good hygiene is recommended when handling these animals. Introduction Expanding human populations coupled with urban/suburban wildlife issues provide increased opportunities for contact with wildlife and associated zoonotic diseases. As a result, the emergence and re-emergence of pathogens for which wild animals act as sources have become increasingly relevant. Bacterial diseases are of particular concern because 54% of emerging human infectious diseases are caused by bacteria, particularly rickettsia (Jones et al., 2008). Odocoileus virginianus Zimmermann (White-tailed Deer, hereafter, Deer) are commonly encountered and handled by professional wildlife veterinarians and biologists as well as the general public, including hunters. Deer can host several bacterial species that cause disease in humans such as anthrax, dermatophilosis, and tularemia (Davidson and Nettles 1988). Despite the frequency of contact with humans and the possibility of zoonotic disease transmission, few studies have addressed the bacteria associated with Deer. In this study, we identified bacteria occurring on the forehead, nasal mucosa, and tongue from hunter-harvested Deer at 2 sites in Georgia in order to provide background data on the bacteria associated with Deer relative to Deer disease and human health. 1Current address - Caesar Kleberg Wildlife Research Institute, Texas A&M University- Kingsville, Kingsville, TX 78363. 2Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602. 3Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, The University of Georgia, Athens, GA 30605. 4Poultry Science Department, University of Georgia, Athens, GA 30602. *Corresponding author - bscohen3@gmail.com. Manuscript Editor: Richard Baird Southeastern Naturalist 489 E.H. Belser, B.S. Cohen, D.A. Osborn, S.P. Keeler, S.M. Russell, and K.V. Miller 2016 Vol. 15, No. 3 Methods From October to December 2011, we sampled 39 hunter-harvested Deer from Cedar Creek Wildlife Management Area (WMA; 33°13'45.1''N 83°31'36.1''W; n = 20) and Berry College WMA (34°19'24.4''N 85°10'46.3''W; n = 19), located in the Piedmont and Ridge/Valley physiographic regions of Georgia, respectively. Although we did not exhaustively examine individuals to assess overall health, we visually inspected Deer for abnormalities associated with common diseases (Davidson and Nettles 1988). We used sterile cotton swabs (Puritan Medical Products, Guilord, ME) to sample the forehead, nasal mucosa, and tongue, which could serve as routes of entry for bacteria. We sampled equally among age classes and between genders (Table 1). We estimated Deer ages using tooth wear and replacement (Severinghaus 1949). We placed each swab in an individual 2.5- mL Cryosaver vial (Hardy Diagnostics, Santa Maria, CA) and kept them on ice until we could transfer them to a freezer on site. We later transported the frozen samples on ice in a cooler for 2 h while on route to the laboratory where they were stored at -20 °C until processed. After thawing, we used each swab to inoculate individual 5% sheep-blood agar plates (University of Georgia College of Veterinary Medicine, Department of Infectious Diseases Media Lab, Athens, GA) using the spread-plate technique. After incubating at 35 °C for a minimum of 48 h, we macroscopically inspected individual colonies for morphological characteristics (color, shape, texture, and size), and re-plated each distinct morphological type onto a separate 5% sheep-blood agar plate and incubated them for a minimum of 24 h to isolate species. After determining the Gram reaction of each isolate by using the potassium hydroxide string test (Agbonlahor et al. 1983), we employed the Vitek 2 automated diagnostic system (bioMérieux, Marcy-l’Etoile, France) to group