Pre-harvest Food Safety Research at Washington State University- in a

Nutshell

Margaret A Davis DVM, MPH, PhD Paul G. Allen School for Global Animal Health

Veterinary Clinical Sciences College of Veterinary Medicine

Washington State University Pullman, WA 99164

I. E. coli O157:H7

– Genotypes

– Seasonality

II. Salmonella Enteritidis – virulence studies

III. Salmonellosis

– Bovine reservoir of MDR Salmonella

– Antibiotic resistance

IV. Campylobacter

• Clinical genotypes (CG) – isolated primarily from human clinical samples

• Bovine-biased genotypes (BBG) – isolated from bovine samples but rarely from human clinical samples

• Bovine isolates are more diverse – include both CG and BBG

• Many subtyping methods correlate well w.r.t. BBG v. CG

I. E. coli O157:H7

Dr. Tom Besser

E. coli O157:H7 subtyping methods • Lineage Specific Polymorphism Assay alleles

(LSPA, 6 loci, 18 potential alleles)

• Stx-encoding Bacteriophage Insertion Sites (SBI, 6 loci, 12 potential alleles)

• Presence or absence of stx2-Q933 and stx2-Q21 alleles

• Tir T255A SNP alleles

• SNP typing – 48 loci

Distribution of SBI genotypes

32%

2%64%

2%

0%

0%0%0%0%0%0%0%0%0%0%

Human Isolates, N=280

Distribution of SBI genotypes

10%

0%

42%

1%0%1%3%

11%1%

1%

3%

1%

19%

6% 1%

Bovine Isolates, N=80

Effect of on-farm interventions that have been studied may have been over- or under-estimated for CG strains of EcO157:

• Vaccines • DFM (Direct fed microbials, probiotics)

• Water trough hygiene • Feed type

• Winter v. summer • Feedlot v. dairy

Comparison of bovine Escherichia coli O157:H7 genotypes by production system (feedlot vs dairy), season (summer vs winter), source (fecal vs water), and sample type (fecal vs recto-anal junction). Woo Kyung Jung, Margaret A. Davis, and Thomas E. Besser Odds of CG Fecal v. RAJ: NS Other factors that will be analyzed: vaccinated v. unvaccinated cattle, probiotic use, grain type, days on feed.

OR 95% Confidence Interval

Feedlot/Dairy 11.3 (6.5 - 19.7)

Summer/Winter 6.5 (2.8 – 15.4)

Water/Fecal 3.0 (1.4 – 6.4)

Figure 1: Seasonal STEC human disease incidence and bovine prevalence. Human: mean reported cases 1998- 2007 (http://www.cdc.gov/ mmwr/mmwr_nd/index.html). Bovine: from Herriot et al. ; nine herds sampled monthly (~60 fecal samples per month) using standardized non-IMS methods.

Seasonality in cattle

• Extrinsic effects?

– Environmental proliferation in warmer temperatures

– Seasonal feed components

• Intrinsic effects?

– Immune responses

– Endocrine effects (day length) Dr. Carolyn Hovde Bohach

Two groups of 10, 9-month-old Holstein steers

Challenge with 109 cfu O157 (mixture of 4 strains):

Rectal challenge Oral challenge

Culture for O157 by Recto-Anal Mucosa Swab (RAMS) Days 1, 4, 7, and weekly, thereafter for 60 days

using SMAC-CTVM selective media

Genotype O157 isolates using a multiplexed PCR to determine the Stx-encoding prophage insertion sites

Trial completed TWICE in the Summer and Winter Seasons Will repeat TWICE more this Summer and Winter Seasons

• Regardless of season, cattle similarly challenged with E. coli O157:H7 shed similar numbers of bacteria for similar durations indicating the effect of season was due to exogenous factors

• No significant differences in duration or the concentration of E.

coli O157:H7 carried resulted from the different oral and rectal challenge methods indicating passage through the gastrointestinal tract was not a factor in these trials.

• Five cattle (ear-tag #s T2, T3, T7, T16, T17) remained culture positive for E. coli O157:H7 for long durations (>42 days) in both seasons, indicating that endogenous factors played a role in O157 carriage in individual animals.

• Bovine-biased genotype 6 (BBG6) predominated among the E. coli O157:H7 shed in both seasons indicating that bacterial factors played a role in bovine colonization.

II. S. Enteritidis virulence

S. Enteritidis in the United States

Sources: http://www.cdc.gov/ and Braden (2006) Clin. Infect. Dis. 43: 512-17

case

rat

e p

er 1

00

,00

0

Number 1 serotype of Salmonella isolated from cases of food borne infections Between 1985 and 1999

997 outbreaks

33,687 cases

328 hospitalizations

82 deaths Dr. Devendra Shah

Cell invasiveness of poultry-associated S. Enteritidis strains

Model: Well-differentiated Caco-2 cells

• Method: Gentamicin protection assay

• Outcome variable: Intracellular CFU

• Total # of strains: 53

3-d

ay o

ld

14

-day

old

Incubate at 37C in 5% CO2 for 2 h

Treat with Gentamicin (200 µg/ml)

