Keeping Foodborne Pathogens Down on the Farm Michael P. Doyle.

Keeping Foodborne Pathogens Down on the Farm

Michael P. Doyle

Leading Bacteriological Causes of Foodborne Illness in USA

Campylobacter jejuni - est. 2 million cases/yr Principal vehicles - poultry, unpasteurized milk

Salmonella sp. - est. 1.5 million cases/yr Principal vehicles - eggs, poultry, beef, pork,

produce E. coli O157:H7 - est. 60,000 cases/yr

Principal vehicles - cattle (handling) and beef, produce, water (recreational and drinking)

Transmission of Foodborne Pathogens

Campylobacter jejuni and Salmonella sp. Carried in intestinal tract of poultry and other

animals Fecal contamination of skin during grow out and

processing Salmonella enteritidis

–Colonize ovarian tissue of poultry

– Internal contents of eggs are contaminated

Transmission of Foodborne Pathogens

E. coli O157:H7 Carried in intestinal tract of cattle Direct or indirect contact with cattle manure is likely

most frequent origin Manure can contaminate food through:

. Use of manure as a soil fertilizer . Polluted irrigation water . Defecation of cattle in vicinity of

produce or

foods of animal origin

Prevalence of Risk Factors for Foodborne Illnesses in the General Population

Approximately 7,500 adults in CA, CT, GA, MN and OR were interviewed by telephone between July 1996 and June 1997 30% ate pink hamburger 18% ate running eggs 1.9% ate raw shellfish 1.5% drank raw milk 7% did not wash cutting board after cutting raw chicken 7% did not wash their hands after handling raw meat or

poultry

B. Shiferaw et al. J. Food Protect. 63:1538 (2000)

Risk Factors for Sporadic Campylobacter Infections in the United States

Case-control study of 6 FoodNet sites from Jan 98 - Mar 99 involving 1463 patients with Campylobacter infection and 1317 controls Risk factors include:

Foreign travel Eating undercooked poultry Eating chicken or turkey cooked outside the home Eating nonpoultry meat cooked outside the home Eating raw seafood Drinking raw milk Living on or visiting a farm Contact with farm animals Contact with puppies

C. Friedman et al. Abstr. Int Conf Emerg Infect Dis 2000 No. 63. P. 149-150

Risk Factors for Fluoroquinolone-Resistant Campylobacter Infections

Case-control study of 7 FoodNet sites from 1998-99 94 of 858 (11%) isolates from Campylobacter infections

were fluoroquinolone-resistant Risk factors include:

Eating chicken or turkey cooked at a commercial establishment

Storing raw chicken in refrigerator without plate to catch drippings

Travel outside the United States H. Kassenborg et al. Abstr. Int Conf Emerg Infect Dis 2000 No. 63, P. 150

Risk Factors Associated with Sporadic Cases of E. coli O157:H7

Infection in U.S.

Eating undercooked ground beef Visiting a farm

U.S. Centers for Disease Control and Prevention, 1998

Risk Factors Associated with E. coli O157:H7 Infections in Scotland

1. Handling / preparing raw food (40%)

2. Involved in gardening / garden play (36%)

3. Lived on / visited farm (20%)

4. Direct / indirect contact with animal manure (17%)

5. Private water supplies (12%)

6. Recent failures with high coliform counts of water supplies (12%)

J. E. Coda et al., J. Infect. 36:317, 1998

Risk Factor Associated with E. coli O157:H7 Infections in Sweden

Risk Factor Contact with farm animals and farms

Swedish Government Recommendations Between June 1 to October 31, farmers with cattle, sheep or

goats should avoid:Visits to animal houses by unauthorized people,

especially children under the age of 5 avoid contact with cattle

Consuming unpasteurized milk and dairy productsContact between grazing animals and humans

sunbathing on nearby beaches

B. de Jong, Smittskydd 4:48, 1998

Risk Factor Associated with Shiga Toxin-Producing E. coli Infections in Canada

Determined spatial relationship between livestock density and human STEC incidence

Based on 3001 cases of STEC (>95% E. coli O157:H7) infection from 1990 - 95 in Ontario, Canada

