Project Number: 6612-32000-004-01
Project Type: Nonfunded Cooperative Agreement

Start Date: Mar 21, 2007
End Date: Mar 01, 2009

Objective:
Whole genomic analysis is currently revealing previously unknown evolutionary trends in Salmonella enterica subsp. I serovar Enteritidis (SE) that impact the ability of this pathogen to grow and colonize hens and to contaminate eggs (http://www.ncbi.nlm.nih.gov/genomes/static/Salmonella_SNPS.html). The objective of this research is to determine critical genetic determinants that impact the ability of SE to contaminate the eggs of hens and to otherwise propagate in the poultry environment.

Approach:
Whole genomic analysis allows high throughput identification of single nucleotide polymorphisms and other subtle small scale genetic differences that evolve between strains that vary in virulence. At least two pools of mutants will be constructed on the basis of new information about subtle genetic variation in SE to help identify those changes that are closely linked with egg contamination. Mutants will be constructed using standard methods of random primer generation, gene manipulation and antibiotic selection. Preliminary tests will involve comparing mutant pools of SE to S. Typhimurium (STM), which is closely related to SE but has evolved associations with carcass contamination rather than egg contamination. Groups of six to twelve immature broiler chickens, between 4 and 12 weeks of age, will be infected orally with pools of mutants of STM and SE in separate experiments. Samples collected for culture after infection will include the crop, cecum, intestine, cloaca and spleen. For technical reasons, the STM pool will be assessed first. Results from each experiment will be compared and a table made of differences that exist between the ability of different mutants to colonize broilers. After completion of studies in broilers, a second round of analysis will be to use the SE mutant pool in egg laying hens to see which mutants survive to contaminate eggs. Two groups of hens will be infected intravenously with mutants at a target dose of 5 x 107 CFU. Other uninfected hens placed in the room will be used to assess if some mutants in the pool are capable of spreading and initiating contact infection and subsequent egg contamination. Egg production will be monitored daily before and after infection to determine if mutation changes the interaction of the pathogen with the reproductive tract of the hen. Eggs will be cultured using methods developed at ARS. Results will be jointly published.