Emerging Options to Control E. coli O157:H7 Pre-Harvest

Guy H. Loneragan Epidemiologist West Texas A&M University Canyon, Texas, USA SteakExpert June 24-25, 2009, Angers, France

Background

• Born and raised in Mudgee, New South Wales, Australia – Parents have a 500 hectare property – Cattle, sheep, crops, ~15 hectares shiraz/cabernet • Veterinary training at the University of Sydney, Australia – Degree awarded 1994 • Epidemiology training at Colorado State University, Fort Collins, Colorado, US – MS and PhD awarded 1998 and 2001, respectively • West Texas A&M University, Canyon, Texas, USA – Focus on food safety, particularly prior to harvest – E. coli O157, Salmonella, antimicrobial drug resistance

Comments Prior to Presentation

• E. coli O157:H7 and non-O157 VTEC are not geographically constrained – Found worldwide • E. coli O157:H7 is the primary cause of HUS in North America, many EU member states, South American countries, Australia, etc.

• Focus of my research has been on the North American production system – Mostly confined feeding – As such, most of the information I present is related to confined cattle feeding operations (i.e., feedlots, dairies) • Some of the options may provide areas for further discussion and opportunities for France and rest of EU

Discussion

• E. coli O157:H7 and non-O157 VTEC should be thought of as commensal organisms of cattle – In other words, they are (likely) beneficial bacteria of cattle and potentially other ruminants • In many instances, we encourage commensals – Historically, we have not had to ponder too much about how to eliminate/reduce commensal bacteria • Over time, we have developed tools to identify and control pathogens – For example, Salmonella Typhimurium, M. bovis, Brucella… • Initial pre-harvest efforts to control E. coli O157:H7 was to approach the problem as if it were a pathogen – Isolation, containment, prevent exposure – Not very effective

Chronology

• From a North American perspective, our initial flawed approach resulted in effective pre-harvest interventions arising relatively late – Most initial control at abattoirs • Highly effective but still failures occurred • Increase in illnesses and recalls because of contamination in 2007 • Finally, we began to understand the ecology of VTEC O157 and understood that it was not a pathogen for cattle • Consequently, interventions were developed that exploit some ‘tool’ or characteristic of the bacterium – Very specific (vaccine) or broad sweeping approaches (chlorate)

Chronology

• There now exists a large collection of scientific literature that provides evidence that it is possible to control E. coli O157 in cattle – Use of appropriate interventions given the production system can reduce the burden of E. coli O157 leaving farms and entering abattoirs • As an epidemiologist, I am most interested in field studies

Interventions

• A variety of interventions have been developed/evaluated: – Biological • DFM • Bacteriophage – Chemical • Neomycin • Sodium chlorate – Immunomodulation • Vaccine 1 0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 0 0.001

to 0.1

0.101

to 0.2

0.201

to 0.3

0.301

to 0.4

0.401

to 0.5

0.501

to 0.6

0.601

to 0.7

0.701

to 0.8

0.801

and in field studies of effect

Prevalence

Observed Expected more – Challenges models do not take into account important determinants of shedding (namely cohort level)

L. acidophilus-based probiotics

• Continuously fed to cattle – Only suitable to confinement operations • In the USA, achieved FDA GRAS status – BUT no FDA-approved label claim for control of E. coli O157 • Most widely evaluated product is that of Nutrition Physiology Corporation – BOVAMINE, includes LAB NP51 (NPC 747) • 9 peer-reviewed manuscripts demonstrating significant reduction using L. acidophilus NP51 – Articles from 4 groups of researchers • Systematic review, Sargeant et al, ZPH 2007;54:260 – Standardized approach – Evidence for efficacy of LAB NP51

Meta-analysis

• Meta-analysis is a method to combined data from different studies – Uncertainty associated with individual studies – Meta-analysis combined data, reduces uncertainty, provides best-estimate of effect • Powerful analytical approach – It is not, however, perfect and can be affected by statistical biases • Performed meta-analysis of NP51 – Both published and unpublished data

