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United States Department of Agriculture

Agricultural Research Service

2011 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
The genetic underpinnings of the ability of Salmonella Enteritidis (S. Enteritidis) to contaminate the internal contents of eggs produced by otherwise healthy hens have been described. Federal agencies that have regulatory authority for the public health such as the Food and Drug Administration (FDA), the Centers for Disease Control (CDC), and the Food Safety and Inspection Service (FSIS) are especially benefited by this research, because it helps them do their work more efficiently and at lower cost. This research contributes to better characterization of outbreaks, improvement of vaccines, and control of the pathogen on farm.

Refrigeration keeps S. Enteritidis from multiplying in eggs. This is a factor that impacts trade practices, because the U. S. refrigerates eggs and some other countries do not. Investigations indicate that refrigeration reduces Salmonella growth in eggs. In addition, refrigeration impedes the bacterium from migrating through the yolk membrane, which is an event that can precede rapid proliferation.

Other Salmonella can colonize the avian reproductive tract, but not necessarily eggs at the efficiency and incidence exhibited by S. Enteritidis. Thus, there is sound scientific evidence for why producers and regulatory agencies are justified in treating S. Enteritidis differently from other Salmonella found on farm.

Evolutionary studies of whole genomes indicated that many typing methods used for other Salmonella are not adequate for analysis of outbreaks caused by S. Enteritidis. For example, Pulsed Field Gel Electrophoresis (PFGE) misses approximately 200 evolutionary events linked to the ability of S. Enteritidis to contaminate eggs and cause disease in people. Thus, analysis of single nucleotide polymorphisms (SNPs) improves typing of S. Enteritidis.

A role for the mouse as an amplifier of S. Enteritidis on farm was delineated through cooperative agreements. Thus, this information helps guide decisions made by producers in regards to best allocation of resources and explains why a rodent control program is an essential ingredient for preventing egg contamination.

In summary, S. Enteritidis is different from other Salmonella pathogens in how it propagates through the food supply and the measures required for control. This pathogen is an extremely important one, in part because it has increased industry focus on the effort required to maintain a safe food supply. Other Salmonella are now monitored for evolution that might facilitate their emergence in a manner similar to S. Enteritidis.

This project was replaced by 6612-32000-007-00D "Genetic Analysis of Poultry-Associated Salmonella enteric to Identify and Characterize Properties and Markers Associated with Egg-Borne Transmission of Illness".

1. Small scale genetic differences occurring with strains of Salmonella Enteritidis that differ in the ability to contaminate eggs were identified. Salmonella Enteritidis is the world’s leading cause of human salmonellosis. It is unique among 2500 Salmonella serotypes, because it is able to colonize and survive in the internal contents of eggs produced by otherwise healthy appearing hens. It does so with an efficiency and persistence that impacts epidemiology of human disease in a manner greater than all the other serotypes. Salmonella Enteritidis presented a puzzle to the research community, because numerous studies indicated that strains varied greatly in their ability to contaminate eggs. However, genetic analysis repeatedly showed that the bacterium had very little genetic difference between strains in comparison to what is seen with other serotypes. Comparing three whole genomes by high-density tiling arrays that generated a mutational map solved the puzzle of detecting genetic differences. Two of the strains were of the same phage type and were known to have no differences in gene content using DNA microarrays. Application of 3 techniques found that 250 Single Nucleotide Polymorphisms (SNPs) differentiated these two strains that varied in the ability to contaminate eggs. This information supports efforts to protect the food supply by improving epidemiological investigations and by providing new gene targets for improving vaccines. Customers benefitting from this information are regulatory agencies such as the Food and Drug Administration (FDA), the Centers for Disease Control (CDC) and the Food Safety and Inspection Service (FSIS); in addition, producers of vaccines and field epidemiologists are benefitted.

2. Salmonella Enteritidis multiplies in egg yolks after inoculation outside, on, and inside vitelline membranes and storage at different temperatures. ARS researchers in Athens, Georgia, determined that Salmonella Enteritidis cells introduced into eggs in the yolk, in the albumen, or on the membrane that separates yolk and albumen were able to reach and grow inside the yolk during storage (and the post-storage levels of Salmonella Enteritidis inside yolks were significantly higher at warmer storage temperatures). Although chickens infected with Salmonella 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. A laboratory egg contamination model was used to assess the ability of an Salmonella Enteritidis strain, introduced into eggs at three different sites (inside and outside of yolks), to grow to higher levels inside the yolk contents during 24 hours of incubation at temperatures ranging from 10 to 25º C. These results demonstrate that substantial Salmonella Enteritidis growth can occur in egg yolks during the first day of storage (even when contamination initially occurs outside of the yolk) and the risk of bacterial growth is higher at warmer storage temperatures, so prompt egg refrigeration is important for protecting consumers against egg-borne transmission of Salmonella Enteritidis. Customers benefitting from this information are regulatory agencies such as the Food and Drug Administration (FDA), the Centers for Disease Control (CDC) and the Food Safety and Inspection Service (FSIS); in addition, commodity industries that aim to produce a high quality safe product and consumers at risk of illness benefit.

3. Salmonella Enteritidis, Salmonella Heidelberg, or Salmonella Hadar colonizing reproductive tissues of experimentally infected laying hens vary in deposition inside eggs. Little is known about if the number of Salmonella cells that reach the reproductive organs of hens influences the likelihood of egg contamination which (especially in the case of Salmonella Enteritidis) poses a significant threat to public health. In an experimental infection study, groups of laying hens were experimentally inoculated with large oral doses of strains of Salmonella Enteritidis, Salmonella Heidelberg, or Salmonella Hadar. ARS researchers in Athens, Georgia, determined that groups of laying hens orally infected with Salmonella Enteritidis produced significantly more contaminated eggs than hens infected with Salmonella, but no significant differences were observed in the numbers of the various Salmonella strains that colonized either the ovary or oviduct. Thus, neither the presence nor level of Salmonella colonization in reproductive tissues consistently predicted the likelihood of egg contamination. This research shows that culturing of the avian reproductive tract is not the best target for locating Salmonella within chickens. It appears that factors other than the ability to colonize the avian reproductive tract contribute to the unique association between Salmonella Enteritidis and eggs. This information is of use to regulatory agencies such as the FDA, which develops protocols for testing eggs going from farm to market.

Review Publications
Guard, J. 2011. Evolutionary Trends Associated with Niche Specialization as Modelled by Whole Genome Analysis of Egg-contaminating Salmonella enterica serovar Enteritidis. In: Porwollik, S., editors. Salmonella: From Genome to Function. San Diego, CA:Caister Academic Press. p. 91-106.

Last Modified: 10/10/2015
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