2009 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.
Completion of the single nucleotide polymorphisms (SNP) database that links phenotype to genotype for Salmonella Enteritidis. 131 putative polymorphisms were sequenced in 3 strains to finish the database. 247 single nucleotide polymorphisms were found that distinguished the genome of egg-contaminating PT13a S. Enteritidis from that of a biofilm-forming strain that has the most common PFGE pattern for this organism. New polymorphisms were reported in Version 7 of the NCBI database on or about August 1, 2009. Each SNP was individually entered into the NCBI dbSNP database and the group will be released sometime in August 2009 in Build 131. The National Center for Biotechnology Information (NCBI) definition of a SNP was applied to the database. This means that contiguous SNPs are considered a single event. Changing the definition impacted the total number of line items in the SNP database. One SNP introduced both an amino acid change and a deletion in one gene, which was noted by having an extra line in the database; thus, there are 248 entries within the database plus an additional 3 that were detected in the large virulence plasmid. Analysis eventually incorporated review of 4 genomes, which means that every SNP difference has been found between the two PT13a strains of interest to within 99% certainty.
Effect of prior passage through laying hens on invasion of reproductive organs by Salmonella Enteritidis. When laying hens were orally infected with a passaged S. Enteritidis strain (obtained from a previously infected group of hens), there were no significant differences between treatments in the ability to colonize internal organs or reproductive tract tissue. Reproductive tract tissue was significantly less colonized than spleen or liver. Colonization of reproductive tract tissue had a wide variance, with the PT4 dimorphic strain yielding both the highest and lowest percent positive samples post-infection. These results suggest that the reproductive tract is resistant to colonization, but that the resistance varies according to the stage of the bird in the hormonal cycle associated with production of eggs.
Site-directed mutagenesis techniques were successful. ORF disruption is of high consequence to gene function. It is necessary to construct defined mutants for further analysis of how phenotype is impacted by singular genetic events. Five mutants out of a collection of 15 genes with ORF disruptions have been constructed.
SUBORDINATE PROJECT REPORT NFCA #58-6612-7-160N: Associated work completed for this agreement. Mice appear to carry higher loads of Salmonella in their gut than do chickens.
SUBORDINATE PROJECT REPORT NFCA Number 58-6612-9-229N: Collaboration with scientists at Washington State, Pullman was started to improve the ability to do transcriptome analysis.
Mixed subpopulations of PT13a colonize the organs of hens as well as does S. enteritidis PT4. Previous research suggested that different subpopulations within the PT13a bacteriophage lineage of S. enteritidis impacted the infection pathway to the egg in different ways and that mixtures of subpopulations were more efficient overall at egg contamination than homogeneous cultures. To further insight into the hypothesis that mixing subpopulations in the PT13a lineage of S. enteritidis contributes to egg contamination, three groups of hens were infected with 3 different culture preparations in 3 trials. The three groups were hens infected with either S. enteritidis PT4 or with one of two mixtures of S. enteritidis PT13a subpopulations given at ratios of 90:10 and 10:90 biofilm-forming to smooth colony type. The two mixtures of the PT13a culture colonized organs of hens similarly to the PT4 strain. The impact of this work is that it suggests phage type diversity of S. enteritidis may impart selective advantage to the pathogen for maintaining its ability to contaminate eggs.
Effect of prior passage through laying hens on invasion of reproductive organs by Salmonella Enteritidis. We determined that passage of Salmonella Enteritidis through infected chickens did not always select for either higher overall invasiveness to internal organs or for a higher ability to colonize reproductive organs. Horizontal transmission of S. Enteritidis between hens is an important mechanism by which infection spreads throughout egg-laying flocks, and the colonization of reproductive tissues in infected laying hens is a pivotal stage in the production of contaminated eggs that can transmit S. Enteritidis to humans. We evaluated how passage through hens affected the ability of an S. Enteritidis strain to invade to various internal tissue sites, including three different locations in the reproductive tract, by infecting groups of hens with a passaged isolate (obtained from a previously infected group of hens) and then comparing the frequencies of colonization of internal organs between the groups given the original and passaged strains. These results demonstrate that the interaction between S. Enteritidis and the multiple environments it encounters within the infected avian host can be highly complex, and the public health consequences of horizontal transmission of infection within and between laying flocks may depend on both the genetically defined characteristics of the original infecting bacterial population and the selective pressures exerted in the tissues of infected chickens.
Publication of the first public access single nucleotide polymorphisms (SNP) database for a microbial genome at NCBI. SNP analysis is routinely used in human clinical biology to find genetic linkages to biology that impacts health and risk of disease. Both humans and S. Enteritidis have a clonal population structure, which increase the strength of linkage between any one SNP and the biological attributes (phenotype) of the organism under investigation. Researchers recognized that S. Enteritidis versus other serotypes could be used to find which SNPs were most strongly linked to phenotype, because other serotypes within Salmonella enterica have a more varied population structure. By focusing on a strategy of locating every SNP difference within a single phage type of S. Enteritidis, genomic “noise” was kept to a minimum so only those SNPs most likely to be strongly linked to phenotype would be found. The need to dampen genetic noise was of paramount importance, because bacteria generally mutate rapidly as compared to eukaryotes and most SNPs are not linked to phenotype. One calculation of the problem represented by bacterial genomic noise suggests that if approximately 250 SNPs distinguish strains that vary in virulence potential within a single phage type, then there will be 2500 SNPs occurring between phage types and 25,000 SNPs between any two serotypes. This means that across 1400 Salmonella enteric subsp. I serotypes, 35 million SNPs could exist. This means that a relatively infinitesimal number of SNPs are the determinants of phenotype and the rest basically prevent recognition of the genomic underpinnings of a particular phenotype. USDA researchers have provided the research community a tool for finding those SNPs of high consequence that have the best predictive power of dangerous trends in the evolution of Salmonella enterica that threaten the safety of the food supply.
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Gast, R.K., Bouldin, J.G., Guraya, R., Holt, P.S. 2009. Effect of prior passage through laying hens on invasion of reproductive organs by Salmonella Enteritidis. International Journal of Poultry Science. (8):116-121.
Gantois, I., Ducatelle, R., Pasmans, F., Haesebrouck, F., Gast, R.K., Humphrey, T., Van Immerseel, F. 2009. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiology Letters. (33):718-738.
Guard, J.G. 2009. Evolutionary trends in two strains of Salmonella enterica subsp. I serovar Enteritidis PT13a that vary in virulence potential. National Center for Biotechnology Information (NCBI). Available:http://www.ncbi.nlm.nih.gov/genomes/static/Salmonella_SNPS.html