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ARS Home » Southeast Area » Fayetteville, Arkansas » Poultry Production and Product Safety Research » Research » Research Project #430483

Research Project: Antibiotic Alternatives for Controlling Foodborne Pathogens and Disease in Poultry

Location: Poultry Production and Product Safety Research

2016 Annual Report

1a. Objectives (from AD-416):
Objective 1: Investigate the use of selected probiotics, natural plant compounds, and bacteriophage, as potential alternatives to antibiotics and mechanisms to reduce the levels of Salmonella and Campylobacter in poultry. Evaluate these products in multiple production/processing systems including conventional, pasture raised, and organic systems. Sub-objective 1A: Reduce the incidence of Campylobacter in preharvest poultry by selecting probiotics that utilize mucin for growth and competitively inhibit Campylobacter colonization in broiler chickens. Sub-objective 1B: Reduce the incidence of Salmonella and Campylobacter in pre and postharvest poultry by utilizing plant extracts and other natural compounds such as chitosan, ß-resorcylic acid, naringenin and rutin hydrate. Sub-objective 1C: Reduce the incidence of Campylobacter in pre and postharvest poultry using mucin-adapted bacteriophage and genome targeting CRISPR-Cas system. Sub-objective 1D: Evaluate the genome wide effect of natural plant compounds and probiotics on Campylobacter especially genes critical for colonization in chicken using high-throughput deep sequencing of mRNA transcripts using RNA-seq. Objective 2: Develop innovative strategies for increasing disease resistance and improving immunity to foodborne pathogens of poultry using egg shell membrane technology. Sub-objective 2A: Determine effects of egg shell membrane on immune indices of chickens. Sub-objective 2B: Develop a proof of concept model for mucosal modulation of immunity by enriching HESM with Salmonella and Campylobacter.

1b. Approach (from AD-416):
Our overall goal is to develop novel natural treatment strategies to reduce or eliminate the incidence of Salmonella and Campylobacter colonization in poultry and contamination in products. Our strategy is to target the site of colonization in the bird, the mucosal lining of the crypts, by evaluating selected probiotic isolates and bacteriophages against Campylobacter that competitively inhibit Campylobacter within the enteric crypt environment. For the studies with natural antimicrobial compounds in feed, the individual effects of ß-resorcylic acid, chitosan, rutin hydrate and naringenin will be tested in broiler chickens, and then the potential additive effects of combining these treatments will be evaluated. Previous results demonstrate that young birds are predictive of efficacy in market age birds (Solis de los Santos et al., 2008a, b, 2009). Use of younger birds reduces the time and expense (e.g., feed costs) so that more isolates or compounds can be tested. Optimal concentrations and combinations will be tested in market-age birds.

