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Long-term goal: To prevent and control enteric diseases of cattle, swine and chickens with a mandate to decrease food and waterborne illness in the USA. <P>Objectives: 1. Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals. 2. Focus on effective interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and food borne disease agents. 3. Focus on disseminating knowledge: We will provide training or continuing education to disseminate new information to students, producers, veterinarians, diagnostic labs and others to implement interventions and preventative measures. 4. Group interaction: The group will interact in a variety of ways to facilitate progress including direct collaborations with joint publications, sharing of resources (pathogen strains, gene sequences, statistical analysis, bioinformatics information/expertise), and friendly feedback and facilitation for all research efforts at annual meetings. <P>Nebraska-Specific Subobjectives: 1. Identify carbohydrates and amino acids regulating differential expression of heat labile enterotoxin (LT) and heat stable enterotoxin-b (STb) in the small intestine. 2. Determine the relationship between LT and STb expression with regard to bacterial colonization and washout from the small intestine.
Non-Technical Summary:<br/>
Diseases result in severe economic losses to the swine industry, resulting from a reduced rate of gain and mortality. Escherichia coli infections cause a significant proportion of the diseases affecting the swine industry. "E. coli diarrhea" (enteric colibacillosis) is the third most common disease diagnosis in pigs, and is caused by enterotoxigenic E. coli (ETEC) strains. Those ETEC strains that express attachment surface proteins known as K88 (F4) fimbria, and toxins known as heat-labile enterotoxin (LT) and heat-stable enterotoxin-b (STb) are the most common causes of fatal disease. Prevention and control of enteric colibacillosis of swine has largely relied on vaccines and antimicrobial agents. Although much research has been done, efficacious vaccines for post-weaned swine have not yet been developed, and production agriculture is under increasing pressure to reduce their use of antibiotics and other antimicrobial agents. Continued research is needed to find alternative ways to prevent and control enteric colibacillosis in swine. For many years, animal scientists have reported that the incidence and severity of enteric colibacillosis can be affected by diet, but the scientific basis for this is yet unknown; hence, more research is needed. This five-year project will employ a porcine ETEC strain and LT-, STb-, and LT- STb- mutant derivatives of this strain that we had constructed in an earlier study. We will genetically construct ETEC reporter strains that will emit light when the LT and STb genes are expressed (i.e., "turned on"), which will allow us to test dietary nutrients that elicit this effect from the bacteria when present in the intestine. We will experimentally inoculate laboratory mice with these strains and feed them different carbohydrates and amino acids, then ultimately different diets to monitor expression of the two enterotoxins. We will search for components that preferably suppress the expression of both enterotoxins, and then will proceed to studies in swine. The initial studies in swine will be done using intestinal loop assays in weaned pigs and orally inoculated gnotobiotic piglets testing the effects of glucose and different amino acids on enterotoxin gene expression using the reporter strains. Subsequent studies will involve the effects of diets in weaned pigs challenged with our isogenic LT- and/or STb- strains. Pigs expressing K88 receptors will be used as described in our previous papers, using PCR-RFLP on blood samples to screen for susceptible pigs and confirm susceptibility post-mortem by testing for K88 receptors and by histopathology and immunohistochemistry. Other methods describing measurement of disease and bacterial colonization and shedding parameters are described in the above-referenced publications.
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Approach:<br/>
This project (five-year plan) will involve a series of studies which will employ a porcine ETEC strain and LT-, STb-, and LT- STb- mutant derivatives of this strain that we constructed in an earlier study (Erume et al. 2008. Infect. Immun. 76:3141-3149). Reporter strains in which a lux operon is integrated into the promoters of either the LT or STb genes of the parent ETEC strain will be constructed. The lux operon encodes for luciferase; insertion of this gene sequence into the promoters of LT and STb will allow us to detect when these genes are transcribed because light will be emitted from these cells when this is occurring. We will inoculate a streptomycin-treated mouse model with these reporter strains and monitor light emission in the live mouse using an in vivo bioluminescence imaging system. The methods for this were recently described by Foucault et al. (Appl. Environ. Microbiol. 2010; 76:264-274) using commensal strains of E. coli. Mice will be fed different carbohydrates and amino acids, then ultimately different diets to monitor expression of the two enterotoxins. Components that suppress the expression of both enterotoxins will be selected, and then studies will be done in swine. A ligated intestinal loop model in 6-8 week-old weaned pigs that we developed will be utilized (Erume et al. 2013. Vet. Microbiol. 161:315-324), as will our gnotobiotic piglet model (Erume et al. 2008. Infect. Immun. 76:3141-3149) to determine if selected components from the mouse studies give the same effects (i.e., activation or suppression of reporter genes). The initial studies in swine will be done using intestinal loop assays in weaned pigs testing the effects of glucose and different amino acids using these different strains. Subsequent studies will involve the effects of diets in weaned pigs challenged with our isogenic LT- and/or STb- strains. Pigs expressing K88 receptors will be used as described in our previous papers, using PCR-RFLP on blood samples to screen for susceptible pigs and confirm susceptibility post-mortem by testing for K88 receptors and by histopathology and immunohistochemistry. Other methods describing measurement of disease and bacterial colonization and shedding parameters are described in the above-referenced publications.