This document summarizes research on the relationship between cattle diets and Shiga toxin-producing E. coli (STEC) infection. It finds that cattle diets can impact STEC populations in cattle, with forage-based diets associated with higher STEC prevalence than grain-based diets. Distillers grains are also linked to higher STEC shedding when included in cattle feed. However, diet-driven changes to the cattle gut microbiome do not clearly correlate with shifts in STEC shedding. While stress and the gut microbiome may influence STEC levels, the specific drivers of these relationships require further investigation.

1. Shiga Toxin-Producing E. coli
and Ruminant Diets: A Match
Made in Heaven?
Todd Callaway
Agricultural Research Service/USDA
Food and Feed Safety Research Unit
College Station, TX

2. Escherichia coli O157:H7 & friends
• N. America has a VERY safe food supply, but it is
still a focus of concern
• Enterhemorrhagic E. coli (EHEC)
– Bloody diarrhea, HUS, children
– O157:H7, O26, O45, O103, O111, O121 and O145 are
adulterants (FSIS) in beef
• O157 evolved with cattle, lives in intestinal tract,
adhering to intestinal mucosa
– Recto anal junction, lymphoid tissue
– 50,000+ years of evolution
• Depending on season 40 % of cattle may have
O157 (non-O157 under investigation)
– May be higher during summer, near 80%
– “Supershedders” >105
CFU/g Source:
ASM

3. phuman infection = panimal infection x pcarcass contamination
x pproduct abuse x peaten by susceptible
= 0.05 x 0.4 x 0.02 x 0.2 = 0.00008 or
24,800 cases/year in a population of 310 million
Thus reductions in pathogens before harvest can yield large returns in
human health and reduced recalls
Focus on food safety “From Farm to Fork”
Processors say “below 103
cells/g, and we can take care of it”
Preharvest prevalence directly affects human illnesses

4. Carcass
Contamination
Hide
Contamination
Fecal
Shedding/
Super-Shedder
Environment
Fecal
Microbiome
Host
Diet

5. Why should we worry about
EHEC in live cattle?
• Several points for improvement
– Food Safety (Reduced pathogen burden)
• Cross contamination of carcasses and foods
• Increased in-plant effectiveness
– Water Issues (indirect)
• irrigation and run-off
– Spinach outbreak; Walkerton, ON
– Agricultural Fair/Open Farms (Petting
zoo)/Farm worker safety (Keen et al., 2003)
• Dust from cattle and O157:H7 outbreak

6. Bovine Gastrointestinal Tract, ‘Generic’ E. coli, and E. coli O157:H7
Total Bacteria consortium (per gram digesta) > 2000 species
1010
106
1010
1011
Total ‘generic’ E. coli (per gram digesta)
104
(104
) (107-8
) 107
E. coli O157:H7, when present (per gram digesta)
<102
<102
<102
<106
Rumen, reticulum, omasum,
and abomasum (fermentation reservoir)
Small Intestine
Large Intestine
Colon
Cecum

7. Bovine Colonization and E. coli O157:H7
Colonization appears to be primarily at RAJ
(rectoanal junction)
Infection in cattle is asymptomatic (lack of
toxin receptors)
Seasonality and variable
Super-shedders (>104
CFU E. coli O157:H7
per gram feces) are a minor proportion
Need >20 animals to sustain levels of E. coli
O157:H7 in the pen (recirculation)
Wells et al., 2008

8. • Management/ecological niche factors
– Diet (including specific feeds and additives)
Effects
Distillers Grains
Orange peel and pulp can reduce (essential oils)
Forage feeding can alter microbiome and EHEC; forage quality
– Microbial Environment/stress
Cross-talk between host and microbes
Norepinephrine role for “microbial organ”
Microbiome and nutrient transporters and EHEC
What factors affect EHEC in
cattle GIT?