Michel et al. Epidemiol. Infect. 122:193 (1999)

Risk Factors Associated with STECInfections in Canada

Results:

Areas with high incidence of STEC cases were situated predominantly in areas of mixed agriculture (high in rural areas compared to urban areas)

Cattle density had a positive association with incidence of STEC cases

Elevated risk of STEC infection in rural populations associated with living in areas with high cattle density

Risk Factors Associated with STECInfections in Canada

Conclusions:

Importance of contact with cattle and cattle manure likely has been previously underestimated as a risk factor for STEC infections

Calf to Human Transmission of E. coli O157:H7

1. 13-month-old boy hospitalized with bloody diarrhea in October 1992; lived on dairy farm in SW Ontario

- E. coli O157:H7 phagetype 23 isolated from stool

2. Boy placed on straw between calves while mother did barn chores

- Boy frequently touched calves and put his fingers in their mouths and his

3. Fecal culture of 1 of 7 calves tested positive from E. coli O157:H7 phagetype 23

Renwick et al. J. Infect. Dis. 168:792 (1993)

E. coli O157:H7 Transmissionvia a Dog

1. Three-year-old U.K. girl developed E. Coli

O157:H7 infection after petting a dog at a farm

visitor center

2. Girl had no other contact with animals or dirt

3. An identical strain of O157:H7 was recovered

from cattle on the farm

Parry et al. 1995. Lancet 346:8974

E. coli O157:H7 Infection from Vegetables

1.39-year-old lacto-ovo-vegetarian woman in Maine had E. coli O157:H7 infection

2.She lived on a farm and her diet consisted almost exclusively of vegetables from her garden

- Garden fertilized with manure from calf and cow

3.E. coli O157:H7 was isolated manured soil from the garden

Cieslak et al., Lancet 342:367 (1993)

Outbreak of E. coli O157:H7 Infections Associated with Farm Visits

51 cases of E. coli O157:H7 infection associated with visiting a petting farm in Pennsylvania during Sept-Nov 2000

16 patients hospitalized, 8 with HUS Case-control study identified physical contact with cattle as

major risk factor (OR = 10.94) Hand washing before eating was protective (OR = 0.23)

O157:H7 isolates from humans, 27 of 216 (13%) cattle and a handrailing all had same PFGE profile

Household survey estimated that 7,000 people developed diarrhea associated with visiting the farm

J. A. Crump et al. Centers for Disease Control and Prevention (2001)

The Manure Glut: A Growing Environmental Threat

Five tons of animal manure is produced annually nationwide for every person living in the United States The amount of animal manure is 130 times greater

than the amount of human waste produced Cattle, hogs, chickens and turkey produced an

estimated 1.36 billion tons of manure in 1997 Democratic Staff of U.S. Senate Agriculture

Committee (1998) “Animal Waste Pollution in America:

An Emerging National Problem”

The U.S. Manure Glut (1997 estimates)

Animal Solid Waste (Tons/yr)

Cattle 1,229,190,000

Hogs 112,652,300

Chickens 14,394,000

Turkeys 5,425,000

TOTAL 1.36 billion

Prevalence of Campylobacter in Manure

Cattle manure Beef cattle at slaughter 89% prevalence

Poultry manure Chickens and turkeys 80-100% prevalence

(depending on flock) Sheep manure

Sheep at slaughter high prevalence

Prevalence of Salmonella in Manure

Cattle manure - 10 to 25% of samples

Poultry manure - 29% of samples

Prevalence of E. coli O157:H7 in Manure

Cattle manure Weaned dairy calves 5% Unweaned dairy calves 2% Cattle at slaughter 13-28%

Reported Levels of Pathogens in Animal Manures

Pathogen Animal

Cattle Poultry Sheep (CFU or Oocysts/g)

