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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
1) Identify environmental influences and management practices that contribute to the colonization of food-borne pathogens in pre-harvest poultry. 2) Identify environmental influences and factors that contribute to necrotic enteritis (NE) including: Clostridium spp. and parasites (Eimeria) that contribute to the onset of disease. 3) Identify prebiotics and symbiotics (lactose, cottonseed, etc.) that can be utilized as pre-harvest intervention strategies, and determine how chlorate and feed additives such as alfalfa control poultry enteropathogen colonization. 4) Identify environmental or management practices that contribute to antibiotic resistance acquisition and dissemination among and between the various pathogenic and commensal microorganisms found in commercial poultry. 5) Characterize the complex interactions between the innate immune and endocrine systems and develop a more fundamental understanding of the role of gastrointestinal endocrinology on the microbial ecology of the gut of food-producing animals.

1b.Approach (from AD-416)
1) Using a newly constructed (i.e., naive) commercial broiler production facility, we will follow bacterial, viral, protozoan, and fungal movement within the facility from prior to the first placement of birds through several successive production cycles. The movement of these organisms within the environment will be mapped using genetic identification and traditional culture methods. 2) Using a necrotic enteritis (NE) in vivo model developed in our laboratory and a primary cell culture model, we will investigate the interactions of Clostridium with other bacterial populations within the gastrointestinal tract of broilers and the development of NE. We will evaluate these bacterial populations using molecular-based techniques (DGGE, PFGE) in order to determine the dynamics between commensal gut bacterial populations and Clostridium. Additionally, we will examine the toxins produced by Clostridium using tissue culture techniques and Multiplex Polymerase Chain Reactions (PCR). 3) The efficacy of lactose, cottonseed and similar prebiotics and symbiotics will be evaluated under commercial conditions for their ability to reduce food-borne pathogens in poultry. Practical feeding trials will be performed to ascertain the ability of chlorate and alfalfa as alternatives to traditional antimicrobials, and the mode of action of these compounds will be determined. We will also study different quorum sensing autoinducers to determine the effects of biological and synthetic bacterial autoinducer inhibitors on poultry enteropathogens. 4) Utilizing an in vitro bacterial conjugation assay, we will identify flavophospholipol-like compounds (flavophospholipol has been shown to reduce horizontal gene transfer between Enterococci in vitro), that inhibit bacterial conjugation and resistance gene acquisition among gut bacteria. 5) We will characterize specific interactions between the immune and endocrine systems that influence enteropathogen colonization in the gastrointestinal tract of poultry. Microarrays will be utilized to assess fluctuations in key avian hormones that correspond to cytokine expression.

3.Progress Report
In FY 2009, sampling of new poultry facilities prior to introduction of any birds has been done. Analysis of these samples, presently underway, will result in a better understanding of the evolution of the poultry environment once birds begin to be reared in these facilities. Work was also focused on the effects of a probiotic (mixture of beneficial microorganisms), when combined with disinfection programs, on the interactions of laying hens and broilers with the disease bacterium Clostridium. The data indicate that the treatment had a positive effect in minimizing bird infection by Clostridium. Epidemiological studies on fungi recovered from commercial poultry operations were largely completed and data analysis is underway. In separate work, we used molecular biological techniques to assess the impact of Salmonella on the endocrine system of poultry; the work has identified several endocrine factors that are likely involved in bird/Salmonella interactions, and ongoing work is focusing on the exact nature and importance of these interactions. In Salmonella and Clostridium vaccine research, progress was made in defining optimum parameters for making the bacteria non-infective while maintaining ability to stimulate a strong antibody response when injected into fertilized eggs subsequently incubated and hatched.

1. Development of new poultry vaccines using high energy electron-beam irradiation: Some microorganisms, and particularly bacteria, cause serious disease in commercial poultry and can be of importance in causing food poisoning in poultry products reaching the consumer. New methods are needed to minimize the effects of such microorganisms, and development of effective vaccines would be of much benefit. In collaboration with scientists at Texas A&M University, we used high energy electron-beam (E-beam) irradiation to render Salmonella non-viable as an infectious agent, but retaining the necessary antigenic properties to stimulate a strong immunological response in poultry. The work established that broiler chickens exposed to E-beam-treated bacteria were much more efficient in fighting off subsequent infections by normal, viable bacteria, meaning that the irradiated bacteria could serve as a vaccine. This accomplishment is important because, although vaccines against viruses are well-known and relatively easy to create, development of effective vaccines against bacteria has historically been much more difficult. E-beam technology appears to be much more effective than X-rays in generating good bacterial vaccines, and should be of great value in development of vaccines to protect poultry from serious diseases while also contributing to enhanced microbial food safety in humans.