9. Dietary factors in cattle
• Co-evolution of host (lack of toxin receptors)
• Fasting increases shedding of EHEC (Buchko) and
Salmonella (Grau)
– Lack of VFA which are toxic to E. coli/Salmonella
• Barley increased EHEC shedding (Buchko)
• Steam flaked corn increased EHEC relative to dry
rolled corn (Fox)
• Monensin generally has no effect on EHEC
(Edrington, McAllister, Van Baale)
– Increased dosage of 44 mg/kg did reduce (Paddock)

10. 0
2
4
6
8
10
-20 0 20 40 60 80 100
Rumen
Colon
E.coli
Grain (%)
Effect of Diet on intestinal coliform and E. coli populations
(Adapted from Diez Gonzalez et al., 1998)
-4 -2 0 2 4 6 8 10 12
Time (d)
Abrupt shift from 90%
grain to
100% alfalfa hay
E. coli
(Log10 cells/g)
9
8
6
4
7
5
3

11. Bovine Diet and E. coli O157:H7: Forage vs Grain Diets
Wells et al, unpublished
In a on-going study, animals fed
forage-based growing rations
over 2 study years had higher
fecal prevalence for E. coli
O157:H7 than animals on corn-
based finishing rations.
Long term feeding of forage-type diets may increase E. coli O157:H7.
May be related to survival in manure and soils (Durso; Berry)
Forage quality appears to play a role in some form

12. DDGS impacts
• Distillers grains are increasingly included in
animal rations
– Significantly alter the gut microbiome (Pinchak)
• Increase fecal shedding of E. coli O157:H7
in cattle (Wells, 2009; Jacob, 2008)
• It appears that there is an effect of DG
compared to corn on O157 populations, but
magnitude is fairly small overall ~1.5
log10, but affects incidence
– Unknown mechanism
– Batch variability/QC issues

13. Fecal Samples
High Moisture/Dry-rolled Corn
Based Diet
(0 or 40% WDGS)Fecal prevalence and percent of fecal
samples with enumerable
E. coli O157:H7 were significantly
different for the pens.
Fecal counts for
E. coli O157:H7 were not
significantly different at any
Bovine Diet and E. coli O157:H7: WDGS in Finishing Diets

14. Finishing Phase (0 or 40% WDGS)
Hide Samples
Hide prevalence and percent of
hide samples with enumerable
E. coli O157:H7 were different
for the pens.
Hide counts for
E. coli O157:H7 were significantly
different from
40 CFU/100cm2
to 480 CFU/100cm2
.
Bovine Diet and E. coli O157:H7: WDGS in Finishing Diets
Wells et al, 2008

15. Finishing Phase
Prior to Shift
(0, 40 or 70% WDGS)
Fecal Samples
Bovine Diet and E. coli O157:H7: WDGS in Finishing Diets
Wells et al, 2010
After Shift

16. Finishing Phase
Time after Shift
Fecal Samples
Reductions in WDGS in
the diet did not result in
reduction of E. coli
O157:H7 in feces by day
28, but did by day 56.
Bovine Diet and E. coli O157:H7: Shift WDGS from Finishing Diets
Wells et al, 2010

17. The Bovine Fecal Microbiome: Corn vs WDGS
Finishing feedlot diets.
N = 4, 0 or 40% WDGS
Shifts in Prevotella and
Bacteroides.
WDGS diet resulted in
more
Sporacetigenium and
Anaerovorax
E. coli O157:H7
shedding not detected
Durso et al, 2010

18. The Bovine Fecal Microbiome: Diet and OTUs Association
A B
Heifer-10
Steer-10
Steer-09
Steer-09
Heifer-10
Steer-10
Principle Coordinate Analysis
Weighted UniFrac
Principle Coordinate Analysis
Unweighted UniFrac
Diet drives the fecal microbial composition in cattle
Heifer-10
30% Alfalfa Haylage
70% Corn Silage
Steer-09
66% Corn
26% Corn Silage
8% Supplement
Steer-10
83% Corn
12% Corn Silage
5% Supplement
Kim et al, submitted JAS

