The document outlines Stephanie Roto's thesis defense presentation on her research investigating the effects of feeding Original-XPCTM on modulating the cecal microbiota and inhibiting Salmonella survival in poultry hosts. Her research included in vitro assays examining Salmonella survival when treated with XPC and in vivo feeding trials analyzing how XPC impacts the cecal microbiota and Salmonella prevalence at different ages. Her results showed that XPC treatment led to a more rapid decrease in Salmonella survival by 24 hours and did not significantly alter the cecal microbial diversity between treatments but did increase with bird maturity.

1. A Molecular Approach to
Understanding the Effects of Original-
XPCTM on the Modulation of the Cecal
Microbiota and the Survival of
Salmonella in the Poultry Host
Department of Food Science
University of Arkansas
Thesis Defense Presentation
Stephanie Roto
Advisor: Dr. Steven C. Ricke
April 8, 2016
1

2. My Credentials
 B.S. in Molecular and Cellular Biology
 Internship at research hospital
 Analytical Microbiologist testing for Legionella
 Incited my interest in foodborne/waterborne
pathogens
2

3. Thesis Project
 Previous research with XPC*:
 GIT morphology
 Immunologic response
 Growth performance
 Pathogen reduction
 Culture-independent analysis
*Jensen et al., 2007; Gao et al., 2008, 2009; El-Husseiny et al., 2010; Osweiler et al., 2010;
3

4. Outline
 Introduction
 Objectives and hypotheses
 Materials and methods
 Results
 Discussion and conclusions
4

5. Chapter 1: Literature Review
 Roto SM, Rubinelli PM, and Ricke SC. (2015) An introduction to the avian gut
microbiota and the effects of yeast-based prebiotic-type compounds as potential
feed additives. Front. Vet. Sci. 2:28. doi: 10.3389/fvets.2015.00028
5

6. Removal of Antibiotics in Food Animals
 Drug resistant bacteria cause two million illnesses
and 23,000 deaths each year in the U.S.*
 “By 2020… elimination of the use of medically-
important antibiotics for growth promotion in food
animals...”
 “...non-traditional therapeutics for treatment of
human disease”
*CDC estimates
6

7. Pathogen Prevention in Poultry
Image borrowed from Dr. R. R. Punuru, NTR College of Veterinary Science
7

8. Prebiotic-Type Compounds
 Term introduction by Gibson and Roberfroid, 1995
 Food ingredient  host health
1. Resistant
2. Fermentable
3. Stimulatory
 Any compound lacking specificities set by Gibson
and Roberfroid
8

9. Goals of Research
 Ch. 2: Evaluate the effect of XPC on the survival of
Salmonella in an in vitro assay
 Ch. 2 & 3: Determine whether XPC has an impact on
the cecal microbiota of the poultry host
 Ch. 2 & 3: Evaluate the effect of bird maturity on
the cecal microbiota of the poultry host
9

10. Research Hypotheses
 Ch. 2: Treatment with XPC will inhibit the survival of
Salmonella in the cecal microbiota
 Ch. 2 & 3: Treatment with XPC would alter the cecal
microbiota diversity
 Cha. 2 & 3: The cecal microbiota diversity would
increase directly with bird maturity
10

11. Chapter 2: InVitro Anaerobic Assay
11

12. Chapter 2: Experimental Design
Variable How
many?
Description
Trial 2 Identical experimental design; different set of birds at
different time of year
Biological replicates 3 Three Cobb 500 male broilers per sampling age
Treatments 4 XPC, Negative control, Cecal only control, and XPC +
Feed Control
Sampling age 3 14, 28, and 42 d
Plating timepoints 3 0 (baseline), 24, and 48 h
Microbiota sampling
timepoints
5 0, 6, 12, 24, and 48 h
 Nalidixic acid resistant Salmonella Typhimurium (ST 97)
 107 CFU/ml
12

13. Chapter 2. Each Biological Replicate
Inoculate
with ST 9724 h pre-
incubation
0 h
24 h
48 h
0 h
6 h
12 h
24 h
48 h
*Donalson et al., 2007
Treatments
13
NGS: Illumina
MiSeq

