Progress 12/10/10 to 12/09/15
Outputs
Progress Report 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. 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. This is the final report for project 5030-32000-101-00D terminated in December 2015 and replaced with 5030-32000-113-00D. The human foodborne pathogen Salmonella can reside in the gut of food- animals for extended periods of time without causing clinical symptoms. This Salmonella carrier-status poses long-term contamination threats throughout the food chain. Thus, implementing Salmonella control strategies at the farm will reduce environmental contamination, and concerns associated with antibiotic usage and transfer of antibiotic resistance via Salmonella. A live Salmonella vaccine was designed that not only protects swine from strains of Salmonella that cause systemic disease in the pig but also reduces the transmission of Salmonella that causes foodborne disease in humans. Furthermore, the vaccine was designed to differentiate between vaccinated and infected animals. This dual purpose Salmonella vaccine is intended to not only protect the health status of the swine herd to reduce production costs, but also support food safety and public health by reducing the spread of human foodborne Salmonella. A potential method for Salmonella intervention is to select animals with improved genetic resistance to Salmonella. ARS researchers at Ames, Iowa have shown that the expression of specific genes in pigs is related to the amount of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) being shed in the feces of the pigs. We revealed that even before pigs were exposed to Salmonella Typhimurium, pigs that would ultimately shed lower levels of Salmonella were already expressing genes associated with the immune response, suggesting that these animals were better prepared for the Salmonella Typhimurium exposure compared to the "will-be" high Salmonella-shedding pigs. These gene expression patterns could potentially be used to identify pigs for genetic selection. Identifying alternative methods for the control and prevention of Salmonella in food animals (such as genetic improvement) is critical for improved profitability for food-animal producers, reduced antibiotic usage, and safer food for consumers. Small ribonucleic acid molecules called microRNA regulate host gene expression involved in a variety of physiological, immunological, and pathological activities. Our investigation of the role of microRNAs in the regulation of the pig�s response to Salmonella Typhimurium identified 50 microRNAs involved in inflammation, immunity and cell lysis. Defining strategic regulatory factors of the host response to Salmonella may identify novel targets for control of bacterial food safety issues. In further support of Objective 2, differences in the swine genome sequence were evaluated to find associations with pigs that had reduced levels of Salmonella. ARS researchers at Ames, Iowa, in collaboration with scientists at Iowa State University, searched greater than 3,000 porcine genes that respond to Salmonella Typhimurium for genetic variations in their deoxyribonucleic acid (DNA) sequence. Thirty-two single nucleotide polymorphisms (SNPs) within the 3,000 Salmonella- responsive pig genes were identified. Four independent pig populations were genotyped for the SNPs, and 13 of the SNPs were associated with Salmonella fecal shedding or tissue colonization. A specific genetic variation in an important activator of the pig�s immune response, was identified more often in pigs that shed low levels of Salmonella in their feces compared to pigs that persistently shed higher levels of Salmonella. These genetic differences in pigs associated with Salmonella shedding provide insight in identifying and selecting for pigs with increased resistance to Salmonella colonization to improve food safety. Research completed during the project determined that the bacteria residing in the intestinal tract of pigs (gastrointestinal microbiota) influences Salmonella shedding. We described 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. The research results identified promising prebiotic and probiotic 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. Antibiotics are used in food animal production for the prevention and treatment of bacterial diseases, but antibiotic usage can have unintended effects. ARS researchers at Ames, Iowa reported that the antibiotics carbadox, enrofloxacin and danofloxacin, which are used in agriculture systems, promote bacterial lysis and the release of bacterial viruses (called bacteriophage or phage) in specific multidrug-resistant (MDR) Salmonella Typhimurium isolates. Horizontal gene transfer of a kanamycin resistance gene occurred when the released phage containing deoxyribonucleic acid (DNA) from the MDR Salmonella Typhimurium infected a kanamycin-sensitive Salmonella