Progress 02/17/06 to 12/15/10
Outputs
Progress Report Objectives (from AD-416) (1) Using optimized sampling strategies, enumeration, and molecular diagnostic, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors. (2) Identify key virulence attributes to differentiate Salmonella and Campylobacter avirulent commensals from those pathogenic strains that pose a public health threat in humans. (3) Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production. Identify microbes associated with gut colonization by, and population shifts of, foodborne pathogens. (4) Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia, in hog carcasses and organs. Approach (from AD-416) Time of entry of Salmonella and Campylobacter will be monitored in turkeys. The study will document flock management practices which affect the prevalence of these foodborne pathogens initially in the brooder period and ultimately throughout live production. Key virulence attributes of C. jejuni and C. coli strains recovered from turkeys will be characterized in vitro (cell invasion assays)and in vivo (day of hatch poult model.) Ultimately, differential gene expression formats will be used to differentiate a virulent from virulent isolates of C. coli and C. jejuni. Bacterial and fungal communities of the ceca of domestic and wild turkeys will be described. This initial survey will provide a measurement of diversity between wild and domestic birds. Analysis of total community flux over time will focus on kinetics of Campylobacter community development and stability in the turkey ceca. Organisms that correlate with Campylobacter colonization or exclusion will be identified. Second generation PCR assays will be developed to detect and quantify Salmonella and Campylobacter on hog and turkey carcasses. Monitoring viscera will serve as an indicator of on-farm versus in-plant sources of contamination. These studies will assist in determining the critical control points of contamination during slaughter. This is the final report for project 3625-32000-080-00D terminated in December 2010 and replaced with 3625-31320-003-00D. Research progress was made to uncover the ecology of foodborne pathogens, Campylobacter, Salmonella, and Yersinia either in poultry or in swine. Investigations were made both on external factors affecting colonization of the animals by these foodborne pathogens and internal (intestinal) factors affecting colonization. Ecological factors within poultry intestines affected the ability of the Campylobacter to colonize that environment. Different Campylobacter species and subspecies were shown to correlate to intestinal bacteria fluctuations and host signals, such as maturation age. Campylobacter coli was found to be a commensal colonizer while Campylobacter jejuni behaved as an opportunistic colonizer implying different intervention strategies will be required to exclude the two species from the poultry intestine. Different Campylobacter species and subspecies were shown to correlate to intestinal bacteria fluctuations and host signals. Megamonas hypermegale (M. hypermegale) type II levels correlated with the exclusion of Campylobacter coli from the turkey intestine. Exclusion of the pathogen from the animal host is the best method to ensure safety of the national food supply. Consequently, the potential of M. hypermegale as an inhibitor of Campylobacter will be examined in the new project. Swine are the major animal reservoir for Yersinia enterocolitica (Y. enterocolitica), a major human foodborne pathogen. We conducted the first attempt to identify risk factors for Y. enterocolitica in the United States hog population. By analyzing feces and tonsilar swabs of hogs from 122 locations, we found three positive risk factors for Y. enterocolitica infections: vaccination for Escherichia coli, percentage of deaths due to scours, and presence of meat/bone meal in grower-finisher diet. Locations in a U.S. midwestern state had reduced risk. We determined that the frequency of detection of Campylobacter on harvested turkeys was three-fold greater when the turkey carcass was swabbed than when neck skin was sampled. These findings indicate that the USDA Food Safety and Inspection Service carcass swab protocol appears a better indicator of Campylobacter status than European neck skin sampling technique. Turkeys are reservoirs for Campylobacter, a major human foodborne pathogen. Transport from farm to abbatoir resulted in a significant increase in Campylobacter numbers in the gall bladders and crops of transported birds. Turkey poults were found to be free of Campylobacter at one day and nine days after hatch. At slaughter (138 days after hatch), Campylobacter prevalence in sampled birds was 92%. These results indicate that this foodborne pathogen is acquired from the environment and colonizes ceca of birds during their growth.
Impacts
(N/A)
Publications
- Scupham, A.J., Jones, J., Rettedal, E.A., Weber, T.E. 2010. Antibiotic Manipulation of Intestinal Microbiota to Identify Microbes Associated with Campylobacter Exclusion in Poultry. Applied and Environmental Microbiology. 76(24):8026-8032.
- Solis-Soto, L., Garcia, S., Wesley, I.V., Heredia, N. 2011. A charcoal- and blood-free enrichment broth for isolation and PCR detection of Campylobacter jejuni and Campylobacter coli. Journal of Food Protection. 74(2):221-227.