2. Rapid identification of fungi in commercial poultry houses: Various fungi are normal components of the microbial ecosystem in commercial poultry houses, but the possible impact (positive or negative) of fungi on the overall health of the birds raised in these facilities is largely unknown. It is known that beneficial bacteria can play a significant role in improving bird health and productivity in commercial rearing facilities, and it is possible that some fungi may play a similar role. Fungi in poultry production ecosystems therefore merit study, but adequate detection/identification techniques and protocols are lacking. We used a modern molecular biological technique called automated rep-PCR to rapidly and accurately identify a large number of fungal and yeast genera taken from the poultry rearing environment. This technique, and the precision it offers, will be critical in facilitating work to better define the presence of fungal/yeast species associated with commercial poultry, and to establish the beneficial or harmful effects these microorganisms may have in the production of healthy and wholesome poultry for human consumption.

6.Technology Transfer

Number of Active CRADAs1

Review Publications
Byrd II, J.A., Burnham, M.R., McReynolds, J.L., Anderson, R.C., Genovese, K.J., Callaway, T.R., Kubena, L.F., Nisbet, D.J. 2008. Evaluation of an experimental chlorate product as a pre-harvest feed supplement to reduce Salmonella in meat producing birds. Poultry Science. 87:1883-1888.

Crippen, T.L., Sheffield, C.L., Andrews, K., Dowd, S.E., Nisbet, D.J. 2008. Planktonic and biofilm community characterization and Salmonella resistance of 14-day old chicken cecal microflora derived continuous-flow cultures. Journal of Food Protection. 71:1981-1987.

Crippen, T.L., Sheffield, C.L., Andrews, K., Bongaerts, R.J. 2008. Bacterial concentration and diversity within repetitive aliquots collected from replicate continuous flow bioreactor cultures. The Open Microbiology Journal. 2:60-65.

Nisbet, D.J., Edrington, T.S., McReynolds, J.L., Callaway, T.R., Byrd II, J.A. 2008. Influence of exogenous melatonin administration on Salmonella enteritidis colonization in molted layers. Poultry Science. 87:1083-1088.

Crippen, T.L., Sheffield, C.L., Esquivel, S.V., Droleskey, R.E., Esquivel, J.F. 2009. The acquisition and internal carriage of Salmonella by lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae). Vector-Borne Zoonotic Diseases. 9:65-72.

Sheffield, C.L., Crippen, T.L., Bischoff, K.M., Andrews, K. 2009. Characterization of planktonic and biofilm communities of day-of-hatch chicks cecal microflora and their resistance to Salmonella colonization. Journal of Food Protection. 72:959-965.

Lee, K.M., McReynolds, J.L., Fuller, C.C., Jones, B., Herrman, T.J., Byrd, J.A., Runyon, M. 2008. Investigation and characterization of the frozen feeder rodent industry in Texas following a multi-state Salmonella typhimurium outbreak associated with frozen vacuum-packed rodents. Zoonoses and Public Health. 55:488-496.

Dunkley, C.S., Kim, W.K., Friend, T.H., Woodward, C.L., McReynolds, J.L., Dunkley, K.D., Kubena, L.F., Nisbet, D.J., Ricke, S.C. 2008. Behavioral responses of laying hens to different alfalfa-layer ration combinations fed during molting. Poultry Science. 87:1005-1011.

Dunkley, C.S., Friend, T.H., McReynolds, J.L., Kim, W.K., Dunkley, K.D., Kubena, L.F., Nisbet, D.J., Ricke, S.C. 2008. Behavior of laying hens on alfalfa crumble molt diets. Poultry Science. 87:815-822.

Poole, T.L., Crippen, T.L. 2009. Conjugative plasmid transfer between Salmonella enterica Newport and Escherichia coli within the gastrointestinal tract of the lesser mealworm beetle, Alphitobius diaperinus (Coleoptera: Tenebrionidae). Poultry Science. 88:1553-1558.

Last Modified: 8/1/2015
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