Typhimurium strain, demonstrating that these agriculturally-used antibiotics can cause dissemination of antibiotic resistance genes from MDR Salmonella Typhimurium to non- resistant bacteria. Antibiotic-induced, phage-mediated gene transfer highlights an unintentional outcome of antibiotic usage that should be considered by veterinarians and producers when selecting an antibiotic for bacterial disease treatment. Infection with multidrug-resistant (MDR) Salmonella is correlated with increased morbidity in humans. One bacterial factor that is associated with morbidity is the ability of Salmonella to attach to host cells using a structure called the fimbriae. In support of Objective 1, researchers at Ames, Iowa measured changes in the production of fimbriae in Salmonella Typhimurium after exposure to various antibiotics and found that the antibiotics tetracycline and chloramphenicol, which are used in swine and humans, significantly increased the production of fimbriae. In addition, the researchers showed that invasion of epithelial cells by some MDR Salmonella Typhimurium isolates was increased by tetracycline. This is important as increases in fimbriae are linked to the persistence of Salmonella in animals, and overall, may play a role in increasing the intensity and severity of MDR Salmonella infections. During completion of Objective 2, researchers at Ames, Iowa identified a novel survival and virulence mechanism in Salmonella Typhimurium that, when mutated, dramatically reduced the ability of the bacterium to survive numerous stress conditions as well as antibiotic and chemical exposures. Furthermore, the gene mutation decreased the ability of Salmonella Typhimurium to colonize the pig. As this genetic system is critical for the ability of the Salmonella Typhimurium to cause disease and resist antibiotics, it offers a novel target for development of intervention strategies against Salmonella.
Impacts
(N/A)
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. Over 35 percent of Salmonella enterica serovar Typhimurium isolates are resistant to three or more different antibiotics. This is a public health concern as multidrug-resistant (MDR) Salmonella are associated with increased disease severity compared to patients infected with antibiotic- sensitive isolates. Bacterial motility is often considered a virulence factor in pathogenic bacteria. To address Objective 1 �Identify molecular mechanisms in Salmonella that impact virulence and antimicrobial resistance,� we conducted tests to determine how several antibiotics affected the motility of MDR Salmonella. We found that most antibiotics either decreased or had no effect on motility. However, the antibiotic kanamycin significantly increased motility. Future studies will focus on identifying the mechanism required for the observed motility differences to determine how different antibiotics can either positively or negatively influence this virulence factor in MDR Salmonella. A major contributor in the development of the host�s immune response to disease-causing agents is microRNA (miRibonucleic Acid). MicroRNAs regulate host gene expression involved in a variety of physiological, immunological, and pathological activities. In support of objective 2 �Characterize the genetic pathways in swine that respond to Salmonella infection,� we investigated the role of microRNAs in the regulation of the pig�s response to the human foodborne pathogen, Salmonella enterica serovar Typhimurium. Fifty microRNAs involved in inflammation, immunity and cell lysis were identified as differentially-regulated in response to Salmonella over a 7-day infection period. Defining strategic regulatory factors of the host response to Salmonella may identify novel targets for control of bacterial food safety issues. Accomplishments 01 Agricultural antibiotics trigger multidrug-resistant Salmonella to transfer genes. Antibiotics are used in food animal production for the prevention and treatment of bacterial diseases, but antibiotic usage can have unintended effects. ARS researchers in Ames, Iowa reported that the agriculturally-used antibiotics carbadox, enrofloxacin and danofloxacin caused specific multidrug-resistant (MDR) Salmonella isolates to lyse and release bacterial viruses (called bacteriophage or phage). The released phage contained a kanamycin (an antibiotic) resistance gene from the MDR Salmonella that infected a kanamycin- sensitive Salmonella strain, resulting in horizontal gene transfer of the antibiotic resistance. This research demonstrates that these agriculturally-used antibiotics can cause dissemination of antibiotic resistance genes from MDR Salmonella to na�ve bacteria. Antibiotic- induced, phage-mediated gene transfer highlights an unintentional outcome of antibiotic usage that should be considered by veterinarians and producers when selecting an antibiotic for bacterial disease treatment.
