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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

2020 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
Sub-objective 1B. ARS researchers are continuing research using plant compounds for reducing foodborne pathogens, Salmonella and Campylobacter in post-harvest poultry and two manuscripts were published in FY 2020. Also, conducted studies testing the efficacy of in-water supplementation of two generally recognized as safe compounds namely, eugenol (EG) and trans-cinnamaldehyde (TC) as nanoemulsion (NE) in reducing C. jejuni colonization in broiler chickens. In addition, the effect of EG and TC on C. jejuni colonization factors (motility, attachment to chicken enterocytes), whole cell proteome and cecal microbiome was investigated. These studies were completed and the manuscript is under internal review for publication. Sub-objective 1C. ARS researchers made substantial progress in conducting studies with Salmonella and/or Campylobacter specific bacteriophages for reducing foodborne pathogens in post-harvest poultry. Revised the sub-objective and were approved to add research on mucin adapted Salmonella phage research to this sub-objective. Sub-objective 1D. ARS researchers successfully completed studies evaluating the efficacy of sub-inhibitory concentration (SIC) of carvacrol in reducing the expression of C. jejuni colonization factors in vitro. Also, investigated the effect of carvacrol on the expression of C. jejuni proteome and the results were published in the journal of Poultry Science. ARS researchers also completed studies investigating the role of serotonin, a gut-derived neurochemical, in affecting growth, motility, and colonization of C. jejuni in an in vitro model of the gut epithelium. Although growth and motility were unaffected, serotonin was found to cause a statistically-significant reduction in adhesion of C. jejuni to the in vitro gut epithelial monolayer. This work provides preliminary evidence that the presence of serotonin in the gut may play a role in the ability of C. jejuni to colonize in the gut. Sub-objective 2B. ARS researchers completed studies on production and characterization of avian crypt-villus enteroids and the effect of chemicals on the villus enteroids. As part of this study, streamlined the procedure to generate and purify avian villus enteroids, characterize them, and study their potential as a screening tool using some selective chemicals and growth factors.

1. Delineate potential mechanisms of action of plant-derived antimicrobials against Campylobacter jejuni. Campylobacter is one of the most commonly reported pathogens causing food borne infections in the United States and epidemiological evidence has implicated raw poultry products as a significant source of human infection. Therefore, it is very important to develop effective strategies for controlling this pathogen in poultry. ARS researchers in Fayetteville, Arkansas, have developed several phenotypic assays, cell culture and gene expression analysis protocols for rapid screening of phytochemicals for efficacy against Campylobacter. Using these assays, we have 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 United States. These plant phytochemicals can potentially provide the poultry industry (both conventional and organic) with economical, effective, and control strategies for Campylobacter.

2. Phytochemicals as natural disinfectants for controlling Campylobacter biofilms in processing areas. Microbial biofilms provide an excellent opportunity for pathogens to survive and is a critical issue for almost all surfaces in food processing facilities. Biofilms are complex bacterial communities with increased resistance to disinfectants and antibiotics. Several studies have shown that Campylobacter can form sanitizer tolerant biofilm leading to product contamination, however, limited research has been conducted to develop effective control strategies against Campylobacter biofilms. ARS researchers in Fayetteville, Arkansas, conducted studies to investigate the efficacy of three generally recognized as safe status phytochemicals namely, trans-cinnamaldehyde, eugenol, or carvacrol in inhibiting Campylobacter (C. jejuni) biofilm formation and inactivating mature biofilm on common food contact surfaces. In addition, the effect of phytochemicals on biofilm architecture and expression of genes and proteins essential for biofilm formation was evaluated. All phytochemicals reduced C. jejuni biofilm formation as well as inactivated mature biofilm on polystyrene and steel surfaces. Electron microscopy studies revealed that exposure to phytochemicals resulted in disruption of biofilm architecture and loss of extracellular polymeric substances. Also, sub-inhibitory concentrations of phytochemicals significantly modulated the expression of genes encoding for motility, cell surface modifications, stress response and quorum sensing. Proteomic analysis showed that phytochemicals significantly modulated the expression of select genes and proteins critical for biofilm formation. These studies provide critical information regarding the anti-biofilm efficacy of phytochemicals against C. jejuni and their potential mechanism(s) of action. This research can be used to develop safe and effective phytochemical based disinfectants and application strategies for controlling the persistence of pathogens in mono and/or mixed biofilms commonly encountered in food processing facilities.