3. Progress Report:
A study was conducted to determine phytochemicals influence on Campylobacter jejuni virulence factors and expression of virulence genes in vitro. Campylobacter jejuni is a major foodborne pathogen that causes severe gastroenteritis in humans. In the human gut, Campylobacter adheres and invades the intestinal epithelium followed by cytolethal distanding toxin mediated cell death, and enteritis. Reducing the attachment and invasion of intestinal epithelium and expression of virulence factors (motility, cytolethal distending toxin production) could potentially reduce Campylobacter infection in humans. This study investigated the efficacy of sub-inhibitory concentrations (SICs, concentration not inhibiting bacterial growth) of five, generally regarded as safe status phytochemicals in reducing the attachment and invasion of C. jejuni on human intestinal cells (Caco-2). The phytochemicals used were trans-cinnamaldehyde, eugenol, naringenin, carvacrol and ß-resorcylic acid. Monolayers of Caco-2 cells were grown in 96-well tissue culture plates at ~105 cells/well, and inoculated with C. jejuni either in the presence or absence of phytochemicals followed by incubation. The cells were lysed using 0.1% triton X and Campylobacter that attached and invaded Caco-2 cells were enumerated. Additionally, the effect of these phytochemicals on Campylobacter motility and toxin production was studied using standard bioassays and gene expression analysis. All experiments had duplicate samples and were replicated three times on three strains of C. jejuni. All phytochemicals reduced C. jejuni adhesion to and invasion of human epithelial cells. In addition, the phytochemicals reduced pathogen motility, and production of cytolethal distending toxin. Real-time PCR revealed that majority of phytochemicals reduced the transcription of C. jejuni genes critical for infection in humans by at least 2 fold as compared to controls. Results suggest that these phytochemicals could potentially be used to control Campylobacter infection in humans. A study investigated the efficacy of eugenol (EG), a generally recognized as safe compound derived from cloves, as an antimicrobial dip and coating to reduce Campylobacter in post-harvest poultry. In the first two trials, skin samples were inoculated with Campylobacter jejuni and allowed to adhere for 30 min. Inoculated skin samples were dipped in treatment solutions (0, 0.25, 0.5, 1 or 2% EG) for 1 min, drip dried for 2 min and then processed at 0, 8, and 24 h (n=5 samples/treatment/time point). In both skin trials, the 1% and 2% EG doses reduced Campylobacter counts by 2 and 3 Log CFU/sample respectively across all time points. In follow-up trials, doses of EG with or without chitosan were evaluated on wingettes. Inoculated wingettes were randomly assigned to controls, EG (0.5, 1 or 2%), chitosan (2% CH) or their combinations in two separate trials. Following 1 min of coating with the given treatments, wingettes were air dried for 1 h and sampled at 0, 1, 3, 5, and 7 days for Campylobacter and aerobic counts (n=5 wingettes/treatment/day). The 2% EG, 2% CH and the combination of either 0.5% or 1% or 2% EG plus 2% CH significantly reduced both Campylobacter and aerobic counts from day 0 through day 7. The 2% EG and 2% CH combination produced a greater reductions than 2% CH across all days. These studies demonstrate the potential of EG as a post-harvest intervention against Campylobacter contamination. A study testing the effect of Hatchery Eggshell Membrane (HESM) feed supplement on chicken performance and immunity was completed. The results show that HESM supplement given during early periods of development may confer resistance to infection. The results are based on the ability of HESM supplement to protect against endotoxin-induced body weight loss and splenic gene expression studies.

4. Accomplishments
1. Proteomic analyses of Hatchery Eggshell Membrane (HESM) extract. ARS scientists in Fayetteville, Arkansas have demonstrated that the HESM given as feed supplement during early growth period to chicks improves immunity and reduce stress variables. The composition of HESM was studied to identify and understand the mechanism of action of protein components using liquid chromatography and tandem mass spectrometry. Cumulatively, over 200 bioactive proteins and peptides and a number of proteins (=65) from a variety of gram negative and positive bacteria were identified. The findings suggest that the bioactive proteins provided exogenously have the potential to modify physiology and immunity and the eggshell membrane byproducts can potentially influence immunity in poultry.

5. Significant Activities that Support Special Target Populations:
Armed to Farm: Training programs for military veterans in agriculture. ARS scientists from the Fayetteville, Arkansas, locations were part of a multi-institutional team that directly supported approximately 300 veterans through workshops, internships, research, and training, and an additional 650 veterans nationally through the Farmer Veteran Coalition (FVC) and helped many of them establish or expand their farming operations. Funded through the NIFA Beginning Farmers and Rancher Development Program, the team developed a program that incorporated ARS research into teaching materials, hands-on activities, mentoring, and networking opportunities for military veterans focused on farming. This program provides specific tools to help veterans during their transition into a rewarding employment option and a fruitful and satisfying civilian life. Through the New Farmer Online Training Program, over 28,000 participants have accessed the training program to date ( This online training course (in English and Spanish) is open to all individuals; however, it is targeted towards the training of veterans, Spanish speaking individuals, women in agriculture, African Americans and economically and educationally disadvantaged groups.

Review Publications
Packialakshmi, B., Rath, N.C., Huff, W.E., Huff, G.R. 2015. Poultry femoral head separation and necrosis: A review. Avian Diseases. 59:349-354.

Makkar, S., Liyanage, R., Kannan, L., Packialakshmi, B., Lay, J., Rath, N.C. 2015. Chicken egg shell membrane associated proteins and peptides. Journal of Agricultural and Food Chemistry. 63(44):9888-9898.

Balamurugan, P., Liyanage, R., Lay, J., Makkar, S., Rath, N.C. 2016. Proteomic changes in chicken plasma induced by Salmonella typhimurium lipopolysaccharides. Proteomics Insights. 7:1-9.

Packialakshmi, B., Liyanage, R., Lay, J.O., Okimoto, R., Rath, N.C. 2016. Proteomic changes in plasma of broiler chickens with femoral head necrosis. Biomarker Insight. 11:55-62.