Campylobacter 104 - 108 104 - 107 up to 105

Salmonella up to 108 - 1010 104 - 107 no information

E. coli O157:H7102 - 105 — 108 Cryptosporidium 105 - 1010 — 107

Fate of Salmonella in Cattle Manure

Storage

Temperature Decimal Reduction Time

(oC) (Days) 4 13-20

20 9-25

37 2-8

S. Himathongkham et al. FEMS Microbiol Lett 178:251 (1999)

Survival of E. coli O157:H7 in Bovine Feces

Detected by

enrichment

37oC

22oC

5oC

Survival of E. coli O157:H7 inSheep Manure

Manure pile (7 m long by 3 m wide by 0.6 m deep) collected from sheep experimentally administered E. coli O157:H7; held undisturbed (not aerated) for 21 months

O157 isolated consistently for 12 months (except for November) from middle and bottom moist layers but not from dry feces at top

O157:H7 counts ranged from <102 to 2.2 x 10

6 cfu/g

O157 detected in 1 of 24 manure samples at 21 months

I. T. Kudva et al. Appl. Environ. Microbiol. 64:3166 (1998)

E. coli O157:H7 Infection Associatedwith Well Water and Infected Cattle

on a Dairy Farm

16-month old child from dairy farm hospitalized with bloody diarrhea

E. coli O157:H7 isolated from: Child's stool 63% of cattle on the farm Well water

Well water was contaminated with cattle manure

S. G. Jackson et al., Epidemiol. Infect. 120:17, 1998

Lake-Associated Outbreak ofE. coli O157:H7 Infection

1.12 cases of E. coli O157:H7 infection during June - July 1995 in Illinois

2.Acquired infection by swimming in a lake at an Illinois State Park

- Case-control study revealed that risk for illness was associated with taking lake water into the mouth and swallowing lake water

CDC, Morbid. Mortal. Weekly Rep. 45(21):437 (May 31, 1996)

Association of E. coli O157:H7 with Water

Drinking and recreational (swimming) waters have been identified as vehicles of E. coli O157Sources of contamination include:

Cattle manure seeping into well water or lakes

Children defecating in a lake and swimming pool

Survival of E. coli O157:H7 in Water

E. coli O157 can survive for a long period of time in water, especially at cold temperaturesSurvival for more than 13 weeks in water at

8EC, with only a 10-to-100-fold reduction Precautions should be taken when using

lake or river waters for drinking or recreational purposes

Association of E. coli O157:H7 with Deer

During July - August 1997, 310 fresh deer fecal samples collected from ground at 5 Georgia wildlife management areasNo isolations of E. coli O157:H7

J. R. Fischer et al., University of Georgia


During autumn 1997, 371 fecal samples collected directly from hunter-killed deer at 6 Georgia wildlife management areasE. coli O157:H7 isolated from 3 deer

All from deer in NW Georgia in vicinity of cattle

Two different PFGE DNA fingerprints



During autumn 1998, 140 fecal samples collected directly from hunter-killed deer at NW Georgia location in vicinity of cattleNo isolations of E. coli O157:H7

During same time period, 231 fecal samples collected directly from cattle present in vicinity of deerE. coli O157:H7 isolated from 12 (5.2%) cattle


Public Health Issues Associated Human Pathogens Carried by Animals

1. Contaminated food from animals Meat, eggs, milk

2. Contaminated food that contacts animal waste Vegetables and fruit grown in soil fertilized with

animal manure or treated with irrigation water with animal waste

3. Contaminated water containing animal waste Untreated drinking water and swimming in

recreational lakes

Methods of Control for E. coli O157

Low infectious dose of E. coli O157 necessitates reducing or eliminating pathogen, rather than solely preventing its growth

HACCP system most effective approach for reducing risk of E. coli O157 infections Most desirable HACCP system includes a step that

kills pathogens For raw foods that do not receive a terminal kill

treatment, HACCP systems must be implemented throughout food continuum, from farm to table

Where Must Food Safety Begin?

