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United States Department of Agriculture

Agricultural Research Service

2008 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
Progress has been made in determining the effects of transport stress on contamination of turkeys with Listeria monocytogenes and in identifying physiological and immunological changes that can be used to identify stressed animals. Research on the pathogenesis of L. monocytogenes has also shown that turkeys can be more easily colonized with environmental L. monocytogenes through the respiratory route than through the oral route. The effects of yeast extracts and vitamin C in transport stress models have been evaluated. (NP 108, Component 1, 1.1.4)

1. Blood parameters for profiling turkey response to transport stress: There is a need to identify turkeys with an unfavorable response to the stressors associated with production. Male and female turkeys from different genetic backgrounds were compared for their response to a respiratory bacterial challenge and transport stress. An important change was that the levels of creatine kinase, an indicator of muscle growth and damage, were over 6-fold higher in challenged and transported birds from a commercial line compared to controls, and iron levels of the transported commercial line males were 3-fold lower than non-challenged male controls. Also, the levels of both iron and alkaline phosphatase were lower in the fast growing lines as compared to the slow growing line. These two blood chemicals were also the only parameters influenced by gender, with males having higher levels of both compared to females. The highly significant differences seen in the commercial turkey line for these blood parameters suggests that they may be useful for selection of turkeys with a favorable response to transport stress. (NP108; Component 1, 1.1.4)

2. Yeast extract modulates transport stress in turkeys: There is a need to develop non-antibiotic methods for controlling pathogens in poultry production. A yeast extract (YE) feed supplement added to the diet of turkeys exposed to transport stress and bacterial challenge improved body weight and gain during week 1, decreased mortality in females, and tended to increase the heterophil/lymphocyte (H/L) ratio, which is a measure of stress in birds. H/L ratio was increased by transport stress and was higher in males as compared to females. An immune assay that indirectly measures the ability of blood cells to kill bacteria was higher in males and in birds fed YE and was lower in challenged and in transported birds. Dietary YE may have different effects in males and females and has potential for modulating the impact of transport stress in turkey production. (NP108; Component 1, 1.1.4)

3. Identification and characterization of functional peptides by mass spectrometric scanning of cell and tissue extracts: Bioactive peptides regulate many physiological functions as hormones, neurotransmitters, antimicrobials, growth, and signaling factors. We were interested to find whether it is possible to identify functional peptides by direct scanning of cells and tissues by mass spectrometry. To test the concept we analyzed macrophages and heterophils of chickens using matrix-assisted laser desorption ionization (MALDI) spectrometry which showed characteristic spectral peaks. We choose only the prominent peaks to isolate respective peptides by HPLC and characterized them. We identified mature thymosin beta 4 peptide (Tb4, involved in wound healing and blood vessel growth) from macrophages, and avian beta 2 defensins (antimicrobial peptide) from heterophils of chickens and turkeys, proving that mature and functional peptides can be identified by direct screening of cells and tissues. This method has potential to identify specific peptides of biological interest from tissues and cells. (NP108; Component 1, 1.1.4)

4. Developing a stress indicator for poultry: Previously we identified ovotransferrin (OVT) as an acute phase protein in chicken. To find whether it can be used as a non-specific indicator of stress in chickens we developed an ELISA method to measure its blood levels. Our results showed that only inflammation and infection up-regulates blood OVT levels, whereas it is not affected by metabolic diseases such as pulmonary hypertension syndrome or skeletal disorders. Measurement of peptides such as ovotransferrin, under different physiological and pathological conditions, will enable study of their regulation, manipulation, and protective effects. (NP108; Component 1, 1.1.4)

5. Real-time PCR detects Listeria monocytogenes in transport stressed turkeys: There is a critical need to determine the sources of L. monocytogenes contamination of poultry processing plants and products. Scientists in the Poultry Production and Product Safety Research Unit at Fayetteville, AR, compared conventional culture methods and Taqman® real time PCR (RTi PCR) for isolation of L. monocytogenes from joints of challenged turkeys. Using pre-enrichment Lm was isolated from 71%, 44% and 23%, and using RTi PCR Lm was isolated from 60%, 40% and 22% of Lm-Dex treated, Lm control, and Lm-transport stressed birds, respectively. No Lm was isolated upon direct plating of swabs. RTi PCR can be used for detection of sub-clinical Lm infection of turkey carcasses. (NP108; Component 1, 1.1.3)

6. Respiratory exposure of turkeys to L. monocytogenes causes more disease than oral exposure: Research is needed to determine the source of product and processing plant contamination with L. monocytogenes. Scientists in the Poultry Production and Product Safety Research Unit at Fayetteville, AR, compared the ability of this bacterium to cause disease when fed directly to turkeys (oral route) with disease caused by placing it in the eye and nose (oculonasal route) and also compared the level of disease under different housing conditions. Challenge by the oculonasal route resulted in higher mortality and lower body weights as compared to both non-challenged controls and to those challenged by the oral route. Birds closely contained in battery brooder cages for 1 week after challenge had higher mortality and higher body weights as compared to floor pen reared birds. These results suggest that day old turkey poults are susceptible to respiratory exposure with L. monocytogenes and this exposure can result in subclinical infection that may lead to carcass contamination. (NP108; Component 1, 1.1.3)

6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings3
Number of Newspaper Articles and Other Presentations for Non-Science Audiences2
Number of Other Technology Transfer1

Review Publications
Rath, N.C., Kannan, L., Liyanage, R., Lay, J.O. 2008. Thymosin beta in macrophages. Journal of Endocrinology and Reproduction. 11(2):1-7.

Bielke, L.R., Higgins, S., Donoghue, A.M., Donoghue, D.J., Hargis, B.M., Tellez, G.I. 2007. Use of wide-host-range bacteriophages to reduce Salmonella on poultry products. International Journal of Poultry Science. 6(10):754-757.

Bielke, L.R., Higgins, S., Donoghue, A.M., Kral, T., Donoghue, D.J., Hargis, B.M., Tellez, G. 2007. Evaluation of alternative host bacteria as vehicles for oral administration of bacteriophages. International Journal of Poultry Science. 6(10):758-761.

Vicente, J.L., Torres-Rodriguez, A., Higgens, S.E., Hernandez-Velasco, X., Pixley, C., Tellez, G., Donoghue, A.M., Hargis, B.M. 2008. Effect of a selected Lactobacillus spp-based probiotic on Salmonella serovar enteritidis-infected broiler chicks. Avian Diseases. 52:143-146.

Huff, G.R., Huff, W.E., Rath, N.C., Anthony, N.B., Nestor, K.E. 2008. Effects of Escherichia coli Challenge and Transport Stress on Hematology and Serum Chemistry Values of Three Genetic Lines of Turkeys. Poultry Science. 87:1-8.

Last Modified: 9/4/2015
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