Review Publications
Wagle, B.R., Arsi, K., Shrestha, S., Upadhyay, A., Upadhyaya, I., Bhargava, K., Donoghue, A.M., Donoghue, D.J. 2019. Eugenol as an antimicrobial wash treatment reduces Campylobacter jejuni in postharvest poultry. Journal of Food Safety. 39:e12704.
Peichel, C., Nair, D., Dewi, G., Donoghue, A.M., Reed, K.M., Kollanoor, J.A. 2019. Effect of lemongrass (Cymbopogon citratus) essential oil on the survival of multidrug-resistant Salmonella enterica serovar Heidelberg in contaminated poultry drinking water. Journal of Applied Poultry Research. 0:1-10.
Upadhyaya, I., Arsi, K., Fanatico, A., Wagle, B.R., Shrestha, S., Upadhyay, A., Coon, C.N., Schlumbohm, M., Trushenski, J., Donoghue, A.M., Owens-Hanning, C., Riaz, M.N., Farnell, M.B., Donoghue, D.J. 2019. Bigheaded carp-based meal as a sustainable and natural source of methionine in feed for ecological and organic poultry production. Journal of Applied Poultry Research. 28(4):1131-1142.
Van De Wouw, M., Lyte, J.M., Boehme, M., Sichetti, M., Moloney, G., Goodson, M.S., Kelley-Loughnane, N., Clarke, G., Dinan, T.G., Cryan, J.F. 2020. The role of the microbiota in acute stress-induced myeloid immune cell trafficking. Brain Behavior and Immunity. 84:209-217.
Wagle, B.R., Marasini, D., Upadhyaya, I., Shreshta, S., Arsi, K., Donoghue, A.M., Carbonero, F., Donoghue, D.J., Maas, K., Upadhyay, A. 2020. Draft Genome sequences of Campylobacter jejuni strains isolated from poultry. Genome Announcements. 9:e01272-19.
Walsh, J., Gheorghe, C.E., Lyte, J.M., Van De Wouw, M., Boehme, M., Dinan, T.G., Cryan, J.F., Griffin, B.T., Clarke, G., Hyland, N.P. 2020. Gut microbiome-mediated modulation of hepatic cytochrome P450 and P-glycoprotein: Impact of butyrate and fructo-oligosaccharide-inulin. Journal of Pharmacy and Pharmacology.
Acharya, M., Rath, N.C., Donoghue, A.M., Arsi, K., Liyanage, R. 2020. Production and characterization of avian crypt-villus enteroids and the effect of chemicals. BMC Veterinary Research.
Gururajan, A., Van De Wouw, M., Boehme, M., Becker, T., O'Connor, R., Bastiaanssen, T.F., Moloney, G.M., Lyte, J.M., Ventura Silva, A.P., Merckx, B., Dinan, T.G., Cryan, J.F. 2019. Resilience to chronic stress is associated with specific neurobiological, neuroendocrine and immune responses. Brain Behavior and Immunity. 80:583-594.
Wagle, B.R., Upadhyay, A., Upadhyaya, I., Shrestha, S., Arsi, K., Liyanage, R., Venkitanarayanan, K., Donoghue, D.J., Donoghue, A.M. 2019. Trans-cinnamaldehyde, eugenol and carvacrol reduce Campylobacter jejuni biofilms and modulate expression of select genes and proteins. Frontiers in Microbiology. 10:1837.
Lyte, J.M., Gheorghe, C.E., Goodson, M.S., Kelley-Loughnane, N., Dinan, T.G., Cryan, J.F., Clark, G. 2020. Gut-brain axis serotonergic responses to acute stress exposure are microbiome-dependent. Neurogastroenterology & Motility. 00:e13881.
Van De Wouw, M., Walsh, A.M., Crispie, F., Van Leuven, L., Lyte, J.M., Boehme, M., Clarke, G., Dinan, T.G., Cotter, P.D., Cryan, J.F. 2020. Distinct actions of the fermented beverage kefir on host behaviour, immunity and microbiome gut-brain modules in the mouse. Microbiome.
Wagle, B.R., Donoghue, A.M., Shrestea, S., Upadhyaya, I., Arsi, K., Gupta, A., Liyanage, R., Rath, N.C., Donoghue, D.J., Upadhyay, A. 2020. Carvacrol attenuates Campylobacter jejuni colonization factors and proteome critical for persistence in the chicken gut. Poultry Science.