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

Location: Poultry Production and Product Safety Research

2019 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
Under Subobjective 1A, we completed the studies on selecting probiotics that utilize mucin for growth and competitively inhibit Campylobacter colonization in broiler chickens. These studies were completed and the manuscript was published in the International Journal of Poultry Science. Under Subobjective 1B, we completed several studies using plant compounds (eugenol, carvacrol, trans-cinnamaldehyde, ß-resorcylic acid) for reducing Campylobacter in post-harvest poultry and the results were either published or under review for publication in peer reviewed journals. We also conducted a study in which we tested the efficacy of in-water supplementation of eugenol (generally recognized as safe status) nanoemulsion in reducing Campylobacter cecal colonization in 14-day-old broiler chickens. Two trials were conducted and the results suggest that eugenol nanoemulsion could potentially be used to control Campylobacter colonization in broiler chickens. We also evaluated the effect of eugenol on cecal microbiome of broiler chickens. We completed the data analysis and the manuscript is under preparation. Under Subobjective 1C, we are conducting studies with Salmonella and/or Campylobacter specific bacteriophages for reducing foodborne pathogens in post-harvest poultry. We have had two SY vacancies since the start of this in-house project and do not have the expertise to efficiently carry out the CRISPR-Cas research planned during the original time frame proposed. We dropped the CRISPR-Cas part and added mucin adapted Salmonella phage research to this subobjective. We made substantial progress in conducting these studies and the proposed milestones can be achieved by the end of the year. Under Subobjective 1D, we successfully completed studies evaluating the genome wide effect of select natural plant compounds on genes critical for Campylobacter colonization in broiler chickens. We also completed studies evaluating the genes critical for the persistence and survival of Campylobacter in the environment and in post-harvest poultry and poultry products. The results were published in peer reviewed journals. Under Subobjective 2A, we completed the studies determining the effects of eggshell membrane on immune indices of chickens and the results are published. Under subobjective 2B, using chicken enterocyte cultures we found that treatment with phorbol myristate acetate (PMA), a protein kinase C activator, produces cachectic changes in the cells which simulates the changes in intestinal their permeability, a condition that makes intestine predisposed to infection and enteritis. The morphological changes in the cells are associated with changes in many cellular proteins particularly associated with nuclear and mitochondrial metabolism. We also developed a method to produce chicken intestinal organoids and characterized them with respect to the types of cells present in those organoids. The organoids are 3 dimensional constructs of tissues which facilitate studying the biological processes such as infection, interactions with chemicals, nutrients, and metabolic processes in culture; therefore, it is considered a better model than the cell line or tissue dissociated cells for different physiological and pathological studies. Using organoids, we studied the effect of some selective chemical and biological factors that provide the glimpse of how these factors may affect intestinal integrity and health. Use of organoids for intestinal studies in chicken may substitute for the use of live animals increasing the sustainability. In the process we also discovered an interesting dynamic of thymosin beta 4 (Tß4), a peptide implicated in tissue healing and regeneration. Whereas the freshly harvested intestinal villi lacked any substantial presence of Tß4, the organoids were Tß4 abound. The enteric organoid model does provide new vista for regeneration research.

1. Developed novel carrier systems for controlling environmental persistence of foodborne pathogens. Salmonella and Campylobacter (C.) jejuni are the leading foodborne bacterial pathogens in the United States responsible for over 2 million illnesses annually. These pathogens colonize the poultry gut thereby leading to contamination of processing environment and carcass during slaughter. Both pathogens are capable of forming biofilms that facilitates their greater survival in the processing environment with increased resistance to antimicrobials and disinfectants. ARS researchers in Fayetteville, Arkansas, developed a safe and effective strategy for controlling Salmonella and C. jejuni biofilms by combining the antimicrobial efficacy of Generally Recognized as Safe (GRAS) status phytochemicals with nanotechnology to develop natural disinfectants with significant antibiofilm efficacy against Salmonella and C. jejuni. We tested several phytochemical nanoemulsions and demonstrated that they are effective in inhibiting the biofilm formation and inactivating mature biofilms on common food contact surfaces. In addition, phytochemicals modulated critical genes and proteins required for C. jejuni biofilm formation. Since C. jejuni can form biofilms in the processing environment leading to contamination of products, phytochemicals could potentially be used for controlling biofilms thereby reducing the risk of human infections.