Summary of LAB NP51 A4 N1 J1 J2 C1 T1 R1 U1 U2 U3 A1 A2 A3 Combined .1

.5

.75 1 Fecal: Relative Risk 2 4

RR Fecal Product Efficacy Lower 95% CL Upper 95% CL P value 0.53

47% 0.44

0.63

<0.01

P value assoc. with Q stat = 0.15; Between study variance = 0.06

Summary of LAB NP51 A1 A2 A3 A4 N1 J1 C1 T1 R1 Combined .1

Hide: Relative Risk .5

.75 1 2 4

RR Hides Product Efficacy Lower 95% CL Upper 95% CL P value 0.60

40% 0.49

0.75

<0.01

P value assoc. with Q stat = 0.46; Between study variance = 0.00

Feedlot studies of NP51

• Meta-analysis; average reduction: – 40% reduction of prevalence in feces (26.5 versus 12.7%) – 47% reduction of prevalence on hides (20.4 versus 11.3%) • Some evidence for an effect on concentration – Fecal load in positive animals reduced by greater 99.5% • NP51 was associated with reduced number of positive animals and reduced number of bacteria in animals that remained positive

LABm and Control of E. coli O157:H7 in Ground Beef

Another Probiotic: PROBIOS FS

Enterococcus, Chr. Hansen • Fed for last 14 days on feed • Average reduction: – 56% feces – Hides not evaluated • Has FDA GRAS status in USA • Limited use – Limited availability???

Direct-Fed Microbials

• Easy adoption within confined feeding operations – Efficacy in a variety of feeding systems – Dairies, feedlots, etc.

• While not dramatic in all studies, evidence of improvements in animal performance • No easy implementation for pastured cattle • No USA FDA label claim for E. coli O157 control – Regulatory agencies in the USA will never ‘endorse’ – Better positioned if had an approved label claim for control/reduction of E. coli O157

Immunomodulation

• Is it possible to ‘trick’ an animal into developing immunity to a commensal?

– In the past, I believed this to be impossible • Three products have been evaluated in commercial settings • Vaccine: 8 peer-reviewed manuscripts – 7 showed significant reduction in shedding/colonization – Data generated from 3 groups of researchers

Bioniche Product

Ontario, Canada • Vaccination against Type III secreted proteins – Tool the bacteria use to colonize the gut • Field work really began with study published by Potter et al., 2004

Year

2002 2003 2003 2004 2004 2005a 2005b 2006

# of cattle

192 608 1003 288 718 20,556 504 168 480

Regimen

3-dose 1-dose 2-dose 3-dose 0-dose 3-dose 3-dose 2-dose 2-dose 2-dose 2-dose 3-dose

Bioniche Product

Ontario, Canada

Outcome

Feces Feces Feces Feces Feces TRM Feces TRM Feces TRM ROPES Feces TRM Hides Feces TRM Hides Feces Feces

Odds ratio 0.36

0.25

0.26

0.20

0.36

0.67

0.81

0.014

0.81

0.07

0.59

0.35

0.71

0.45

0.40

0.73

0.70

0.66

0.34

Vaccine efficacy

59% 68% 67% 73% 59% NS NS 98% NS 83% 27% 62% NS 54% 58% NS 28% 33% 65%

P-value

0.04

0.0001

0.0001

0.0001

0.0003

>0.10

>0.10

0.0001

0.56

0.0008

0.004

0.002

0.65

0.005

0.005

0.48

0.06

0.09

0.002

Comments

“bench-top” vaccine herd immunity?

ranch vaccination, low prevalence low prevalence 19 NE feedlots Between pens Within pens Dose effect

Bioniche Product

Ontario, Canada • Available in Canada as Econiche – Soon to be ‘Conditionally licensed’ in the USA • Cannot use trade name • In studies in USA, it has resulted in significant reductions in burden of E. coli O157:H7 – In feces – At TRM (RAMS, RAJ) – On ROPES (an indication of animal to contact-surface transfer) – On hides (both at the feedlot and at the packing plant) • Canadian study (not Bioniche product) found no effect – Van Donkersgoed et al., CVJ 2005 – Likely manufacturing issues rather than an efficacy issue

