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The specific objective of the proposed research is to analyze transcriptional profiles in the ceca of neonatal chicks following treatment with a Lactobacillus-based probiotic culture derived from poultry with and without Salmonella enteritidis challenge. The overall goal is to determine genes and gene networks that may be involved in mediating the observed reduction of Salmonella infection in chicks within 24h of treatment. Outputs will include quantitation of Salmonella reduction, lists of differentially expressed genes, and output from gene expression analyses.
NON-TECHNICAL SUMMARY: Beneficial bacteria (probiotics) have been shown effective for reducing Salmonella in neonatal chicks in a number of studies. As Salmonella is a food-borne pathogen that can infect humans, it is important for non-antibiotic treatments for this pathogen to be available in the poultry industry. Previous research at the University of Arkansas resulted in a Lactobacillus-based probiotic culture that is capable of reducing Salmonella within 24h in newly hatched chicks. This experiment will use the 24h model used for testing this probiotic culture, and take tissue samples (cecae) at 12 and 24h after treatment for microarray analysis. Microarray analysis will allow us to determine levels of gene expression for 20,000 genes simultaneously, and provide information as to the mechanism by which probiotics mediate the Salmonella-reducing effects. The knowledge of the genes and networks involved may lead to more effective future treatments for reduction of Salmonella.
<P>APPROACH: Day-of-hatch chicks will be obtained from a commercial broiler breeder hatchery. Chicks will be randomly assigned to one of 4 treatment groups (n=80/group): 1. Control 2. Probiotic treated 3. Salmonella enteritidis (SE) challenged 4. SE challenged and probiotic treated. Challenge with SE will be administered upon arrival at the laboratory by oral gavage (10,000 cfu in 0.25 mL) using an animal feeding needle. Groups 1 and 2 will receive sterile saline by gavage. One h post-challenge, probiotic treatment will be administered (100,000 cfu in 0.25 mL) to groups 2 and 4 by oral gavage. At this time, groups 1 and 3 will receive vehicle. Twelve h and 24h post-treatment, 40 chicks will be humanely killed, and both ceca will be removed. Four ceca (1 ceca each from four chicks) will be pooled in a sterile sample bag for recovery of Salmonella. Four rinsed ceca samples will be pooled (ten pools per treatment group) and immediately frozen in liquid nitrogen. Four sample pools per group will be utilized for microarray analysis, but all ten pools will be utilized for qRT-PCR following microarray analysis to confirm the microarray results. The frozen samples will be subjected to RNA isolation, fluorescent dye incorporation and reverse transcription, and hybridization to microarray slides. 32 microarray slides total (16 per time point) will be hybridized and scanned to provide gene expression levels for 21,120 genes. The data will be analyzed to determine significant differences between groups. Selected differentially expressed genes will be selected for qRT-PCR analysis to confirm the microarray data. Additionally, genes that are significantly different will be subjected to self-organizing maps (SOMS) analysis to organize the genes into groups exhibiting similar. Genes in clusters exhibiting specific and reciprocal responses to SE and probiotic will be studied further. Gene Ontology (biological process) analysis will be completed using the Bioresource for Array Genes database and Pathway Miner databases.