14. Chapter 2:Treatments
Treatment Cecal
slurry
Poultry
feed
XPC ST 97 Rationale
XPC ✓ ✓ ✓ ✓ Effects of XPC
Negative Control (NC) ✓ ✓ ✓ Ability of cecal microbial
populations to inhibit ST 97
with no XPC
Cecal Only Control
(CO)
✓ Baseline; birds were not
infected with Salmonella
prior to study
XPC + Feed Control ✓ ✓ ✓ Determine if the ability of
XPC to inhibit ST 97 is
dependent on the presence
of the cecal microbiota
24 h pre-
incubation
14

15. Results: Salmonella Survival
ST 97 survival in 28 d old birds treated
with XPC in Trial 1
ST 97 survival in 28 d old birds treated
with XPC in Trial 2
0.00
2.00
4.00
6.00
8.00
0 24 48
LogCFU/ml
Plating Timepoint (hours)
XPC
NC
CO
N.S. A
B
C
A
A
C
B
0.00
2.00
4.00
6.00
8.00
0 24 48
LogCFU/ml
Plating Timepoint (hours)
XPC
NC
CO
N.S.
A
A
A
B
B BC
 Both trials revealed similar trends in treatment with XPC
 Large variation in reduction
15

16. Results: Salmonella Survival
ST 97 survival in 42 d old birds treated
with XPC in Trial 1
ST 97 survival in 42 d old birds treated
with XPC in Trial 2
 Both trials revealed similar trends
 No significance among any treatments by 48 h
16

17. Results: XPC + Feed Control
 Trial 2: 42 days
 ~6.0 log reduction of ST 97 across
time with inclusion of cecal slurry
 ~3.5 log reduction of ST 97 across
time with absence of cecal slurry
 Trial 1: 28 days
 ~2.5 log reduction of ST 97 across
time with inclusion of cecal slurry
 Log fluctuation, yet no overall
reduction in ST 97 with absence of
cecal slurry
17

18. Results: Phylum Level Analysis
Bacteroidetes
Firmicutes
Proteobacteria
Phylum
NCXPC
14 28 42
Sampling Age (days) Sampling Age (days)
 Firmicutes most abundant in both trials
 Proteobacteria much greater percentage of bacteria in trial 1
 Bacteroidetes much greater percentage of bacteria in trial 2
Trial 1 Trial 2
14 28 42
18

19. Results: Salmonella Survival Recap
Trial 1: 28 days Trial 2: 28 days
0.00
2.00
4.00
6.00
8.00
0 24 48
LogCFU/ml
Plating Timepoint (hours)
XPC
NC
CO
N.S. A
B
C
A
A
C
B
0.00
2.00
4.00
6.00
8.00
0 24 48
LogCFU/ml
Plating Timepoint (hours)
XPC
NC
CO
N.S.
A
A
A
B
B BC
Trial 1: 42 days Trial 2: 42 days
19

20. Results: Family Level Analysis
Enterobacteriaceae
Lachnospiraceae
Ruminococcaceae
CONCXPC
Family14 28 42
Sampling Age (days) Sampling Age (days)
Trial 1 Trial 2
14 28 42
 Relative abundances of the most abundant families balance out
20

21. Results: Shannon Diversity Index
A
AB
B
N.S.
N.S.
 Shannon diversity index combines
species richness with species
abundance
Shannon
A) Trial 1
and B)
Trial 2
B
AB
A
N.S.
N.S.
 Both trials show similar trends in
Shannon diversity:
 Increases directly with age in XPC
 No significance among NC and CO
Trial 1
Trial 2
ShannonDiversityIndex
ShannonDiversityIndex
Treatment/ Age
Treatment/ Age
21