Impacts
(N/A)
Publications
- Bearson, B.L., Bearson, S.M., Kich, J.D., Lee, I.S. 2014. An rfaH mutant of Salmonella enterica serovar typhimurium is attenuated in swine and reduces intestinal colonization, fecal shedding, and disease severity due to virulent Salmonella Typhimurium. Frontiers in Veterinary Science. Available:
- Brunelle, B.W., Bearson, B.L., Bearson, S.M. 2015. Chloramphenicol and tetracycline decrease motility and increase invasion and attachment gene expression in specific isolates of multidrug-resistant Salmonella enterica serovar Typhimurium. Frontiers in Microbiology. 5(801). Available:
- Kommadath, A., Bao, H., Arantes, A.S., Plastow, G.S., Tuggle, C.K., Bearson, S.M., Guan, L., Stothard, P. 2014. Gene co-expression network analysis identifies porcine genes associated with variation in Salmonella shedding. Biomed Central (BMC) Genomics. 15(452). Available:
- Knetter, S.M., Bearson, S.M., Huang, T., Kurkiewicz, D., Schroyen, M., Nettleton, D., Berman, D.J., Cohen, V., Lunney, J.K., Ramer-Tait, A.E., Wannemuehler, M.J., Tuggle, C.K. 2014. Salmonella enterica serovar Typhimurium-infected pigs with different shedding levels exhibit distinct clinical, peripheral cytokine and transcriptomic immune response phenotypes. Innate Immunity. 21(3):227-241.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. Salmonella shedding in pigs is controlled by pig gene expression. The human foodborne pathogen, Salmonella can reside in the gut of food- animals for extended periods of time without causing clinical symptoms. This Salmonella carrier-status poses long term contamination threats throughout the food chain. Thus, implementing Salmonella control strategies at the farm will reduce environmental contamination and concerns associated with antibiotic usage. One potential method of intervention is to select animals with improved genetic resistance to Salmonella. ARS Researchers in Ames, IA in collaboration with Iowa State University and the University of Alberta, Canada have shown that the expression of specific genes in pigs is related to the amount of Salmonella being shed in the feces of the pigs. These collaborators revealed that even before pigs were exposed to Salmonella, pigs that would ultimately shed lower levels of Salmonella were already expressing immune response genes, suggesting that these animals were better prepared for the Salmonella exposure compared to the "will-be" high Salmonella- shedder pigs. Identifying alternative methods for the control and prevention of Salmonella in food animals (such as genetic improvement) is critical for improved profitability for food-animal producers, reduced antibiotic usage, and safer food for consumers. Antibiotics enhance factors associated with persistence in multidrug- resistant Salmonella. More than 27 percent of Salmonella enterica serovar Typhimurium isolates from humans in the United States are resistant to three or more antibiotics. This presents an important food safety concern as multidrug-resistant (MDR) Salmonella is correlated with increased morbidity in humans. One bacterial factor that is associated with morbidity is the ability of Salmonella to attach to host cells using a structure called the fimbriae. Our goal was to measure changes in the production of fimbriae in Salmonella after exposure to antibiotics using next-generation high-throughput ribonucleic acid sequencing (RNA-seq). We found that the antibiotics tetracycline and chloramphenicol significantly increased the production of fimbriae. This is important as such increases are linked to the persistence of Salmonella in the host. Reduced Salmonella fecal shedding in pigs given an immune stimulator. Key objectives of pre-harvest food safety programs are to detect asymptomatic Salmonella carriage in food animals, reduce colonization, and prevent transmission of Salmonella to other animals and into the environment. One possible on-farm intervention is the use of immune modulators to boost the animal�s immune response prior to periods of stress, such as weaning and transportation, to reduce Salmonella shedding and transmission. ARS Researchers in Ames, IA evaluated the use of a porcine immune stimulator (granulocyte-colony stimulating factor; G-CSF) to increase a specific immune cell in the blood (neutrophils) that is important in the control of Salmonella infections. Pigs that were given the immune stimulator shed less Salmonella in their feces and carried less Salmonella in their tonsils compared to pigs that did not receive the immune stimulator. This data suggest that the prophylactic use of the porcine immune modulator should be explored as a potential alternative to antibiotics in food animals. Accomplishments 01 Salmonella 'alarmed' by the antibiotic tetracycline. 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 multidrug-resistant Salmonella Typhimurium (greater than 30 percent of isolates in humans and livestock are resistant). Because antibiotic exposure can affect the ability of multidrug-resistant bacteria to cause disease (also known as virulence), ARS Researchers in Ames, IA examined the effect of tetracycline on the virulence of multidrug-resistant Salmonella Typhimurium. We have found that, in some multidrug-resistant Salmonella Typhimurium, tetracycline promotes the ability of Salmonella to enter (invade) cells of a host. The impact of this virulence-promoting effect of tetracycline is that the antibiotic may play a role in increasing the intensity and severity of multidrug- resistant Salmonella infections. Therefore, prudent use of tetracycline in animals would limit the potential for Salmonella to cause severe disease, resulting in a healthier food supply.