- Muraoka, W.T., Zhang, Q. 2011. Phenotypic and genotypic evidence for L- fucose utilization by Campylobacter jejuni. Journal of Bacteriology. 193(5) :1065-1075.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) (1) Using optimized sampling strategies, enumeration, and molecular diagnostic, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors. (2) Identify key virulence attributes to differentiate Salmonella and Campylobacter avirulent commensals from those pathogenic strains that pose a public health threat in humans. (3) Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production. Identify microbes associated with gut colonization by, and population shifts of, foodborne pathogens. (4) Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia, in hog carcasses and organs. Approach (from AD-416) Time of entry of Salmonella and Campylobacter will be monitored in turkeys. The study will document flock management practices which affect the prevalence of these foodborne pathogens initially in the brooder period and ultimately throughout live production. Key virulence attributes of C. jejuni and C. coli strains recovered from turkeys will be characterized in vitro (cell invasion assays)and in vivo (day of hatch poult model.) Ultimately, differential gene expression formats will be used to differentiate a virulent from virulent isolates of C. coli and C. jejuni. Bacterial and fungal communities of the ceca of domestic and wild turkeys will be described. This initial survey will provide a measurement of diversity between wild and domestic birds. Analysis of total community flux over time will focus on kinetics of Campylobacter community development and stability in the turkey ceca. Organisms that correlate with Campylobacter colonization or exclusion will be identified. Second generation PCR assays will be developed to detect and quantify Salmonella and Campylobacter on hog and turkey carcasses. Monitoring viscera will serve as an indicator of on-farm versus in-plant sources of contamination. These studies will assist in determining the critical control points of contamination during slaughter. We examined the effects on Campylobacter prevalence of feed supplements and probiotics in commercially reared birds. In the first study, dried distillers grain and solubles (0, 17%, 35%, 50%) were fed to four groups of 60 birds per group. By chi-square test of equal proportions, no differences were noted between the groups. Next, a commercial farm was located in which half of the birds in the house were fed a commercial all natural microbial probiotic whereas control cohorts again were fed the unsupplemented diet. No differences in the Campylobacter prevalence based on cloacal swabs were evident between the two groups. This indicates that significant differences between treatment groups may only be recognized by quantifying the levels of Campylobacter, an approach we are now pursuing. USDA methods for sampling turkey carcasses rely on swabbing a 100cm2 prescribed area whereas the European Union prefers neck skin sampling. We compared the recovery of Campylobacter, swabs of the external carcass (Food Safety and Inspection Service (FSIS) protocol) and the internal abdominal cavity versus homogenized neck skins. Cecal prevalence of Campylobacter was determined in order to estimate levels in live birds entering the abattoir (95%, 143/150). For carcasses sampled immediately prior to the final chlorinated chiller, Campylobacter is recovered more frequently from external (35%, 106/305) and the internal abdominal (30%, 73/240) surfaces than from the neck skin (12%, 29/257). Thus, the carcass swab may be a better indicator of Campylobacter status than the neck skin. Methods to detect and identify Campylobacter are tedious. We have developed a modified blood free enrichment broth (M-BFEB) , which does not incorporate oxygen quenchers, such as blood or activated charcoal, does not require microaerobic incubation, is as sensitive as routinely used enrichments, such as Bolton�s broth, and is free of Polymerase Chain Reaction (PCR) inhibitors. In collaboration with APHIS, explored molecular approaches to replace conventional Salmonella serotyping. A comparison of 23 serotypes from five livestock species (~160 samples) indicates that the Luminex bead-based protocol is more sensitive than a European microarray-based format. Arcobacter is a close relative to Campylobacter, is a zoonotic agent of �Significant Importance� and is frequently misidentified. With APHIS colleagues we reviewed nine clinical cases from non-livestock species to illustrate the difficulties in distinguishing Arcobacter from Campylobacter fetus, which causes livestock infertility and abortion. PCR and 16S ribosomal ribonucleic acid sequencing were used for identification. These cases are of potential significance because of the clinical presentation, the impact on the industry, and the challenges of differentiating Arcobacter by routine culture. Significant Activities that Support Special Target Populations USDA collaborator on USDA 1890 Capacity Building Grant awarded to Tuskegee University (�Attenuated Salmonella mutants as live vaccine�; Amin Fadl, College of Veterinary Medicine). Co-author with colleagues from Tuskegee University a manuscript describing a real-time PCR assay to detect Campylobacter in poultry (Debretsion et. al., 2009). Accomplishments 01 Comparison of Neck Skins Versus Swabs for Campylobacter Detection. ARS researchers at Ames, Iowa, are comparing the Food Safety and Inspection Service (FSIS) carcass swab and European neck skin protocols for the detection of Campylobacter on turkey carcasses. For carcasses sampled immediately prior to the final chlorinated chiller, Campylobacter is recovered more frequently from external (35%, 106/305) surface swabs tha from the excised neck skin (12%, 29/257). This indicates that the FSIS carcass swab protocol may be a better indicator of Campylobacter status than neck skin sampling.
Impacts
(N/A)
Publications
- Debretsion, A., Habtemariam, T., Wilson, S., Wesley, I.V., Yehualashet, T. 2009. Comparative Assessment of Standard Culture and Real-Time PCR to Detect Campylobacter jejuni in Retail Chicken Samples. Journal of Food Safety. 29(4):588-600.
- Wang, B., Wesley, I.V., Mckean, J.D., O'Connor, A.M. 2010. Sub-iliac Lymph Nodes at Slaughter Lack Ability to Predict Salmonella-enteric Prevalence for Swine Farms. Foodborne Pathogens and Disease. 7(7):795-800.
- Wesley, I.V., Miller, W.G. 2010. Arcobacter: An Opportunistic Human Foodborne Pathogen? In: Scheld, W.M., Grayson, M.L., Hughes, J.M., editors. Emerging Infections 9. Washington, DC: American Society of Microbiology Press. p. 185-211.
- Byeonghwa, J., Muraoka, W.T., Zhang, Q. 2010. Advances in Campylobacter Biology and Implications for Biotechnological Applications. Microbial Biotechnology. 3(3):242-258.
