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:
Progress has been made on both objectives focused on strategies to control foodborne pathogens and disease in poultry. Objective 1: A study was conducted testing the efficacy of in-water supplementation of carvacrol (phytochemical derived from oregano) nanoemulsion in reducing Campylobacter cecal colonization in chickens. Nanoemulsions improve solubility, a challenge associated with plant phytochemicals. Two trials were conducted in which day of hatch broiler chickens were supplemented with carvacrol (normal or nanoemulsion form) in drinking water at 0, 0.0625, 0.125, 0.25% level for 14 days. On day 7, the birds were challenged with a four-strain cocktail of Campylobacter by oral gavage. On day 14, ceca were collected and processed for Campylobacter colonization in the cecum of broiler chickens. Administration of 0.125% or 0.25% carvacrol nanoemulsion in drinking water consistently reduced Campylobacter colonization. Administration of carvacrol or its nanoemulsions in water did not show any reduction in feed consumption, water intake or body weight gain when compared to controls. Results suggest that carvacrol nanoemulsion could potentially be used to control Campylobacter colonization in broiler chickens. Follow up analysis on the effect of carvacrol on cecal microbiome of broiler chickens is currently underway. In another study under Objective 1, the efficacy of peracetic acid and zinc in reducing Campylobacter jejuni on chicken skin was evaluated. Peracetic acid is extensively used as an antimicrobial treatment in the U.S. commercial poultry. However, the approved dose of peracetic acid produces inconsistent/low pathogen reductions. Metal ions have been used as effective antimicrobials for centuries. Zinc is a GRAS (Generally Recognized As Safe by the U.S. FDA), status metal used in meat and packaging to prevent microbial spoilage. However, the efficacy of zinc for poultry carcass disinfection has not been investigated. This study evaluated the efficacy of peracetic acid either alone or in combination with zinc as an antimicrobial dip treatment in a simulated chiller for reducing Campylobacter and Pseudomonas spp. (an important meat spoilage bacteria) on chicken skin. The highest dose of peracetic acid reduced counts by ~0.5 - 1 log CFU/sample for both pathogens. Zinc reduced C. jejuni counts by ~1 – 1.5 log CFU/sample, however, no reduction was observed for Pseudomonas spp. compared with control. Select combination treatments synergistically reduced C. jejuni at 24 hours of storage when compared with individual treatments. The combination of 220 ppm peracetic acid with 125 ppm zinc was the most effective treatment and reduced C. jejuni by ~3.5 log CFU/sample and Pseudomonas spp. by ~2 log CFU/sample. Results suggest that combination of zinc and peracetic acid could be an effective strategy to control C. jejuni in post-harvest poultry. Further, we are also evaluating the efficacy of these compounds in reducing Salmonella in postharvest poultry. We investigated the antibiofilm effect of plant compounds, trans-cinnamaldehyde, eugenol or carvacrol on poultry plant processing surfaces. It has been proposed that C. jejuni survives in the processing environment by forming biofilms. Biofilms are complex bacterial communities with increased resistance to disinfectants and antibiotics. Studies have shown that Campylobacter can form sanitizer tolerant biofilms, but limited research has been conducted to develop effective control strategies against C. jejuni biofilms. This study investigated the efficacy of three plant phytochemical compounds, trans-cinnamaldehyde, eugenol or carvacrol (derived from cinnamon, cloves and oregano; respectively) on inhibiting or inactivating C. jejuni biofilms on common food contact surfaces. For the inhibition study, C. jejuni was grown in either the presence or absence (control) of sub-inhibitory concentrations of the phytochemicals for 48 hours. For the inactivation study, C. jejuni biofilms were exposed to the phytochemicals and surviving C. jejuni in the biofilm were enumerated. All phytochemicals reduced C. jejuni biofilm formation as well as inactivated mature biofilm on polystyrene and steel surfaces. The genes encoding for C. jejuni motility systems were downregulated by all phytochemicals. In addition, the expression of stress response and cell surface modifying genes were reduced by eugenol. Proteomic analysis showed that phytochemicals significantly downregulated the expression of a proteins required for signaling pathway during oxidative stress and biofilm formation. Scanning electron microscopy revealed disruption of