Solutions are complex but must begin at the farm

Food producers must consider and treat their products as foods rather than as commodities

Intervention or Control Points

Food Producers

Examples of CP’s for preharvest foods Probiotics and competitive exclusion bacteria

– Use of beneficial microorganisms that prevent colonization or eliminate pathogens from animals used for food products

Bacteriophage Innovative vaccines Dietary and feeding practices

Control of E. coli O157:H7 in Cattle by Competitive Exclusion Bacteria

Competitive exclusion involves use of microbial cultures that out-compete pathogens from colonizing specific niches Principal sites of E. coli O157 localization in cattle

are the animal’s three forestomachs and the large intestine

Isolates of E. coli that produce antimicrobials to E. coli O157 and localize in the same sites of bovine GI tract as E. coli O157 can eliminate or reduce carriage of E. coli O157 in ruminating calves

Recovery of E. coliO157:H7 at necropsy (13 to 27 days postinoculation) from experimentally infected calves

No. Range Meana

Sample positive/ (CFU/gram) (CFU/gram) Site total contents contents Rumen 9 / 9 <0.5 X 101 - 3.2 X 103 3.8 X 102

Recticulum 7 / 9 <0.5 X 101 - 2.5 X 103 4.1 X 102

Omasum 9 / 9 <0.5 X 101 - 2.5 X 103 2.9 X 102

Abomasum 0 / 9 0 0Duodenum 2 / 9 <0.5 X 101 <0.5 X 101

Ileum 4 / 9 <0.5 X 101 - 4.0 X 101 1.4 X 101

Distal cecum 7 / 9 <0.5 X 101 - 2.5 X 101 0.8 X 101

Spiral colon 7 / 9 <0.5 X 101 - 6.3 X 102 1.2 X 102

Descending 5 / 9 <0.5 X 101 - 2.5 X 102 6.8 X 101

colon

Protocol 20 adult steers (weight 980-1160 lbs) were fed

production diet containing monensin (30g/ton)

Each administered by gavage 1010 E. coli O157 (5-strain mixture) at day 0

10 steers administered at 48 and 72 h post-challenge 1010 probiotic E. coli (3 strains) E. coli O157 and probiotic bacteria fecal

shedding monitored until day 33

E. coli O157:H7 (log CFU/g) in feces of cattle administered E. coli O157:H7 only

Steer No. Day 2 Day 12 Day 21 Day 30 1 4 5 8111316172124

4.43.33.04.85.65.23.53.32.93.1

3.41.61.64.02.63.02.53.12.52.6

5.32.61.94.74.01.52.01.41.31.3

5.1 1.1<1.0 5.4 1.8 2.7 2.5 4.0 1.3 1.1

E. coli O157:H7 (log/g) at necropsy (day 33) in cattle administered E. coli O157:H7 only

Steer No.

Rumencontent

Rumentissue

Coloncontent

Colontissue Feces

1 4 5 8111316172124

3.6 1.5<1.1 3.1<1.1<1.1<1.1 3.4<1.1<1.1

3.1 1.5 1.1 2.6 1.2 1.5 2.5 1.9 1.5 1.2

4.8 1.6<2.4 4.9<1.1 2.4<1.1 1.2 1.4 1.4

5.0<2.4<2.4 5.2 4.3 4.8 3.4<2.4 3.1<2.4

5.1 1.3<1.1 5.7<1.1<1.1<1.1<1.1 1.0<1.1

E. coli O157:H7 (log/g) in feces of cattle administered E. coli O157:H7 and probiotic bacteria

Steer No. Day 2 Day 12 Day 21 Day 30

2 3 6 7 91015182022

4.74.74.93.65.55.34.43.43.75.0

<1.1<1.1<1.1<1.1<1.1<1.1<1.1<1.1<1.1<1.1

<2.4<2.4 2.1<2.4<2.4<2.4<2.4<2.4<2.4<1.1

<1.1<1.1<1.1<1.1<1.1<1.1<2.4<1.1<2.4<1.1

E. coli O157:H7 (log/g) at necropsy (day 33) in cattle administered E. coli O157:H7 and probiotic bacteria

Steer No. Rumencontent

Rumen tissue

Colon content

Colon tissue Feces

2 3 6 7 91015182022

<1.1 2.5<1.1<1.1<1.1<1.1<1.1<1.1<1.1<1.1

<1.1 1.6<1.1<1.1<1.1<1.1<1.1<1.1<1.1<1.1

<1.1<1.1<1.1<1.1<1.1<1.1<1.1<1.1<1.1<1.1

<2.4<2.4<2.4<2.4<2.4<2.4<2.4<2.4<2.4<2.4

<1.1<1.1<1.1<1.1<1.1<1.1<2.4<1.1<2.4<1.1

Bacteriophage Treatment of E. coli O157:H7 Infection of Calves

Six bacteriophage (1011 pfu) capable of lysing most E. coli O157:H7 strains were orally administered at -7, -6, 0 and 1 day to 6-week old calves (5 per group) per orally administered 3x109 E. coli O157:H7 on day 0 Phage-treated calves shed fewer E. coli O157 on day 2,

