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United States Department of Agriculture

Agricultural Research Service

2010 Annual Report

1a.Objectives (from AD-416)
The objectives of this proposed research will be to identify naturally occurring genetic variation in Salmonella enterica that correlates with the evolution of egg contamination and enhanced growth of the bacterium in the hen, the egg and in the on-farm environment and to then determine how different genetically defined strains vary in their pathobiology within the hen and how these differences affect the risk of egg contamination and the control of disease.

1b.Approach (from AD-416)
Our approach will be to identify single nucleotide polymorphisms (SNPs) that occur naturally in the genome of Salmonella enterica, link them to the phenotypic attributes of the pathogen that are relevant to causation of egg contamination and growth to high cell density, and develop a phylogenetic database that aids cost effective screening of the Salmonellae for these traits. We will then characterize the processes by which bacteria are deposited inside eggs laid by infected laying hens and assess the significance of these processes for proposed cost effective and feasible disease control measures such as egg refrigeration, diagnostic egg culturing, and assay of shell quality.

3.Progress Report
Impact of SNPs on the phenotype and virulence of Salmonella Enteritidis. Mutational analysis is required to explore the linkage between naturally occurring genetic variation and phenotype of the bacteria. ARS researchers in Athens, GA constructed three mutants. Mutation of dsdA (D-serine dehydratase) altered phenotype microarray profiles as evidenced by the unexpected loss of resistance to salts, a sefD mutant impacted virulence in hens as measured by suppression of egg production but did not cause a phenotype microarray change, and a diguanylate cyclase mutant abolished biofilm, but did not alter phenotype microarray results. Construction of mutants in genes that were previously linked in surprising ways to the egg contamination pathway is a first step towards improving vaccine design.

Impact of overexpression of sefD on reproductive tract function in hens: Whereas a sefD mutant and its parent strongly suppressed egg production and blood calcium levels, overexpression of the gene produced results similar to uninfected control hens and mitigated any sign of infection. These results have implication for epidemiological monitoring of virulent Salmonella and for design of vaccines. ARS researchers in Athens, GA contacted the FDA about sefD being a biomarker of virulence in S. Enteritidis.

In Vitro Penetration of Salmonella Enteritidis through Yolk Membranes of Eggs from Six Genetically Distinct Commercial Lines of Laying Hens. After inoculation onto the exterior surface of vitelline membranes in a laboratory egg contamination model, S. Enteritidis penetrated into the interior yolk contents of eggs from six genetically different lines of laying hens during 24 hours of warm temperature storage, although some significant differences between eggs from different lines were observed. This research suggests that certain breeds of birds may have eggs that resist egg yolk contamination more than others. It is important to discern host factors that protect against food borne illness.

1. Identification of natural mutation occurring in gene sefD as a biomarker of Salmonella virulence. The SefD protein may be contributing to egg contamination by mitigating disease in hens so that farmers cannot tell that hens are infected using observation of flock health parameters. Spontaneous loss of SefD by naturally-occurring mutation could then set off outbreaks by rapidly increasing virulence. SefD is now a second molecule suspected of mitigating disease, the first of which was capsular LPS O-antigen. The project leader from USDA-Athens-GA contacted FDA about sefD being a biomarker of virulence in S. Enteritidis, because lack of sefD enhanced virulence and NCBI reference genome databases are dominated by sefD mutants.

2. In vitro penetration of salmonella enteritidis through yolk membranes of eggs from six genetically distinct commercial lines of laying. ARS researchers in Athens, Georgia determined that S. Enteritidis was able to penetrate through the yolk (vitelline) membrane to reach the yolk contents in eggs from six genetically different lines of laying hens in a laboratory egg contamination model, with eggs from one chicken line allowing significantly more penetration than two other lines. Although chickens infected with S. Enteritidis do not deposit this pathogen inside egg yolks very often, bacteria from the surrounding albumen might penetrate through the vitelline membrane that surrounds the yolk and begin rapid and extensive growth in the nutrient-rich interior contents of the yolk prior to egg refrigeration. We used a laboratory egg contamination model to assess the ability of an S. Enteritidis strain to penetrate into the yolk contents during 24 hours of incubation at 30° C in eggs from six genetically distinct lines of commercial laying hens (simulating potential conditions under federal S. Enteritidis control regulations that allow unrefrigerated storage of eggs on farms for up to 36 hours). These results demonstrate that penetration of S. Enteritidis to reach the yolk contents and multiply to more dangerous levels can occur during unrefrigerated storage of eggs from diverse lines of laying hens (although some differences between lines are apparent), reinforcing the importance of prompt refrigeration of eggs for protecting consumers from egg-borne transmission of S. Enteritidis infections.

Review Publications
Gast, R.K., Jones, D.R., Anderson, K.E., Guraya, R., Guard, J.Y., Holt, P.S. 2010. In Vitro Penetration of Salmonella Enteritidis through Yolk Membranes of Eggs from Six Genetically Distinct Commercial Lines of Laying Hens. Poultry Science: 89:1732-1736.

Guard, J.Y., Gast, R.K., Guraya, R. 2010. Colonization of avian reproductive tract tissues by variant subpopulations of Salmonella Enteritidis. Avian Diseases. 54:857-861.

Line, J.E., Hiett, K.L., Guard, J.Y., Seal, B.S. 2010. Differential carbon source utilization by Campylobacter jejuni strain 11168 in response to growth temperature variation. Journal of Microbiological Methods. 80:198-202.

Last Modified: 8/4/2015
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