22. Results: Chao1 Index
 Chao1 index estimates the total number of species present in a community
Trial 1 Trial 2
*
14 days
28 days
42 days600
400
200
0
0 2500 5000 7500 10000
Sequences Per
Sample
SpeciesRichness
0 2500 5000 7500 10000
Sequences Per
Sample
 Both trial exhibit similar trends
600
400
200
0
22
14 and 42 days  P > 0.05
28 and 42 days  P > 0.05
14 and 28 days  P > 0.05
14 and 42 days  P = 0.003
28 and 42 days  P = 0.003
14 and 28 days  P > 0.05

23. Results: Observed OTUs
 OTUs use ≥ 97% sequence identity to group sequences into OTUs
NumberofOTUs
*
300
200
100
0
Trial 1 Trial 2
14 days
28 days
42 days
0 2500 5000 7500 10000
Sequences Per
Sample
0 2500 5000 7500 10000
Sequences Per
Sample
300
200
100
0
 Both trial exhibit similar trends
23
14 and 42 days  P > 0.05
28 and 42 days  P > 0.05
14 and 28 days  P > 0.05
14 and 42 days  P = 0.003
28 and 42 days  P = 0.003
14 and 28 days  P > 0.05

24. Chapter 3: InVivo FeedingTrial
24

25. Chapter 3: Experimental Design
Variable How
many?
Description
Biological replicates 24 Twenty-four Ross 708 male broilers per treatment per
sampling age
Treatments 4 Negative control, XPC, SAL, and XPC + SAL
Sampling ages
• Plating timepoints
• Growth
performance data
• Microbiota
sampling
3 16, 28, and 42 d
Salinomycin (SAL) 0.044 g/kg added only to grower diet ( 16 to 28 d)
XPC added at 1.25 g/kg in starter (0 to 16 d) and grower diet (16 to 28 d), decreased to 0.625 g/kg in finisher diet
(28 to 42 d)
25

26. Pooled
Samples
Chapter 3. Each Biological Replicate
M G1 G2 G3 G4
Library
Preparation
DNA
Extraction DNA
Extraction
Sequence
Analysis
PCR- Based
DGGE Analysis
Cecal microbial analysis via DGGE Cecal microbial analysis via NGS
26

27. Treatment Treatment Description XPC SAL
T1 Negative control (NC) -- --
T2 XPC 0.125% (starter & grower);
0.0625% (finisher)
--
T3 SAL -- 0.0044% (grower)
T4 XPC + SAL 0.125% (starter & grower);
0.0625% (finisher)
0.0044% (grower)
Chapter 3:Treatments
SAL = Salinomycin
27

28. The following two slides contain data
that I did not conduct and therefore is
not contained in my thesis. However,
this data is the first part of what will
(hopefully) be a two-part publication.
28

29. Results: Growth Performance
 T2 and T4 exhibited significantly increased BW compared to both T1 and T3 while
inclusion rate was 0.125%
 Reduction of XPC inclusion to 0.0625% rid of significant differences in
FCR
29

30. Results: Salmonella prevalence
 Salmonella prevalence was most reduced in T4 at all three sampling ages
0.000
0.005
0.010
0.015
0.020
0.025
16 days
28 days
42 days
N.S.
N.S.
A
A
AB
B
B
AB
Group 16 d 28 d 42 d
T1 1 5 10
T2 0 3 6
T3 3 4 5
T4 0 1 1
Total 4/96 13/96 22/96
Frequency
30

31. Results: DGGE Analysis 16 days
T1 T2 T3 T4
31

32. T1 T2 T3 T4
T2 &
T4
Results: DGGE Analysis 42 days
32

33. Results: Shannon Diversity Index
 Shannon diversity index most
influenced by bird maturity
Treatment Description
2.50
2.35
2.30
2.25
2.40
2.45
ShannonDiversityIndex
16 days 28 days 42 days
33

34. 700
600
500
400
300
200
100
0
16 and 42 days  P = 0.003
28 and 42 days  P = 0.003
16 and 28 days  P > 0.05
Results: Chao1 Index
SpeciesRichness
 Chao1 index estimates the total number of species present in a community
0 200 400 600 800 1000
Sequences per Sample
16 d
28 d
42 d
*
34

