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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

2013 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:
As part of our work to identify management procedures that increase or decrease foodborne pathogens in poultry, studies were conducted in FY 2013 to establish the impact of vaccination programs on the colonization of pathogenic bacteria in the gastrointestinal tract of preharvest poultry. The work showed that Marek-vaccinated day-of-hatch chickens were more likely to be contaminated with Salmonella than were broiler embryos vaccinated in the egg at day 18 of incubation. In other work, sampling is continuing 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. Blow flies use bacterial quorum sensing to detect food. Insects locate food nutrients by a host of variables and are dependent on the stimulus and the age of the insect. Volatile compound attractants to which primary colonizers, such as blow flies, respond are the same signaling molecules used by bacteria found on the feed source. ARS scientists at College Station, Texas, working with scientists at Texas A&M University, established that the mechanism used by blow flies for detecting food can be associated with quorum sensing (communication among individual bacterial cells) and that the physiological state of the insect influences its response. The work also identified several differences in volatile compounds produced by the bacteria that could explain blow fly response. This work has important food safety implications for understanding how insects, potential vectors for spreading pathogens, are attracted to a contaminated food source. Identifying the attractant could lead to the development of non-pesticide insect control technologies that will have as the end result less Salmonella colonization of food-producing animals and microbiologically safer meat products for the consumer.

2. Darkling beetles retain Salmonella through life stages. Many sources of foodborne pathogens within poultry production facilities currently go unrecognized. The darkling beetle is a serious pest in poultry facilities and is known to carry pathogens affecting both human and animal health. Darkling beetle mobility, indiscriminate feeding habits, and potential to then be eaten by poultry within the grow-out house provide a way for the dissemination of Salmonella. ARS scientists at College Station, Texas, working with scientists at Texas A&M University and Huazhong Agricultural University (China), exposed darkling beetles to Salmonella and evaluated the insect for shedding of the bacterium. Exposed larvae were followed through pupation, and newly emerged adults were examined for Salmonella. Exposed adult and larval beetles produced Salmonella-positive feces for up to 12 days. Current poultry management programs involve the reuse of poultry litter, these beetles survive between flock rotations on the reutilized litter, and this accomplishment has shown that Salmonella can survive with them. The work is useful to poultry producers because it has identified an important foodborne pathogen reservoir that affects food animals and thus the microbiological safety of poultry meat products.

Review Publications
Stanley, V.G., Hickerson, K., Daley, M.B., Hume, M.E., Hinton Jr, A. 2012. Single and combined effects of organic selenium and zinc on egg, fertility, hatchability, and embryonic mortality of exotic cochin hens. Agrotechnology. 2(1):106. doi:10.4172/2168-9881.1000106.

Tomberlin, J.K., Crippen, T.L., Tarone, A.M., Singh, B., Adams, K., Rezenom, Y.H., Benbow, M.E., Flores, M., Longnecker, M., Pechal, J.L., Russell, D.H., Beier, R.C., Wood, T.K. 2012. Interkingdom responses of flies to bacteria mediated by fly physiology and bacterial quorum sensing. Animal Behaviour. 84:1449-1456.

Stanley, V.G., Shanklyn, P., Daley, M., Gray, C., Vaughan, V., Hinton Jr., A., Hume, M.E. 2012. Effects of organic selenium and zinc on the aging process of laying hens. Agrotechnology. 1:103-105.

Martynova-Van Kley, M.A., Oviedo-Rondon, E.O., Dowd, S.E., Hume, M.E., Nalian, A. 2012. Effect of Eimeria infection on cecal microbiome of broilers fed essential oils. International Journal of Poultry Science. 11:747-755.

Escarcha, J.F., Callaway, T.R., Byrd II, J.A., Miller, D.N., Edrington, T.S., Anderson, R.C., Nisbet, D.J. 2012. Effects of dietary alfalfa inclusion on Salmonella Typhimurium populations in growing layer chicks. Foodborne Pathogens and Disease. 9:945-951.

Zheng, L., Crippen, T.L., Singh, B., Tarone, A.M., Dowd, S., Yu, Z., Wood, T.K., Tomberlin, J.K. 2013. A survey of bacterial diversity from successive life stages of black soldier fly (Diptera: Stratiomyidae) by using 16S rDNA pyrosequencing. Journal of Medical Entomology. 50:647-658.

Demirok, E., Veluz, G., Stuyvenberg, W.V., Castaneda, M.P., Byrd, J.A., Alvarado, C.Z. 2013. Quality and safety of broiler meat in various chilling systems. Poultry Science. 92:1117-1126.

Sheffield, C.L., Crippen, T.L., Poole, T.L., Beier, R.C. 2013. Destruction of single-species biofilms of Escherichia coli or Klebsiella pneumoniae subsp. pneumoniae by dextranase, lactoferrin, and lysozyme. International Microbiology. 15:185-189.

Last Modified: 4/19/2014
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