2009 Annual Report
1a.Objectives (from AD-416)
The host/pathogen interaction is poorly understood because establishment of an infection in livestock by a microorganism is a dynamic event. The complex interaction between the host and the pathogen can now be explored in more detail using microarrays. In this project, we will evaluate the genetic interaction of livestock and pathogenic bacteria during colonization in an attempt to elucidate and interpret the mechanistic roles of gene regulation in pathogenesis. Our goal is to develop a comprehensive knowledge of host/pathogen interactions during colonization and to elucidate how these interactions are affected by stress. These studies will lead to new and/or improved diagnostic techniques, new therapeutics and vaccines, and improved health management strategies for livestock.
1b.Approach (from AD-416)
Objective 1: Utilize microarray, microbiologic, and bioinformatic approaches in a ligated-intestinal model to elucidate host/pathogen genetic regulatory interactions during infection. Approach: We hypothesize that the pathogen, when introduced into the host environment, will regulate its virulence factors to adapt to the intestinal environment to achieve a colonized state. In turn, we expect the host will regulate its immune response at the site of infection to prevent or respond to the pathogen’s attempts to establish a foothold. We will utilize various molecular methods such as microarrays, in conjunction with a ligated intestinal model and directed colonization approach, to evaluate these interactions. Objective 2: Using two stress models (transportation and neuroendocrine) in conjunction with Salmonella challenge in swine, develop an understanding of the relationship between various forms of stress and food borne pathogen susceptibility and shedding. Approach: Our hypothesis is that an animal has differential physiological and immunological responses to various stressors. These responses are unique to the type of stress encountered or experienced. Therefore, different stressors differentially affect a host’s susceptibility to, and shedding of, food borne pathogens. We will expose pigs to two types of stress. We hypothesis the initiation of a stress response via peripheral CRH should have a negative influence that will lower the natural immunity of the host and increase susceptibility to pathogens. If our hypothesis is correct, then a lower infectious dose of pathogen will be required to induce infection and shedding. Objective 3: Model on-farm management practices to evaluate pathogen transmission in swine herds. Approach: The hypothesis is that there is a difference in pathogen transmission rates within swine herds which is dependent upon the type of housing system (group versus individual pens). This objective will be accomplished by experimentally infecting one individual within each type system and monitoring the spread of infection among the group. Individually, these separate transmission rates (pen or stall housing) can provide specific information on its particular system, but together these rates can be used in the future to design and generate more universal models and more appropriate rates for on-farm Salmonella herd transmission.
This will serve as the final report for this project, as it was combined with the 6208-32000-006-00D project within the Livestock Issues Research Unit during FY2009. Following the resignation of the lead scientist associated with this project, completion of the outlined studies for FY2009 was not feasible. Major accomplishments by ARS and collaborating scientists throughout the life of this project include:.
1)Identification of genetic factors that influence the pathogenesis of Salmonella;.
2)Identification of genes that control the pathogenic communication system in pathogens;.
3)Elucidation of genetic evidence that different pathogens can communicate with each other;.
4)Identification of physiological changes in the bacteria that promote antibiotic-resistance;.
5)Providing evidence that the control of virulence genes in pathogens can be influenced by their environment; and.
6)Results that demonstrated that a variety of Escherichia coli O157:H7 suspected of having less potential to be transmitted and cause disease in humans, expresses significantly less toxin than the more virulent variety of this bacteria. Collectively, these findings provide important information that will be necessary in the implementation of future studies to further elucidate important host-pathogen interactions in economically important livestock species.
