2013 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.
ARS researchers in Athens, GA developed, tested and applied a new database for assigning serotype to Salmonella, and it lowered cost of analysis by 80%. Less expensive and simpler laboratory assays facilitate testing by industry and aids the ability of smaller or disadvantaged producers and food operations to assay for the presence of dangerous Salmonella serotypes.
ARS researchers at Athens, GA tested a new vaccine for reducing persistence of Salmonella Enteritidis in the spleens of hens. Whole genome of analysis of Salmonella Enteritidis was used to obtain information on genes contributing to the ability of the bacteria to contaminate eggs. Four experiments were completed to test a new vaccine designed from this information. Results indicated that the new killed vaccine (bacterin) significantly reduced egg-contaminating Salmonella Enteritidis in the spleens of hens. A grant was submitted to obtain support for pursuing additional research.
Progress report and termination of trust with Columbian Veterinarians and Poultry Specialists (AMEVEA) [ARIS accession # 45218]. Funds for this trust were used to facilitate analysis of samples received from South America and to help educate visiting scientists on the technique called dkgB-linked intergenic sequence ribotyping (ISR). This project is finished and funds have been depleted ($7,500). This trust supported research objectives by providing a source of samples from outside the United States to examine and by facilitating transfer of knowledge of the technique to other scientists.
USDA ARS Athens applied Intergenic Sequence Ribotyping to aid trade partners from South America in Brazil, Columbia and Peru. In Peru, collaboration between USDA-ARS and local veterinary consultants and pediatricians revealed that illness in children was likely associated with a high prevalence of Salmonella Infantis in chickens, and pediatricians and veterinarians now have the respective information they need to educate the public and to vaccinate poultry. In Columbia, a high death rate in chickens was observed on farms where the bird pathogen S. Gallinarum co-circulated with the human pathogen S. Enteritidis, thus illness in birds may have an unrecognized association with the emergence of illness in humans. In Brazil, S. Enteritidis was the only serotype recovered from 1-day old chicks, whereas S. Heidelberg was prevalent in cloacal and carcass swabs, thus two important Salmonella pathogens appear to have different niches within poultry flocks. These results show Intergenic Sequence Ribotyping (ISR) generates new information about local problems in a timely manner that can be used to improve the health of people and animals.
Intergenic Sequence Ribotyping (ISR) slashed the cost and complexity of obtaining serotype for Salmonella and it can be made broadly available at low cost. ARS scientists at Athens, GA determined that a short region of the Salmonella genome contained hundreds of variations that can be sequenced and used to accurately assign serotype. The new method, called Intergenic Sequence Ribotyping (ISR), was compared to traditional methods of serotyping used in Europe and the US. The new method was as specific and sensitive as older methods, and it had the added advantages of discovering new serotypes and detecting mixtures of serotypes in culture. Because the method reduced cost of serotyping by 80% from traditional methods, hundreds of samples can be processed to explore the on-farm epidemiology of Salmonella using streamlined cost-effective analysis.
Guard, J.Y., Sanchez-Ingunza, R., Morales, C., Stewart, T.E., Liljebjelke, K., Van Kessel, J.S., Ingram, K.D., Jones, D.R., Jackson, C.R., Cray, P.J., Frye, J.G., Gast, R.K., Hinton Jr, A. 2012. Comparison of dkgB-linked Intergenic Sequence Ribotyping to DNA Microarray Hydridization for Assigning Sterotype to Salmonella enterica. FEMS Microbiology Letters. 337(1):61-72.
Shah, D., Zhou, X., Kim, H., Call, D., Guard, J.Y. 2012. Transposon mutagenesis of Salmonella Enteritidis identifies genes that contribute to invasiveness in human and chicken cells and survival in egg albumen. Infection and Immunity. 80(12)4203-4215.
Pulido-Landinez, M., Sanchez-Ingunza, R., Guard, J.Y., Pinheiro Do Nascimen, T. 2013. Assignment of serotype to Salmonella enterica isolates obtained from poultry and their environment in Southern Brazil. Letters in Applied Microbiology. Available: http://www.ncbi.nlm.nih.gov/pubmed/23734786.
Pulido-Landinez, M., Laviniki, V., Sanchez-Ingunza, R., Guard, J.Y., Pinheiro Nascimento, V. 2012. Use of FTA Cards for the Transport of DNA Samples of Salmonella spp. from Poultry Products from Southern Brazil. Acta Scientia Veterinariae. 40(4):1073.