19. Red: Top 10% Average
Enumerable E. coli O157:H7
Blue: Lowest 10% Average
Enumerable E. coli O157:H7
Community structure may
not be driving shedding
Principle Coordinate Analysis
Weighted UniFrac
Heifers
30% Alfalfa Haylage
70% Corn Silage
Steers
66% Corn
26% Corn Silage
8% Supplement
Steers
83% Corn
12% Corn Silage
5% Supplement
Microbiome OTU Composition and Shedding of E. coli O157:H7
Kim et al, in preparation

20. Specific Microbiome Taxa Association with E. coli O157:H7
30% Alfalfa Haylage
70% Corn Silage
66% Corn
26% Corn Silage
8% Supplement
83% Corn
12% Corn Silage
5% Supplement
No obvious single taxa group driving
shedding
No community association for heifers.
Weak separation for steers diets
Community structure not driving
supershedding
Few OTUs associated supershedding
for any of the three diets
Kim et al, in preparation

21. The Bovine Fecal Microbiome and E. coli O157:H7
Propensity to shed E. coli O157:H7 appears to associate with
groups of OTUs within diet for animals fed finishing diets that
are corn-based.
In contrast, there appeared to be little association between
shedding E. coli O157:H7 and any single OTU or groups of
OTUs for animals fed the corn silage/haylage diet.
Shedding high levels of E. coli O157:H7 appears to have low
association with any OTU or groups of OTUs for any diet.

22. Tasco, tannins, and essential oils
• Produced from brown seaweed
• Reduced growth of
EHEC and Salmonella in
In vitro fermentations
• Improves meat quality and shelf life
• Polyphenolics (tannins) can reduce EHEC
– Naturally found in plants (Min; Lacombe)
• Essential oils can be bactericidal (spices)
– Limonene, terpenes, thymol, oregano (Varel)

23. Dried orange peel/pulp
• By-product, competitively prices into least-
cost ration formulation
– Used widely in dairy and feedlot rations in
Florida and Southern California
• Orange oils (e.g., limonene) improve animal
health and productivity (esp. in swine)
• Source of pectin and fermentable fiber to
ruminal microbial population
– 9% CP, TDN 82%, DE 1.64, NEm 0.88, and NEg 0.59
Mcal/lb (CSU)

24. Sheep fed 20% orange
peel consumed less peel
than did sheep fed 10%
0
1
2
3
4
5
6 0
10
20
SalmonellaentericaTyphimurium
(log
10
CFU/ml)
Rumen Cecum Rectum
0
5
10
0
1
2
3
4
E.coliO157:H7CFUlog
10
/gdigesta
Rumen Cecum Rectum
a b b ba aa b b
5 and 10% OP reduced
E. coli O157:H7 in rumen, cecum
and rectum
Final pH , TVFA, and A:P ratio
were not affected
Doesn’t work with Campylobacter

25. Dowd et al., 2006
Stress can affect microbial populationsStress can affect microbial populations
Bailey et al., 2011
Cecal community structure
changes following
social disruption (SDR)
Inverse relationship bet.
cytokines (IL-6) and
bacterial populations

26. Oral administration of
Norepinephrine
Pullinger et al., 2010
Stress is a complicated issue
It’s like pornography
Hard to replicate
Host/bacterial crosstalk (Lyte)
“Feed Me” signals
Microbial organ as a driver
of homeostasis and disease

27. Conclusions
• E. coli O157:H7 (and other EHEC?) co evolved
with cattle, so combatting it is neither easy, nor
straightforward
• Diet impacts O157:H7 populations
– Forage quality, WDGS vs corn, dry rolled vs steam-flake
• Diet changes the microbiome population
• Diet-driven microbiota changes are not linked to
O157:H7 shedding shifts
– Generic E. coli yes…
• What is driving the microbiome/EHEC change?
– Future avenues for exploring this linkage are evolving
rapidly

28. 9th
International Symposium on
Shiga Toxin (Verocytotoxin)
Producing Escherichia coli
Infections
Boston, Massachusetts
Todd Callaway
Jeff LeJeune
31 May- 3 June, 2015