35. 0 200 400 600 800 1000
Sequences per Sample
16 and 42 days  P = 0.003
28 and 42 days  P = 0.003
16 and 28 days  P > 0.05
350
300
250
200
150
100
50
0
Results: Observed OTUs
NumberofOTUs
16 d
28 d
42 d
*
 OTUs use ≥ 97% sequence identity to group sequences into OTUs
35

36. Discussion of results
 Ch. 2: Cecal microbiota maturity appears to be critical in
the reduction of Salmonella
 Ch. 2: XPC treatment indicated a more rapid decrease
(by 24 h timepoint) in Salmonella at 42 d sampling age
 Ch. 2 & 3: Neither in vitro assay or in vivo feeding trial
revealed significant differences in species diversity
among treatments
 Ch. 2 & 3: In vitro assay and in vivo feeding trial indicated
similar species diversity and richness results in relation
to bird maturity
36

37.  In vitro assay reveals similar results to the in vivo
feeding trial, attesting to the validity of the
methodology
 DGGE indicated treatment attributing to the
stability of the cecal microbial populations
 NGS: Illumina MiSeq revealed little detectable
contribution from treatment in the modulation of
cecal microbial populations
Discussion of methods
37

38. Conclusions
 Poultry producers may want to consider using
interactive effects of both bird maturity in
collaboration with feed additives to improve host
health
38

39. Prepared Manuscripts for Publication
 Chapter 3:
Roto SM, Park SH, Lee SI, Kaldhone P, Pavidis HO, McIntyre DR, Frankenbach SB, Striplin K,
Brammer L, and Ricke SC. Effects of feeding Original XPCTM to broilers with a live coccidiosis-
vaccine under industry conditions: Part 1. Growth performance and Salmonella inhibition.
Prepared for Poultry Science.
Roto SM, Park SH, Pavidis HO, McIntyre DR, Striplin K, Brammer L, and Ricke SC. Effects of
feeding Original XPCTM to broilers with a live coccidiosis-vaccine under industry conditions: Part
2. Cecal microbiota analysis. Prepared for Poultry Science.
 In ovo literature review:
Roto SM, Kwon YM, and Ricke SC. Potential applications of in ovo technique for optimal
development of the gastrointestinal ract and establishment of its microbiome in the poultry.
Preparing for submission.
39

40. Acknowledgments
 Committee: Drs. Steven Ricke, Jeff Lewis, Steven
Foley
 Drs. Si Hong Park, Peter Rubinelli, and Sun Ae Kim
 My Biomass lab members
 Diamond V
40

41. References Used in the Presentation
 Donalson LM, Woo-Kyun Kim VI, Chalova VI, Herrera P, Woodward CL, McReynolds JL, Kubena LF, Nisbet DJ, Ricke SC.
2007. In vitro anaerobic incubation of Salmonella enterica serotype Typhimurium and laying hen cecal bacteria in
poultry feed substrates and a fructooligosaccharide prebiotic. Anaerobe 13: 208-214.
 Gao HJ, Zhang HJ, Yu SH, Wu SG,Yoon I, and others. 2008 Effects of yeast culture in broiler diets on performance and
immunolmodulatory functions. Poult Sci. 87:1377-1384.
 Gao HJ, Zhang HJ, Wu SG, Yu SH, Yoon I , and others. 2009. Effects of Saccharomyces cerevisiae fermentation products
on immune functions of broilers challenged with Eimeria tenella. Poult Sci. 88:2141-2151.
 Jensen GS, Patterson KM, Yoon I. 2007 Yeast culture has anti-inflammatory effects and activates NK cells. Comp,
Immunol, Micro, Infect Dis. 31:487-500.
 Gibson R, Roberfroid M. 1995. Dietary modulation of the human colonic microbiota: introducing the concept of
prebiotics. J Nutr 125:1401-1412
 Osweiler GD. 2010. Evaluation of XPC and prototypes on aflatoxin-challenged broilers. Poult Sci 89:1887-1893.
 Roberfroid M. 2007. Prebiotics: The concept revisited. J Nutr 137:830-837
41

42. Thank you for listening!
42