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United States Department of Agriculture

Agricultural Research Service

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Location: Livestock Issues Research

2008 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.

3.Progress Report
Research conducted within the Livestock Issues Research Unit during FY2008 was associated with National Program 108, Component 1, Problem Statement 1.1.3 Ecology and Host Pathogen Relationships. Only with the accumulation of functional genetic regulation data that is specific for a particular host/pathogen interaction can development of improved therapeutic and management practices that reduce persistence, shedding, and contamination of these pathogens be developed in a logical manner. Research was conducted to evaluate the genetic interaction between pigs and the foodborne pathogen Salmonella enterica DT104. Microarray data was analyzed which showed that early infection with S. enterica DT104 may be regulated by IL-27 providing a potential target to enhance the immune response of young pigs and prevent colonization with this noted pathogen. A collaborative study was begun to evaluate pigs as a model for Escherichia coli O157:H7 early infection. Results showed valuable changes in the regulation of genes in this important foodborne pathogen highlighting the utility of this system as a model for O157:H7 infectivity. Collaborative studies were conducted generating data using an in-vitro model of O157:H7 infection of bovine neutrophils. Research was conducted to evaluate a model system for the transmission of Salmonella enterica within a swine nursery facility. Research to develop a more sensitive method for the evaluation of Salmonella and E. coli infection for use in stress and transmission studies was conducted. Finally, research was conducted evaluating the effects of 3 different physiological stressors on Salmonella enterica infection in pigs. These research projects allowed us to fully meet our milestones for the year. Collectively, these studies allowed us to make significant progress with regard to all of our stated objectives within this CRIS project for the FY2008 period, and provided important information that will be necessary in the implementation of future studies. (NP 108, Component 1, Problem Statement 1.1.3).

1. Genetic factors of Salmonella that affect pathogenesis: Collaborative studies continued with scientists from the Livestock Issues Research Unit in Lubbock, Texas, and Texas A&M University to evaluate genetic interactions in Salmonella enterica that influence pathogenesis of this important foodborne pathogen. Bacterial communication via quorum sensing (QS) has been reported to be important in the production of virulence factors, antibiotic sensitivity, and biofilm development. High-density oligonucleotide microarrays for the Salmonella enterica Typhimurium genome were used to investigate global gene expression patterns of Salmonella Typhimurium luxS mutant modulated by in vitro synthesized AI-2. In the presence of synthetic AI-2, 708 genes were differentially expressed (350 up-regulated and 358 down-regulated). Although the only known role of AI-2 in Salmonella Typhimurium is the regulation of the lsr operon which helps to internalize AI-2, the microarray analysis reveals that AI-2 also represses virulence genes in Salmonella Typhimurium. Our results suggest that AI-2 could be a master regulator of genes in the pathogenicity of Salmonella Typhimurium. (NP108, Component 1, Problem statement 1.1.1)

2. Communication system in pathogens influences pathogenesis: Collaborative studies continued with scientists from the Livestock Issues Research Unit in Lubbock, Texas, and Texas A&M University to evaluate genetic interactions in Salmonella enterica that influence pathogenesis of this important foodborne pathogen. The luxS gene, which is conserved in several Gram-positive and Gram-negative bacteria, is involved in the synthesis of the autoinducer molecule-2 (AI-2). Genes controlled by luxS in S. Typhimurium were identified using microarrays from wild type Salmonella Typhimurium and its isogenic luxS mutant, in two growth conditions (presence and absence of glucose), and at two different time points. Minimal differential gene expression was observed in the presence of glucose. In the absence of glucose, a total of 1560 genes were differentially expressed; 1361 genes were identified as luxS/AI-2-regulated at the mid-log phase and 199 genes at the early-stationary phase. The results suggest that although the expression of the luxS gene in Salmonella Typhimurium is independent of the growth condition, its role in the production of AI-2 and the regulation of genes depends on the growth condition. It was found that luxS/AI-2 plays a vital role in a variety of processes such as metabolism, virulence gene expression, motility, transcription, and translation. AI-2 was observed to control several key virulence genes. (NP108, Component 1, Problem Statement 1.1.1)