isolates by biochemical properties and make preliminary identifications to genus or species. We did not record the hemolytic phenotype of individual colonies because we used the Vitek 2 for preliminary identification. We retained at least 1 sample of each confirmed species and any isolates that could not be identified by the Vitek 2 and stored them at -20 °C in CryoBank vials (Copan Diagnostics, CA) until further processing . We thawed samples and extracted DNA from 3–6 beads in the CryoBank vials of each isolate using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). We amplified a 600-bp region of the 16S ribosomal-RNA (rRNA) gene using E334F and E939R primers (Rudi et al. 1997). The PCR amplification was performed in 25-μl-reaction mixtures, each containing 11 μl of DNA-free PCR water (MO BIO Laboratories, Inc., Carlsbad, CA), 0.25 μl of dNTP, 7.75 μl of GoTaq Flexi DNA Polymerase (Promega Corporation, Madison, WI), 0.5 μl of each primer, and 5-μl volume of DNA sample. Amplifications were performed in a thermal cycler (BioRad DNA Engine, Hercules, CA) with the following protocol: initial 5-min denaturation at 95 °C with 35 cycles, each consisting of 1-min denaturation at 94 °C, 1-min annealing at 55 °C, 1-min extension at 72 °C, and a final 5-min extension at 72 °C. We analyzed the PCR products via electrophoresis on 1.5% agarose gel with ethidium bromide at 100 V for 20 min with a 100-bp DNA ladder (Promega Southeastern Naturalist E.H. Belser, B.S. Cohen, D.A. Osborn, S.P. Keeler, S.M. Russell, and K.V. Miller 2016 Vol. 15, No. 3 490 Corporation, Madison, WI) as a DNA marker. To avoid contamination of samples, we maintained separate areas for DNA extraction and PCR preparation, changed our gloves frequently, and used DNA-away (Molecular BioProducts, Inc., San Diego, CA) to clean equipment. All PCR cycles included negative controls of DNA-free PCR water (MO BIO Laboratories, Inc.) without DNA template to ensure there was no contamination in the reaction mixture. We extracted each sample from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and sent them Table 1. Sex, age, and number of bacteria species isolated from the forehead, nose, and tongue of each White-tailed Deer at Cedar Creek (CC) and Berry College (BC) Wildlife Management areas, GA, September–January 2011 and 2012. Location Sex Age Forehead Nose Tongue BC F 2.5 4 3 5 BC F 2.5 2 3 5 BC F 2.5 3 2 6 BC F 2.5 2 2 4 BC F 2.5 4 2 2 BC M 0.5 3 3 3 BC M 1.5 4 4 5 BC M 1.5 8 2 6 BC M 1.5 3 6 4 BC M 1.5 5 2 5 BC M 1.5 3 5 4 BC M 2.5 3 2 4 BC M 2.5 8 3 7 BC M 2.5 4 1 4 BC M 2.5 5 5 4 BC M 2.5 4 3 5 BC M 3.5 4 4 4 BC M 3.5 1 2 2 BC M 4.5 3 5 2 CC F 0.5 3 4 5 CC F 2.5 4 2 4 CC F 2.5 3 1 1 CC F 5.5 3 1 4 CC F 6.5 4 2 6 CC M 1.5 6 4 5 CC M 1.5 4 4 5 CC M 1.5 4 2 6 CC M 1.5 4 2 3 CC M 1.5 5 6 5 CC M 2.5 3 6 5 CC M 2.5 3 2 3 CC M 2.5 3 3 6 CC M 2.5 4 2 7 CC M 2.5 4 2 5 CC M 3.5 4 2 4 CC M 4.5 5 3 3 CC M 4.5 3 2 5 CC M 5.5 3 6 4 CC M 6.5 2 2 3 Southeastern Naturalist 491 E.H. Belser, B.S. Cohen, D.A. Osborn, S.P. Keeler, S.M. Russell, and K.V. Miller 2016 Vol. 15, No. 3 for genetic sequencing to the Georgia Genomics Facility (University of Georgia, Athens, GA). We conducted a basic local alignment search tool (BLAST) search of the results to identify each sample to species (T ables 2; Hall 1999). Results and Discussion We obtained a total of 435 bacterial isolates from 117 samples from 39 Deer, consisting of 60 species of bacteria. Most (n = 342) were Gram-positive; 93 isolates were Gram-negative (Table 2). Many of the bacterial genera and species isolated in this study were previously reported from the tarsal tufts of hunter-harvested Deer in Georgia (Alexy et al. 2003). The genera and species identified using the Vitek system