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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Analysis of Salmonella Virulence, Antibiotic Resistance, and Host Response
2013 Annual Report


1a.Objectives (from AD-416):
1)Identify molecular mechanisms in Salmonella that impact virulence and antimicrobial resistance in the gastrointestinal environment of cattle and swine. 2)Characterize the genetic pathways in swine that respond to Salmonella infection, their relationship to carrier status, and the ability to predict and control Salmonella prevalence and ultimately food safety. 3)Investigate interactions between Salmonella and host microbiota that influence Salmonella colonization and persistence, alter the commensal composition in the host, and provide novel intervention strategies to decrease Salmonella in swine populations.


1b.Approach (from AD-416):
1) Use phenotypic assays, recombineering, and global transcriptional analysis to identify the genes and genetic pathways involved in regulating Salmonella DT104 hyper-invasion in response to a) rumen protozoa and b) sub-inhibitory concentrations of antibiotics..
2)Use an attenuated, LPS/flagella-deficient S. Typhimurium strain to re-focus the pig’s immune response from serovar-specific antigens to conserved antigens during vaccination against Salmonella, thereby providing cross-protection against multiple Salmonella serovars..
3)Classify the porcine gene expression differences between low and high shedders of Salmonella during colonization..
4)Compare the gastrointestinal microbial communities of pigs before, during and after Salmonella colonization to assess correlations with Salmonella shedding status in the pigs.


3.Progress Report:
The antibiotic tetracycline promotes disease-causing factors in Multidrug-resistant (MDR) Salmonella Typhimurium. Multidrug-resistant Salmonella is an important food safety concern because it is associated with increased morbidity in humans compared to antibiotic sensitive strains. The antibiotic tetracycline is used in humans and livestock, and resistance to this antibiotic is very common among isolates of MDR Salmonella Typhimurium. Because antibiotic exposure can affect the ability of MDR bacteria to cause disease (also known as virulence), we are examining the effect of tetracycline on the virulence of MDR Salmonella Typhimurium. In support of objective 1B, we have found that, in some MDR Salmonella Typhimurium, tetracycline can promote the expression of virulence factors that are linked to increased colonization and persistence of Salmonella in the host. This virulence-promoting effect of tetracycline may play a role in increasing the duration and intensity of a MDR Salmonella infection.

Variation in the DeoxyriboNucleic Acid (DNA) sequence of a key immune activator is linked to Salmonella shedding in pigs. Salmonella has the ability to survive and thrive in a variety of different environments, thereby creating significant challenges for food-producing industries in controlling Salmonella in food chain products. Swine are an important reservoir of Salmonella because colonization and shedding of this human pathogen occurs in asymptomatic pigs, imposing elevated risks to public and animal health. Thus, diverse intervention strategies are needed to control the transmission of Salmonella from pigs to humans and to the environment. One potential control approach is through genetic improvement by identifying and selective breeding for pigs with decreased Salmonella colonization and shedding. In support of objective 2B, ARS researchers in Ames, Iowa collaborated with researchers at Iowa State University and Embrapa in Brazil searched for DNA sequence variations in an important activator of the pig’s immune response, Toll-like receptor 4 (TLR4). Genetic variations, called single nucleotide polymorphisms (SNPs), in the TLR4 gene have been associated with different infectious diseases in humans and with infection and disease in cattle, chicken and pigs. The investigators identified eighteen SNPs in the TLR4 gene of two pig populations. Furthermore, a specific sequence was found more often in pigs that shed low levels of Salmonella in their feces compared to pigs that persistently shed higher levels of Salmonella. Our results highlight the importance of linking genetic variations that may influence the function of a key immune regulator (TLR4) with a desirable trait - low Salmonella shedding in swine.


4.Accomplishments
1. Gastrointestinal microbiota influences Salmonella shedding in pigs. Salmonella causes the most bacterial foodborne disease and death in the United States. ARS researchers in Ames, Iowa examined the influence of the bacteria residing in the intestinal tract of pigs (gastrointestinal (GI) microbiota) on Salmonella shedding. The results of the study indicate that during swine production, the gastrointestinal microbiota could be influenced by sub-clinical Salmonella infections with potential impacts on animal health, growth performance and production profits. Furthermore, the research results have identified promising approaches to reduce Salmonella fecal shedding which will improve food safety because Salmonella-carrier pigs can contaminate the environment at numerous points along the farm-to-fork continuum.

2. Salmonella vaccine protects pigs against disease and reduces transmission. ARS researchers in Ames, Iowa have designed and demonstrated effectiveness of a vaccine for Salmonella. The live vaccine can both protect swine from Salmonella that cause systemic pig disease and also reduce the transmission of Salmonella that cause foodborne disease in humans. Furthermore, the vaccine was designed to be able to tell the difference between vaccinated and infected animals. Because most foodborne disease agents are transferred to humans from animals/animal products, interventions in the animal reservoir are necessary to fully optimize control strategies. This is especially important as livestock production systems continue to increase in size and complexity, and antibiotic usage in feed becomes increasingly controversial. This dual use Salmonella vaccine is intended to not only protect the health status of the herd to reduce production costs, but also support food safety and public health by reducing the spread of human foodborne Salmonella. We anticipate that other livestock industries will also benefit from the cross-protective vaccine.


Review Publications
Bearson, S.M., Allen, H.K., Bearson, B.L., Looft, T.P., Brunelle, B.W., Kich, J.D., Tuggle, C.K., Bayles, D.O., Alt, D.P., Levine, U.Y., Stanton, T.B. 2013. Profiling the gastrointestinal microbiota in response to Salmonella: low versus high Salmonella shedding in the natural porcine host. Infection, Genetics and Evolution. 16:330-340.

Mullins, M., Register, K.B., Brunelle, B.W., Aragon, V., Galofre-Mila, N., Bayles, D.O., Jolley, K.A. 2013. A curated public database for multilocus sequence typing (MLST) and analysis of Haemophilus parasuis based on an optimized typing scheme. Veterinary Microbiology. 162(2-4):899-906.

Last Modified: 9/29/2014
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