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United States Department of Agriculture

Agricultural Research Service

2010 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 2010, we conducted monitoring studies of a new commercial poultry facility after completion of 10 broiler poultry rearing cycles, including a facility disinfection and new litter placement. Extensive analyses of fungal and environmental samples for pathogenic bacteria are underway; the data will result in a better understanding of the evolution of the poultry environment in commercial production facilities. Project scientists also evaluated a probiotic (mixture of beneficial microorganisms) administered to growing birds and that was combined with disinfection programs to establish if these approaches reduced Clostridium (a pathogenic bacterium) in laying hens and broilers. Data analysis to date indicates that the treatment had a positive effect in minimizing bird infection by Clostridium; the work is providing a better understanding of how to reduce disease in growing birds. To better understand the relationship between fungi and foodborne pathogens, project scientists used a modern molecular biology technique known as rep-PCR to identify fungi recovered from commercial poultry operations. Results from this work will allow the rapid identification of fungi and will lead to the development of assays to select fungi that have antibacterial activity against foodborne pathogens. In other work, we used molecular biology techniques to assess the impact of Salmonella on the endocrine system of poultry; the work identified several factors that are likely involved in bird/Salmonella interactions; ongoing work is focusing on the exact nature and importance of these interactions. In Salmonella and Clostridium vaccine research, we defined optimum parameters for making the bacteria non-infective while maintaining the ability to stimulate a strong antibody response when injected into fertilized eggs that were subsequently incubated and hatched. These results are very supportive of ongoing work to develop safer and more effective products for reducing pathogens in poultry and poultry products and which in turn will minimize incidences of food poisoning in U.S. consumers. Work under this project has made significant advances in understanding the ecology and management practices that impact on poultry health and food safety issues. Project work has made major strides in identifying dietary additives that can reduce the spread of foodborne pathogens in poultry products that ultimately reach the consumer. Scientific information developed by project scientists allow us to better understand the changes that occur within microbial populations that are associated with commercial rearing of poultry, and are very supportive of our ongoing work to develop new technology and protocols that will be practical for use in poultry production to improve the microbiological safety of the U.S. poultry and egg supply.

1. Broiler house lighting affects Salmonella in poultry. The intensity of lighting, and also the light/dark cycle (total hours in light and dark during a 24-hour period) are factors used in commercial broiler grow-out to ensure bird health and improve production (growth rate, etc.). There is limited information on how lighting might affect how Salmonella colonizes or otherwise affects the growing birds. ARS researchers at College Station, Texas, working with colleagues at Mississippi State University, showed that different lighting protocols do in fact affect Salmonella colonization of the birds. This finding is important because it indicates that appropriate lighting protocols can reduce the Salmonella problem. If such protocols are confirmed to be compatible with necessary production parameters, they can be implemented simply and with little if any added costs to produce microbiologically safer birds.

2. Darkling beetles are a factor in potential antibiotic resistance associated with poultry production. The lesser mealworm (known generally by poultry producers as the darkling beetle) is a nuisance pest that can live in poultry houses by the millions. These insects have been implicated in poultry disease transmission and spread, and can also serve as a reservoir for human food poisoning bacteria such as Salmonella. ARS researchers in College Station, Texas, used modern molecular biology techniques to show that both the larvae and adults of the darkling beetle can harbor in their guts many different types of bacteria, some of which have important human health significance. The work also showed that if antibiotic resistant bacteria are present in the beetle, the genetic elements (plasmids) that are associated with the resistance can be transferred within the beetle gut to other species of bacteria that would otherwise not be resistant to antibiotics. This work is important because it is further evidence that darkling beetles are a serious issue in commercial poultry production, and from several perspectives including bird health, ultimate microbial safety of poultry products reaching the consumer, and in the possible enhancement and spread of antimicrobial resistance among different bacterial types.