biofilm architecture and loss of extracellular polymeric substances after phytochemical treatment. Results suggest that these phytochemicals could serve as a natural disinfectant for controlling C. jejuni biofilms. To understand the mechanism of pathogenesis of Campylobacter, we conducted a study where whole genome sequencing and virulence characterization of Campylobacter jejuni strains isolated from poultry were performed. Chickens act as the host for C. jejuni, wherein the pathogen colonizes the ceca thereby leading to contamination of the carcass during slaughter and subsequent human infections. In the human gut, the pathogen attaches and invades the intestinal epithelium followed by toxin mediated cytopathy and enteritis. Little is known about how this pathogen is able to colonize multiple hosts while competing with specialist microbiota in the gut. Comprehensive characterization of C. jejuni strains could facilitate in better understanding of their pathophysiology and development of effective intervention strategies. Whole genome sequencing is a powerful technology that provides in-depth genetic information and is increasingly being utilized to study the evolution, epidemiology, virulence, and detection of foodborne pathogens. Herein, we report the complete genomic sequence of four wild-type C. jejuni strains isolated from poultry. In addition, mechanistic analysis was conducted to test the 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. In addition, genes imparting resistance to antibiotics such as beta-lactams and toxic compounds such as copper and arsenic were also found in all four strains. The C. jejuni genome of tested strains also revealed CRISPR sequences imparting adaptive immunity against phage invasion. Follow up mechanistic analysis showed that all strains were capable of motility at 42°C and 37°C, biofilm formation and attachment to epithelial cells from chickens and humans. Taken together, the genomic data from these potentially virulent strains should provide a better understanding of the colonization and pathogenesis mechanisms of C. jejuni leading to better control strategies in poultry. Comparative analysis outside the conserved core genomes of these four strains is currently underway. Objective 2: During the past year, 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. We studied the proteomics changes induced by sodium butyrate on chicken enterocytes cell culture model. Butyrate is a short-chain fatty acid synthesized by gut microbiota. In the human gut, butyrate regulates epithelial transport, inflammatory pathways, and barrier function. However, a paucity of information exists on the effect of butyrate on chicken gut physiology. The objective of this study was to develop a chicken primary epithelial cell culture model and to study the effect of butyrate on various physiological/biochemical pathways proteins using liquid chromatography tandem-mass spectrometry (LC-MS/MS). Intestinal villi were harvested from day old broiler chicks and enterocytes were cultured to semi-confluency and treated with sodium butyrate for 24 hours. The treated cells were lysed and proteins subjected to electrophoresis. Gel segments were reduced/alkylated followed by trypsin digestion. Peptides were evaluated to identify and quantify proteins using LC-MS/MS. Differentially regulated proteins were determined using Scaffold software. 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 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. Protein-protein interaction analysis (STRING) is currently underway to understand the effect of butyrate on major protein groups critical for gut health in poultry.
1. Novel prebiotics and probiotics reduce stress/pathogen induced mortality and production losses in chickens. Stress, specifically cold stress can significantly decrease performance and increase susceptibility to infections in poultry flocks. Prebiotics may alter intestinal ecology modulating inflammation through the production of short-chain fatty acids. Probiotics can directly alter the intestinal microbiome resulting in similar effects. ARS researchers at Fayetteville, Arkansas, investigated the potential of prebiotics/probiotics to protect intestinal health under cold stress and pathogen (E. coli) challenge. While the prebiotic studies did not prevent effects of cold stress or E. coli challenge, the probiotic treatment was consistent in reducing production losses. Continuous inclusion of probiotic increased body weight and decreased mortality. Research demonstrates effectiveness of probiotics and their beneficial effects on performance and protecting chicks from infections and various stressors.
