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United States Department of Agriculture

Agricultural Research Service

2010 Annual Report

1a.Objectives (from AD-416)
The objectives of this work include: 1)Determine the impact of stress on the immune response and on colonization of foodborne pathogens in turkeys; 2)Optimize strategies for decreasing the impact of stress on colonization of turkeys with pathogens of food safety importance.

1b.Approach (from AD-416)
We hypothesize that the response to common stressors of commercial turkey production, including Escherichia coli respiratory disease (airsacculitis), moving and transport, and temperature extremes, can increase pre-harvest contamination of turkeys with pathogens of food safety importance, and that basic understanding of how turkey immunity is affected by stress will lead to the development and application of practical strategies to improve product safety. Because stress has been shown to both increase disease resistance at low levels and decrease disease resistance at high or sustained levels, its effects on food safety have been difficult to quantify. We have developed transport stress and cold stress models which result in repeatable levels of stress-induced infection of turkeys with E. coli. We will use these models to study the colonization of turkeys with other bacteria of food safety importance. Cell culture studies of the interaction between bacterial pathogens and primary turkey synovial cells, macrophages, and heterophils, from normal and stressed animals will provide basic data and systems for testing the efficacy of therapeutic and prophylactic products to modulate the stress response, improve disease resistance, and decrease carcass contamination with pathogens. Novel non-antibiotic interventions will include antimicrobial peptides and acute phase proteins that will be developed using exploratory analysis of physiological reactions in our stress models as described in the previous section. These products will be incorporated into stress models; however variations in both dosage and timing relative to stressor will be emphasized in multiple experiments to maximize production gains while minimizing pathogen contamination.

3.Progress Report
We evaluated the effects of vitamin D3 (VD) and yeast extract (YE) supplementation in two stress models, dexamethasone (Dex) injection and a concurrent heat stress/ transport stress model (HTS). YE significantly decreased early mortality and nominally increased body weight through week 6. After the stress challenges, YE decreased overall mortality. Both HTS and VD decreased cecal Salmonella colonization, while both HTS and VD increased cecal Campylobacter colonization. The combination of YE, VD, and HTS eliminated Salmonella colonization from 87.5% in untreated controls to 0%, while increasing Campylobacter colonization from 62.5% in untreated controls to 100% in those treated with YE, VD, and HTS. We also measured the changes in the quality, strength, and growth of bone in these turkeys. Our results showed that stress negatively affects bone growth, quality, and strength and these changes were not mitigated by dietary supplementation of either YE or VD. We are developing a stress model in genetic lines of Japanese quail that differ in corticosterone response to restraint stress. In collaborative work with USDA-ARS-NADC, we observed an increased incidence in Salmonella isolation in quail subjected to heat stress, suggesting that these quail lines can be used to study stress-related differences in susceptibility to food-borne pathogens. Birds were subjected to heat stress and challenged with an aerosol spray of E. coli. At necropsy the entire intestinal tract was screened for both Salmonella and Campylobacter. While Campyobacter was not recovered, we observed a differential effect of heat stress on Salmonella isolation from male and female quail. Both males and females had significantly more Salmonella isolation after heat stress; however, there tended to be subtle gender differences in the effect of genetic line. We speculate that the stress of coming into lay may have confounded the female isolation data. The proposed work using these quail lines will lead to an understanding of the influence of sex and stress hormones on both host immunity and pathogen virulence. The failure to isolate Campylobacter from these birds suggests that they will be useful for Campylobacter challenge studies. Avian beta defensins have potential as antimicrobials to improve food safety of poultry. A method to measure avian beta defensin-2 release from heterophils in response to pathogen-associated molecules was developed. We also determined that thymosin beta 4, a peptide responsible for resolving inflammation, was released upon cell death. These studies were done using peptidomic approaches such as mass spectrometry, HPLC, and chemical modifications. We continue developing strategies for organic poultry production that can reduce pathogens focusing on natural antimicrobials, such as medium chain fatty acids and essential oils. In the past year we have developed a state-of-the-art organic/pasture poultry research facility. This facility is one of the very few organic certified poultry research facilities in the U.S. It is the only one to our knowledge in which both large-scale and small-scale production can be studied.

