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United States Department of Agriculture

Agricultural Research Service

Research Project: Microbial Interactions and Management Approaches to Reduce Pathogenic Bacteria in Poultry

Location: Food and Feed Safety Research

2012 Annual Report

1a.Objectives (from AD-416):
Objective 1: Identify/characterize management practices and environmental factors that will reduce fitness characteristics of foodborne pathogens in poultry as related to persistent colonization, survival growth, virulence, and antimicrobial resistance. Sub-objective 1A: Determine the effects of vaccine management programs on the viability of pathogenic bacteria in poultry. Sub-objective 1B: Effect of wood extracts on the viability of pathogenic bacteria in poultry litter. Sub-objective 1C: Identify attractants and spatial distribution of the lesser mealworm. Sub-objective 1D: Determine if Bambermycin in animal feed prevents recipient bacteria from accumulating multiple resistance plasmids, and to determine if multidrug resistant Salmonella lose resistance plasmids when there is no selection pressure. Sub-objective 1E: Determine changes in poultry microbial/fungal digestive populations and identify potential protective isolates against human enteropathogen colonization. Objective 2: Develop/evaluate new intervention strategies that prevent or reduce enteric colonization and other fitness characteristics that will be integrated into existing management practices and decrease shedding of foodborne pathogens. Sub-objective 2A: Determine the bacteriocidal effects of chitosan as a feed additive against human foodborne enteropathogen colonization in poultry. Sub-objective 2B: Determine the bacteriocidal and anticoccidial effects of chlorate as a feed additive against human foodborne enteropathogen colonization in poultry. Sub-objective 2C: Determine the bacteriocidal effects of clay montomorillonite and calcium formate as feed additives against human foodborne enteropathogen colonization in poultry. Objective 3: Understand the effect of waste management conditions in poultry (extrinsic and intrinsic) under which foodborne microorganisms exist, and determine the complex interactions among waste management practices on survival and dispersion of pathogens within the poultry facility and the surrounding environment. Sub-objective 3A: Determine the fate and transport into the environment of poultry litter bacteria and the effects of lesser mealworm as a vector. Sub-objective 3B: Modifications of poultry litter composting to increase efficiency and efficacy with respect to reducing pathogen levels. Objective 4: Determine the complex interactions among fungi/protozoa/microbial population within the gastrointestinal tract of poultry and how it affects food safety. Specifically, research will be focused on understanding the interactions and developing strategies that reduce foodborne pathogens, including antimicrobial resistance. Sub-objective 4A: Identify fungi that will reduce or control the growth of Salmonella and Campylobacter. Sub-objective 4B: Define the role of broiler and layer fungal digestive populations in poultry production and protection against colonization by human foodborne enteropathogens.

1b.Approach (from AD-416):
Objective 1: Management practices and environmental factors and their interactions on the growth of foodborne pathogens in poultry will be evaluated. Broiler chickens will be vaccinated with commercially available vaccines (e.g., Marek's) using several routes of administration and evaluated for Salmonella within the gastrointestinal tract. Beneficial bacteria will be identified from broilers infected with Salmonella and compared to the bacteria found in broilers without Salmonella using a sophisticated DNA technology called pyrosequencing. Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle which will allow the identification of hundreds of bacteria from a single sample. This will lead to the development of new interventions to help control numerous foodborne pathogens. Another approach will investigate the spread of antibiotic resistance from bacteria to bacteria, using an antibiotic (bambermycin) that prevents the transfer of genetic information. Within the environment of broilers, lesser mealworms will be exposed to numerous natural and artificial attractants for the development of new control programs. Poultry are raised on wood flakes that may be contaminated with foodborne pathogens, numerous wood products will be evaluated for their antimicrobial activity against these pathogens using pyrosequencing technology. Objective 2: New intervention strategies will be evaluated to reduce foodborne pathogen colonization in poultry that can be incorporated into existing management practices. Several compounds, including chitosan, chlorate, calcium formate, and clay montomorillonite, will be incorporated into existing commercial management programs. Broilers will be provided the products and challenged with foodborne pathogens and evaluated for the recovery of the pathogens. These compounds utilize different mechanisms for controlling foodborne pathogens, including absorption of the pathogen, targeting enzymatic pathways within the pathogens, and activation of the host immune system. Objective 3: Existing management practices will be evaluated on survival and dispersion of pathogens within the poultry facility and the surrounding environment. Comparisons of management practices on their ability to prevent the spread of pathogenic bacteria and lesser mealworm into the environment will be made using conventional bacterial culture methodology, PCR-based technology, mineral identification, and minimum inhibitory concentration (MIC) analysis. Objective 4: The role of broiler and layer fungal digestive populations in poultry production and protection against colonization by human foodborne enteropathogens will be defined. Using conventional fungal culture methodology, PCR methodology, and sequencing technology fungal species will be identified that have ability to reduce Salmonella and Campylobacter. Building on the information gained in Objective 1, fungal populations recovered in the gastrointestinal tract of broilers taken from Salmonella positive or negative farms will be compared for changes in the fungal profiles using pyrosequencing.