2. Outdoor structural enrichments positively impact the behavior of organic meat chickens. Chickens provided with outdoor access often do not fully use the range area. Natural cover, such as trees, can provide shelter and increase range use, but may not be practical for use in all free-range operations. ARS researchers at Fayetteville, Arkansas, conducted a study to determine whether constructed enrichments that simulate natural structures increase range use and impact behavior of meat chickens. Birds were randomly assigned to one of two treatments: No Range Enrichment or Range Enrichment which had roosts made of plastic pipe or screened shelters placed at various distances from the poultry house. The numbers of birds inside the poultry house and in the 4 quadrants of the range were counted multiple times daily. Various behaviors were also evaluated during these evaluatiohns. On average, only 12.9% of birds used the range at any given time. The behavior of the birds was strongly affected by location (Inside vs. Outside), with birds mainly foraging and walking while Outside and feeding, standing and sitting while Inside. Overall, birds using the range were most often observed in the quadrant nearest house. However, in the Enrichment treatment, more birds were observed in the furthest two quadrants than in the control group. Although the presence of enrichments outside did not encourage more birds to go out, it did result in better range utilization. In hot weather, shade is likely a key factor in the quality of the range environment. Structural enrichments may provide useful options in ecological production, especially if natural cover, such as trees and woody perennials, is not possible.
3. Implications of management strategies on meat quality of birds reared under organic and pasture production systems. Currently there is little information regarding the effect of different housing types, production methods and bird genotype on the final quality/quantity of broiler meat. Fast growing broilers have greater breast meat yields, a uniform carcass and higher feed efficiency than heritage or slower growing breeds however are not generally utilized in pasture based systems. ARS researchers at Fayetteville, Arkansas, evaluated the production performance and quality of meat from birds of different genotype, raised on portable versus fixed housing, with or without access to pasture in two different seasons (spring and fall). Forage consumption by poultry is influenced by several factors and may affect the yields and meat quality parameters. Access to pasture did not alter meat quality parameters such as moisture, protein, fat percentage, pH, color, texture, cooking loss, shear energy when compared to birds without pasture access. Since forages contribute to the nutritive value of the meat and may help in reducing costs, the type and quality of forage choices in pastures are important considerations for these production methods and need to be explored further for pasture production systems. Results from these studies can help the producers in taking informed decisions regarding the genotype of birds and management strategies adaptable for alternative production systems. Specifically, many pasture producers have switched to fast growing, broiler chickens because of these research findings as their yields/profits have improved.
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Fanatico, A.C., Mench, J.A., Archer, G.S., Liang, Y., Brewer-Gunsailus, V.B., Owens-Hanning, C.M., Donoghue, A.M. 2016. Effect of outdoor structural enrichments on the performance, use of range area, and behavior of organic meat chickens. Poultry Science. 95:1980-1988. https://doi.org/10.3382/ps/pew196.
Nguygen, P.H., Green, E., Ishola, P., Huff, G.R., Donoghue, A.M., Dridi, S. 2015. Chronic mild cold conditioning modulates the expression of hypothalamic neuropeptide and intermediary metabolic-related genes and improves growth performances in young chicks. PLoS One. 10(11):e0142319. https://doi.org/10.1371/journal.pone.0142319.
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Huff, G.R., Huff, W.E., Rath, N.C., El-Gohary, F.A., Zuoyong, Z.Y., Shini, S. 2015. Efficacy of a novel prebiotic and a commercial probiotic in reducing mortality and production losses due to cold stress/Escherichia coli challenge in broiler chickens. Poultry Science. 94:918-926. https://doi.org/10.3382/ps/pev068.
Johny, A., Frye, J.G., Donoghue, A.M., Donoghue, D.J., Porwollik, S., Mcclelland, M., Venkitanarayanan, K. 2017. Gene expression response of Salmonella enterica Serotype Enteritidis phage type 8 to subinhibitory concentrations of the plant-derived compounds trans-cinnamaldehyde and eugenol. Frontiers in Microbiology. 8:1828. https://doi.org/10.3389/fmicb.2017.01828.
Woo-Ming, A., Arsi, K., Wagle, B.R., Shrestha, S., Donoghue, A.M., Donoghue, D.J. 2018. Probiotic cultures of Lactobacillus spp. isolates reduce the foodborne pathogen, Campylobacter jejuni on post-harvest chicken. International Journal of Advances in Science Engineering and Technology. 6(2):40-44.