FDA Grant FD-U-001603-01

Evaluation and Use of FDA/BAM and Rapid Methods for On-Farm Survey (of Salmonella, Campylobacter, E. Coli O157:H7 and Yersinia enterocolitica)

Ann Draughon, David Golden, Stephen Oliver and Alan Mathew

Food Safety Center of Excellence

The University of Tennessee

Knoxville, TN 37996

Dale Hancock, CVM

Washington State University

Background:

Improved methodology for detection of foodborne pathogens in on-farm environments is needed to obtain reliable baseline data on the occurrence of common zoonotic pathogens.  Official methods published in the BAM and USDA methods manuals for food microbiologists have focused quite appropriately on detection of pathogens in food products in the past.  These methods must be modified or adapted for accurate and reliable detection of pathogens in environmental or animal samples.

Numerous studies have been conducted to compare and improve microbiological techniques for detection and isolation of Salmonella, Campylobacter, shigatoxin-producing E. coli, and Yersinia enterocolitica from foods throughout the world.  These methods were each evaluated and a minimum of 16 methods were evaluated for each of the above pathogens in on-farm samples using ten replications for each method.  On-farm samples evaluated included both animal and environmental samples such as cow, swine and poultry fecal samples, hair or feather samples from animals, oral samples, walls, fans, other surfaces, insects, wild animals, worker boots, air samples, bedding materials, water supplies, feed supplies both before and after contact with animals, samples associated with milking of dairy cattle (teat, bulk tank, filters, equipment, etc.).

The optimal methods for isolation of each of the above pathogens from this wide variety of sample types are included in this summary.  The summary also provides information on types of pathogens found on dairy farms by use of the methodology previously developed.

Results:

Methodology for detection of Salmonella, Campylobacter, E. coli O157:H7 and Yersinia

enterocolitica was critical for consistent and reliable isolation of target microorganisms from on-farm samples.  Due to the wide variety and diversity of samples, enrichment and differential plating media combinations gave recoveries of 0 to 100% depending on appropriate choice of methodology.  The recommended methods for the isolation of the pathogens in study from the different animal and environmental sources are presented in Tables 1, 2, 3, and 4.

Salmonella spp. were isolated with high frequency from all types of environmental samples associated with farms.  The percentage isolation of Salmonella over a two year period in environmental farm samples ranged from: 0 to 82% positive in grain samples, 0 to 80% positive in insects, 0 to 88% in trough water, 0 to 68% positive in soil, 0 to 83% positive in air and 0 to 75% positive in wild birds.  The highest seasonal incidence of Salmonella overall in the farm environment was in the summer months (July, August and September, Southeastern United States).

E. coli O:157:H7 was isolated infrequently in dairy farm environments and infrequently from animals.  Incidence was highest in the summer months with isolation rates ranging from 0% in grain to 32% in soil (8% positive in insects, 8% positive in trough water, and 8% positive in wild birds).  Fecal samples from cows showed 14% positive in the summer months, 3% positive in the fall (October, November & December) and 2% positive in the spring (April, May, June).  E. coli O:157:H7 was not isolated from animals in the winter months (January, February, March).

Incidence of Campylobacter jejuni in the farm environment was highly seasonal with the majority of positive isolations occurring in the fall months for grain (up to 83% positive) and soil (up to 42% positive).  However, Campylobacter in insects and wild birds was highest in the winter months (up to 63% and 70% positive, respectively.  Strangely enough, positive Campylobacter samples for water and air were highest in the spring months (83% and 44% positive).  Campylobacters were found infrequently in farm environments in the summer months. 

We can infer from these data that campylobacters behave in the farm environment in a way which is somewhat similar to their behavior in foods.  Campylobacter jejuni is not an organism which grows or competes well in most environments except in the intestine or tissue of animals due to its thermophilic nature and intolerance to oxygen.  However, research in our lab and others has shown that C. jejuni survives quite well in cooler environments in which it does not attempt to grow, merely survive.  This would explain its prevalence in wild birds in the winter and its high occurrence in water, grain and air in the winter or spring months.  The infrequent isolation of campylobacters in the summer might be attributed both to stress on the organism due to its attempts to grow in the environment and the active competition of mesophiles such as coliforms, Salmonella, and common intestinal microflora associated with animals.

Y. enterocolitica was found at very low incidence in environmental samples but occurrence was statistically higher (p<0.05) in the colder months of the year in the winter.  Of the animals evaluated, cows, poultry and swine were all found to harbor Y. enterocolitica but the highest incidence by far was in swine which averaged approximately 25% positives for mature hogs.  The psychrotrophic nature of Y. enterocolitica probably contributes to its predominance in the winter months in the southeastern United States.  Locations which have more temperate climates and longer winters may be at risk for higher incidence of Y. enterocolitica in other seasons as well.