- Eicher, S.D., Wesley, I.V., Sharma, V.K., Johnson, T.R. 2010. Yeast Cell- Wall Products Containing Beta-Glucan plus Ascorbic Acid affect Neonatal Bos-Taurus Calf Leukocytes and Growth after a Transport Stressor. Journal of Animal Science. 88(3):1195-1203.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) (1) Using optimized sampling strategies, enumeration, and molecular diagnostic, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors. (2) Identify key virulence attributes to differentiate Salmonella and Campylobacter avirulent commensals from those pathogenic strains that pose a public health threat in humans. (3) Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production. Identify microbes associated with gut colonization by, and population shifts of, foodborne pathogens. (4) Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia, in hog carcasses and organs. Approach (from AD-416) Time of entry of Salmonella and Campylobacter will be monitored in turkeys. The study will document flock management practices which affect the prevalence of these foodborne pathogens initially in the brooder period and ultimately throughout live production. Key virulence attributes of C. jejuni and C. coli strains recovered from turkeys will be characterized in vitro (cell invasion assays)and in vivo (day of hatch poult model). Ultimately, differential gene expression formats will be used to differentiate a virulent from virulent isolates of C. coli and C. jejuni. Bacterial and fungal communities of the ceca of domestic and wild turkeys will be described. This initial survey will provide a measurement of diversity between wild and domestic birds. Analysis of total community flux over time will focus on kinetics of Campylobacter community development and stability in the turkey ceca. Organisms that correlate with Campylobacter colonization or exclusion will be identified. Second generation PCR assays will be developed to detect and quantify Salmonella and Campylobacter on hog and turkey carcasses. Monitoring viscera will serve as an indicator of on-farm versus in-plant sources of contamination. These studies will assist in determining the critical control points of contamination during slaughter. Significant Activities that Support Special Target Populations Transport Stress: To determine the impact of transportation on Campylobacter, six commercial turkey flocks were examined on the farm and after transport to the slaughterhouse. Of the six intestinal sites examined, Campylobacter prevalence increased post transport only in the enlarged gall bladder and in the crop. The increase in Campylobacter in the crop was correlated with statistically significant changes in six of 13 carboxylic acids monitored. This indicates that unlike Salmonella, subtle changes occur in market-weight turkeys during transport which influence Campylobacter. Foodborne Pathogen Colonization of Hatchlings: We determined the time of entry of Campylobacter and Salmonella in two commercial turkey flocks. Whereas Salmonella was detected in day of hatch poults, Campylobacter was not. By day 9, Salmonella was frequently isolated from the intestine (45%); Campylobacter was not. In contrast, at slaughter (138 days), Salmonella prevalence in the ceca had declined (4. 5%); Campylobacter prevalence (92%) increased. PFGE analysis of Salmonella isolates indicated fluctuating populations as the birds matured. Comparisons of the bacterial community compositions in turkey feces were shown to differ from communities colonizing the mucosal epithelium. Commensal Bacteria Affecting Campylobacter: Bacterial intestinal community shifts around 11 weeks post-hatch were corroborated for commercially produced animals. The foodborne pathogens C. jejuni and C. coli respond to the community instability, indicating a host component in Campylobacter colonization. A subspecies of the poultry intestinal anaerobe Megamonas hypermegale was identified as a candidate for competitive exclusion studies to protect against colonization by Campylobacter. Carcass Locations of Foodborne Pathogens: Collaboration with ARS and international colleagues generated Multi Locus Sequencing Typing (MLST) profiles of Arcobacter, which will facilitate epidemiological tracebacks of foodborne outbreaks. The distribution of Campylobacter, Salmonella and indicator microbes can be determined by �mapping� the turkey carcass. The goal of this �carcass mapping� project is to identify �hot spots� of pathogens and indicators, which can be used by FSIS to monitor the efficiency of turkey plant HACCP protocols. Whereas the post-harvest status can be estimated by sampling the carcass, the on-farm status of the flock can be approximated by sampling the ceca. Field trials have compared conventional and PCR-based detection assays, demonstrated variation attributed to season, flock, number of birds slaughtered, and day of sampling, suggesting that a larger sample size will be needed to minimize the variation. In general, carcass swabs (taken with synthetic sponge) have fewer microbial competitors than either skin samples or ceca. Thus, we have adapted the RX microtiter format to screen turkey carcass swabs for Salmonella. By incorporating antimicrobials and shortening the incubation, the RX format is a good predictor of overall Salmonella flock status. Significant Activities that Support Special Target Populations USDA collaborator on USDA 1890 Capacity Building Grant awarded to Tuskegee University (�Attenuated Salmonella mutants as live vaccine�; Amin Fadl, College of Veterinary Medicine).
Impacts
(N/A)
Publications
- Wesley, I.V. 2009. Food Safety Issues and the Microbiology of Poultry. In: Heredia, N., Wesley, I., Garcia, J.S., editors. Microbiologically Safe Foods. Hoboken, NJ: Wiley & Sons, Inc. p. 169-183.
- Jeon, B., Muraoka, W., Scupham, A.J., Zhang, Q. 2009. Roles of Lipooligosaccharide and Capsular Polysaccharide in Antimicrobial Resistance and Natural Transformation of Campylobacter jejuni. Antimicrobial Chemotherapy. 63(3):462-468.
- Patton, T.G., Scupham, A.J., Bearson, S.M., Carlson, S.A. 2009. Characterization of Fecal Microbiota from a Salmonella Endemic Cattle Herd as Determined by Oligonucleotide Fingerprinting of rDNA Genes. Veterinary Microbiology. 136(3-4):285-292.