4 and 6 than calves fed E. coli O157 only, and E. coli O157 was not shed after day 8

4 of 5 calves fed E. coli O157 shed O157 for 10 to 16 days

T. Waddell et al. Abstr. VTEC 2000, No. 179 (2000)

Control of E. coli O157:H7 in Cattle by Vaccination

Vaccination involves exposing animal to attenuated pathogen or antigen of a virulent microorganism to produce immunity Traditional approaches to vaccinate cattle against E. coli

O157 are not likely to be successful Innovative vaccines may be useful

Example, insert genes of virulence factors of E. coli O157 into alfalfa to stimulate production of IgA in GI tract

Edible Vaccine in Potatoes

Potatoes were genetically engineered to produce B subunit of E. coli heat labile enterotoxinResults of volunteers who ingested transgenic

potatoes: 10 of 11 had a 4-fold increase in serum (IgG)

antibodies 6 of 11 had a 4-fold increase in intestinal

(IgA) antibodies

Control of E. coli O157:H7 in Cattle by Farm Management Practices

Water troughs are on-farm sources of E. coli O157 contamination from cattle manure and cudNeed frequent cleaning of water troughs and

improved design of cattle water reservoirs to reduce contamination

Effect of Diet on Carriage of E. coli O157:H7 by Cattle

Eight 1- to 2-year-old Holstein steers were fed finishing diets of 82 to 90% grain (barley/corn), 100% alfalfa hay or 100% timothy grass (modified crossover design)

Administered 1010 E. coli O157:H7 via gastric tube into rumen to each steer 3 weeks after adaption to a particular diet

C. J. Hovde et al. Appl. Environ. Microbiol. 65:3233 (1999)

Effect of Diet on Carriage of E. coli O157:H7 by Cattle

Average duration of fecal shedding of E. coli O157:H7:

Grain diet 4 days

Alfalfa diet 39 days

Timothy grass diet 42 days Acid resistance of E. coli O157:H7 was unaffected by

diets

C. J. Hovde et al. Appl. Environ. Microbiol. 65:3233(1999)

Influence of Feed Rations on Fecal Shedding of Shiga Toxin-Producing E. coli by Cattle

Cattle were fed rations of grain or hay in 2- to 3-week increments or solely grain or hay Irrespective of the feeding regime, STEC excretion was

significantly reduced during the first week after changing the feed

Thereafter, STEC fecal excretion increased STEC isolates from feces possessed high acid tolerance

which was not influenced by feeding regime

H. Richter et al. Abstr. Shiga Toxin-Producing E. Coli 2000, No. 8 (2000)

Approach to Addressing Foodborne Pathogens on the Farm

1. Prioritize foodborne pathogens of greatest concern in livestock and poultry

2. Identify vehicles of transmission of specific foodborne pathogens in production environment

3. Identify points of intervention that will have the greatest impact on reducing the occurrence of foodborne pathogens at production

Approach to Addressing Foodborne Pathogens on the Farm

4. Develop practical and effective intervention strategies that will reduce or eliminate selected foodborne pathogens from principal animal source

5. Develop Good Agricultural Practices for producing livestock and poultry

Prioritize Foodborne Pathogens of Greatest Concern in Livestock and Poultry Epidemiologic case-control studies of human illness indicate

strong correlations with:Human Disease Principal Risk FactorsE. coli O157:H7 infections - Eating undercooked

ground beef- Visiting a farm- Contact with cattle

Campylobacter infections - Handling raw poultry- Eating undercooked

poultrySalmonella enteritidis - Eating uncooked or lightly infections heated egg products