Overall Conclusions:

Distribution of bacterial pathogens associated with on-farm samples varied significantly (p<0.05) by season for both animal and environmental samples.  Salmonella and E. coli predominated in the summer months; Y. enterocolitica occurrence was predominantly in the winter and occurrence of C. jejuni varied with sample type and season.  Occurrence of both Salmonella and Campylobacter isolation in dairy cows was highly correlated with positive occurrence in grain, soil and insects.  E. coli O157:H7 incidence in animals was highly correlated with occurrence in soil and water.

Table 1.  Isolation methods recommended for Salmonella spp. in farm samples

                (level of detection < 1 CFU/g)

Type of Sample	Best Recovery Method
Mouth - Cow, Calf/ Teat-Cow	LB-T35-BS / XLT4   or  LB-T42-BS1
Hair	LB-T42-BS   or   DE-RV-BS
Air Samples	LB-T35-BS   or   LB-T42-BS
Fecal - Cow, Calf, Poultry Swine	LB-T35-BS
Mixed Grain	LB-RV-XLT4  / HE
TMR	LB-T35-BG /BS /HE
Silage	LB-SC-BS   or   LB-T42-BS
Poultry Feed	DE-T35- BS/ HE/ XLT4
Trough Water, Sippers	LB-RV-BG / BS
Soil, Bedding,	DE-T42-BS        or     DE-T42-HE
Litter	LB-T35-HE/XLT4 or LB-RV-HE
Insects - Environment Swabs	LB-RV-BS or  LB-T42-HE/XLT4
Milk-Bulk Tank Liners	LV-RV-BS         or      LB-SC-BS
Milk	LB-T42-BS

1
  Abbreviations and shorthand used in the above table are as follows: LB = lactose broth pre-enrichment, DE = no pre-enrichment step, T42 = tetrathionate broth at 42 C for 24 h, T35 = tetrathionate broth at 35 C for 24 h, RV = rappaport vassiliadis broth, SC = selenite cysteine broth.  Differential plating media are noted using the following abbreviations: BS = bismuth sulfite agar, HE = hektoen enteric agar, BGA = brilliant green agar.  See FDA BAM for preparation of culture media.   The pH adjustments are critical for these media.  Removal of solid materials from enrichment after 4 hours is strongly recommended (i.e., grains, silage, bedding,  etc.).

2
  10 ml of 1-M sodium thiosulfate should be added to chlorinated water or samples which may have been exposed to sanitizer (i.e., equipment) to inactivate oxidizing agents

Table 2.  Recommended methods for recovery of Campylobacter jejuni from farm samples

               (level of detection 10 cfu/25 g)

Type of Sample	Best Recovery Method
Mouth , Teat, Bedding, Soil	CEB-R1:10- CCDA
2 Hair, Water	CEB-CCDA
Fecal	CEB1:10-AHB of CEBR1:10-CCDA
Mixed Grain	CEB1:10-CCDA
TMR	BB-R-AHB
Silage	BB1:10-AHB or  BB-CCDA
Insects	B-R-CCDA
Milk	CEB-R-AHB
Miscellaneous Environmental	CEB 1:10-CCDA

1
   Abbreviations and shorthand used in the above table are as follows: CEB = Campylobacter Enrichment Broth, CEB-R = CEB plus rifampicin, CEB1:10 = a 1 to 10 dilution of CEB, BB = Bolton’s Broth, BB-R = Bolton’s Broth plus rifampicin, BB1:10 = a 1 to 10 dilution of BB.  Differential plating media included CCDA = modified Campylobacter Blood-Free Selective agar with supplements, AHB = Abeyta Hunt Bark agar with supplements.  The pH adjustments are critical for these media.  Removal of solid materials from enrichment after 4 hours is strongly recommended (i.e., grains, silage, bedding, etc.).

2
   10 ml of 1-M sodium thiosulfate should be added to chlorinated water or samples which may have been exposed to sanitizer (i.e., equipment) to inactivate oxidizing agents

Table 3.  Recommended methods for recovery of E. coli O157:H7 from farm samples

               (level of detection 10 cfu/25 g)

Type of Sample	Best Recovery Method
Mouth , Teat, Bedding,  Soil	mTSB+N-CTSMAC (18 h @ 37 C)
2 Hair, Water, Environmental	mTSB+N-CTSMAC or mTSB+N+IMS (18 h @ 37 C)
Fecal - Cow	mTSB+N-CTSMAC (18 h @ 37 C)
Fecal - Grower Finisher  Pigs	EEB + IMS - HC/SMAC OR EEB - HC (6 h @ 37 C)
Fecal - Sow	EEB-SMAC or mEC+N-SMAC/EMB (14 h @ 37 C)
TMR, Mixed Grains, Dry Feeds	mTSB+N-CTSMAC (16 h @ 37 C)
Litter	EEB-CTSMAC (6 or 16 h @ 37 C)
Insects	EEB-CTSMAC (16 h @ 37 C) or mTSB+N-CTSMAC (18 h @ 37 C)
Milk	mTSB+N-CTSMAC or SMAC(18 h @ 37 C)
Miscellaneous Environmental	EEB-SMAC (16 h @ 37 C)

1
   Abbreviations and shorthand used in the above table are as follows: mTSB+N = trypicase soy broth with novobiocin (BAM), EEB = E. coli enrichment broth, MEB = modified E. coli broth, mTSB-+N = TSB with immunomagnetic separation, EEB-IMS = EEB with immunomagnetic separation, CTSMAC = cefixime tellurite sorbitol MacConkey agar, SMAC = sorbitol MacConkey agar, HC = Hemorrhagic Colitis agar.  The pH adjustments are critical for these media.  Removal of liquid from solid materials in from enrichment after 4 hours is strongly recommended for optimal recovery (i.e., grains, silage, bedding, etc.).  Stomacher bags with filters incorporated are ideal for this purpose.

