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Colibacillosis, caused by APEC, is a multimillion dollar annual problem for this country's poultry industry. Also, recent recognition that APEC may be a food-borne source of E. coli causing human UTIs, sepsis, and neonatal meningitis, suggests that control of avian colibacillosis may be highly desirable for reasons of both animal and human health. Unfortunately, APEC's molecular pathogenesis is poorly understood, thus hampering identification of rational control targets. A promising area of study in this regard is APEC autotransporters.
<P> Here, we explore an area of great promise for vaccine development, as we have identified APEC genes which encode proteins bearing strong similarity to trimeric autotransporters that enable bacterial pathogens to cause disease in their hosts. Theoretically, blocking the functions of these proteins should decrease the disease-causing potential of APEC. Supporting this premise are the results of genetic analyses which reveal that these putative autotransporters are located on APEC's surface where they are accessible to the body defenses; all characterized members of the trimeric-autotransporter subfamily contribute to adhesive activity; and adhesins have proved good candidates for vaccines. Thus, we believe this study will ultimately lead to better understanding of APEC pathogenesis and enhanced control of APEC-caused peritonitis in laying hens.
NON-TECHNICAL SUMMARY: Colibacillosis is one of the three most significant infectious diseases affecting turkeys, layers, and broilers. Avian pathogenic Escherichia coli (APEC) infections in poultry take many forms with peritonitis being the layer industry's chief disease concern. Collectively, these diseases result in annual multimillion dollar losses to the poultry industry due to mortality, lost production, and condemnations. Unfortunately, control of these diseases has been elusive, since the virulence mechanisms used by APEC are poorly understood, hampering identification of rational control targets. However, recent completion of the APEC genome has provided researchers with an unparalleled opportunity to apply cutting-edge approaches to the study and control of colibacillosis. Using comparative genomic analysis, we have identified APEC-specific genomic regions that may encode candidate vaccine targets. Using bioinformatic techniques, these regions have been evaluated in order to identify all putative open reading frames (ORFs). Among the identified ORFs, three putative, but novel, autotransporters were identified based on specific motifs present in the protein sequences that allow export or secretion through the type V secretary pathway. All three candidate autotransporters possess a similar C-terminal translocator domain and a typical long signal peptide, while their passenger domains showed no homology to any known autotransporter. Based on knowledge of characterized autotransporters and their functions, we hypothesize that these three candidates 1) are novel autotransporters; 2) contribute to APEC virulence by facilitating adhesion, invasion and/or resistance to host serum proteins such as complement; and 3) are potentially useful targets for effective APEC vaccines. In the previous two years of funding we proved that all three candidates were novel autotransporters and two of them AatA and AatB contributed to virulence of APEC. For this coming year, we will evaluate the immunogenicity of AatA and AatB. Such studies will improve our understanding of APEC's molecular pathogenesis, which will enable development of novel approaches for preventing colibacillosis.
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APPROACH: Evaluate the immunogenicity of AatA and AatB in APEC-caused peritonitis. The autotransporters AatA and AatB will be expressed and purified as His-tagged recombinant proteins. The contaminated endotoxin will be removed from purified recombinant proteins. The two purified protein antigens will be each biochemically cross-linked to the adjuvant cholera toxin (CT) (Sigma) at a ratio of 10:1 using 3-(2-pyridyldithio) propionate (SPDP) (Pierce) according to the manufacturer's recommendations. Four groups of chickens at age of 5 days old will be immunized intranasally (mucosal immunization) with 200 microgram of 1) crosslinked AatA, 2) crosslinked AatB, 3) crosslinked AatA plus crosslinked AatB, or 4) CT alone. One boost of 50 microgram antigen will be given intranasally on day 21, and chickens will be challenged with APEC O1 at days 28 using the E. coli-peritonitis model developed by Dr. Trampel. E. coli will be inoculated into the vagina of chickens immediately after oviposition when the oviduct is empty and no albumen is being discharged into the lumen of the magnum. The inoculum will consist of 1 ml of PBS containing 2 X 106 bacteria. All chickens will be examined at necropsy for severity of peritonitis and salpingitis (oviduct infection). These results will be used to evaluate if these recombinant proteins can serve as protective immunogens. Four groups of chicken will be also immunized for purpose of Objective 2. Examine antigen-specific antibodies in chicken serum and secretions of reproductive tract. To evaluate the systemic antibody response to these two vaccine candidates, the levels of antigen-specific serum antibodies generated by vaccinated chickens will be compared to those of CT control chickens. Using ELISA, all isotypes of antibody in chicken serum will be examined in a time course. Mucosal immunization is considered the most effective means to prevent certain bacterial infections because secretion of antigen-specific antibody can block bacterial infection at a primary infection site. APEC are thought to enter into chicken reproductive tract to cause salpingitis and peritonitis. Since local immunization of the vagina is not practical, we propose use of intranasal inoculation, which we hypothesize, will generate a distant mucosal response. Therefore, the level of antigen-specific SIgA in secretions from the reproductive tract of vaccinated chickens will also be compared to those from CT control chickens.