Epitopix Product

Minnesota, USA

E. coli

O157 • Targets the mechanism by which some bacteria acquire iron from environment – E. coli O157, Salmonella • Iron largely acquired via siderophore receptor & porin proteins (SRP) – Passive, gradient dependent – At low concentrations, energy dependent

SRP Technology

• Research suggests it is possible to restrict iron acquisition – via immunity against cell-surface SRP proteins – Competitive disadvantage • Vaccine consists of purified SRPs as antigens (Epitopix, 2004) • Technology developed for Salmonella in turkeys, then cattle, and now E. coli O157

E. coli

O157

Epitopix Product

Minnesota, USA • Initial field study suffered from low prevalence so hard to make statistical comparisons • • Significant reductions on hides or cattle positive at any site Subsequent study changed from 2 dose to 3-dose regimen • Feces – 39% • RAJ – 48% • Hides – 70%

Epitopix Product

Minnesota, USA P=0.28

P=0.13

P<0.01

Epitopix Product

Minnesota, USA P=0.01

98% reduction in MPN P=0.28

P=0.13

P<0.01

Epitopix Product

Minnesota, USA • Available in USA – Conditionally licensed • In studies in USA, it has resulted in significant reductions in burden of E. coli O157:H7 – In feces – On hides – In concentration of E. coli O157 • Ongoing research in production system in USA – Cows > suckling calves > feedlot • Opportunity for further research in pastured cattle outside of North America

Ft. Dodge

• Evaluated once (and published) in commercial settings FPT 2006;26:393-400

– Only 3 pens per exposure and unusual analysis – Unclear what to conclude except appeared beneficial • Feces – 68% • Hides – 50% • Other vaccines… UNM technology • Licensed to a Colorado company

Immunomodulation

• Strong evidence that it is possible to ‘trick’ an animal into developing immunity to a commensal • Bioniche’s Econiche licensed in Canada – Pending conditional license in the USA • Epitopix product conditionally licensed in the USA • Dose response – 3 doses appears to provide greatest response • 2 doses may be more effective when whole herds vaccinated • Easily administered to cattle in almost all production systems – More doses associated with more handling of animals • Appears to be slightly more efficacious than LAB

Sodium Chlorate

• Generated a substantial amount of interest in North America – Suitable for confined animal feeding operations • Acts as a suicide substrate for bacteria that respire using nitrate reductase • Chlorate >>> Chlorite – E. coli O157, Salmonella • Very effective in challenge studies – No slaughter authorization from USA’s FDA so not field studies to report – Currently within the regulatory process requesting label claim approval

Sodium Chlorate

• Very little field work with E. coli O157 • Challenges of making inferences from these challenge models – Needs field studies Callaway et al.,

JAS

2002

Bacteriophage

• Warrant consideration • Potential application – In feed – On hide – On meat surface • Example is Gangagen’s or Ivy Natural Solution’s products • Applied data of in-field effects is limited – Experimental evidence provides some promise • Callaway et al., FPD 2008 – challenge study, sheep • Sheng et al., AEM 2006 – challenge study, cattle

Expectations of Interventions?

Smith DR

, et al. 2001. J Food Prot 64 (12) 1899-1903 • None will provide 100% control – May not need 100% USA Feedlot Study • 73 feedlots visited twice • Summer 15.8% positive • Winter 5.6% positive Dave Smith, JFP 2001;64:1899 • Summer 30% positive • Winter 6.1% positive Dr. Smith’s project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number #0002501.

Expectations of Interventions?

