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:
Objective 1: During the past year we conducted four studies with the overall aim of developing safe, effective and environmentally friendly strategies for controlling Campylobacter in poultry and poultry products. In the first study, we tested the effect of three plant derived compounds (trans-cinnamaldehyde, derived from cinnamon bark; carvacrol, an antimicrobial ingredient in oregano oil; and eugenol, the active ingredient in the oil from cloves) on Campylobacter virulence factors (motility, attachment to intestinal cells) that are critical for colonization in chickens. The results revealed that all plant compounds reduced Campylobacter motility, adhesion to chicken intestinal cells and the transcription of critical chicken colonization genes. Results suggest that trans-cinnamaldehyde, carvacrol, and eugenol could potentially be used to control Campylobacter colonization in chickens and reduce the incidence of human foodborne illnesses. As a follow-up of the above investigation, we conducted our second study in which we tested the efficacy of in-water supplementation of trans-cinnamaldehyde (generally recognized as safe status) nanoemulsion in reducing Campylobacter cecal colonization in 14-day-old broiler chickens. In two separate trials, day of hatch broiler chickens were supplemented with trans-cinnamaldehyde (normal or nanoemulsion form) in drinking water at 0, 0.0625, 0.125, 0.25, 0.5, and 1% level for 14 days. On day 7, the birds were challenged with a four-strain cocktail of Campylobacter by oral gavage. On day 14, the birds were sacrificed and Campylobacter colonization in cecal contents were quantified. Administration of trans-cinnamaldehyde nanoemulsion in drinking water at 0.25% reduced Campylobacter colonization by ~1 or 2 logs CFU/mL in trial 1 or trial 2 as compared to respective controls. No reduction in feed consumption, water intake or body weight gain was observed in 0.25% or lower concentration treatments as compared to controls. Results suggest that trans-cinnamaldehyde nanoemulsion could potentially be used to control Campylobacter colonization in broiler chickens. Follow up analysis on the effect of trans-cinnamaldehyde on Campylobacter transcriptome, proteome and cecal microbiome of broiler chickens is currently underway. In our third study, we investigated the efficacy of trans-cinnamaldehyde nanoemulsion as an antimicrobial wash treatment for reducing Campylobacter on chicken skin as a first step before conducting a large-scale study on poultry carcass or products. In addition, the effect of trans-cinnamaldehyde treatments on chicken skin color was analyzed. The nanoemulsion form of trans-cinnamaldehyde at 0.5 and 1% significantly reduced Campylobacter counts by ~1.8 log and 2 log CFU/ml after 1 min of dipping treatment. Increasing the contact time of trans-cinnamaldehyde nanoemulsions from 1 to 3 or 5 min did not further reduce the counts of Campylobacter on skin. Shaking of skin samples in antimicrobial treatments enhanced the antimicrobial efficacy of trans-cinnamaldehyde. By 5 min of treatment time, both normal and nanoemulsion form of trans-cinnamaldehyde at 0.5 and 1% reduced Campylobacter counts by at least 2.6 and 2.7 log CFU/ml respectively. There was no change in the color of skin treated with trans-cinnamaldehyde. Results suggest that trans-cinnamaldehyde (normal and nanoemulsion forms) can be used as antimicrobial wash treatments for reducing Campylobacter survival on chicken skin. Follow-up analysis testing the efficacy of trans-cinnamaldehyde on chicken wingettes is currently underway. Our fourth study was based on our results from the first study. We observed that eugenol was effective in reducing the critical colonization factors (motility, attachment) of Campylobacter. Therefore, we investigated the effect of eugenol on the proteome of Campylobacter. We identified more than 600 proteins in Campylobacter with many virulent proteins modulated by eugenol. The major groups of proteins that were identified contribute to physiological process and virulent attributes in Campylobacter. Eugenol reduced the expression of major virulence proteins contributing to biological adhesion (PorA, CadF), motility system (MotA, MotB, FliA, FliD, FliF, fliL, fliY), energy taxis (IlvH, CetA, CetB), molecular transport (TatA, TatB, TolB) and Quorum sensing (LuxS) when compared to controls. Overall, these results delineate the prospective mechanism of action of eugenol on Campylobacter and the potential of using this phytochemical to control Campylobacter colonization in chickens. Objective 2: We conducted experiments using hatchery eggshell matrix (HESM) which was enriched with E. coli. We fed these and control diets to the chickens, monitored their growth and specific antibody response against E. coli proteins measuring blood levels of IgG, IgM, and IgA in chickens that were either challenged or nonchallenged with E. coli LPS for 24 h. The results are being analyzed. We optimized a novel chicken enterocyte culture to screen chemicals, dietary antigens, an alternative to antibiotics as well as studied the mechanisms and pathways of these effects using biochemical and proteomic approaches.
1. Elucidating the mechanisms of action of plant-derived antimicrobials against the food borne pathogen Campylobacter. 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 scientists at 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 three 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. A plant compound, Beta-resorcylic acid, reduces Campylobacter in post-harvest poultry. 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. Reduction of Campylobacter counts on poultry products would greatly reduce the risk of subsequent infections in humans. ARS scientists at Fayetteville, Arkansas, investigated the potential of a plant-derived compound, Beta-resorcylic acid, to reduce Campylobacter counts on post-harvest poultry (chicken skin or meat). Found in Brazilian Wood, Beta-resorcylic acid is a secondary metabolite that protects plants against pathogens, and is classified under “Everything added to Food in the US” by the FDA. Trials were conducted in which chicken skin or meat samples were inoculated with Campylobacter then treated with Beta-resorcylic acid. In addition to evaluating whether Campylobacter was reduced in the wash solution, the change in the expression of survival and virulence genes of Campylobacter exposed to Beta-resorcylic acid was evaluated. Treatments significantly reduced Campylobacter populations on both chicken skin or meat samples when compared with non-treated washed controls. Beta-resorcylic acid treatment down regulated expression of select genes coding for motility and attachment in a majority of Campylobacter strains evaluated and stress response genes were up regulated. This research has tremendous potential since Campylobacter is responsible for causing an estimated 1.3 million foodborne illnesses in the United States. Beta-resorcylic acid could be effectively used as antimicrobial dip treatment during poultry processing for reducing Campylobacter on chicken carcasses.
3. A novel chicken intestinal cell culture to identify factors that may improve gut health was developed. Significant research is being conducted to develop antibiotic alternatives (e.g. phytochemicals, probiotics, prebiotics) to maintain poultry health. However, previous to this work, no in vitro poultry enteric model was available to understand the interaction between antibiotic alternatives and host gastrointestinal factors that contribute to bird health. Mammalian cell cultures cannot be substituted since they lack poultry specific intestinal receptors. ARS scientists in Fayetteville, Arkansas, developed an intestinal cell culture that can be used to screen and understand the mechanisms of action of the factors that affect gut health including dietary, abiotic, and microbial factors. The cell culture system will help screen and identify these factors in a more rapid manner without the use of a large number of birds. Cell culture systems that can facilitate rapid and accurate screening of antibiotic alternatives are highly sought after technology. 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.
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