3. Natural plant oils in poultry diets reduce harmful bacteria. Digestive bacteria populations are key components for sustained healthy broiler chicken production. Certain plant oils and beneficial bacteria used in feed mixtures for chickens have been shown to promote healthy digestive bacteria which results in improved poultry production. ARS researchers at College Station, Texas, working with colleagues at the University of Colombia, used modern molecular biology techniques to show changes in the bacterial populations in the gastrointestinal tracts of birds that were infected with Eimeria (the protozoan parasite that causes coccidiosis). Eimeria-infected chickens that were fed plant oils had gastrointestinal bacterial populations similar to chickens provided beneficial bacteria. The oils likewise caused reductions in the populations of harmful bacteria in the bird gut. This finding is important because it indicates that some plant oils can shift bacterial populations in a way to reduce the levels of harmful bacteria, and thus can be good candidates to replace antibiotics in commercial poultry production.

4. Defining the production ecosystem to produce microbiologically safer poultry. Harmful bacteria such as Salmonella and Campylobacter are routinely associated with poultry rearing facilities and the birds reared in those facilities. In order to rear birds that will carry a minimum of harmful bacteria into the processing plant, it is important to understand the dynamics of bacteria/bird/facility interactions from a holistic perspective. ARS researchers at College Station, Texas, conducted a very extensive analysis of the occurrence and spread of both pathogenic and non-pathogenic bacterial types among many components of the overall broiler production facility/environment including the broilers, litter, feed, water, insects, rodents, wild birds, and others. The work generated detailed multifactorial analyses of how these harmful bacteria interact with relevant components of the production ecosystem, and will be the basis for development of predictive models to facilitate the production of poultry that are not colonized/contaminated by bacterial species harmful to human health.

Review Publications
Stringfellow, K., McReynolds, J.L., Lee, J., Byrd II, J.A., Nisbet, D.J., Farnell, M.B. 2009. Effect of bismuth citrate, lactose, and organic acid on necrotic enteritis in broilers. Poultry Science. 88:2280-2284.

Hassan, S.M., Haq, A.U., Byrd, J.A., Berhow, M.A., Cartwright, A.L., Bailey, C.A. 2010. Haemolytic and Antimicrobial Activites of Saponin-Rich Extracts from Guar Meal. Food Chemistry. 119:600-605.

Crippen, T.L., Poole, T.L. 2009. Conjugative transfer of plasmid-located antibiotic resistance genes within the gastrointestinal tract of lesser mealworm larvae, Alphitobius diaperinus (Coleoptera: Tenebrionidae). Foodborne Pathogens and Disease. 6:907-915.

Dowd, S.E., Wolcott, R., Crippen, T.L., Sun, Y., Callaway, T.R. 2010. Microarray analysis and draft genomes of two Escherichia coli 0157:H7 lineage II cattle isolates FRIK966 and FRIK2000 investigating lack of Shiga toxin expression. Foodborne Pathogens and Disease. 7:763-773.

Guerin, M.T., Sir, C., Sargeant, J.M., Waddell, L., O'Connor, A.M., Wills, R.W., Bailey, H.R., Byrd II, J.A. 2010. The change in prevalence of Campylobacter on chicken carcasses during processing: A systematic review. Poultry Science. 89:1070-1084.

Larsen, E., Byrd II, J.A., Davis, M. 2010. Effects of litter amendments on broiler growth characteristics and Salmonella colonization in the crop and cecum. Journal of Applied Poultry Research. 19:132-136.

Anderson, P., Hume, M.E., Byrd II, J.A., Hernandez Jr, C.A., Stevens, S., Stringfellow, K., Caldwell, D. 2010. Evaluation of repetitive extragenic palindromic-PCR and denatured gradient gel electrophoresis in identifying Salmonella serotypes isolated from processed turkeys. Poultry Science. 89:1293-1300.

McReynolds, J.L., Genovese, K.J., He, H., Swaggerty, C.L., Byrd II, J.A., Ricke, S.C., Nisbet, D.J., Kogut, M.H. 2009. Alfalfa as a nutritive modulator in maintaining the innate immune response during the molting process. Journal of Applied Poultry Research. 18:410-417.

Last Modified: 8/31/2015
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