E. coli O157:H7 Summer season prevalence 0.4

0.3

0.2

0.1

0 1 0.9

0.8

0.7

0.6

0.5

Summer: EVERY pen positive but highly variable prevalence (1-80%) Very few pens near the mean. Mostly far greater or far lower prevalence than the mean Doesn’t follow expected distribution Smith et al. 0 0.001

to 0.1

0.101

to 0.2

0.201

to 0.3

0.301

to 0.4

0.401

to 0.5

0.501

to 0.6

0.601

to 0.7

0.701

to 0.8

0.801

and more

Prevalence

J Food Prot

. 2001, 64 (12) 1899-1903 Observed Expected

Model to Compare Prevalence of Cattle During Summer, Winter, and with intervention (Summer) Data-driven Simulation. Source, Dave Smith, UNL • Stochastic simulation model of the prevalence of E. coli O157:H7 in live cattle with vaccination (@risk 4.5)

Distribution of prevalence

• 5,000 pen simulations

Mean, 5th and 95th Percentile

1 (500,000+ cattle) – Intervention: Summer (vaccine) – No intervention: Summer – No intervention: Winter 0.8

• Intervention efficacy 65% – Pert(50, 65, 80) 0.6

Pert(0.5, 0.65, 0.8) 0.4

7 6 1 0 5 4 3 2 0.2

0 90.0% 5.0% 0.5568

0.7432

5.0% No interv.

Interv.

No interv.

Model to Compare Prevalence of Cattle During Summer, Winter, and with intervention (Summer) Data-driven Simulation. Source, Dave Smith, UNL Summer, no intervention 1 0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 Lower risk Higher risk 0 0.001 to 0.1

0.101 to 0.200

0.201 to 0.300

0.301 to 0.400

0.401 to 0.500

0.501 to 0.600

0.601 to 0.700

0.701 to 0.800

0.801

and more

Prevalence

Winter, no intervention 1 0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 Lower risk Higher risk 0 0.001 to 0.1

0.101 to 0.200

0.201 to 0.300

0.301 to 0.400

0.401 to 0.500

Prevalence

0.501 to 0.600

0.601 to 0.700

0.701 to 0.800

0.801

and more

Model to Compare Prevalence of Cattle During Summer, Winter, and with intervention (Summer) Data-driven Simulation. Source, Dave Smith, UNL Summer, no intervention 1 0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 Lower risk Higher risk 0 0.001 to 0.1

0.101 to 0.200

0.201 to 0.300

0.301 to 0.400

0.401 to 0.500

0.501 to 0.600

0.601 to 0.700

0.701 to 0.800

0.801

and more

Prevalence

Winter, no intervention 1 0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 Lower risk Higher risk 0 0.001 to 0.1

0.101 to 0.200

0.201 to 0.300

0.301 to 0.400

0.401 to 0.500

Prevalence

0.501 to 0.600

0.601 to 0.700

0.701 to 0.800

0.801

and more 1 0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 Predicted intervention distribution (summer) Lower risk Higher risk 0 0.001 to 0.1

0.101 to 0.200

0.201 to 0.300

0.301 to 0.400

0.401 to 0.500

Prevalence

0.501 to 0.600

0.601 to 0.700

0.701 to 0.800

0.801

and more

Expectations of Interventions?

• No intervention is 100% – Need not be • Effective turns peak-season shedding patterns into low-season shedding patterns – 30 to 40% of cases in North America attributed to beef • In low season, observe: – Less frequently contaminated beef • Fewer recalls • Less product diverted to cooking – Fewer human illness

Expectations of Interventions?

Seasonal Occurrence of Human Illnesses • Repeatable overrepresentation of cases during May-October • 70% of reported cases in 6 months of the year • In winter, prevalence in cattle, percentage ground beef samples positive, and human cases are all substantially lower compared to summer • Target winter-time burdens. Supported by data.

Expectations of Interventions?

• In winter, prevalence in cattle, percentage ground beef samples positive, and human cases are all substantially lower compared to summer • Target winter-time burdens. Supported by data.

Expectations of Interventions?

• If not 100% effective, then what is its purpose – What do we want it to do?

– What can we expect it to do? • Issues: – Purpose of pre-harvest interventions – Different measures of prevalence in different studies (e.g., feces versus hides versus RAMs) – Cross-contamination and interventions – What can pre-harvest do for us

Expectations of Interventions?

