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Advancing the Understanding of the Zoonotic Risk of Expecs and Evaluation of a Vaccine for the Control of these Contaminants in Poultry Products


<p>We propose to advance the understanding of the zoonotic risk posed by Extraintestinal pathogenic Escherichia coli (ExPECs) in poultry products (meat and eggs) to fill the existing gaps in our knowledge regarding ExPEC biology and transmission; moreover, our project proposes to evaluate a safe, easy to use Salmonella-based vaccine, to protect against ExPEC infections in chickens and reduce or eliminate the risk of contamination of poultry products. Our specific research objectives are to</p>
<p>(i) identify the presence of different sub-pathotypes of ExPEC from healthy chicken intestines (a potential reservoir of ExPEC) and poultry meat and eggs from retail markets (potential vehicles of ExPECs to humans);</p>
<p>(ii) ascertain if subset(s) of these ExPECs are able to cause different human ExPEC infections in animals; and</p>
<p>(iii) develop and evaluate a Salmonella-based vaccine expressing ExPEC antigens to protect against ExPEC infections in chickens and eradicate their presence in chicken products to eliminate their transmission through the food chain to humans. Success will provide improved human and poultry health, an economic benefit to producers, and will enhance food safety.</p>

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<p>NON-TECHNICAL SUMMARY:<br/> Extra-intestinal pathogenic E. coli (ExPEC) cause a number of different diseases in humans and costs billion of dollars annually to the US health care system. People affected are mostly women (urinary tract infections), newborns (meningitis), elderly and immuno-compromised people (abdominal sepsis and septicemia). This group of E. coli also causes diseases in chickens that are suspected to be a source of the E. coli that cause disease in humans. The antibiotics used to treat these infections often fail because of the resistance of these bacteria to the antibiotics used in their treatment. New treatments are needed to avoid the increase of cost and mortality related to these infections. We propose to increase our understanding on how these bacteria are transmitted from chickens to humans by analyzing chicken food-products, including meat and eggs (vehicles) and chicken intestines (reservoir), for the presence of human-like ExPEC bacteria and evaluate these strains to cause human diseases in mice. Moreover, our project proposes to evaluate a safe, easy to use Salmonella-based vaccine, to protect against ExPEC infections in chickens and reduce or eliminate the risk of contamination in poultry products. Success will provide improved human and poultry health, an economic benefit to producers, and will enhance food safety.
<p>APPROACH: <br/>The proposed studies will first focus on genomic and phenotypic characterization of E. coli isolates from feces of healthy chickens, and chicken-food products (meat and eggs), the eventual vehicles of ExPECs to humans, to identify the different subpathotypes of ExPEC on these products (Aim 1). PCR will be performed using multiplex PCR primer sets to examine the presence of representative genes known for their association with human ExPEC or APEC virulence as well to determine their phylogenic group (A, B1, B2, or D). The different subpathotypes of ExPEC from poultry will then undergo a set of in vitro tests, including their ability to survive in body fluids (serum, urine), biofilm formation, internalization by host cells, and efficient iron uptake. To test the potential of ExPEC isolates to invade human cells, we will test the ability of bacteria to attach and invade three human cell lines. Human bladder cell lines: 5637 and T24 will be used for bacterial urethral infections. The Human BMEC cell line will be used for bacteria capable of causing meningitis infection. For the first time subsets of ExPECs from poultry products will be tested in four different mouse strains, proven efficient in mimicking human ExPEC infections (Aim 2). Finally, we will continue to develop and evaluate a Salmonella-based vaccine expressing ExPEC antigens to protect against their infections in chickens and eliminate their presence in chicken products or transmission through the food chain to humans (Aim 3). Chicks will be immunized with our engineered Salmonella vaccine strains delivering multiple ExPEC antigens. At 3.5 weeks post vaccination, chicks will be challenged with homologous APEC orally, intratracheally and via caudal airsac inoculation. Protection against ExPECs will be monitored in birds with determination of quantitative bacterial titers in organs and cecal contents. Heterologous protection will be measured by challenging with Salmonella and APEC from different serogroups.
<p>PROGRESS: 2012/03 TO 2013/03<p>Target Audience:<br/> Poultry veterinarians; Consumers; Physicians <p> Changes/Problems: In our proposal, we suggested using pAPEC-1, a ColV-plasmid of APEC ?7122 (O78:K80:H9), as vector in Salmonella vaccines to protect against multiple APEC strains. Unfortunately, in in vivo conditions, the plasmid was unstable, as the Salmonella strain host loses the plasmid when in internal organs (spleen and liver of mice). This could probably be due to its Inc region that is truncated. We however decided to test other similar ColV plasmids from both Salmonella and APEC, to select the plasmid that is more stable in in vivo conditions to use in our vaccine strains. What opportunities for training and professional development has the project provided? Research technician positions (in Drs. Curtiss and Mellata�s Labs). Jacob Maddux, Assistant
Research Technician (full-time), Biodesign Institute, ASU (08/2011-06/2012) Natalie Mitchell, Research Technologist (full-time), Biodesign Institute, ASU (11/2011-present) Timothy Nam, Assistant Research Technician (full-time), Biodesign Institute, ASU (09/2012-present) Undergraduate students trained Alyssa Stacy (2010-2012); Alec Zamarripa (2011-2012); Jason Clark (2012-present); Brittany Chou (2012-present); Taylor Webb (2012-present); and Riley McDougall (2012-present) have been trained by Dr. Melha Mellata (Co-director). Jacob Maddux and Alyssa Stacy presented research posters at the General Meeting of the American Society for Microbiology in June of 2012 in San Francisco. The training has significantly influenced and fostered the careers of these young scientists. After graduating and working for a year as a full-time technician in Dr. Mellatas lab, Jacob Maddux is now a
