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

Location: Poultry Production and Product Safety Research

2021 Annual Report

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.

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.

Progress Report
The new Project 6022-32420-001-00D, Multi-hurdle Approaches for Controlling Foodborne Pathogens in Poultry is replacing this old Project 6022-31230-001-001D, Antibiotic Alternatives for Controlling Foodborne Pathogens and Disease in Poultry. Objective 1. Mechanisms of action of plant-derived antimicrobials against Campylobacter jejuni. We developed several phenotypic assays, cell culture and gene expression analysis protocols for rapid screening of phytochemicals for efficacy against Campylobacter. We identified plant-derived antimicrobials with significant anti-Campylobacter efficacy. These compounds (trans-cinnamaldehyde, derived from cinnamon bark; carvacrol, an antimicrobial ingredient in oregano oil; and eugenol, the active ingredient in the oil from cloves) are effective in reducing Campylobacter colonization in chickens and survival on poultry products (chicken skin, wings). In addition, using proteomic analysis, our team has been successful in delineating the potential mechanism of action of these compounds. This research has tremendous potential since Campylobacter is responsible for causing an estimated 1.3 million foodborne illnesses in the US. These plant phytochemicals provide the poultry industry (conventional & organic) with economical, effective control strategies for Campylobacter however more work on solubility and treatment methods are critical as outlined in new project plan. Objective 1. Mechanism of pathogenesis of Campylobacter. To understand the mechanism of pathogenesis of Campylobacter, whole genome sequencing and virulence characterization of C. jejuni strains isolated from poultry were performed. Mechanistic analysis was conducted to test colonization potential and virulence attributes of the strains. Multiple genes coding for virulence factors such as motility, toxin production, and stress tolerance were observed in all tested strains. The genomic data from these potentially virulent strains provides a better understanding of the colonization/pathogenesis mechanisms of C. jejuni leading to better control strategies in poultry. Objective 2. Proteomic analyses of Hatchery Eggshell Membrane (HESM) extract. We 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 from a variety of Gram negative and positive bacteria were identified. The findings suggest that the bioactive proteins provided exogenously modify physiology and immunity and the eggshell membrane byproducts can influence immunity. Objective 2. Developed a novel chicken intestinal cell culture to identify factors that may improve gut health. We developed an intestinal cell culture to screen and understand the mechanisms of action of the factors that affect gut health including dietary, abiotic, and microbial factors. Cell culture systems that can facilitate rapid and accurate screening of antibiotic alternatives are highly sought-after technologies without the use of a large number of birds. Successful identification of novel antibiotic alternatives will directly translate into improved poultry health, enhanced microbiological safety of poultry products, and enhanced sustainability of the U.S. poultry industry. Objective 2. Proteomics changes induced by sodium butyrate on chicken enterocytes cell culture model. Butyrate is a short-chain fatty acid synthesized by the gut microbiota. Studies focused on developing a chicken primary epithelial cell culture model and evaluating the effect of butyrate on various physiological and biochemical pathways proteins using liquid chromatography tandem-mass spectrometry (LC-MS/MS). A total of 234 proteins were identified. At least 30 proteins were uniquely present in control cells, 11 proteins in butyrate treated cells and over 176 proteins were common in both. Gene ontology revealed that a majority of proteins contribute to cellular activity, biological regulation and metabolic function. Butyrate increased the expression of proteins contributing to critical biological functions including cell integrity and physiological response. The butyrate treatments reduced the expression of proteins contributing to actin binding, pro-inflammatory response, and signal transduction. Results suggest that butyrate may have impact on cytoskeletal changes, energy metabolism in chicken epithelial cells and also have potential to inhibit histone deacetylase that plays a role in cancer. To determine the signaling pathway of proteins, the differentially regulated proteins were analyzed using software such as Protein Analysis through Evolutionary Relationships software (PANTHER) and STRING protein association network (FDR 0.05). We completed the study and the results were published in Plos One.

