Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety

<p>Non-Technical Summary: Infection by Campylobacter spp. is one of the leading causes of bacterial gastroenteritis, causing an estimated 1,322,000 cases annually in the U.S., resulting in health care costs of $0.8-5.6 billion per year. Campylobacteriosis is a food-borne disease, with the handling and consumption of poultry considered the most significant risk factor in transmission. Due to the emergence and persistence of antibiotic resistance, coupled with increasing regulatory restrictions on the poultry industry, control strategies such as vaccination are urgently needed. To date, there is no intervention method or vaccine available to the producer to effectively reduce numbers of Campylobacter from poultry going to processing. The development of an efficacious vaccine to reduce the Campylobacter load in chickens would be an innovative breakthrough for the control of this significant food-borne disease. Additionally, very little is known about the various presentations of campylobacteriosis. The most common clinical presentation of Campylobacter gastroenteritis presents as severe acute watery diarrhea, with variable fever, myalgia, and headache. A less common presentation is a dysenteric illness with blood and mucus in the feces. Virulence factors for Campylobacter adherence and invasion associated with the dysenteric illness have been identified, but those factors that induce the frequently observed watery diarrhea are unknown. Identification of a putative toxin will give a more detailed understanding of the fundamental, underlying mechanisms that contribute to the common pathology of acute watery diarrhea resulting from campylobacteriosis. Furthermore, a lesser known risk factor in transmission of Campylobacter is from beef cattle. Sequencing the genomes of unique strains of Campylobacter will improve our understanding of the bacterium as it interacts with the host, and significantly enhance understanding of the disease process. We expect that these discoveries will shift the current research of Campylobacter pathogenesis, leading to new diagnostic tests and targets for anti-campylobacter therapeutics and vaccines.</p>
<p>Approach: CONDUCTING VACCINE STUDIES IN POULTRY: We will continue with our efforts to develop an efficacious vaccine using an attenuated Salmonella vector to express C. jejuni proteins shown to be involved in the colonization of broilers. Currently, we have identified and extracted three proteins involved in C. jejuni biofilm production. Two of the three genes encoding these proteins have been cloned and mutated in C. jejuni by insertion of a chloramphenicol cassette into the center of each gene. The vector containing the CAT gene and the flanking bases of each gene was then introduced by electroporation into C. jejuni and the mutation transferred into the genome via a double crossover. Both of the mutant strains have demonstrated a significant reduction in the colonization of inoculated birds when compared to the wild type strain. These genes will be cloned individually into the attenuated Salmonella vector and examined for immunogenicity in vaccination studies in broilers.
<br/>IDENTIFICATION OF A PUTATIVE TOXIN IN NON-INVASIVE C. JEJUNI POULTRY STRAINS: We have demonstrated in the past that 78% of C. jejuni poultry strains are non-invasive or invade epithelial cells at a very low frequency when compared to human clinical or bovine isolates. In addition, the non-invasive poultry isolates produce a high amount of intestinal fluid in the inoculated piglet model. Three proteins with very high homology to the C. jejuni genome have been identified in the intestinal fluid of pigs infected with the bacterium. We will mutate the genes encoding these proteins and examine their role in fluid production in the piglet model.
<br/>COMPARISON OF GENES IN C. JEJUNI ISOLATES SHOWING DIFFERENT FITNESS TRAITS IN BEEF CATTLE: In conducting a longitudinual cohort study for the presence of C. jejuni in calves from range through the feedlot to processing, we identified different C. jejuni isolates in the same calf at a different time point during their stay in the feedlot. In one instance, we identified three different isolates by PFGE in the same calf. We will sequence the genomes of these unique strains and make comparisons.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: Campylobacter jejuni 1. Vaccination of chicks with the attenuated Salmonella vector expressing the CjLAJ3 protein. We are continuing with our efforts to develop an efficacious vaccine using an attenuated Salmonella vector to express C. jejuni proteins shown to be involved in the colonization of broilers. The Salmonella strain used as the vector was developed by Curtiss and has three different mutations which results in its attenuation. Gene CjLAJ3 was inserted into an expression plasmid, cloned and amplified in an E. coli shuttle vector, extracted, and electroporated into the Salmonella vector. Chicks were orally vaccinated with the vector vaccine or empty vector at 10 and 16 days. Chicks were orally challenged with the homologous C. jejuni strain NCTC11168 at 10 days after the final vaccination. Ten days post challenge the chickens were necropsied, the ceca were removed, and the cecal contents serially diluted and direct plated for enumeration of C. jejuni on Campy Cefex plates.
<br/>2. Challenge studies. Challenge studies were conducted in chickens with several strains of C. jejuni.
<br/>3. Focus on disseminating knowledge. Dr. Joens organized the University of Arizona Food Safety Conference on October 12, 2012, held at the Omni Tucson National Resort. Approximate attendance was 108 attendees, with 20% of attendees coming from food safety industries or government agencies. The Food Safety Conference consisted of 10 hours of contact time over one day, with seven invited speakers followed by a poster session and a dinner to facilitate dialogue and collaborative work.
<br/>PARTICIPANTS: Lynn Joens, Professor; Bibiana Law, Associate Research Professor; Alexandra Armstrong, Assistant Research Scientist; Alix McCloskey, Doctoral Student; Shivanna Johnson, Master's student.
<br/>TARGET AUDIENCES: The research provided laboratory and field training for students. The Food Safety Conference targeted food safety researchers, students, industry professionals, public health members and government agency representatives.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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Impact:<br/>
Campylobacter jejuni 1. Vaccination of chicks with the attenuated Salmonella vector expressing the CjLAJ3 protein. There was not a significant reduction of C. jejuni in cecal contents of chicks vaccinated with the vector expressed CjLAJ3 protein following challenge. Although another trial needs to be conducted, these initial results demonstrate that the CjLAJ3 protein is not effective as a vaccine candidate in reducing the numbers of C. jejuni in chickens.
<br/>2. Challenge studies. Generally, poultry derived isolates colonize at consistently high levels with relatively low challenge doses (<105), while human clinical isolates colonize poorly or variably even at relatively high challenge doses (106-107). These challenge studies will assist us in selecting dosages for future challenges of chicks in vaccine studies.
<br/>3. Focus on disseminating knowledge. In the post-conference evaluations, attendees found the ability to network with food safety researchers, students, industry professionals, public health members and government agency representatives to be the most valuable part of attending the Food Safety Conference. Researchers who attended the workshop had the opportunity to interact with stakeholders and learn of current food and water safety concerns and research needs; food safety professionals and funding agencies came away from the conference with a greater understanding of what food safety research is being done by University of Arizona Food Safety Consortium members and who they could collaborate with on future projects; students discovered different areas of food and water safety through the presentations as well as in discussions with fellow attendees which they found beneficial in assisting with determining their future educational goals and career paths.