student at the University of Arizona College of Medicine in Tucson. Alyssa Stacy was recently awarded a fellowship and is currently pursuing her research at the National Institutes of Health. Graduate student Experiments done by Benoit Lanthier, a member of Dr. John Fairbrothers lab, are part of a Master degree project How have the results been disseminated to communities of interest? Press and news releases Dealing with stress: new research highlights the survival skills of disease-causing E. coli. By Richard Harth, January 30, 2012.…
<p>PROGRESS: 2011/03/15 TO 2012/03/14 <p>OUTPUTS:<br/> This past fiscal year, we have covered many objectives cited in Aims 1 and 3 of the proposal. As suggested in the Aim 1, Drs. John Fairbrother and James Johnson collected a large number of ExPEC isolates from chicken intestines and poultry products (meat and eggs) respectively: Dr. John Fairbrother had made available for this project 21 intestine isolates of healthy 29-day-old chickens from his collection; these isolates were previously defined by the presence of one or more of the virulence genes papC, iucD, and tsh commonly associated with APEC isolates. For this study, Dr. Fairbrother's team has isolated 191 E. coli from pooled fecal samples of both antibiotic-free broiler flocks and conventionally raised chickens from 9 different sites of industrial rearing located in the Quebec area. Enriched samples were examined by
PCR for the presence of ExPEC virulence genes and preselected isolates for further analysis. Dr. James Johnson has provided us with 124 archived ExPEC isolates from retail chicken meat samples, previously isolated and characterized in his laboratory as part of various retail food surveillance projects. For the present study, Dr. Johnson has extended the screening by PCR of a selected subset of these isolates of chicken meat (n=124) and intestine (n=21) for five additional plasmid-linked markers that are statistically associated with APEC. Moreover, Dr. Johnson has obtained from another USDA investigator, Michael Musgrove (USDA, Athens, GA), 179 E. coli isolates from eggs that were characterized in the same manner as the chicken-meat isolates. All isolates have been further tested for the major E. coli phylogenetic groups (A, B1, B2, and D) in Dr. Johnson's lab. <br/>After full genotypic and
phylogenetic analysis, Dr. Johnson provided us with 124 chicken-meat, 21 chicken-intestine, and 5 egg presumptive ExPEC isolates. We have analyzed the 124 meat-isolates phenotypically and for their plasmid profiles. The plasmid content and size of selected isolates have been determined using large plasmids extraction and using computer software respectively. Phenotypic analysis of the isolates included survival in serum-complement, biofilm formation, colicin production, and iron-uptake using techniques described in our proposal. Regarding the objectives of Aim 3, which consist to complete construction and evaluation of S. Typhimurium vaccine strains to deliver protective APEC antigens; we have so far generated seven vaccine strains that are genetically modified to carry large plasmids of other enteric bacteria and contain different combinations of mutations for attenuations,
bio-containment and immunogenicity-enhancing. The plasmids pAPEC-1-1 from an avian pathogenic E. coli and pYA3337 containing E. coli common pilus genes were selected as vector antigens to use in our vaccine strains. Our project was reported by our University's media outlet and presented to Dr. Jeanette Thurston, USDA National Program Leader, at the Biodesign Institute and at the Food Safety Programs Project Director's Meeting.<p> PARTICIPANTS: <br/>Roy Curtiss, Principal Investigator-experiment design and supervised; Melha Mellata, Co-Principal Investigator- experiment design, supervised, performed experiments, and communicated with collaborators; Jacob Maddux, Assistant Research Technologist-assisted in strains construction, animal experiments, and preparation of media and solutions; Natalie Mitchell, Research Technologist-assisted in strains construction, animal experiments, and preparation of
media and solutions; Tina Hartig, Lab-coordinator-provided sterile materials and managed dishwashing facilities Jacquelyn Kilbourne, Veterinary Tech Coordinator-assisted in animal experiments Vanessa Chesi, Glassware Attendant-assisted in providing sterile materials Yingqin Luo, Bioinformatics Post doc-provided information on the genome of Salmonella Typhimurium Angela Jansen, Project Manager Research- Orientation to laboratory procedures and ensured that laboratory equipment is fully functional. <p>Collaborators: 1) University of Minnesota, Infectious Diseases, VA Medical Center James Johnson-collaborator (supervised and designed experiments) and Brian Johnston (performed experiments). 2) University of Montreal, Faculty of Veterinary Medicine: John Fairbrother-collaborator (supervised and designed experiments); Ghyslain Vanier- research associate (supervised technical aspects of
experiments and performed some experiments); Benoit Lanthier-Master's graduate student (performed experiments). Opportunities for training or professional development: 1. Jacob Maddux who was an undergraduate student-worker in our lab for almost three years, was recruited after his graduation as an assistant research technologist to work full time on this project. 2. Two undergraduate students Alyssa Stacy and Alec Zamarripa have been trained in our lab. 3. Experiments done by Benoit Lanthier, a member of Dr. John Fairbrother's lab, are part of a Master degree projectTARGET AUDIENCES: Poultry veterinarians, consumers, human physicians PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

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