- Wesley, I.V., Rostagno, M.H., Hurd, S.H., Trampel, D. 2009. Prevalence of Campylobacter jejuni and Campylobacter coli in Market-Weight Turkeys On- Farm and at Slaughter. Journal of Food Protection. 72(1):43-48.
- Krueger, N.A., Anderson, R.C., Krueger, W.K., Horne, W.J., Callaway, T.R., Edrington, T.S., Harvey, R.B., Nisbet, D.J., Cartsens, G.E., Wesley, I.V. 2008. Prevalence and concentration of Campylobacter in rumen contents and feces in pasture and feedlot-fed cattle. Foodborne Pathogens and Disease. 5:571-577.
- Bhaduri, S., Wesley, I.V., Richards, H., Draughon, A., Wallace, M. 2009. Clonality and Antibiotic Susceptibility of Yersinia enterocolitica Isolated From U.S. Market Weight Hogs. Foodborne Pathogens and Disease. 6(3):351-356.
- Scupham, A.J. 2009. Campylobacter Colonization of the Turkey Intestine in the Context of Microbial Community Development. Applied and Environmental Microbiology. 75(11):3564-3571.
- Wesley, I.V. 2009. Public Health Impact of Foodborne Illness: Impetus for an International Food Safety Effort. In: Heredia, N., Wesley, I., Garcia, J.S., editors. Microbiologically Safe Foods. Hoboken, NJ: Wiley & Sons, Inc. p.3-11.
- Wesley, I.V., Muraoka, W.T. 2009. Time of Entry of Salmonella and Campylobacter into the Turkey Brooder House. Food and Bioprocess Technology. Available: http://www.springerlink. com/content/986482v5v5621k68/fulltext.html.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) (1) Using optimized sampling strategies, enumeration, and molecular diagnostic, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors. (2) Identify key virulence attributes to differentiate Salmonella and Campylobacter avirulent commensals from those pathogenic strains that pose a public health threat in humans. (3) Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production. Identify microbes associated with gut colonization by, and population shifts of, foodborne pathogens. (4) Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia, in hog carcasses and organs. Approach (from AD-416) Time of entry of Salmonella and Campylobacter will be monitored in turkeys. The study will document flock management practices which affect the prevalence of these foodborne pathogens initially in the brooder period and ultimately throughout live production. Key virulence attributes of C. jejuni and C. coli strains recovered from turkeys will be characterized in vitro (cell invasion assays)and in vivo (day of hatch poult model.) Ultimately, differential gene expression formats will be used to differentiate a virulent from virulent isolates of C. coli and C. jejuni. Bacterial and fungal communities of the ceca of domestic and wild turkeys will be described. This initial survey will provide a measurement of diversity between wild and domestic birds. Analysis of total community flux over time will focus on kinetics of Campylobacter community development and stability in the turkey ceca. Organisms that correlate with Campylobacter colonization or exclusion will be identified. Second generation PCR assays will be developed to detect and quantify Salmonella and Campylobacter on hog and turkey carcasses. Monitoring viscera will serve as an indicator of on-farm versus in-plant sources of contamination. These studies will assist in determining the critical control points of contamination during slaughter. Significant Activities that Support Special Target Populations We determined the prevalence of Campylobacter and Salmonella in two commercial turkey flocks reared with either natural (NV) or mechanical (MV) ventilation. For summer, Campylobacter was isolated from cloacal swabs of NV (86%) and MV (94%) birds; Salmonella was detected in drag swabs of the NV (75%) and MV (12%) flocks. Salmonella prevalence in ceca of NV (5%) was significantly higher (P < 0.05) than in MV (0%) flock. For winter, when NV and MV houses are closed, Salmonella prevalence in ceca of the NV (7.5%) and MV (8%) flocks was comparable. For both seasons, Campylobacter prevalence in ceca of the NV and MV birds was comparable. This indicates that MV may reduce Salmonella during the summer months, when the curtains in the NV house are raised but not in the winter, when the NV and MV houses remain closed. To compare colonization of conventional turkeys, three-week-old birds were inoculated with either C. jejuni, C. coli or diluent. C. coli persisted for a longer interval in the ceca, and like C. jejuni, the prevalence declines to zero. This indicates that birds are not colonized for life. A description of the microeukaryotic intestinal populations indicated the 'normal' intestinal fungal populations in wild turkeys is substantially different from those in commercially raised turkeys. Comparisons of the bacterial community compositions in turkey feces were shown to differ from communities colonizing the mucosal epithelium. Bacterial intestinal community shifts around 11 weeks post-hatch were corroborated for commercially produced animals. The foodborne pathogen C. jejuni responds to the community instability by colonizing the intestine around the week 11 shift, transiently displacing C. coli. These population dynamics indicate the microbiota is responding rapidly to host-derived signals and different intervention strategies are required for exclusion of the two species from the food supply. In addition, fungi Malassezia and Trichosporon were identified as active colonizers of the ceca of three-week-old turkeys. The poultry intestinal anaerobe Megamonas hypermegale was identified as a candidate for competitive exclusion studies to protect against colonization by Campylobacter. PCR-based formats identified isolates of Campylobacter fetus derived from bovine abortions and C. jejuni recovered from feces of cattle raised on grass and in feedlots, suggesting that diet did not impact Campylobacter prevalence. Collaboration with ARS resulted in Multi Locus Sequencing Typing (MLST) profiles of Arcobacter; MLST analysis of Campylobacter hyointestinalis, which colonizes livestock and infects humans, is in progress. We have adapted the RX microtiter format to screen poultry ceca. Despite incorporating antimicrobials and shortening the incubation, nonspecific blackening of the wells yielded false-positive reactions. Studies are in progress to adapt the RX format to screen turkey excision neck samples and carcass swabs, which harbor fewer microbial competitors. This work aligns with Component 1.1 of the National Program 108 Food Safety Action Plan and addresses Problem Statement: 1.1.3 (Ecology, Host Pathogen and Chemical Contaminants Relationships). Significant Activities that Support Special Target Populations USDA collaborator on USDA 1890 Capacity Building Grant awarded to Tuskegee University (�Attenuated Salmonella mutants as live vaccine�; Amin Fadl, College of Veterinary Medicine). Technology Transfer Number of Other Technology Transfer: 6
Impacts
(N/A)
Publications
- Kegode, R.B., Doetkott, D.K., Khaitsa, M.L., Wesley, I.V. 2008. Occurrence of Campylobacter species, Salmonella species, and generic Escherichia coli in meat products from retail outlets in the Fargo metropolitan area. Journal of Food Safety. 28(1):111-125.
