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United States Department of Agriculture

Agricultural Research Service

Related Topics

Research Project: CONTROLLING EGG CONTAMINATION WITH SALMONELLA ENTERICA BY UNDERSTANDING ITS EVOLUTION AND PATHOBIOLOGY

Location: Egg Safety and Quality

2008 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
Confirmation of small scale polymorphisms within the genome of Salmonella enterica serovar Enteritidis (S.enteritidis). New polymorphisms were reported in Version 5 of the National Center for Biotechnology Information database on or about March 1, 2008. To date, a total of 319 single nucleotide polymorphisms (SNPs) and other small polymorphisms have been confirmed by forward/reverse sequencing of 3 key strains as differentiating phenotypes of S. enteritidis. This is an increase of 102 over last year. There are another 131 putative polymorphisms remaining to be confirmed.

In vitro multiplication of Salmonella enteritidis on the egg yolk membrane and penetration to the yolk contents. After inoculation onto the exterior surface of vitelline membranes in a laboratory egg contamination model, four S. enteritidis were all able to penetrate into the yolk contents during as little as 12 hours of incubation at 30 deg. C, but the concentration of S. enteritidis after incubation was significantly higher in whole yolks (including the vitelline membrane) than in yolk contents at both 12 hours and 36 hours of incubation.

Epidemiological analysis of ORF disrupting small scale polymorphisms in Salmonella spp. Approximately 1000 BLAST searches were conducted, gene sequences of interest were downloaded and translated, and SeqMan and MegAlien analyses were applied to determine if a subset of genes with ORF altering mutations that changed predicted gene transcripts were present or absent in all available Salmonella enterica and other relevant bacterial genome databases. Genes were chosen on the basis of having an ORF-altering disruption that distinguished PT13a isolates of S. enteritidis, which vary in the ability to contaminate eggs.

Site directed mutagenesis techniques were successful. Fourteen of 319 confirmed polymorphisms disrupt the open-reading-frame (ORF) of genes that differentiate subpopulatiions of PT13a S. enteritidis. In the hierarchy of types of genetic change that is likely to impact phenotype, especially the ability to colonize chickens and to contaminate eggs, ORF disruption is of hight consequence. In order to link phenotype to genotype with the stringency required to establish a causal effect, it is necessary to construct defined mutants for further analysis of how phenotype is impacted by singular genetic events. Two mutants out of a collection of 14 genes with ORF disruptions have been constructed. Thus, this year we have successfully introduced the Datsenko and Wanner method of constructing mutants to the laboratory.

This research addresses National Program 108 (Food Safety), Component 1.1 Pathogens, Toxins and Chemical Contaminants Preharvest, Problem Statements 1.1.2 (Epidemiology) and 1.1.3 (Ecology, Host Pathoghen and Chemical Contaminants Relationships).


4.Accomplishments
1. In vitro multiplication of Salmonella enteritidis on the egg yolk membrane and penetration to the yolk contents. We determined that four S. enteritidis were all able to penetrate from the exterior of the yolk (vitelline) membrane into the yolk contents during as little as 12 hours of incubation at 30°C in a laboratory egg contamination model, but the concentration of S. enteritidis after incubation was significantly higher in whole yolks (including the vitelline membrane) than in yolk contents at both 12 hours and 36 hours of incubation. Although chickens infected with Salmonella do not deposit this pathogen inside egg yolks very often, bacteria from the surrounding albumen might penetrate through the membrane that surrounds the yolk, resulting in rapid and extensive Salmonella 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 four S. enteritidis strains to multiply on the vitelline membrane or to penetrate this membrane and multiply inside yolks during incubation at warm temperatures (simulating the conditions under a proposed national S. enteritidis control program that would allow unrefrigerated storage of eggs on farms for up to 36 hours). These results demonstrate that extensive bacterial multiplication on the yolk membrane may occur in addition to (and before) penetration into the yolk contents, further supporting an emphasis on rapid refrigeration of egg for protecting consumers from egg-borne transmission of S. enteritidis infections.