Impacts
(N/A)
Publications
- Kich, J.D., Uthe, J.J., Benavides, M.V., Cantao, M.E., Zanella, R., Tuggle, C.K., Bearson, S.M. 2014. TLR4 single nucleotide polymorphisms (SNPs) associated with Salmonella shedding in pigs. Journal of Applied Genetics. 55(2):267-271.
- Brunelle, B.W., Bearson, S.M., Bearson, B.L. 2013. Tetracycline accelerates the temporally-regulated invasion response in specific isolates of multidrug-resistant Salmonella enterica serovar Typhimurium. BMC Microbiology. DOI: 10.1186/1471-2180-13-202.
- Bearson, S.M., Bearson, B.L., Lee, I., Kich, J.D. 2013. Polynucleotide phosphorlyase (PNPase) is required for Salmonella enterica serovar Typhimurium colonization in swine. Microbial Pathogenesis. 65:63-66.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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.
Impacts
(N/A)
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.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. The incidence of multidrug-resistant (MDR) Salmonella is an important health and food safety concern in humans as 33.1 percent of Salmonella Typhimurium isolates are resistant to at least one class of antibiotics, and 20.8 percent are resistant to five or more classes. Because the response of drug resistant bacteria exposed to antibiotics can affect virulence, we investigated the effect of tetracycline (a commonly used antibiotic in animal feed) on isolates of MDR Salmonella Typhimurium. We found that exposure to tetracycline induced invasion in several Salmonella isolates. These results indicate that the invasive capabilities of a subset MDR Salmonella Typhimurium are enhanced in the presence of tetracycline. Salmonella-shedding pigs contaminate other animals, meat products during processing, and crops when Salmonella-containing manure is used as a soil fertilizer. To identify factors that may influence Salmonella shedding in pigs, we are examining the bacteria residing in the intestinal tract of pigs, referred to as commensal bacteria or the intestinal microbiota. We have sequenced a common gene within this �community� of bacteria in pigs to determine which bacteria are present before and after Salmonella challenge that might affect Salmonella shedding levels. Our goal is to identify Salmonella antagonists (or their bio-products) that reduce Salmonella shedding in pigs to decrease environmental contamination and increase food safety. Accomplishments 01 Linking genetic variations in porcine genes to colonization and shedding of Salmonella in pigs. To address this strategy, ARS researchers at Ames IA and collaborators at Iowa State University searched greater than 3,00 porcine genes to identify genetic variations in pig genes that respond t Salmonella. DNA segments know as single nucleotide polymorphisms (SNPs) were identified in 31 pig genes whose expression is altered in response Salmonella. To determine if the identified porcine SNPs were associated with tissue colonization or fecal shedding of Salmonella, four independe pig populations were genotyped for the SNPs. Statistical analysis reveal 13 SNPs that associated with Salmonella fecal shedding or tissue colonization. These genetic differences in pigs associated with Salmonel shedding provide insight in identifying and selecting for pigs with increased resistance to Salmonella colonization to improve food safety (for example, development of diagnostic tests to screen pig lines for th desired trait).
Impacts
(N/A)
Publications
- Uthe, J.J., Bearson, S.M., Qu, L., Dekkers, J.C., Nettleton, D., Y Rodriguez, T., O'Connor, A., Mckean, J., Tuggle, C.K. 2011. Integrating comparative expression profiling data with association of SNPs to Salmonella shedding for improved food safety and porcine disease resistance. Animal Genetics. 42(5):521-534.
