Reducing Salmonella Enteritidis and Campylobacter Jejuni in Chickens by Dietary Supplementation of Plant-Derived Antimicrobial Molecules

NON-TECHNICAL SUMMARY: S. Enteritidis and C. jejuni are major foodborne pathogens transmitted through poultry products. Chickens are the reservoir host of S. Enteritidis and C. jejuni, with intestinal colonization being the most significant factor causing contamination of meat and eggs. Effective on-farm strategies for reducing colonization of birds with these pathogens are critical to improve the microbiological safety of poultry products. Plant-derived antimicrobials are natural, generally regarded as safe molecules used to preserve foods and enhance food flavor. Our preliminary research revealed that plant molecules, including trans-cinnamaldehyde, carvacrol, thymol, and eugenol were bactericidal on S. Enteritidis and C. jejuni in chicken cecal contents in vitro. Additionally, trans-cinnamaldehyde and eugenol reduced significant cecal populations of these pathogens in chickens. Trans-cinnamaldehyde and eugenol also decreased motility in S. Enteritidis, a potential virulence factor aiding colonization. The proposed research will investigate (1) effect of trans-cinnamaldehyde, carvacrol, thymol, and eugenol as dietary supplements to reduce colonization of S. Enteritidis and C. jejuni in broiler chickens, (2) effect of plant molecules on gene expression in S. Enteritidis and C. jejuni using DNA microarrays, and (3) their safety in chickens by histopathological analyses of internal organs. The proposed work will potentially lead to decreased outbreaks of salmonellosis and campylobacteriosis, thereby improving public health and economic opportunities for poultry farmers. It is also expected to provide critical information on the antimicrobial mechanism(s) of plant molecules at gene level in S. Enteritidis and C. jejuni.

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APPROACH: The effect of trans-cinnamaldehyde, carvacrol, thymol, and eugenol on the colonization of S. Enteritidis and C. jejuni in broiler chickens will be studied in day-old chicks. The chicks will be randomly divided into seven treatment groups, including 1. Negative control (no pathogen challenge, no antimicrobial) 2. Positive S. Enteritidis control (pathogen challenge, no antimicrobial) 3. Positive compound control (no pathogen challenge, feed supplemented with compound at 1%) 4. Pathogen challenged and feed supplemented with compound at 0.25% 5. Pathogen challenged and feed supplemented with compound at 0.5% 6. Pathogen challenged and feed supplemented with compound at 0.75% 7. Pathogen challenged and feed supplemented with compound at 1% On day 1, all the chicks will be weighed and their fecal samples tested for the pathogens. The birds belonging to different treatments will be provided feed containing respective concentrations of the antimicrobial and water ad libitum. On day 5, birds will be infected orally with 0.25 ml of PBS containing ~ 7 log CFU of a 5-strain mixture of nalidixic acid-resistant S. Enteritidis/C. jejuni. The birds not challenged with pathogen will be given 0.25 ml of sterile PBS. Following challenge, ten birds from each treatment group will be sacrificed on weeks 1, 2, 3, 4, 5, and 6. The ceca, liver and spleen from each bird will be collected for bacteriologic analysis. Further, pH and pathogen populations in the cecal contents will also be determined. The feed consumption and body weight of the birds will be determined. Finally, histopathological examination of internal organs will be done to rule out any toxicity of the compounds on birds. To determine the effect of plant antimicrobials on gene expression in S. Enteritidis and C. jejuni, each pathogen will be grown to mid-log phase and treated separately with sub-inhibitory concentrations of each compound. RNA from untreated cultures will be used as controls. Sub-inhibitory concentration of each compound will be determined for S. Enteritidis and C. jejuni by standard broth dilution assay. The bacteria will be sampled and RNA extracted prior to treatment (Time 0, T0), and then at appropriate time points after treatment (10 min, 30 min, 60 min). RNA from each time point will be compared to RNA from T0 via whole genome microarray analysis to determine changes in gene expression induced by trans-cinnamaldehyde, carvacrol, thymol, and eugenol. S. Enteritidis and C. jejuni DNA microarrays constructed by Dr. Frye and collaborators will be used. Data will be analyzed to determine what pathways are activated by the compounds in each bacterium, and differences in bacterial response to the different compounds will also be determined. These results will be compared to published data on S. Enteritidis and C. jejuni responses to stress, toxins and antimicrobial compounds to identify pathways that could be used by these bacteria to develop resistance to the effects of trans-cinnamaldehyde, carvacrol, thymol, and eugenol.