include Acinetobacter spp., Bacillus cereus, Bacillus megaterium, Bacillus sp., Cellulomonas sp., Enterobacter sp., Escherichia hermanii, Hafnia alvei, Micrococcus luteus, Pseudomonas sp., Serratia marcescens, Staphylococcus cohnii, Staphylococcus Table 2. Gram-positive and Gram-negative bacteria isolated from forehead, nasal, and lingual samples of White-tailed Deer from Cedar Creek (n = 20) and Berry College (n = 19) Wildlife Management areas, GA, September–January 2011 and 2012. [Continued on next page.] Number of positive samples Cedar Creek WMA Berry College WMA Bacteria Forehead Nasal Lingual Forehead Nasal Lingual Total Gram-positive Arthrobacter arilaitensis 1 1 1 3 2 2 10 nicotinovorans 1 - - - - - 1 non-speciated - 2 - 3 1 - 6 Bacillus cereus 1 1 1 4 2 2 11 cibi - - - - - 1 1 firmus - 1 2 - - - 3 gibsonii 1 - - - - - 1 lichenformis - - - - - 1 1 megaterium - - - - 1 - 1 non-speciated 13 7 15 14 13 14 76 Cellulomonas spp. - - - - 1 2 3 Curtobacterium spp. - 1 - 2 1 - 4 Exiguobacterium spp. 9 2 4 6 4 7 32 Microbacterium chocolatum - 1 - - - - 1 oleivorans 1 - - - - - 1 non-speciated 1 - - 1 - 1 3 Micrococcus spp. - 1 1 1 1 1 5 Paenibacillus lautus 2 1 1 - - 1 5 polymyxa - - 1 - - - 1 non-speciated 1 - - - - - 1 Planomicrobium sp. - - - 1 - - 1 Rhodococcus sp. - - - - - 1 1 Southeastern Naturalist E.H. Belser, B.S. Cohen, D.A. Osborn, S.P. Keeler, S.M. Russell, and K.V. Miller 2016 Vol. 15, No. 3 492 Table 2, cont. Number of positive samples Cedar Creek WMA Berry College WMA Bacteria Forehead Nasal Lingual Forehead Nasal Lingual Total Rothia nasimurium - 1 1 - - 1 3 terrae 1 - 1 - - - 2 non-speciated - - 1 - - - 1 Sporosarcina sp. 1 - - - - - 1 Staphylococcus agnetis 4 1 2 6 - 1 14 aureus 9 2 6 4 2 3 26 cohnii - - 1 - 1 2 4 epidermidis - - - 2 - - 2 kloosii 3 1 - - - - 4 saprophyticus - - - 2 8 - 10 sciuri 5 - 1 - - - 6 simulans 1 - - - - - 1 non-speciated 10 5 4 10 3 10 42 Streptococcus gallolyticus - 4 5 - - 4 13 merionis - - 3 - - - 3 pseudoporcinus - 1 - - - - 1 non-speciated 1 4 10 3 4 8 30 Streptomyces spp. - 1 2 2 1 2 8 Trueperella pyogenes 1 1 - - - - 2 Gram-negative Acinetobacter spp. - 1 1 - 1 1 4 Bosea sp. - - - - 1 - 1 Chryseobacterium spp. 1 5 8 2 2 6 24 Enterobacter spp. 1 1 3 - - 1 6 Escherichia hermanii - - - - - 1 1 Gibbsiella quercinecans - - 2 1 2 3 8 Hafnia alvei - - 1 - - - 1 Leclercia adecarboxylata - - - - 1 - 1 Moraxella spp. - 6 - - 4 2 12 Neisseria weaveri - - 2 - - - 2 non-speciated 2 1 1 - - 1 5 Pantoea ananatis - - - - - 1 1 non-speciated 1 - - 1 - - 2 Pseudomonas boreopolis - - 1 - - - 1 non-speciated 2 2 4 3 1 1 13 Serratia marcescens - 1 1 - - - 2 non-speciated - - 1 - - - 1 Solibacillus silvestris - - - 2 - - 2 Stenotrophomonas spp. - 2 1 - 2 - 5 Southeastern Naturalist 493 E.H. Belser, B.S. Cohen, D.A. Osborn, S.P. Keeler, S.M. Russell, and K.V. Miller 2016 Vol. 15, No. 3 sciuris, Staphylococcus sp., and Streptococcus sp. (Alexy et al. 2003). Two other studies (Karns et al. 2009, Turner et al. 2013) that employed traditional bacterialidentification techniques isolated many of the same genera and species from hunter-harvested Deer in Maryland, including Trueperella pyogenes, Bacillus sp., Staphylococcus sp., Streptococcus sp., Acinetobacter sp., Enterobacter sp., and Moraxella sp., suggesting that these genera of bacteria are likely widely associated with Deer. Staphylococcus species made up the largest group of isolates (25.1%; 109/434) identified from Deer in this study. To some extent, Staphylococcus species have evolved along with their host species and are intimately associated with animals (Hermans et al. 2010). Staphylococcus aureus had the highest culture recovery both within this genus (17.9%; 26/145) and among all the bacteria identified