1. Developed a chicken enteroid model to understand host-pathogen interactions, and screen antibiotics alternatives to prevent poultry zoonotic diseases. The organoids are 3-dimensional cellular structures that resemble organs in structure and function. Thus, the model can facilitate studying the biological processes such as infection, interactions with chemicals, nutrients, and metabolic processes in culture. Typically, the organoids are generated from primary tissues, progenitor stem cells, or induced pluripotent cells, and maintained using a variety of growth and differentiation factors, and extracellular matrix supports. In avian species, enteric organoid (enteroid) research is very limited. In this regard, ARS researchers in Fayetteville, Arkansas, have developed a streamlined procedure to purify and characterize avian villus enteroids from mucosal tissues in a relatively simple and cost-effective manner. The enteroids structurally resemble the villi and are used to study the physiology, metabolism, and pathology of the intestinal villi; can be useful for preliminary screenings of the factors that may affect gut health; and can potentially reduce the use of live animals in research.

2. Neurochemical-based host-microbe interaction as a novel strategy to control foodborne pathogen colonization in pre-harvest poultry. Stress during the pre-harvest production cycle plays a major role in determining the susceptibility of the chicken gut to colonization by foodborne pathogens (e.g., Campylobacter jejuni). However, the mechanisms by which production stress generates this vulnerability are largely unknown thereby limiting the ability to develop antibiotic alternatives that ameliorate or prevent stress-related foodborne pathogen carriage. ARS scientists at Fayetteville, Arkansas, have identified that stress-related ‘fight-or-flight’ neurochemicals (e.g., serotonin) not only are increased in the avian gut following stress but directly cause changes in Campylobacter colonization of the gut. Using cell culture, gene expression, and high-performance liquid chromatography we have identified that serotonin is present at sites in the chicken gut that are typically colonized by Campylobacter, and that serotonin can partially inhibit Campylobacter colonization in the gut. Using proteomic analyses, we have identified a likely mechanistic pathway by which serotonin affects Campylobacter ability to attach to the gut. We have also identified that change in serotonin in the gut is related to the bird’s gut microbiome. Thus, the intersection of stress-related neurochemicals and the gut microbiome represents a new frontier in antibiotic alternative research and has immense potential to offer the poultry industry safe, effective, and inexpensive approaches to control foodborne pathogens.

Review Publications
Bastiaanssen, T.F., Gururajan, A., Van De Wouw, M., Moloney, G.M., Ritz, N., Long-Smith, C.M., Wiley, N.C., Murphy, A.B., Lyte, J.M., Fouhy, F., Stanton, C., Claesson, M., Dinan, T., Dinan, T.G., Cryan, J.F. 2020. Volatility as a concept to understand the impact of stress on the microbiome. Psychoneuroendocrinology.
Lyte, J.M., Keane, J., Eckenberger, J., Anthony, N., Shrestha, S., Marasini, D., Daniels, K.M., Caputi, V., Donoghue, A.M., Lyte, M. 2021. Japanese quail (Coturnix japonica) as a novel model to study the relationship between the avian microbiome and microbial endocrinology-based host-microbe interactions. Microbiome.
Acharya, M., Liyanage, R., Gupta, A., Arsi, K., Donoghue, A.M., Lay, J.O., Rath, N.C. 2020. Thymosin Beta 4 dynamics during chicken enteroid development. Molecular and Cellular Biochemistry. 476:1303–1312.
Liu, J., Stewart, S., Robinson, K., Yang, Q., Lyu, W., Whitmore, M.A., Zhang, G. 2021. Linkage between the intestinal microbiota and residual feed intake in broiler chickens. Journal of Animal Science and Biotechnology.
Lyte, J.M., Shrestha, S., Wagle, B.R., Liyange, R., Martinez, D.A., Donoghue, A.M., Danels, K.M., Lyte, M. 2021. Serotonin modulates Campylobacter jejuni physiology and in vitro interaction with the gut epithelium. Poultry Science. 100(3). Article 100944.
Sorouri, M., Chang, T., Jesudhasan, P., Pinkham, C.L., Elde, N., Hancks, D.C. 2020. MISTR: A conserved MItochondrial STress Response network revealed by signatures of evolutionary conflict. PLoS Biology.
Jesudhasan, P., Bhatia, S.S., Sivakumar, K.K., Praveen, C., Genovese, K.J., He, L.H., Droleskey, R.E., McReynolds, J.L., Byrd II, J.A., Swaggerty, C.L., Kogut, M.H., Nisbet, D.J., Pillai, S.S. 2021. Controlling the colonization of Clostridium perfringens in broiler chickens by an electron-beam-killed vaccine. Animals. 11(3). Article 671.