3.Progress Report:
In FY 2012, major focus was on implementing protocols for the control of foodborne pathogens in poultry. As part of project work to identify management practices that increase or decrease foodborne pathogens in poultry, studies were conducted to define the impact of vaccination programs on the colonization of pathogenic bacteria in the gastrointestinal tract of preharvest poultry. The work showed that infectious bronchitis-vaccinated day-of-hatch chickens were more likely to be contaminated with Campylobacter than broilers not vaccinated. In other FY 2012 work, sampling was begun on commercial broiler farms from four states for the comparison of bacterial and fungal microflora from broilers contaminated with Salmonella and Campylobacter. The objective of the work is to identify changes within the gastrointestinal tract of broilers that can then be manipulated using dietary and bacterial beneficial cultures to reduce the spread of foodborne pathogens.

1. Bactericidal effects of chitosan in poultry. A number of supply, production, and environmental sources can serve as Salmonella contamination points during food animal production. Prebiotic feed additives offer potential alternatives to antibiotic use against gut pathogens in poultry production. ARS researchers at College Station, Texas, showed that incorporation of chitosan into the diet significantly reduced Salmonella cecal contamination in young broilers. Chitosan is a derivative of chitin, which is the structural element of the exoskeleton of insects, shrimp, etc. Chitosan is totally safe to higher animals. This discovery has important food safety implications because chitosan as a feed additive offers potential application against Salmonella contamination during pre-harvest poultry production. Reduced pre-harvest pathogen load will result in fewer Salmonella in live broilers entering the processing plant and will lessen the potential for carcass cross-contamination.

Review Publications
Crippen, T.L., Zheng, L., Sheffield, C.L., Tomberlin, J.K., Beier, R.C., Yu, Z. 2012. Transient gut retention and persistence of Salmonella through metamorphosis in the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae). Journal of Applied Microbiology. 112:920-926.

Ma, Q., Fonseca, A., Liu, W., Fields, A.T., Pimsler, M.L., Spindola, A.F., Tarone, A.M., Crippen, T.L., Tomberlin, J.K., Wood, T.K. 2012. Proteus mirabilis interkingdom swarming signals attract blow flies. The ISME Journal: Multidisciplinary Journal of Microbial Ecology. 1:1-11.

Volkova, V.V., Byrd II, J.A., Hubbard, S.A., Magee, D., Bailey, R.H., Wills, R.W. 2010. Lighting during grow-out and Salmonella in broiler flocks. Acta Veterinaria. 52:46-52.

Volkova, V.V., Wills, R.W., Hubbard, S.A., Magee, D.L., Byrd II, J.A., Bailey, R.H. 2010. Risk factors associated with detection of Salmonella in broiler litter at the time of new flock placement. Zoonoses and Public Health. 58:158-168.

Crippen, T.L., Poole, T.L. 2012. Lesser mealworms on poultry farms: A potential arena for the dissemination of pathogens and antimicrobial resistance. In: Callaway, T.R., Edrington, T.S., editors. On-Farm Strategies to Control Foodborne Pathogens. New York, NY: Nova Science Publishers, Inc. p. 233-272.

Crippen, T.L., Esquivel, J.F. 2012. Improved visualization of Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) - Part II: Alimentary canal components and measurements. Psyche. 2012:Article 607609.

Sheffield, C.L., Crippen, T.L. 2012. Invasion and survival of Salmonella in the environment: The role of biofilm. In: Kumar, Y., editor. Salmonella-A Diversified Superbug. Rijeka, Croatia: InTech Publishers. p. 3-28.

Hume, M.E., Barbosa, N.A., Dowd, S.E., Sakamura, N.K., Nalian, A.G., Kley, A.M., Oviedo-Rondon, E.O. 2011. Use of pyrosequencing and denaturing gradient gel electrophoresis to examine the effects of probiotics and essential oil blends on digestive microflora in broilers under mixed Eimeria infection. Foodborne Pathogens and Disease. 8:1159-1167.

Esquivel, J.F., Crippen, T.L., Ward, L.A. 2012. Improved visualization of Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) - Part I: Morphological features for sex determination of multiple stadia. Psyche. 2012:Article 328378. doi 1155-12-328478.

Last Modified: 4/20/2014
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