3. Different pathogens can communicate with each other: Collaborative studies continued with scientists from the Livestock Issues Research Unit in Lubbock, Texas, and Texas A&M University to evaluate 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. DNA microarray experiments were conducted on luxS mutant of Salmonella Typhimurium to study its gene regulation in the presence of condition media (CM) of its own wild type and CM of Escherichia coli. It was observed that in the presence of CM of Salmonella Typhimurium, 1143 genes were differentially expressed (504 repressed and 639 expressed). In the presence of CM of E. coli, 392 genes were observed to be differentially expressed (133 repressed and 259 expressed). It was found that all the genes in the pathogenicity island of luxS mutant of Salmonella Typhimurium were turned down in the presence of CM of E. coli, and CM of Salmonella Typhimurium. These experiments not only suggest that AI-2 acts as a master controller of genes in the pathogenicity island of Salmonella Typhimurium, but also that Salmonella Typhimurium could sense the AI-2 molecules produced by E. coli in a multi-species environment. (NP108, Component 1, Problem Statement 1.1.1)

4. Importance of 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. (NP108, Component 1, Problem Statement 1.1.5)

5. Control of virulence genes in the foodborne pathogen Listeria monocytogenes are influenced by their environment: Collaborative studies continued with scientists from the Livestock Issues Research Unit in Lubbock, Texas, and University of Arkansas to evaluate genetic interactions in Listeria monocytogenes (L.m) that influence pathogenesis of this important foodborne pathogen. The effects of an inhibitor of proton motive force (PMF) and an inhibitor of pyruvate dehydrogenase (PD) on transcriptional changes in L.m and its sigB mutant were compared. 2,4-dinitrophenol (DNP) was used to inhibit PMF while sodium arsenite (SAs) was used to inhibit PD. These inhibitors were added individually in separate experiments to L.m 10403S wild type and a sigB mutant strain growing at log phase (23 deg C) in Brain Heart Infusion broth. The resulting transcriptomes were analyzed using Pathogen Functional Genomics Resource Center (PFGRC) microarrays and protocols. During growth in the presence of DNP, 193 and 223 genes were differentially expressed for the wild type and sigB mutant, respectively. The data shows that cellular processes involving cell division or DNA metabolism appear to be PMF-dependent while those involving the phosphotransferase system appear to be PMF-independent. Repression of genes related to cell division or synthesis/degradation of cellular components occurred following PMF loss. During growth in the presence of SAs, differential expression of 742 and 134 genes were exhibited by the wild type and sigB mutant, respectively. The results indicate that most of the genes involved in regulation during growth of L. m may be sigB-dependent. Induction of protein synthesis genes also occurred following the addition of SAs. Interestingly, the reaction catalyzed by SAs may provide an energy source for protein synthesis. In addition, energy stress induced by SAs led to induction of the fur and prfA genes. (NP108, Component 1, Problem Statement 1.1.1)

6. Comparison of Shiga-like Toxin II expression between two diverse lineages of Escherichia coli O157:H7: 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 Shiga-like Toxin II (stx2) expression between the two lineages. It was conclusively shown that a variety of this bacteria suspected of having less potential to be transmitted and cause disease in humans, 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. (NP108, Component 1, Problem Statement 1.2.1)

6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings4

Review Publications
Dowd, S.E., Sun, Y., Secor, P.R., Rhoads, D.D., Wolcott, B.M., James, G.A., Wolcott, R.D. 2008. Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing. BMC Microbiology. 8:article 43.

Dowd, S.E., Thurston Enriquez, J.A. 2008. Evaluation and guidelines for use of polymerase chain reaction-computer database homology comparison (PCR-CDHC) for detection and species determination of human pathogenic microsporidia. African Journal of Microbiology Research. 2(1):001-007.

Dowd, S.E., Callaway, T.R., Sun, Y., McKeehan, T., Edrington, T.S. 2008. Evaluation of the bacterial diversity in the feces of cattle using bacerial tag-encoded FLX amplicon pyrosequencing (bTEFAP). BMC Microbiology. 8:article 125.

Last Modified: 4/18/2014
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