to species. This bacterium is an opportunistic pathogen that causes a wide variety of diseases and infections, some of which include dermatitis, enterotoxemia, septicemia, and toxic-shock syndrome in humans, mastitis in Bos taurus L. (Cattle) and small ruminants, and mastitis and septicemia in Sus scrofa domestica Erxleben (Swine) (Hermans et al. 2010). The 2nd-largest group of isolates identified was Bacillus sp., making up 21.6% (94/434) of total isolated bacteria in this study. Most Bacillus species are common soil saprophytes. Therefore, the high frequency we obtained could be explained by hunters pulling Deer across the ground to the check station prior to swab samples being taken. Although most Bacillus sp. are nonpathogenic, some can be opportunistic pathogens. For example, Bacillus cereus can cause food poisoning in humans and occasionally septicemia, meningitis, and ocular infections (Granum and Baird- Parker 2000). We detected only 2 isolates of Trueperella pyogenes in our study; this bacterium is commensal and opportunistic in nature, and it is commonly found on the mucosal surfaces of healthy Cattle and Swine, as well as other domestic and freeranging species (Jost and Billington 2005). Trueperella pyogenes is known to cause a variety of purulent infections, including post-partum endometritis and mastitis in Cattle, abortions in Ovis aries L. (Sheep), and udder abscesses in Swine (Jost and Billington 2005). This organism can also cause infections such as pneumonia, mandibular osteomyelitis, peritonitis, and hepatic, pulmonary, renal and subcutaneous abscesses in captive wildlife, including Antilope cervicapra L. (Blackbuck Antelope; Portas and Bryant 2005). Trueperella pyogenes is the pathogen mostassociated with intracranial abscesses in Deer (Baumann et al. 2001, Davidson et al. 1990). Other bacteria identified in this study have been isolated from intracranial abscesses in Deer, including Serratia marcescens, Hafnia alvei, Staphylococcus aureus, and S. sciuri (Baumann et al. 2001, Davidson et al. 1990). Many of the bacteria isolated from hunter-harvested Deer in this study are nonpathogenic environmental bacteria. For example, Enterobacter species normally exist as saprophytes in the gastrointestinal tract of warm-blooded animals. They are widely distributed throughout the environment in water, sewage, soil, and plants (Stiles 2000). Other genera, such as Serratia, are commensals of warm-blooded Southeastern Naturalist E.H. Belser, B.S. Cohen, D.A. Osborn, S.P. Keeler, S.M. Russell, and K.V. Miller 2016 Vol. 15, No. 3 494 animals and rarely cause infection in humans (Stiles 2000). Infection by these organisms is primarily associated with patients with compromised immune systems. Therefore, clinically healthy individuals are at low risk of infection. Understanding the bacterial species associated with Deer is important to understanding Deer health and may provide some insights for disease conditions in people who frequently come in contact with Deer. Although this study detected a few potential pathogens associated with Deer, none likely would be considered a high risk for humans. The aerobic isolation-methods used in this study limited the potential to detect all bacterial species present. Thus, it is recommended that individuals that come into physical contact with Deer should take appropriate hygiene measures, such as wearing gloves when field-dressing Deer and thorough handwashing immediately after contact. Acknowledgments Funding for this project was provided by the Georgia Wildlife Resources Division through the Wildlife Restoration Program, which derives monies through an excise tax on sporting arms and ammunition, paid by hunters and recreational shooters. We thank Charlie H. Killmaster and John W. Bowers for helping organize sample collection. We also thank the student volunteers who assisted us in the laboratory . Literature Cited Agbonlahor, D.E., T.O. Odugbemi, and P.O. Udofia. 