Trypsinize cells

Determine the intracellular CFUs

Infect cells at 10 MOI

Poultry-associated strains of SE vary in cell invasiveness

• Low invasive strains show lowered virulence in a mouse model

• Low invasive strains have impaired motility

• Low invasive strains have impaired tolerance to acid

• Low invasive strains show impaired survival in chicken macrophages

III. Cattle-associated salmonellosis and antibiotic resistance

Dairy cattle are a reservoir for human MDR Salmonella infections

– Cornell collaboration

– Two-part comparison

Washington v. New York State 2004 - 2005

Odds Odds Ratio 95% CIb

bovine vs. human Newport 18.2 6.3 - 52.6

bovine vs. human Typhimurium 3.5 1.7 – 7.1

bovine Newport vs. bovine Typhimurium 6.1 2.1 - 17.9

human Newport vs. human Typhimurium 1.2 0.6 - 2.2

Northwest vs. Northeast isolates 2.0 1.2 - 3.4

Odds ratio estimates for odds of multidrug resistance based on nominal logistic regression model

Hoelzer et al. AEM 2010;76(17):5947-59.

Northeast

N (%)

Northwest

N (%)

Χ2 P-value

Newport % Suscept 36 (52.2) 60 (36.8) <.05

% MDR 33 (47.8) 99 (60.7) .07

Typhimurium % Suscepta 154 (72.0) 51 (16.0) <.0001

%MDRb 47 (22.0) 223 (69.9) <.0001

Bovine Human

Newport % Suscept 36 (31.6) 60 (50.9) <.05

% MDR 76 (66.7) 56 (47.5) <.05

Typhimurium % Suscept 60 (35.7) 145 (39.7) .4

% MDR 100 (59.5) 170 (46.6) <.05

Table 2. Proportions of Salmonella enterica serotypes Newport and Typhimurium

that were susceptible to all antibiotics and that were multi-drug resistant, by region

and species affected.

a Suscept, susceptible to all antibiotics tested. bMDR, multi-drug resistant. Resistant to two or more classes of antibiotics.

For both projects:

• All isolates were compared using – Pulsed-field gel electrophoresis (PFGE)

– Multilocus variable number of tandem repeats analysis (MLVA)

– Similarities combined cluster analysis in Bionumerics (Applied Maths, Austin, TX)

• Isolates indistinguishable by both methods were detected from different host species and regions:

Salmonella Newport isolates 2004-2005

Proportions of human Salmonella isolates indistinguishable from

bovine isolates by PFGE and MLVA combined

• Human isolates more likely to match bovine isolates in

the NW than in the NE (2004-2009):

Serotype NY WA P-value

NEWPORT 5/65 (7.7%) 28/118 (23.7%) <0.01

TYPHIMURIUM 3/191 (1.6%) 19/294 (6.5%) <0.01

Direct transmission between cattle and humans > foodborne route

– Case-case study

– Cummings KJ et al, EID (In press)

• Case=salmonellosis associated with bovine PFGE type – Had to match PFGE profile from >=2 bovine

animals

• Control=salmonellosis associated with non-bovine PFGE type – Serotype or PFGE profile never found in

Salmonella from bovine animals

• Interviews conducted by local health department staff with more precise questions about animal exposures

Variable Odds Ratio 95% Confidence

Interval

p-value

Farm animal contact 3.1 (1.6, 6.0) 0.0005

Undercooked ground beef 1.7 (0.9, 3.5) 0.1

Unpasteurized milk 0.5 (0.1, 3.9) 0.5

1

Variable Odds Ratio 95% Confidence

Interval

p-value

Bovine contact 5.2 (2.0, 13.3) 0.0005

Undercooked ground beef 1.8 (0.9, 3.5) 0.1

Unpasteurized milk 0.6 (0.1, 4.7) 0.6

1

• Are bovine-associated salmonella infections more likely to be MDR?

Washington cases and controls

Case Control

Resistant to 3 or more drug classes)

11 23

Resistant to 0 to 2 drug classes 39 197

50 220

Odds ratio = 2.3 (1.1, 5.3)

Summary

• Salmonella enterica are more likely to be MDR

– From cattle than from humans

– In the NW compared to the NE

– In serotype Newport than Typhimurium (sometimes)

• Cattle and humans are more likely to have the same subtypes

– In the NW compared to the NE

Summary (cont’d)

• Direct transmission may be a more important route of exposure for humans than the foodborne route

• Human infections with bovine-associated strains are more likely to be MDR in WA

How do new MDR Salmonella strains get introduced into herds?

Adhikari B, et al. The role of animal movement, including off-farm rearing of heifers, in the interherd transmission of multidrug-resistant Salmonella. J Dairy Sci. 2009 Sep;92(9):4229-38. PubMed PMID: 19700684.

Dr. Dale Hancock

• 59 dairy herds X 7 visits e 2 – 4 months

• Samples cultured for Salmonella

• Subtyping by PFGE

Factors not associated with the introduction of new MDR Salmonella strains were housing of heifers and cows in the same close-up pen, a common hospital-maternity pen, and the number of purchased cattle. This study highlights the role of animal movement in the interherd transmission of MDR Salmonella spp.