- Scupham, A.J., Patton, T.G., Bent, E., Bayles, D.O. 2008. Comparison of the cecal microbiota of domestic and wild turkeys. Microbial Ecology. 56(2) :322-31. Available: http://www.springerlink. com/content/x2kr73p8l4837555/fulltext.html.
- Wesley, I.V., Bhaduri, S., Bush, E. 2008. Prevalence of Yersinia enterocolitica in market weight hogs in the United States. Journal of Food Protection. 71(6):1162-1168.
- Wesley, I.V., Larsen, S., Hurd, H.S., Mckean, J.D., Griffith, R., Rivera, F., Nannapaneni, R., Cox, M., Johnson, M., Wagner, D., De Martino, M. 2008. Low prevalence of Listeria monocytogenes in cull sows and pork. Journal of Food Protection. 71(3):545-549.
- Kawasaki, S., Fratamico, P.M., Wesley, I.V., Kawamoto, S. 2008. Species- specific identification of campylobacters by PCR-restriction fragment length polymorphism and PCR targeting of the gyrase b gene. Applied and Environmental Microbiology. 74(8):2529-2533.
- Avdelbaqi, K., Buissoniere, A., Prouzet-Mauleon, V., Gesser, J., Wesley, I. V., Megraud, F., Menard, A. 2007. Development of a real-time fluorescence resonance energy transfer PCR to detect Arcobacter species. Journal of Clinical Microbiology. 45(9):3015-3021.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) (1) Using optimized sampling strategies, enumeration, and molecular diagnostic, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors. (2) Identify key virulence attributes to differentiate Salmonella and Campylobacter avirulent commensals from those pathogenic strains that pose a public health threat in humans. (3) Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production. Identify microbes associated with gut colonization by, and population shifts of, foodborne pathogens. (4) Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia, in hog carcasses and organs. Approach (from AD-416) Time of entry of Salmonella and Campylobacter will be monitored in turkeys. The study will document flock management practices which affect the prevalence of these foodborne pathogens initially in the brooder period and ultimately throughout live production. Key virulence attributes of C. jejuni and C. coli strains recovered from turkeys will be characterized in vitro (cell invasion assays and in vivo day of hatch poult model). Ultimately, differential gene expression formats will be used to differentiate a virulent from virulent isolates of C. coli and C. jejuni. Bacterial and fungal communities of the ceca of domestic and wild turkeys will be described. This initial survey will provide a measurement of diversity between wild and domestic birds. Analysis of total community flux over time will focus on kinetics of Campylobacter community development and stability in the turkey ceca. Organisms that correlate with Campylobacter colonization or exclusion will be identified. Second generation PCR assays will be developed to detect and quantify Salmonella and Campylobacter on hog and turkey carcasses. Monitoring viscera will serve as an indicator of on-farm versus in-plant sources of contamination. These studies will assist in determining the critical control points of contamination during slaughter. Accomplishments Development of a method for identifying functional microbial species in vivo: It is important for studies of food pathogen ecology to understand how the microbes respond to changes in the intestinal environment, however to date no method was available to perform these experiments. We demonstrated that the thymidine analog bromodeoxyuridine (BrdU), when supplied in the drinking water of turkey poults, will be incorporated into the DNA of actively dividing intestinal microbes. Thus BrdU can be used for identification of turkey intestinal microbes that grow in response to in vivo environmental changes resulting from feed withdrawal or other stressors experienced by the fowl host. This method will allow examination of microbial and host-microbe interactions affecting animal health, nutrition and food safety. This work aligns with Component 1.1 of the ARS NP 108 Food Safety Action Plan and addresses Problem Statement: 1.1.3 (Ecology, Host Pathogen and Chemical Contaminants Relationships). Showed how commercial bird housing systems impact the prevalence of bacterial foodborne pathogens. Ceca derived from hens raised under three different commercial systems were screened for Campylobacter and Salmonella. When the three housing types were compared during the winter, the prevalence of Campylobacter was highest in the non-caged birds. In contrast, for the summer sampling, there was no difference in either Campylobacter or Salmonella prevalence for the three housing types. This indicates that commercial bird housing systems evaluations should consider animal welfare issues as well as microbial food safety impact. This work aligns with Component 1.1 of the ARS NP 108 Food Safety Action Plan and addresses Problem Statement: 1.1.2 (Epidemiology). Significant Activities that Support Special Target Populations Presented a seminar (�Foodborne Pathogens of Public Health Significance�) to faculty and veterinary students and reviewed USDA Capacity Building Grants at Tuskegee University, Tuskegee, Alabama, February 14, 2007. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 18 Number of Newspaper Articles,Presentations for NonScience Audiences: 2
Impacts
(N/A)
Publications
- Harbaugh, E., Trampel, D., Wesley, I.V., Hoff, S., Griffith, R., Hurd, H.S. 2006. Rapid aerosol transmission of Salmonella among turkeys in a simulated holding-shed environment. Poultry Science. 85(10):1693-1699.