This research addresses National Program 108 (Food Safety), Component 1.1 Pathogens, Toxins and Chemical Contaminants Preharvest, Problem Statement 1.1.3 (Ecology, Host Pathoghen and Chemical Contaminants Relationships).

2. Genes that vary between PT13a subpopulations also vary among other Salmonella spp. We determined that 14 genes of PT13a S. enteritidis have ORF-disrupting mutations that distinguish strains from each other, even if they at first appear clonal by DNA-DNA microarray hybridization. It is important to be able to tell bacterial strains from each other, because only some of them cause food borne illness. Analysis of the amino acid sequences of these 14 genes within other gram-negative bacteria and Salmonella spp. by BLAST analysis of public genomic databases available from National Center for Biotechnology Information and Sanger Institute indicate that they can vary greatly from serotype to serotype. In one gene, the introduction of a termination codon coincides with deletion of the 3’ end of the gene in other Salmonella serotypes, which suggests that introduction of small genetic mistakes might make DNA more fragile and prone to converting to a larger irreversible mistake. Another gene known to be important for survival of Salmonella in white blood cells is missing or has coding errors in 26 of 29 Salmonella genomes, which suggests that it is very hard for Salmonella to maintain this gene even though it facilitates disease potential. These results suggest that small genetic change (genetic drift) impacts the biology of Salmonella as much as does large genetic change (genetic shift). These results provide new epidemiological information about Salmonella serotypes that periodically emerge as food-borne pathogens.

This research addresses National Program 108 (Food Safety), Component 1.1 Pathogens, Toxins and Chemical Contaminants Preharvest, Problem Statements 1.1.2 (Epidemiology) and 1.1.3 (Ecology, Host Pathoghen and Chemical Contaminants Relationships).


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of Web Sites Managed1
Number of Non-Peer Reviewed Presentations and Proceedings8

Review Publications
Olson, A.B., Andrysiak, A.K., Tracz, D.M., Bouldin, J.G., Demzcuk, W., Ng, L., Maki, A., Jamieson, F., Gilmour, M.W. 2007. Limited genetic diversity in Salmonella enterica Serovar Enteritidis PT13. BMC Microbiology.(7)p.87.

Bouldin, J.G., Morales, C., Frye, J.G., Gast, R.K., Musgrove, M.T. 2007. Detection of Salmonella enterica subpopulations by phenotype microarray antibiotic resistance patterns. Applied and Environmental Microbiology. 73(23):7753-7756.

Gast, R.K. 2007. Serotype-specific and serotype-independent strategies for pre-harvest control of foodborne Salmonella in poultry. Avian Diseases. 51:817-828.

Gast, R.K. 2008. Paratyphoid Infections. Diseases of Poultry, 12 edition. Blackwell Publishing, Ames, iowa, pp.636-655.

Waltman, W.D., Gast, R.K., Mallinson, E.T. 2008. Salmonellosis. Isolation and Identification of Avian Pathogens, 5th edition. American Association of Avian Pathologists, Jacksonville, FL, pp.3-9.

Gast, R.K. 2008. Salmonella Infections: Diseases of Poultry, 12th edition. Blackwell Publishing, Ames, Iowa, pp.619.

Guard, J.G. 2008. Evolutionary trends in two strains of Salmonella enterica subsp. I serovar Enteritidis PT13a that vary in virulence potential: Version 5 NCBI database. National Center for Biotechnology Information (NCBI)Available:http://www.ncbi.nlm.nih.gov/Salmonella_SNPs.html

Bouldin, J.G., Morales, C. 2008. Nucleotide sequence of a predicted diguanylate cyclase unique to egg contaminating Salmonella enteritidis that does not form biofilm.. National Center for Biotechnology Information (NCBI). Available:http://www.ncbi.nlm.nih/entrez/viewer.fcgi?db=nuccore&id=170516891

Gast, R.K., Guraya, R., Bouldin, J.G., Holt, P.S. 2008. Multiplication of Salmonella Enteritidis on the yolk membrane and penetration to the yolk contents at 30 c in an in vitro egg contamination model. Journal of Food Protection. 71:1905-1909.

Last Modified: 7/24/2014
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