2. Developed natural and environmentally friendly strategies to improve the post harvest food safety and shelf life of poultry products. One of the missions of the Fayetteville, Arkansas, research unit has been to provide the poultry industry with antibiotic alternatives for the control of Campylobacter and Salmonella in conventional and the organic poultry sectors. The use of phytochemicals and probiotic bacteria as antimicrobials and bio-preservatives in food products is one such technology that is safe, effective and environmentally friendly. Our results indicate that plant-based, GRAS (Generally Regarded as Safe) status compounds (eg. carvacrol, caprylic acid, eugenol, Beta-resorcylic acid, trans-cinnamaldehyde) and probiotic cultures from Bacillus and Lactobacillus sp. are very effective in reducing Campylobacter or Salmonella on poultry meat and eggs. Further, we also evaluated edible coatings such as chitosan, gum Arabic or pectin fortified with phytochemicals. Antimicrobial edible coatings, due to their presence on products, reduces the chance of cross-contamination during storage and handling. Results from these studies demonstrate that edible coatings fortified with phytochemicals consistently reduced C. jejuni and modulated several genes critical for survival and virulence of C. jejuni. This research has tremendous potential since Campylobacter is responsible for causing an estimated 1.3 million foodborne illnesses in the United States. These plant phytochemicals can potentially provide the poultry industry (both conventional and organic) with economical, effective, and control strategies for effective control of Campylobacter at various stages of poultry production and processing.

3. A method to study the mechanism of the changes in intestinal permeability. Abnormal changes in intestinal permeability can lead to gastrointestinal (GI) problems since it can permeate toxins, antigens, pathogens, and parasites into the system creating problems such as inflammation, autoimmunity, and enteritis. With the restrictions in the antibiotic usage, the chances of poultry flocks prone to GI problems has significantly increased. Hence it is imperative to understand the mechanism of the disease and its therapy. ARS researchers in Fayetteville, Arkansas, developed a method to culture intestinal epithelial cells (enterocytes) and used it to study intestinal permeability problems. Using a chemical named phorbol myristate acetate (PMA) that is found in croton oil, we showed that this chemical profoundly affects the cell’s health that resembles the change in intestinal permeability. We went on to find how this chemical affects the proteomics of the cells that may be relevant to their dystrophy and found that PMA affects the energy metabolism and nuclear activities of the enterocytes that may explain why these cells become cachectic. We also used the enterocyte culture to screen what other factors may affect intestinal cells and make them amenable to gastrointestinal problems or may improve intestinal health. This cell culture system can be used to study intestinal problems in poultry in a cost effective and sustainable manner particularly for screening assays. The observations from this system have led to the development of chicken intestinal organoids, a 3D culture model, to study similar phenomenon such as intestinal disease pathogenesis, host pathogen interaction, and antibiotic alternative screening which is novel to poultry research.

Review Publications
Upadhyay, A., Arsi, K., Upadhyaya, I., Donoghue, A.M., Donoghue, D.J. 2019. Natural and environmentally friendly strategies for controlling Campylobacter jejuni colonization in poultry, survival in poultry products and infection in humans. In: Venkitanarayanan K., Thakur S., Ricke S. (eds) Food Safety in Poultry Meat Production. Food Microbiology and Food Safety. Springer, Cham. p. 67-93.
Arsi, K., Donoghue, D.J., Venkitanarayanan, K., Donoghue, A.M. 2019. Reducing foodborne pathogens in organic poultry: Challenges and opportunities. In: Venkitanarayanan K., Thakur S., Ricke S. (eds) Food Safety in Poultry Meat Production. Food Microbiology and Food Safety. Springer, Cham. p. 25-46.
Wagle, B.R., Upadhyay, A., Shrestha, S., Arsi, K., Upadhyaya, I., Donoghue, A.M., Donoghue, D.J. 2019. Pectin or chitosan coating fortified with eugenol reduces Campylobacter jejuni on chicken wingettes and modulates expression of critical survival genes. Poultry Science. 98:1461-1471.
Lyte, J.M. 2019. Eating for 3.8x10^13: Examining the impact of diet and nutrition on the microbiota-gut-brain axis through the lens of microbial endocrinology. Frontiers in Endocrinology. 9:796.
Shrestha, S., Wagle, B.R., Upadhyay, A., Arsi, K., Upadhyaya, I., Donoghue, D.J., Donoghue, A.M. 2019. Edible coatings fortified with carvacrol reduce Campylobacter jejuni on chicken wingettes and modulate expression of select virulence genes. Frontiers in Microbiology. 10:583.
Rath, N.C., Liyanage, R., Gupta, A., Packialakshmi, B., Lay, J. 2018. A method to culture chicken enterocytes and their characterization. Poultry Science. 0:1-8.
Arsi, K., Donoghue, A.M., Metcalf, J.H., Donoghue, D.J. 2019. Effect of dietary supplementation of essential oils, eugenol or trans-cinnamaldehyde, on enteric colonization of campylobacter in broiler chickens. International Journal of Advances in Science Engineering and Technology. 7:(1-2):30-32.
Rath, N.C., Gupta, A., Liyanage, R., Lay, J. 2019. Phorbol 12-myristate 13-acetate induced changes in chicken enterocytes. Proteomics Insights. 10:1-13.
Shrestha, S., Wagle, B.R., Upadhyay, A., Arsi, K., Donoghue, D.J., Donoghue, A.M. 2019. Carvacrol antimicrobial wash treatments reduce Campylobacter jejuni and aerobic bacteria on broiler chicken skin. Poultry Science. 98:(9)4073-4083.