2007 Annual Report
1a.Objectives (from AD-416)
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.
1b.Approach (from AD-416)
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.
This report documents research conducted under a non-funded cooperative agreement between ARS and the Sidney Kimmel Cancer Center, San Diego, CA. Additional details of research can be found in the report for the in-house associated project 6612-3200-004-00D entitled “Controlling egg contamination with Salmonella enterica by understanding its evolution and pathobiology. The objective of this research is to determine critical genetic determinants that impact the ability of Salmonella enteritidis (SE) to contaminate the eggs of hens and to otherwise propagate in the poultry environment. To do this, a large number of mutations were first introduced by the cooperator into the genome of Salmonella typhimurium, which does not efficiently contaminate eggs. In one experiment, 11 hens were infected by USDA with a bank of 960 mutants and sampled on days 3, 7 and 14 post-infection. In another experiment, 4 hens were infected by USDA with a randomly generated cultured mutated by transposons. Samples collected by USDA were modified as listed in the NFCA to include culturing of whole spleens, total cecal contents and total intestinal contents, because literature indicated that these samples were more likely to supply a threshold concentration of cells (founder population) that was required for downstream processing. Numerous emails spanning April 2007 document progress and the exchange of scientific information. Information on processing of samples were sent by email as a powerpoint presentation on 5/1/2007. Isolates from chickens for downstream processing were shipped during May 2007 in 3 batches to the collaborator per agreement. Results from enumeration of bacteria in samples were sent April 18, 2007 and updated with other communications during May. Shipment of isolates completed the work agreed to by USDA for this phase.