1983. Differentiation of Gram-positive and Gram-negative bacteria and yeasts using a modification of the “string” test. American Journal of Medical Technology 49(3):177–178. Alexy, K.J., J.W. Gassett, D.A. Osborn, K.V. Miller, and S.M. Russell. 2003. Bacterial fauna of the tarsal tufts of White-tailed Deer (Odocoileus virginianus). American Midland Naturalist 149:237–240. Baumann, C.D., W.R. Davidson, D.E. Roscoe, and K. Beheler-Amass. 2001. Intracranial abscessation in White-tailed Deer of North America. Journal of Wildlife Diseases 37:661–670. Davidson, W.R., and V.F. Nettles. 1988. White-tailed Deer. Pp. 22–92, In W.R. Davidson (Ed.). Field Manual of Wildlife Diseases in the Southeastern United States. Southeastern Cooperative Wildlife Disease Study, Athens, GA. 448 pp. Davidson, W.R., V.F. Nettles, L.E. Hayes, E.W. Howerth, and C.E. Couvillion. 1990. Epidemiologic features of an intracranial abscessation/suppurative meningoencephalitis. Journal of Wildlife Diseases 26:460–467. Granum, P.E., and T.C. Baird-Parker. 2000. Bacillus species. Pp. 1029–1039, In B.M. Lund, T.C. Baird-Parker, and G.W. Gould (Eds.). The Microbiological Safety and Quality of Food. 1st Edition. Aspen Publishers, Inc., Gaithersburg, MD. 2024 pp. Hall, T.A. 1999. BioEdit: A user-friendly biological sequence-alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41:95–98. Hermans, K., L.A. Devriese, and F. Haesebrouck. 2010. Staphylococcus. Pp. 75–89, In C.L. Gyles, J.F. Prescott, J.G. Songer, and C.O. Theon (Eds.). Pathogenesis of Bacterial Infections in Animals. 4th Edition. Wiley-Blackwell, Ames, IA. 664 pp. Jones, K.E., N.G. Patel, M.A. Levy, A. Storeygard, D. Balk, J.L. Gittleman, and P. Daszak. 2008. Global trends in emerging infectious diseases. Nature 451:990–993. Southeastern Naturalist 495 E.H. Belser, B.S. Cohen, D.A. Osborn, S.P. Keeler, S.M. Russell, and K.V. Miller 2016 Vol. 15, No. 3 Jost, H.B., and S.J. Billington. 2005. Arcanobacterium pyogenes: Molecular pathogenesis of an animal opportunist. Antonie van Leeuwenhock 88:87–102. Karns, G.R., R.A. Lancia, C.S. DePerno, M.C. Conner, and M.K. Stoskopf. 2009. Intracranial abscessation as a natural mortality factor for adult male White-tailed Deer (Odocoileus virginianus) in Kent County, Maryland, USA. Journal of Wildlife Diseases 45:196–200. Portas, T.J., and B.R. Bryant. 2005. Morbidity and mortality associated with Arcanobacterium pyogenes in a group of captive Blackbuck (Antilope cervicapra). Journal of Zoo and Wildlife Medicine 36:286–289. Rudi, K., O.M. Skulberg, F. Larsen, K.S. Jakobsen. 1997. Strain characterization and clas - sification of oxyphotobacteria in clone cultures on the basis of 16s-RNA sequences from the variable regions V6, V7, and V8. Applied and Environmental Microbiology 63:2593–2599. Severinghaus, C.W. 1949. Tooth development and wear as criteria of age in White-tailed Deer. Journal of Wildlife Management 13:195–215. Stiles, M.E. 2000. Less-recognized and suspected foodborne bacterial pathogens. Pp. 1394–1419, In B.M. Lund, T.C. Baird-Parker, and G.W. Gould (Eds.). The Microbiological Safety and Quality of Food. 1st Edition. Aspen Publishers, Inc., Gaithersburg, MD. 2024 pp. Turner, M.M., C.S. DePerno, M.C. Conner, T.B. Eyler, R.A. Lancia, R.W. Klaver, and M.K. Stoskopf. 2013. Habitat, wildlife, and one health: Arcanobacterium pyogenes in Maryland and Upper Eastern Shore White-tailed Deer populations. Infection Ecology and Epidemiology. Available online at 3:10.3402/iee.v3i0.19175. Accessed 1 March 2016.