2
   10 ml of 1-M sodium thiosulfate should be added to chlorinated water or samples which may have been exposed to sanitizer (i.e., equipment) to inactivate oxidizing agents

Table 4.  Recommended methods for recovery of Yersinia enterocolitica 0:3 and 0:8 from

                farm samples (level of detection 10-100 CFU/25 g)

Type of Sample	Best Recovery Method
All Farm Animal and Environmental Samples   Serotype 0:8	1TSBPN - 24 h @ 18 C, plating on CIN agar
All Farm Animal and Environmental Samples   Serotype 0:3	2ITC Broth - 24 h or 48 h @ 22 C, plating on                 CIN agar

1
  TSBPN Broth formulation: Tryptic Soy Broth with Polymyxin and Novobiocin (Landgraf et al., 1993, J. Food Prot. 40: 56: 447-450).  Incubation temperature modification is critical for farm samples.  Long-term low temperature incubation is not recommended due to extremely poor recovery in farm samples.

2
  ITC Broth formulation: Irgasan Ticarcillin Potassium Chlorate (Wauters et al., Appl. Envrion. Microbiol.  54: 851-854.).  Incubation temperature modification is critical for farm samples.  Long-term low temperature incubation is not recommended due to extremely poor recovery in farm samples.

Abstracts and Publications Based on the FDA Supported Research:

Pangloli, P., Y. Dje, S.P. Oliver, A. Mathew, D.A. Golden, W.J. Taylor and F.A. Draughon.  2002, Evaluation of methods for recovery of Salmonella from dairy cattle, poultry and swine farms.  Manuscript Submitted to FDA for review.

Dje, Y., F.A. Draughon, D.A. Golden, S.P. Oliver and W. Taylor.  2000.  Isolation of Salmonella from air samples in a dairy calf barn.  Proceedings,  Annual Meeting of the Institute of Food Technologists (Poster 78F-17), Dallas, TX, June 18-21, 2000.

Dje, Y., F.A. Draughon, D.A. Golden, S.P. Oliver and W. Taylor.  2000.  Recovery of Salmonella from on-farm environmental surfaces and hair from lactating cows.  Proceedings,  Annual Meeting of the Institute of Food Technologists (Poster 78F-18), Dallas, TX, June 18-21, 2000.

Dje, Y., F.A. Draughon, D.A. Golden, S.P. Oliver and W. Taylor.  2000.  Recovery of Salmonella from artificially contaminated dairy feeds.  Proceedings, Annual Meeting of the International Association for Food Protection (Poster P125), Atlanta, GA, August 6-9, 2000.

Kiefer, C.A., D.A. Golden, F.A. Draughon, A. G. Mathew and S.P. Oliver, 2000.  Evaluation of FDA/BAM and rapid methods for enumeration and detection of Escherichia coli O157:H7 from farm animal environments.   Proceedings,  Annual Meeting of the International Association for Food Protection (Poster P117), Atlanta, GA, August 6-9, 2000.

Taylor, W.J., F.A. Draughon, D.A. Golden, S.P. Oliver and M. Saul.  2000.  Detection of Campylobacter jejuni in dairy silage.  Proceedings, Annual Meeting of the International Association for Food Protection (Poster P127), Atlanta, GA, August 6-9, 2000.

Taylor, W.J., F.A. Draughon, D.A. Golden, S.P. Oliver and M. Saul.  2000.  A comparison of isolation protocols for recovery of Campylobacter jejuni from cattle feces.    Proceedings,  Annual Meeting of the International Association for Food Protection (Poster P128), Atlanta, GA, August 6-9, 2000.

Pangloli, Philipus, Draughon, F.A., Oliver, S.P., and D. Golden.  2001.  Recovery of Salmonella from dairy cattle and their environment.   Presented at the Annual Meeting of the International Association for Food Protection (Presentation P136), Minneapolis, MN, August 4-9, 2001.

Pangloli, Philipus, Draughon, F.A., Oliver, S.P., and D. Golden.  2001. Escherichia coli O157:H7 in dairy cows and their environment.  Presented at the Annual Meeting of the International Association for Food Protection (Presentation P137), Minneapolis, MN,  August 6-9, 2001.

Lamar, Kimberly, Oliver, S.P. and F.A. Draughon.  2001.  GIS and epidemiology of Salmonella on dairy farms.  Presented at the Annual Meeting of the International Association for Food Protection (Presentation P138), Minneapolis, MN, August 4-9, 2001.