Potential Vehicles of Transmission of Foodborne Pathogens in the

Production Environment

Manure Drinking water Feed Environment Rodents, insects, wildlife

What Points of Intervention Will Have Greatest Impact on Reducing Occurrence of

Foodborne Pathogens at Production

Case-control studies to identify major risk factors associated with occurrence/transmission of foodborne pathogens at production

Case-Control Study of E. coli O157:H7 Fecal Shedding in Dairy Calves

1. Determined that calves are more likely to shed E. coli O157:H7 after weaning (4.8%) than before weaning (1.4%)

2. Shedding in calves was associated with:

a. Grouping calves before weaning

b. Sharing buckets and bottles among unweaned calves without washing or rinsing

c. Feeding grain to calves less than 5 days old

Case-Control Study of E. coli O157:H7 Fecal Shedding in Dairy Calves

3. Shedding was negatively associated with:a. Feeding calves whole cottonseedb. Pasturing in clover

Garber et al. 1995. JAVMA 207:46-49.

Possible Intervention Strategies to Reduce Pathogens at Production in

Livestock and Poultry Probiotics/competitive exclusion bacteria Bacteriophage Innovative vaccines Genetically modified feeds (e.g., introduce antimicrobials

into grain) Genetically modified animals resistant to colonization by

pathogens Modify farm management practices Manure treatment

Example of Approach to

Reduce E. coli O157:H7

On Farm

Example of Approach to ReduceE. coli O157:H7 at Farm

Principal contributing factor to human illness is exposure to cattle feces based on principal risk factors (eating undercooked ground beef, visiting a farm, contact with cattle)


Principal points of intervention to reduce human exposure: Reduce/eliminate intestinal carriage and fecal shedding

of E. coli O157 Reduce contamination of drinking water vessels used by

cattle Reduce E. coli O157 contamination of manure in the

farm environment Good personal hygiene of animal handlers


Develop practical and effective intervention strategies such as:Reduce intestinal carriage and fecal shedding

by: Competitive exclusion bacteria Innovative vaccines Bacteriophage Farm management practices


Develop practical and effective intervention strategies such as:Reduce contamination of drinking water vessels

Redesign vessels that remove cud sediment Cleaning regimes to remove biofilms that

entrap E. coli O157


Develop practical and effective intervention strategies such as: Reduce contamination of manure in farm environment

Develop biological competitive approaches to kill E. coli O157 in manure in farm yard

Develop methods to kill E. coli O157 in cow manure-based compost

Develop farm management practices for manure application to soil to reduce E. coli O157 contamination of produce and feeds


Develop practical and effective intervention strategies such as: Educate animal handlers in good personal hygiene

Hand washing Avoid touching mouth CDC guidelines

Farm visitors avoid contact with animals and manure Use good personal hygiene CDC guidelines


Develop Good Agricultural Practices for producing cattleNeed to identify control points and intervention

treatmentsPrepare GAP guidelines for control food safety

aspects of beef cattle production

Program Needs

1. Develop Good Agricultural Practices for livestock and poultry production– Should include input of experts in food safety

with experience in animal production

Program Needs

2. Develop HACCP/GAPs for composting/manure treatment– Should include input of experts in food safety

with experience in animal waste handling

Program Needs

3. Research program (case-control studies) to identify principal risk factors associated with transmission of target pathogens in livestock and poultry production

Program Needs4. Research program to develop effective intervention

strategies to:a. Reduce carriage/fecal shedding of pathogens by

livestock and poultry● EHEC 0157:H7 - Cattle● Campylobacter - Poultry, Cattle● Salmonella - Poultry, Swine, Cattle● Cryptosporidium - Cattle

b. Treat manure to kill pathogens before used for soil application, or contaminates irrigation or processing water

Program Needs5. Develop educational program for producers/farmers

a. Should include involvement of:

b. Extension Service at Land Grant Universities

c. Veterinary Schools/Veterinarians

d. Veterinary Pharmaceutical Companies

e. Representatives of food service, food retail and food processing industries

f. Public relations firm to simplify messages and present them for effective communication

Keeping Foodborne Pathogens Down on the Farm Michael P. Doyle.

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