- Scupham, A.J. 2007. Examination of the microbial ecology of the avian intestine in vivo using bromodeoxyuridine. Environmental Microbiology. 9(7) :1801:1809.
- Rostagno, M.H., Wesley, I.V., Trampel, D.W., Hurd, H.S. 2006. Salmonella prevalence in market-age turkeys on-farm and at slaughter. Poultry Science. 85(10):1838-1842.
- Borneman, J., Becker, J.O., Bent, E., Lanoil, B., Gardener, B.M., Olatinwo, R., Presley, L., Scupham, A.J., Valinsky, L., Yin, B. 2007. Identifying microorganisms involved in specific in situ functions: experimental design considerations for rRNA gene-based population studies and sequence- selective PCR assays. In: Hurst, C.J. editor. Manual of Environmental Microbiology. 3rd edition. Washington, D.C.: ASM Press. p.748-757.
- Wesley, I.V. 2007. Listeriosis in Animals. In: Listeria, Listeriosis, and Food Safety, 3rd edition. Boca Raton, FL:CRC Press. p.55-84.
- Andersen, M.E., Wesley, I.V., Nestor, E., Trampel, D.W. 2007. Prevalence of Arcobacter species in market-weight commercial turkeys. Antonie Van Leeuwenhoek. 92:309-317.
- Scupham, A.J. 2007. Succession in the intestinal microbiota of pre- adolescent turkeys. Federation of European Microbiological Societies Microbiology Ecology. 60(1):136-147.
- Bhaduri, S., Wesley, I.V. 2006 Isolation and characterization of yersinia enterocolitica from swine feces recovered during the National Animal Health Monitoring System Swine 2000 Study. Journal of Food Protection. Vol. 69:1552-1560.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Food animals are the primary source of bacterial human foodborne pathogens. Following USDA-Food Safety and Inspection Service (FSIS)- Hazard Analysis and Critical Control Points (HACCP) regulation and consumer concerns, livestock producers are attempting to reduce the on- farm prevalence of these pathogens. The newly drafted project has four objectives: (1) Using optimized sampling strategies, enumeration, and molecular diagnostics, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors; (2) Identify key virulence attributes to differentiate avirulent commensal Salmonella and Campylobacter from those pathogenic strains that pose a human public
health threat; (3) Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production; identify microbes associated with gut colonization by, and population shifts of, foodborne pathogens; (4) Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia, in hog carcasses and organs. In the U.S., approximately 5 million cases of bacterial foodborne illness occur annually at a cost of approximately $3.2 billion. The U.S. turkey industry is valued at $7.8 billion with per capita consumption ~ 18 pounds annually. An estimated 458,000 pounds of whole or cut-up turkey parts are exported annually. Thus, international embargoes against U.S. poultry attributed to Salmonella contamination impact the industry. Turkeys are a natural reservoir for C. jejuni, C. coli and Salmonella. The recent ARS Salmonella summit was convened in response to CDCs reports that although E. coli
O157:H7, Campylobacter and Listeria- associated enteritis have declined and are approaching HealthyNBPeople 2010 goals, Salmonella contamination in poultry is increasing. Salmonella reduction in poultry, meat animals, and produce is now a priority for FSIS and other regulatory agencies. ARS has been advised of the need for data to support hazard evaluation and risk assessment of bacterial pathogens on-farm. Further, in the U.S., over 300,000 swine are slaughtered daily. Approximately 3% to 50% of those animals harbor Salmonella when they reach the kill floor. Hogs are also a major animal reservoir for Yersinia enterocolitica, a bacterial agent which is which is transmitted to humans by pork, is the sixth most frequent cause of hospitalizations ascribed to foodborne illness, and is under FoodNet surveillance. Thus, reducing the prevalence of these bacteria in turkeys and hogs may lower human foodborne illnesses and deaths. These studies are relevant to regulatory agencies as they
conduct risk assessments, to the swine and turkey industries, and to the consumers in the global market. Sampling methods for isolation, identification, characterization, and quantification of foodborne pathogens in livestock are major research priorities for ARS pre-harvest food safety efforts. Project Plan focuses on understanding the ecology and epidemiology of foodborne pathogens in swine and turkey production processes under Sections 1.1.2 (Epidemiology); 1.1.3 (Ecology, host pathogens and chemical residue relationships); and 1.2.3 (Production and processing ecology), improved sampling methods under Section 1.1.1 (Methodology); and recommendations for on-farm production practices under Section 1.1.4 (Intervention Strategies). This research is central to achieving effective intervention strategies to reduce on-farm transmission (Action Plan Priority Objective 1.4.1). In- house efforts are complemented by access to farms and processing plants facilitated by the Iowa Turkey
Federation and extramural collaborations. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2006) Objective 1: Using optimized sampling strategies, enumeration, and molecular diagnostics, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors. Subobjective 1.1 Begin flock sampling. Subobjective 1.2 With FSIS design questionnaire. Objective 2-Identify key virulence attritubes to differentiate Salmonella and Campylobacter avirulent commensals from those pathogenic strains that posse a public health threat in humans. Subobjective 2.1 Map Campylobacter in crop, intestine, ceca. Subobjective 2.4 Recruit 0.5 SY. Objective 3- Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production. Identify microbes associated with gut colonization by, and