Cattle Operations Packing Plant HACCP/PR Various Interventions Ground Beef Trim for off site grinding Not a fail-safe system In-coming load can overwhelm the system

Expectations of Interventions?

Cattle Operations Packing Plant HACCP/PR Various Interventions Ground Beef Trim for off site grinding Not a fail-safe system In-coming load can overwhelm the system

Expectations of Interventions?

Cattle Operations Packing Plant HACCP/PR Various Interventions Ground Beef Trim for off site grinding Not a fail-safe system In-coming load can overwhelm the system

Expectations of Interventions?

• Purpose not simply ‘more is better’ • To ensure burden of E. coli O157 on cattle presented for harvest is within manageable limits – The in-plant series of HACCP/PR interventions effectively mitigate the burden on incoming cattle • Efficacy need not be (or even approach) 100% • Desired efficacy depends on the burden within groups of cattle AND on the pathogen-mitigation capacity of the plant – Linear of threshold? I suspect the latter.

Expectations of Interventions?

67% Product Efficacy: (RR-1)*100 50% 30% 50% Plant HACCP/PR Threshold Incoming burden without intervention Incoming burden with intervention

Expectations of Interventions?

67% Product Efficacy: (RR-1)*100 50% 30% 50% Plant HACCP/PR Threshold Incoming burden without intervention Incoming burden with intervention

Summary: Immunomodulation as an Intervention for E. coli O157:H7

• No intervention will be 100% effective E. coli O157 Pre-harvest Harvest Consumers • Scientific evidence, simulation models, and empirical data indicate pre-harvest interventions are effective – Ultimately should reduce consumer exposure to E. coli O157

Summary: Immunomodulation as an Intervention for E. coli O157:H7

• No intervention will be 100% effective E. coli O157 Pre-harvest Harvest Consumers • Scientific evidence, simulation models, and empirical data indicate pre-harvest interventions are effective – Ultimately should reduce consumer exposure to E. coli O157

Summary: Immunomodulation as an Intervention for E. coli O157:H7

• No intervention will be 100% effective E. coli O157 E. coli O157 Pre-harvest Harvest Consumers • Scientific evidence, simulation models, and empirical data indicate pre-harvest interventions are effective and fit the purpose – Should reduce consumer exposure to E. coli O157

Summary: Immunomodulation as an Intervention for E. coli O157:H7

• No intervention will be 100% effective E. coli O157 E. coli O157 Pre-harvest Harvest Consumers • Scientific evidence, simulation models, and empirical data indicate pre-harvest interventions are effective and fit the purpose – Should reduce consumer exposure to E. coli O157

Summary: Immunomodulation as an Intervention for E. coli O157:H7

• No intervention will be 100% effective E. coli O157 E. coli O157 Pre-harvest Harvest Consumers • Scientific evidence, simulation models, and empirical data indicate pre-harvest interventions are effective and fit the purpose – Should reduce consumer exposure to E. coli O157

Summary: Immunomodulation as an Intervention for E. coli O157:H7

• No intervention will be 100% effective E. coli O157 E. coli O157 Pre-harvest Harvest Consumers • Scientific evidence, simulation models, and empirical data indicate pre-harvest interventions are effective and fit the purpose – Should reduce consumer exposure to E. coli O157

Issue: How Much is Enough?

• Provide cattle with burden below threshold Either by: – Reducing burden from some problematic level – Preventing groups of cattle reaching problematic burden • Threshold is uncertain • But empirical data suggest warmer months associated with increased risk – Shedding in cattle and human illnesses

Summary

• Pre-harvest control of E. coli O157 (and potentially other non-O157 VTEC) is possible – Not 100% but no need to be 100% • Purpose is within a system of multiple controls – Pre-harvest > harvest > packaging > consumers • Effectively turn peak-season shedding patterns into low season shedding patterns • Unclear what numerical contribution this has on risk of final product contamination • Opportunities to evaluate products in pasture settings

Acknowledgements

• SteakExpert organizers for invitation • Patrice Arbault for co-ordination • Your hospitality • You for listening to me in English rather than in French • Colleagues for sharing data • Contact Information: [email protected]

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