2011 Annual Report
1a.Objectives (from AD-416)
1. Use the Salmonella Enteritidis (SE) SNP database to link specific genes and regulatory circuits to phenotypes associated with egg contamination, virulence, pathogenicity, host preference, or environmental persistence of subpopulations.
2. Determine whether recurring subsets or patterns of SE SNPs can be identified that are causally linked to outbreak potential of other Salmonella serotypes, and determine if SNPs can be used for risk profiling and cluster detection.
3. Apply information about genomic variation occurring across Salmonella enterica to improve serotyping schemes and vaccines that can be used to develop rapid and sensitive diagnostic and epidemiological testing methodologies for SE in poultry flocks and eggs.
1b.Approach (from AD-416)
Sixteen genes have been identified that are disrupted by SNPS SNPs in strains that vary in virulence potential. These genes will be mutated in a parent strain with a characterized genome and will be assayed by phenotype microarray and hen infection studies for function in virulence pathways. New SNPs will be identified that differentiate prominent phage types that vary in geographical incidence using whole genome comparisons. SNPs that have been linked to virulence or geographical incidence will be analyzed across other Salmonella serotypes by primer-directed sequencing for the purpose of evaluating epidemiological trends associated with SNPs.
Forty three new Salmonella serotypes were identified and verified by a method developed at ARS. New sequences were filed with The National Center for Biotechnology Information (NCBI). New information indicates that Salmonella biology is more complex than thought and this knowledge about the biology of the pathogen may help to reduce contamination of the egg supply and infection in people.
Testing of a new type of vaccine to protect hens from colonization by Salmonella Enteritidis was begun. This vaccine is in the first phases of analysis. Industry partners will be informed of results. Absence of a protein called “SefD” in Salmonella Enteritidis makes hens sick. We want to know if “SefD” has to be present to make an effective vaccine. Salmonella serotypes that lack the protein SefD are overrepresented in the NCBI databases, which suggest that the absence of this protein may be an important biological marker of its ability to cause disease.
A set of Single Nucleotide Polymorphisms (SNPs) in Salmonella Enteritidis facilitates tracking phage type viruses called bacteriophages which introduce new genetic material into bacteria and produce distinctive phage types. Analysis of phage type revealed that pyrosequencing can miss small deletions and ribosomal gene differences. Use of high throughput pyrosequencing for investigation of whole genome differences in epidemiological outbreaks requires application of controls and an understanding of method limitations.
Collaborative work was conducted with Washington State University, Pullman, WA, which included assays on the disease causing potential of mutants of Salmonella Enteritidis. Collaborations with other government scientists at the Food and Drug Administration (FDA), The Food Safety and Inspection Service (FSIS), and The Centers for Disease Control (CDC) were established. Collaborators submitted 5 strains for SNP analysis as a first test for applying information contained within the Salmonella Enteritidis SNP database. Within the U.S. Department of Agriculture at The Agricultural Research Service, collaborations with 4 other research groups are in progress.
Subordinate Project Report: Completed work associated with Non-Funded Cooperative Agreement (NFCA) Number 58-6612-9-164FN. Isolates and substantial scientific information on the background of isolates were submitted last year to this project.
The principal investigator is still automating robotics for high throughput processing. Communications were maintained by email exchange.
Subordinate Project Report NFCA Number 58-6612-9-229N. This is the final report on this NFCA, which continues as collaboration. Mutants and complemented mutants of Salmonella Enteritidis were submitted.
Progress was monitored via email communications to update collaborators of results. A collaborative manuscript was published (May 2011).
This project replaces 6612-32000-004-00D “ Controlling Egg Contamination with Salmonella Enterica by Understanding its Evolution and Pathobiology.”
Cameras can detect changes in the droppings of chickens infected with pathogenic Salmonella. Bile excretion is sometimes copious during the early stages of infection of hens with Salmonella Enteritidis. Studies so far indicate that bile has discernable signals that can be detected at concentrations within fecal droppings that are below visual detection levels. This research may facilitate development of a rapid monitoring system of large hen houses for the early emergence of infection. The goal is to have producers set up cameras at collection sites for droppings, which would signal if something is not normal. This would help protect consumers, because hens that have acquired infection with Salmonella Enteritidis are difficult to detect because they have few signs of diseases. If the farmer has some indication that the flock has changed, management decisions could be made to vaccinate hens or to divert eggs for pasteurization. These types of actions help reduce the risk to consumers of being exposed to contaminated eggs.
A new method for serotyping Salmonella enterica that greatly reduces cost of the assay was developed. The method is called Intragenic Sequence Ribotyping (ISR). ISR was validated against the microarray method certified in Europe called PremiTest. ISR avoids the need for submission of live culture, it greatly reduces the cost of serotyping by at least 4 fold and it is expected that robotics will attain even greater efficiencies of scale. It requires only commonly used laboratory equipment. Data requirements are modest and do not need vastly complicated computer systems. Veterinarians from Peru and Columbia visited to learn the technology. The ISR method facilitates exchange of information to the poultry industry and food regulatory agencies about Salmonella serotypes across international boundaries. Having less expensive methods for serotyping Salmonella helps protect the American consumer, trade partners and the safety of the food supply by encouraging frequent testing.
Shah, D.H., Zhao, X., Addwebi, T., Davis, M., Orfe, L., Call, D., Guard, J.Y., Besser, T.E. 2011. Cell invasion of poultry-associated Salmonella Enteritidis isolates is associated with pathogenicity, motility and secretion of type-three secretion system secreted proteins. Microbiology. 157(5):1428-1445.