Microbial Characterization of Deer Intestinal Communities in Texas Emily C. Martin, Emily B. Hollister, and Terry J. Gentry

Texas A&M University, College Station, TX

Bacterial contamination is a significant source of water impairment and US EPA

303(d) listings in the state of Texas and across the US. Gut microbial communities

have been shown to be host specific and amenable to source tracking efforts, especially

through the creation of molecular markers that target bacterial taxa specific to each

host organism, including humans and cattle.

In Texas, wildlife sources, such as deer, have been implicated as major contributors of

bacterial impairment, but our fundamental knowledge of wildlife gut communities and

thus ability to track them as specific contamination sources is considerably lacking.

To this end, microbial communities from 11 deer fecal samples were collected from

southern and central Texas and characterized using 16S rRNA tag-encoded

pyrosequencing. A total of 32,163 sequences were analyzed with an average library

size of 2,924 members and an average sequence length of 260 bp.

Among our eleven libraries, two phyla, the Firmicutes and Bacteroidetes, were found

to be most abundant. Firmicutes accounted for 58-90% of the membership of each

community, while Bacteroidetes accounted for an additional 3-42% of each library.

This trend continued at the order level with Clostridiales and Bacteroidales accounting

for an average of 95% of the sequences in each library.

Analysis revealed 8,956 unique OTUs in the overall dataset with no single OTU

accounting for greater than 3.0% of the entire dataset. However, some OTUs,

classified as Clostridiales and Bacteroidales, were found in common across most

samples and locations and will serve as the target for future efforts to develop deer-

specific markers for bacterial source tracking.

Ab

stract

Questions? Comments? Please contact:

Emily Martin, emartin@ag.tamu.edu

Department of Soil & Crop Sciences, TAMU 2474, College Station, TX 77843-2474

Su

mm

ary

Rati

on

ale Bacteria are the primary cause of stream impairment on EPA 303(d) list in Texas.

Wildlife, including deer, have been implicated as major contributors.

We lack source-specific targeted markers to track deer fecal contamination, unlike

humans and cattle.

Deer intestinal communities are poorly characterized.

Ob

ject

ives

To characterize bacterial communities from 11 deer intestinal

samples collected from south and central Texas and identify

potential targets for deer-specific marker development.

Co

mm

un

ity

ric

hn

ess

an

d d

iver

sity

A

• Average sequence library size = 2,924 members

• 8,956 unique OTUs (97% similarity cutoff)

• Individual sample OTUs ranged from 525 to 1,559.

- Welder 2008 samples (n=4) Average 1,303 OTUs

- Welder 2009 samples (n=4) Average 1,033 OTUs

- Leon 2009 samples (n=3) Average 1,165 OTUs

• Community richness and diversity indices show little variation over time or location.

Sampling

Site/YearSample ID Sequence

Library Size

Number of

OTUs

Chao I Richness

Estimate

Shannon

H’

Simpson

D

Welder 2008 81 3825 1388 2595 6.63 0.99

84 2304 1168 2893 6.59 0.99

87 2825 1358 3092 6.73 0.99

88 3262 1298 2916 6.55 0.99

Welder 2009 91 1948 903 2014 6.30 0.99

92 3508 1559 3593 6.78 0.99

93 2047 525 1269 4.97 0.97

95 3368 1145 2301 6.17 0.99

Leon L1 3198 1327 3029 6.51 0.99

L2 2752 1019 1929 6.33 0.99

L3 3126 1148 2317 6.42 0.99

Overall 32 163 8956 - -

Ph

ylo

gen

etic-Based

Resu

lts

Figure 1: Heatmap depicting family-level relative

abundances. Abundances for each taxonomic group (i.e., rows)

were scaled relative to the mean across all samples and are

depicted by color (red = above average, green = below average)

Firmicutes and Bacteroidales dominate

communities at the phylum level.

On average, Firmicutes account for 80% of library

membership and members of the Bacteroidales

account for an additional 17%.

Sample 93 was the least diverse of all samples

showing a depletion of Ruminococcaceae and

enrichment of Bacteroidales members.

Samples did not cluster by sampling location or

time (Figure 1, Table 2).

Met

hod

s

11 Deer intestinal samples

Samples collected from fresh deer kills (2008, 2009)

- Welder Wildlife Foundation, Sinton, TX

- Comanche, TX in Leon County

Direct fecal sampling from lower intestine

Samples frozen at -80°C

DNA extraction using Mo-Bio® PowerSoil kit

16S rRNA bacterial tag-encoded amplicon pyrosequencing

Research and Testing Laboratory, Lubbock, TX

Roche Titanium chemistry

Forward reads from 27F

Sequence analysis and community comparisons

Mothur (version 1.18.1)

Quality trimmed (Q=25, rolling window average=50)

Chimera checked

Average length post quality trim and alignment - 260 bp

OTU (97% similarity cutoff), average neighbor clustering

Neighbor-joining trees with nearest neighbor BLAST hits

MEGA (v5)

This research was supported by the Texas Water Resources Institute – USGS Graduate Research Program and the Texas State Soil and Water Conservation

Board. The authors would like to thank the staff at Welder Wildlife Foundation and Whitt Weems, Commanche County Texas AgriLife Extension Service, for

assistance in collecting samples, as well as Heidi Mjelde, SAML Technician.

• Deer intestinal samples yielded communities similar to other mammals, and were dominated by

members of the Firmicutes and Bacteroidetes.

• Despite similarities at high-levels of taxonomy, very few OTUs were shared across the entire

sample set.

• Communities did not segregate by gender, harvest time, or location.

• Overlapping OTUs, including sequences from the Bacteroidaceae and Ruminococcaceae, will

serve as the basis of a more in-depth look at possible deer-specific markers for use in future

source tracking efforts.

Sampling

Site/YearSample

IDSex

Age

(yrs)

Live Wt

(kg)

Welder 2008 81 Male 4.5 47

84 Female 5.5 42

87 Female 7.5 46

88 Female 1.5 23

Welder 2009 91 Female 4.5 44

92 Female 3.5 44

93 Female 4.5 48

95 Male 1.5 38

Leon L1 N/A N/A N/A

L2 N/A N/A N/A

L3 N/A N/A N/A

Table 1: Summary of community diversity based on OTUs

Table 2: Deer Physical Characteristics

Ph

ylo

gen

etic-B

ased

Resu

lts

• Two OTUs, members of the Ruminococcaceae and

Veillonellaceae, were shared across all 11 sites (Figure 2).

- Three additional OTUs were shared across 10 of the 11

samples, and another 5 were shared across 9 of the 11

and included Ruminococcaceae as well as

Clostridiales.

• These same OTUs were also the most abundant and will

serve as the basis for additional studies (Figure 3).

Figure 2: Neighbor-joining tree of overlapping OTUs and

nearest neighbor Blast hits.

( Ruminococcaceae; Veillonellaceae)

Figure 3: Neighbor-joining tree of most abundant OTUs and

their nearest neighbor Blast hits

( Rikenellaceae; Porphyromonadaceae; Bacteroidales;

Ruminococcaceae)

Poster N-2725