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
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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
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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
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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
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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
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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
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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
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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 .
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