OR (95% CI)

Off-farm heifer raising, including commingling

8.9 (2.4, 32.8)

Herd size per 100-animal increment

1.04 (1.01 – 1.05)

Calf health and antibiotic resistance

Association between antimicrobial use and diarrhea in preweaned calves

Berge AC, Moore DA, Besser TE, Sischo WM. Targeting therapy to minimize antimicrobial use in preweaned calves: effects on health, growth, and treatment costs. J Dairy Sci. 2009 Sep;92(9):4707-14.

Dr. Berge Dr. Sischo

Average daily gain

Current project: USDA-NIFSI funded Minimizing antimicrobial resistance

transmission: The dairy farm as a model system

• Communication model – What is the direction of information flow on large

dairies?

– How can we effectively deliver messages about minimal antimicrobial use?

• Calf housing – Exploring the effect of hutch row configuration on

transmission of antibiotic resistant bacteria

• Wm Sischo, Dale Moore, Cornell Collaborators

Possible mechanisms for resistance transmission

Prevalence of resistance to cephalosporins; S. Dublin

Davis et al. 2007

Adhikari 2009

Signature for selection from ceftiofur is limited in the gut

Daniels et al. 2009

Ceftiofur

• Used to treat respiratory disease and foot-rot in cattle

• Injectable, converted to ‘CFM’ within minutes

• 70% excreted in urine; 30% in feces

• Metabolite is inactivated in feces

Dr. Doug Call

Bioavailability of different antibiotics can vary dramatically in soil

Subbiah et al. 2011b

IV. Campylobacter

2006 15.6/100,000

Methods

• Case-control study

– Cases interviewed by Whatcom and Yakima Counties Health Department personnel

– Controls identified by RDD and interviewed by staff of WSU Social and Economic Sciences Survey Center (SESRC)

– Matched by age category and county of residence

– Asked about food, recreation and animal related exposures in the 2 weeks prior to interview/illness

Methods

• Analysis: Conditional logistic regression analysis in SAS/PC v. 9 (Cary, SC)

• Period of study: February, 2009 – Aug, 2010

• 176 cases and 214 controls

Exposures not found to have a significant association

• Outdoor recreation (camping, hiking, yard work, hunting)

• Recreational water exposure

• Drinking water source (well v. public water supply)

• Pets at home

• Contact with sheep, goats, pigs, camelids

• Eating undercooked poultry*

Results of conditional logistic regression analysis

Multivariate conditional regression analysisa

Exposure Odds

ratio

95% C.I.

Male gender 0.6 (0.3, 1.2)

Hispanic ethnicity 7.0 (3.5, 14.3)

Lived/worked on a dairy farm 7.3 (1.6, 33.0)

Ate poultry 0.2 (0.1, 0.5)

Live poultry 2.4 (0.9, 6.3)

Handled raw poultry 0.2 (0.1, 0.7)

Ate restaurant food 2.3 (1.0, 4.9)

a Consumption of unpasteurized dairy products was removed from the

model by the stepwise procedure.

Genetic subtyping of Washington State Campylobacter isolates from cattle, humans and retail chicken

• Multilocus sequence typing

– Dingle et al. Multilocus sequence typing system for Campylobacter jejuni. J Clin Microbiol 2001;39:14-23.

– Submission of allele data to PubMLST

• http://pubmlst.org/campylobacter/

– Sequence types (ST) and clonal complexes (CC) are assigned

Host association (Washington) of human isolates’ specific sequence types (ST)

BOVINE 23

NEITHER 33

POULTRY 9

Total human isolates 65

Bovine Human Poultry

Clonal

Complex

WA PubML

ST

WA PubML

ST

WA PubML

ST

ST-21 40 38 17 20 21

ST-22 6 4 1 1

ST-283 1 1 8 1

ST-353 9 13 9

ST-443 14 4 5 5

ST-45 3 8 2 7 11 7

ST-48 4 7 6 9 2 2

ST-61 7 10 1 1

Total 95 63 65 56 27 45

Frequency of C.jejuni MLST clonal complexes from WA compared to PUBMLST databasea 2008-2011.

Thank you

MLST Results Clonal Complex Bovine Retail

chicken

Human clinical Total

- 11 7 18

PENDING 2 2

ST-179 COMPLEX 1 1

ST-21 COMPLEX 40 17 57

ST-22 COMPLEX 6 4 10

ST-257 COMPLEX 7 7

ST-283 COMPLEX 8 1 9

ST-353 COMPLEX 9 9

ST-354 COMPLEX 2 2

ST-403 COMPLEX 1 1

ST-42 COMPLEX 14 4 18

ST-443 COMPLEX 5 1 6

ST-45 COMPLEX 3 11 2 16

ST-460 COMPLEX 2 2

ST-464 COMPLEX 1 1

ST-48 COMPLEX 4 2 6 12

ST-49 COMPLEX 1 1

ST-508 COMPLEX 2 2 4

ST-52 COMPLEX 1 1

ST-607 COMPLEX 2 2

ST-61 COMPLEX 7 1 8

TOTAL 95 27 65 187