population shifts of, foodborne pathogens. Subobjective 3.1 Begin bacterial, fungal OFRG. Subobjective 3.2 Complete on-farm flock data collection. Objective 4-Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia in hog carcasses and organs. Subobjective 4.1 Evaluate single tube Salmonella MPN. Year 2 (FY 2007) Objective 1 Subobjective 1.1 - Evaluate data; redesign sampling strategy, if needed. Subobjective 1.2 - With risk assessors analyze questionnaire. Objective 2 Subobjective 2.2 - Begin in vivo virulence assays with C. coli. Subobjective 2.2 - Begin in vitro virulence assays with C. coli. Subobjective 2.4 Begin C. coli microarray analysis. Objective 3 Subobjective 3.1 - Complete bacterial, fungal OFRG. Subobjective 3.2 - Complete analysis. Subobjective 3.3 - Begin antibiotic dissection of gut community. Objective 4 Subobjective 4.1 - Evaluate DNA extraction protocols. Subobjective 4.2 - Evaluate DNA and
real-time PCR forms with various organs. Subobjective 4.3 - Plan studies with FSIS; secure plant access. Year 3 FY 2008 Objective 1 Subobjective 1.1 - Data analysis; retest if needed. Subobjective 1.2 - Present data and survey responses to risk assessors. Objective 2 Subobjective 2.2 - Continue screening in vivo models. Subobjective 2.3 - Continue screening cell lines. Subobjective 2.4 Select genes for quantitative PCR. Objective 3 Subobjective 3.1 - Present and publish results. Subobjective 3.3 - OFRG and validation. Objective 4 Subobjective 4.1 - Evaluate DNA extraction protocols. Subobjective 4.3 - Begin in-plant sampling. Year 4 FY 2009 Objective 1 Subobjective 1.1. - Present results. Objective 2 Subobjective 2.2 Analyze data. Subobjective 2.3 - Analyze data. Subobjective 2.4 Select genes for quantitative PCR. Objective 3 Subobjective 3.3 Examine naturally occurring gradients. Objective 4 Subobjective 4.1 - Evaluate real time PCR formats. Sobobjective 4.3 - Continue in-plant
sampling. Year 5 FY 2010 Objective 1 Subobjective 1.1 Publish results. Objective 2 Subobjective 2.2 Publish results. Subobjective 2.3 Publish results. Objective 3 Subobjective 3.3 Isolation of microbes. Objective 4 Subobjective 4.2 Publish data. Subobjective 4.3 Complete plant sampling; analyze data. 4a List the single most significant research accomplishment during FY 2006. This accomplishment focuses on understanding the ecology and epidemiology of foodborne pathogens in swine and turkey production processes under Sections 1.1.2 (Epidemiology); 1.1.3 (Ecology, host pathogens and chemical residue relationships); and 1.2.3 (Production and processing ecology). Total analysis of intestinal bacteria communities: We have shown that intestinal bacterial communities differ between domestic and wild turkeys; that significant community differences emerge during the life of a turkey from day of hatch to market-weight; that intestinal communities may be flock-specific; and that intestinal
communities of non-transported and transported turkeys differ. Description of the intestinal microbiota offers the potential to determine the molecular basis for colonization of turkeys with Camplyobacter and Salmonella. 4b List other significant research accomplishment(s), if any. These accomplishments focus on improved sampling methods under Section 1. 1.1 (Methodology); and recommendations for on-farm production practices under Section 1.1.4 (Intervention Strategies) and address effective intervention strategies to reduce on-farm transmission (Action Plan Priority Objective 1.4.1). In-house efforts are complemented by access to farms and processing plants facilitated by the Iowa Turkey Federation and extramural collaborations. We screened for Campylobacter in three types of egg layer housing systems: a) buildings in which hens are confined but kept on the ground (n=4 flocks; non-caged birds); b) high-rise buildings with chickens in cages and a manure pit beneath the chickens (n=4
flocks) and c) buildings with chickens in cages and a manure belt beneath the chickens (n=4 flocks) in collaboration with Iowa State University (Xin et al). When the three housing types were compared, the prevalence of Campylobacter spp., especially C. coli, was significantly higher in non-caged birds which were confined but kept on the ground (p<0.05). No such differences were seen among flocks in the distribution of either C. jejuni or in Salmonella. This indicates that the consequences of management practices should include consideration of foodborne pathogens. We have miniaturized Salmonella isolation and detection into a microtiter deep-well format. Validation studies with field samples of turkey ceca and hog feces in side-by-side comparisons with traditional conventional isolation have shown agreement between the two assays. The microtiter format has the potential of streamlining detection of Salmonella. 5. Describe the major accomplishments to date and their predicted or
actual impact. This accomplishment focuses on understanding the ecology and epidemiology of foodborne pathogens in swine and turkey production processes under Sections 1.1.2 (Epidemiology); 1.1.3 (Ecology, host pathogens and chemical residue relationships); and 1.2.3 (Production and processing ecology). Total analysis of intestinal bacteria communities: Molecular analysis of bacteria and fungi from livestock intestines begins with rigorous testing of DNA extraction protocols and selection of methods to avoid bias inherent in template preparation. Using these optimized methods, we have subsequently shown that intestinal bacterial communities differ between domestic and wild turkeys; that significant community differences emerge during the life of a turkey from day of hatch to market-weight; that intestinal communities may be flock-specific; and that intestinal communities of non-transported and transported turkeys differ. These accomplishments focus on improved sampling methods under