- Uthe, J.J., Qu, L., Couture, O., Bearson, S.M., O'Connor, A., Mckean, J., Rodriguez, T.Y., Dekkers, J.C., Nettleton, D., Tuggle, C.K. 2011. Use of bioinformatic SNP predictions in differentially expressed genes to find SNPs associated with Salmonella colonization in swine. Journal of Animal Breeding and Genetics. 128(5):354-365.
- Brunelle, B.W., Sensabaugh, G.F. 2012. Nucleotide and phylogenetic analyses of the Chlamydia trachomatis ompA gene indicates it is a hotspot for mutation. BMC Research Notes [serial online]. 5(53). Available:
- Huang, T., Uthe, J.J., Bearson, S.M., Demirkale, C.Y., Nettleton, D., Knetter, S.M., Christian, C.M., Ramer-Tait, A.E., Wannemuehler, M.J., Tuggle, C.K. 2011. Distinct peripheral blood RNA responses to Salmonella in pigs differing in Salmonella shedding levels: intersection of IFNG, TLR and miRNA pathways. PLoS One [serial online]. 6(12). Available:
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. Within a host, salmonellae are exposed to a variety of different environmental conditions; however, it is not known how antibiotics affect the virulence of drug-resistant Salmonella Typhimurium DT104 under these various conditions. In order to test the effect each antibiotic has on invasion under a wide range of different growth conditions, we developed a high-throughput screening tool. A fluorescent reporter plasmid was constructed to determine the relative level of salmonella invasiveness by measuring the key invasion-regulating protein HilA. This allows us to quickly screen a multitude of different combinations of antibiotics, antibiotic concentrations, antibiotic exposure durations, and growth conditions, thereby determining which combination of antibiotics and environmental conditions affect Salmonella Typhimurium DT104 invasion. Since over 2,500 serovars of Salmonella exist, a live Salmonella vaccine was designed to improve vaccination strategies by providing broad protection against Salmonella. The Salmonella vaccine was tested in pigs and provided protection against subsequent exposure to a pathogenic strain of Salmonella. The goal is to provide pig producers with a vaccine that will improve both animal health and food safety. Accomplishments 01 Identification of a novel survival and virulence mechanism in Salmonella Salmonella Typhimurium is a human foodborne pathogen and is one of the most prominent Salmonella serovars isolated from swine production farms. Unfortunately, Salmonella Typhimurium can undetectably reside in pigs without causing noticeable infection. These Salmonella-carrier pigs are food safety problem for humans through contamination of penmates, the environment and slaughter plants that process pork for consumption. In searching for improved intervention strategies against Salmonella on the farm, ARS researchers in Ames, IA, have identified a gene (poxA) in Salmonella Typhimurium that, when mutated, dramatically reduces the ability of the bacterium to survive numerous stress conditions as well a antibiotic and chemical exposures. Furthermore, the gene mutation decreased the ability of Salmonella Typhimurium to colonize the pig. As this genetic system is critical for the ability of the Salmonella to cau disease and resist antibiotics, it offers a novel target mechanism for intervention development against Salmonella.
Impacts
(N/A)
Publications
- Bearson, B.L., Bearson, S.M. 2011. Host specific differences alter the requirement for certain Salmonella genes during swine colonization. Veterinary Microbiology. 150:215-219.
- Bearson, S.M., Bearson, B.L., Brunelle, B.W., Sharma, V.K., Lee, I. 2011. A mutation in the poxA gene of Salmonella enterica serovar Typhimurium results in altered protein production, elevated susceptibility to environmental challenges, and decreased swine colonization. Foodborne Pathogens and Disease. 8(6):725-732.
- Tuggle, C.K., Bearson, S.M., Uthe, J.J., Huang, T.H., Qu, L., Couture, O.P. , Wang, Y., Kuhar, D.J., Lunney, J.K., Nettleton, D., Honavar, V., Dekkers, J.C. 2010. Methods for transcriptomic analyses of the porcine host immune response: application to Salmonella infection using microarrays. Veterinary Immunology and Immunopathology. 138(4):280-291.
- Brunelle, B.W., Bearson, S.M., Bearson, B.L. 2011. Salmonella enterica serovar Typhimurium DT104 invasion is not enhanced by sub-inhibitory concentrations of the antibiotic florfenicol. Journal of Veterinary Science and Technology. 2:104. Available:
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