Bacteria communicate or "Bac Talk": Collaborative studies with scientists from the Livestock Issues Research Unit in Lubbock, Texas, and Texas A&M University evaluated genetic interactions in Salmonella enterica that influence pathogenesis of this important foodborne pathogen. Quorum sensing, or cell-to-cell communication, is made possible by the production and sensing of small, extracellular chemical signals called autoinducers (AI). These autoinducers accumulate as the population density increases, and thereby help bacteria to regulate their behavior by promoting or repressing gene expression. Results from these experiments suggest not only that AI-2 acts as a master controller of genes for pathogenicity of Salmonella Typhimurium, but also that Salmonella Typhimurium could sense the AI-2 molecules produced by E. coli in a multi-species environment. This information will be especially beneficial in future research endeavors attempting to elucidate the complex host-pathogen interactions as it highlights the importance of the bacterial environment and the communication that takes place between diverse bacterial populations.
Understanding Antibiotic Resistance in Foodborne Pathogens: Antibiotics are important tools used to control infections. Unfortunately, microbes can become resistant to antibiotics, which limits the drugs' usefulness for clinical and veterinary use. ARS scientists collaborating with Texas Tech University, Texas Tech University Health Sciences Center, and Washington State University derived a multi-drug-resistant mutant of Salmonella and utilized modern molecular genetics techniques to study how such resistance develops. Results indicate a variety of physiological changes in the bacteria which promote elimination of the antibiotics from the pathogen's cells and other mechanisms that prevent antibiotics from entering the cell. These results will allow scientists to better understand the molecular, genetic, and physiological changes that contribute to antibiotic resistance in these pathogens. Such understanding will improve our ability to prevent the development of antibiotic resistance and our ability to design better treatments for these pathogens.
Are All Escherichia coli O157:H7 toxic? ARS scientists from the Livestock Issues Research Unit in Lubbock, Texas, examined a defined collection of two lineages of Escherichia coli O157:H7 for genetic factors that cause infection in humans and transmission from food to human hosts. Molecular and biochemical assays were utilized to investigate the difference in toxin expression between the two lineages. It was conclusively shown that a variety of this bacteria previously suspected of having less potential to be transmitted and cause disease in humans, actually expresses significantly less toxin. A tremendous amount of research will still be needed in order to fully evaluate the existence of a less dangerous variety of O157:H7, and studies will be needed to ensure that these less pathogenic lineages cannot convert to more virulent forms. The future of this research, if one of these lineages can be conclusively proven not to be a public health risk, is of great significance to beef producers and could prevent billions of dollars in meat recalls.
Genes that Control Communication in Bacteria: Collaborative studies with scientists from the Livestock Issues Research Unit in Lubbock, Texas, and Texas A&M University evaluated genetic interactions in Salmonella enterica that influence pathogenesis of this important foodborne pathogen. Through these studies, researchers revealed an important gene that is associated with the communication within bacterial that dictates whether or not toxins will be expressed by a particular strain of bacteria. This gene (luxS) was shown to be a master regulator of the autoinducer molecule-2 (AI-2) that controls pathogenicity of bacteria. The expression of this important communication gene was also shown to be regulated by the environmental conditions in which the bacteria were grown. Collectively, these data demonstrate that luxS/AI-2 plays a vital role in a variety of processes such as metabolism, virulence gene expression, motility, transcription, and translation. Understanding this critical control genes associated with toxin production by bacteria is essential in further efforts to prevent foodborne illnesses from contaminated meat products.
Crippen, T.L., Sheffield, C.L., Andrews, K., Dowd, S.E., Nisbet, D.J. 2008. Planktonic and biofilm community characterization and Salmonella resistance of 14-day old chicken cecal microflora derived continuous-flow cultures. Journal of Food Protection. 71:1981-1987.
Chiang, H., Swaggerty, C.L., Kogut, M.H., Dowd, S.E., Li, X., Pevzner, I.Y., Zhou, H. 2008. Gene expression profiling in chicken heterophils with Salmonella enteritidis stimulation using a chicken 44 K Agilent microarray. Biomed Central (BMC) Genomics. 9:Article 526.
Acosta Martinez, V., Dowd, S.E., Sun, Y., Allen, V. 2008. Tag-encoded pyrosequencing analysis of bacterial diversity in a single soil type as affected by management and land use. Soil Biology and Biochemistry. 40(11):2762-2770.