Section 1. 1.1 (Methodology); and recommendations for on-farm production practices under Section 1.1.4 (Intervention Strategies) and address effective intervention strategies to reduce on-farm transmission (Action Plan Priority Objective 1.4.1). In-house efforts are complemented by access to farms and processing plants facilitated by the Iowa Turkey Federation and extramural collaborations. We screened for Campylobacter in three types of egg layer housing systems: a) buildings in which hens are confined but kept on the ground (n=4 flocks; non-caged birds); b) high-rise buildings with chickens in cages and a manure pit beneath the chickens (n=4 flocks) and c) buildings with chickens in cages and a manure belt beneath the chickens (n=4 flocks) in collaboration with Iowa State University (Xin et al). When the three housing types were compared, the prevalence of Campylobacter spp., especially C. coli, was significantly higher in non-caged birds which were confined but kept on the ground
(p<0.05). No such differences were seen among flocks in the distribution of either C. jejuni or in Salmonella. This indicates that the consequences of management practices should include consideration of foodborne pathogens. We have miniaturized Salmonella isolation and detection into a microtiter deep-well format. Validation studies with field samples of turkey ceca and hog feces in side-by-side comparisons with traditional conventional isolation have shown agreement between the two assays. The microtiter format has the potential of streamlining detection of Salmonella. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Fundamental knowledge on the kinetics of the microbiota in the turkey ceca is critical in understanding the impact of on-farm
interventions, including administration of pre- and probiotics. Basic knowledge on the dynamics of cecal microbiota was emphasized as a research need by the Iowa Turkey Federation at the Iowa State University-USDA Poultry Science Day. Details of the effect of feed withdrawal, transport, and holding on the intestinal populations of Campylobacter jejuni and C. coli in the market weight turkey have been published and presented to various user groups, including attendees at the annual meeting of the National Turkey Federation, Iowa State University-USDA Poultry Science Day, and the ARS Salmonella summit. Similarly, the lack of effect of perimarketing events on Salmonella shedding in turkeys in contrast to what occurs in market weight hogs is being presented to regulatory, scientific, and commodity groups. Despite the FSIS emphasis on Salmonella reduction, although specific on-farm interventions are well known, the economics of such undertaking may be cost prohibitive. The finding of
higher levels of Campylobacter in non-caged hens indicates that the consequences of management practices should include consideration of foodborne pathogens. These findings will be available to commodity groups following publication of these results in the scientific literature. The microtiter format has the potential of streamlining detection and ultimately enumeration of Salmonella. Rapid screening of livestock may be critical in evaluating on-farm Salmonella reduction strategies. These findings will be available to commodity groups following publication of these results in the scientific literature. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Presented, Report on the National Advisory Committee on Microbiological Criteria for Foods. Annual meeting of the National Turkey Federation in Orlando, Florida. February 14, 2006. Presented,
Turkey Trips dont aggravate contamination. Tristate Food Safety Consortium Spring Newsletter. 2006. Presented, Do feed withdrawal, livehaul and holding influence the prevalence of Salmonella and Campylobacter in turkeys. Annual meeting of the National Turkey Federation, in Orlando, Florida. February 14, 2006.
Impacts
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Publications
- Bearson, S.M., Gerbyshak, H.A., Harbaugh, E., Hurd, H.S., Jones, J., Muraoka, W.T., Rostagno, M.H., Scupham, A.J., Wesley, I.V. 2006. An update of research at the National Animal Disease Center. Available: http://www. ars.usda.gov/research/programs/programs.htm?np_code=108&docid=13576.
- Briggs, R.E., Register, K.B., Tabatabai, L.B., Tatum, F.M., Scupham, A.J., Wesley, I.V. 2006. An update of turkey research at the National Animal Disease Center. Poultry Science Day, May 25, 2006, Ames, Iowa. 2006 CDROM.
- Scupham, A.J. 2006. Microbial community changes in the intestine of the pre-adolescent turkey [abstract]. RRI-INRA Gut Microbiology 2006. 46(1) :S29.
- Wesley, I.V., Harbaugh, E., Trampel, D.W., Rivera, F., Hurd, H.S. 2006. The effect of perimarketing events on the prevalence of salmonella in market weight turkeys. Journal of Food Protection. 69(8):1785-1793.
- Rostagno, M., Trampel, D., Rivera, F., Harbaugh, E., Wesley, I.V., Hurd, H. S. 2006. Preslaughter events do not impact Salmonella prevalence in turkeys. Poultry Science Day, May 25, 2006, Ames, Iowa. 2006 CDROM.
- Wesley, I.V., Muraoka, W.T., Trampel, D., Hurd, H.S. 2006. The effect of preslaughter events on the prevalence of Campylobacter jejuni and Campylobacter coli in turkeys. Poultry Science Day, May 25, 2006, Ames, Iowa. 2006 CDROM.
- Andersen, M., Nestor, E., Wesley, I.V., Trampel, D.W. 2006. Prevalence of Arcobacter in commercial turkey production. Poultry Science Day, May 25, 2006, Ames, Iowa. 2006 CDROM.
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