1. Factors regulating poultry immunity and health. Many low molecular weight proteins and peptides provide immediate defense against disease-causing organisms and help recuperation of damaged tissues. These factors are produced by the white blood cells. Analyzing these factors and measuring their production under different physiological and pathological conditions will contribute to understanding their mechanism of action and their potential use in developing pathogen control and food safety strategies. Using proteomic technologies, we measured avian defensin-2 (AvBD2), an antimicrobial peptide produced by a type of white blood cells called heterophils. By manipulating heterophils to respond to various bacteria- and virus-associated molecules we found that these cells show discrete responses to certain stimulants but not all. Similarly another peptide, thymosin beta 4, is also released by another type of white blood cell called macrophages upon stimulation with different pathogen associated factors. Our research demonstrated that thymosin beta 4 is released upon the death of these white blood cells, neutralizing pathogens and contributing to wound healing.

2. Organic certification awarded to poultry research facility. Despite a 20% growth in organic poultry production/year, few research and outreach programs target the needs of these producers. Organic poultry production has unique challenges; the lack of safe, approved, and effective treatments for diseases can adversely influence bird health and the wholesomeness of poultry products. As part of our research targeting organic and small farm poultry production we have developed a state-of-the-art organic/pasture poultry research facility collaboratively with the University of Arkansas. The facility meets the livestock requirements of the USDA National Organic Program, the animal welfare recommendations for poultry by the National Organic Standards Board and the Organic Poultry Guidance Document of the Accredited Certifiers Association, both of which encourage a high amount of outdoor access. This facility is one of the very few organic certified poultry research facilities in the U.S. It is the only one to our knowledge in which both large-scale and small-scale production can be studied.

5.Significant Activities that Support Special Target Populations
Scientists have participated in activities targeting veterans, minorities and historically underserved small farmers including:.
1)serving as Principal Investigator on a SARE grant in cooperation with the ARS Dale Bumpers Small Farms Research Center, Booneville AR; The Kerr Center for Sustainable Agriculture, Poteau, OK; NCAT, Fayetteville, AR; Louisiana State University, Baton Rouge, LA, as well as several small poultry producers in Arkansas;.
2)serving as a co-investigator on a Beginning Farmer and Rancher Development Program NIFA grant on a project that provides bilingual English and Spanish whole-farm learning and experiential opportunities for new farmers interested in adopting integrated poultry, small ruminant, and agroforestry production practices, cooperating with the Booneville Unit, the Kerr Center, NCAT, and the University of Arkansas, Fayetteville and Pine Bluff (the 1890 institution for the State of Arkansas); and.
3)the Unit partnered with ARS laboratories at Lane, OK; Booneville, AR; and El Reno, OK, to develop and staff an exhibit for the Know Your Farmer, Know Your Food Conference and Gala Dinner in El Reno in July 2010. The Conference was attended by over 200 persons, the majority being small- to mid-sized farmers, institutional food professionals, and extension or outreach specialists.

Review Publications
Huff, G.R., Huff, W.E., Rath, N.C. 2010. Nutritional immunomodulation as an approach to decreasing the negative effects of stress in poultry production. Arkansas Academy of Science. 63:87-92.

Rath, N.C., Anthony, N.B., Kannan, L., Huff, W.E., Huff, G.R., Chapman, H.D., Erf, G.F., Wakenell, P. 2009. Serum Ovotransferrin as a Biomarker of Inflammatory Diseases in Chickens. Poultry Science. 88(10):2069-2074.

Huff, G.R., Huff, W.E., Farnell, M.B., Rath, N.C., De Los Santos, F.S., Donoghue, A.M. 2010. Bacterial clearance, heterophil function, and hematological parameters of transport stressed turkey poults supplemented with dietary yeast extract. Poultry Science. 89(3):447-456.

Shini, S., Huff, G.R., Kaiser, P. 2010. Understanding stress-induced immunosupression: Exploration of cytokine and chemokine gene profiles in chicken peripheral leukocytes. Poultry Science. 89(4):841-851.

Rath, N.C., Kannan, L., Liyanage, R., Lay, J.O. 2009. Evaluation of beta defensin 2 production by chicken heterophils using direct MALDI mass spectrometry. Molecular Immunology. 46(15)3151-3156.

Last Modified: 11/28/2015
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