Source: IOWA STATE UNIVERSITY submitted to
ADVANCING THE UNDERSTANDING OF THE ZOONOTIC RISK OF EXPECS AND EVALUATION OF A VACCINE TO CONTROL THESE CONTAMINANTS IN POULTRY PRODUCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1008837
Grant No.
2011-67005-25080
Project No.
IOW05464
Proposal No.
2015-11629
Multistate No.
(N/A)
Program Code
A4141
Project Start Date
Sep 15, 2015
Project End Date
Mar 14, 2017
Grant Year
2016
Project Director
Mellata, M.
Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
Food Science & Human Nutrition
Non Technical Summary
Extraintestinal pathogenic E. coli (ExPEC) is the leading cause of sepsis infections in humans, costing the US heath care system billions of dollars each year. Deaths due to ExPEC infections exceed those caused by E. coli O157 by a factor of 500. ExPEC also infect chickens causing economic losses for the poultry industry. Chicken products are now suspected as vehicle sources of ExPEC to humans. The increase in numbers of immunocompromised populations coupled with multidrug-resistance among ExPECs has challenged the treatment of their infections. One difficulty in developing an effective vaccine against ExPEC is related to their antigenic diversity. Thus, a polyvalent vaccine is required. Studies to advance our understanding of the zoonotic risk of avian ExPECs and determining strategies for their reduction in chicken food-products are needed. Therefore, we propose to extend the understanding of the zoonotic mechanisms of ExPEC and develop a strategy to prevent their carriage in chickens and their presence in poultry products. Our objectives are to (i) identify the presence of different sub-pathotypes of ExPEC from chicken intestines and poultry meat and eggs from retail markets; (ii) ascertain that different subset(s) of these ExPEC are able to cause different human ExPEC infections in mice models; and (iii) develop and evaluate a Salmonella-based vaccine expressing ExPEC antigens to protect against ExPEC infections in chickens to eliminate their presence in chicken products and/or transmission through the food chain to humans. Success will provide improved human and poultry health, an economic benefit to producers and enhanced food safety.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
35%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7223299110040%
3114010104010%
3154099202010%
7124010109040%
Knowledge Area
722 - Zoonotic Diseases and Parasites Affecting Humans; 311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
4099 - Microorganisms, general/other; 3299 - Poultry, general/other; 4010 - Bacteria;

Field Of Science
2020 - Engineering; 1090 - Immunology; 1100 - Bacteriology; 1040 - Molecular biology;
Goals / Objectives
We propose to advance the understanding of the zoonotic risk posed by ExPEC in poultry products 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 in poultry products.Our specific research objectives are to:(i) identify the presence of different sub-pathotypes of ExPEC from healthy chicken intestine (a potential reservoir of ExPEC) and poultry meat and eggs from retail markets (potential vehicles of ExPEC to humans);(ii) ascertain if subset(s) of these ExPEC are able to cause different human ExPEC infections in animal models; and(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.
Project Methods
We have previously tested extraintestinal pathogenic E. coli (ExPEC) isolates from healthy chickens (feces) and their products (eggs and meat) in animal models for human sepsis (BALBc) and urinary tract infection (CBA/j and C3HeN). Our future studies will determine the virulence of selected isolates in day-old chicks by inoculating birds subcutaneously. Morbidity/Death will be recorded for 6 days after inoculation.We have succeeded in genetically engineering Salmonella vaccine strains expressing E. coli iron uptake antigens (IutA and IroN) and E. coli common pilus. The strains were fully characterized in vitro for antigens expression and plasmid stability. Similarly, we will evaluate antigen expression and plasmid stability in the newly constructed Salmonella vaccine expressing E. coli antigens (OmpT, TraT, and EtsC). Vaccine strains deemed to have satisfactory performance criteria will be analyzed for capacity to elicit appropriate immune responses to multiple APEC antigens in chickens. Groups of five chicks will be orally immunized with the vaccines. Sera from wing web bleeds and intestinal secretions will be assessed by enzyme-linked immunosorbent assay (ELISA) for IgA and IgY antibodies to APEC antigens. Sera will also be used for western blot analyses to reveal antibody responses to outer membrane proteins (OMPs) and iron-regulated outer membrane proteins (IROMPs) and for agglutination assays with a diversity of E. coli strains. Sera will also be analyzed for avian pathogenic E. coli (APEC) neutralization and growth restriction titers.The protection ability of vaccines against avian APEC (O1, O2, and O78) will be evaluated using airsacs challenges on both vaccinated and vaccinated chickens.All generated data will be statistically analyzed. All statistical tests will be performed using GraphPad Prism 6.0 software. Data will be included in manuscripts, posters, and oral presentations.

Progress 09/15/15 to 03/14/17

Outputs
Target Audience:Microbiologists; Poultry veterinarians; Poultry product consumers; Physicians Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?ost-doctoral fellows, researcher technicians, and students were involved in this project and were trained by Dr. Mellata at Arizona State University (ASU) or Iowa State University (ISU). One master student was trained in Dr. John Fairbrother's lab. At Arizona State University (ASU) in Drs Mellata and Curtiss' labs (PIs) Research technician positions Jacob Maddux, Assistant Research Technician (full-time), Biodesign Institute, ASU (08/2011-06/2012) Natalie Mitchell, Research Technologist (full-time), Biodesign Institute, ASU (11/2011-06/2015) Timothy Nam, Assistant Research Technician (full-time), Biodesign Institute, ASU (09/2012-08/2013) Undergraduate students Alyssa Stacy (2010-2012); Alec Zamarripa (2011-2012); Jason Clark (2012-2013); Brittany Chou (2012-2013); Tylor Webb (2012-2013); Riley McDougall (2012-2013); Cyrus Zandi-Bami (2013); Paul Haddad (2013); Collin Coelet (2014); Kristen Morrow (2013-2015), Megan Mcfeely (2014-2015). At University of Montreal in Dr. Fairbrother Graduate student Jocelyn Bernier-Lachance (2011-2012) At Iowa State University (ISU) in Dr. Mellata's lab (PI) Post-doctoral fellows Dr. Zachary Stromberg, FSHN, Iowa State University (2/2016-present) Dr. Angelica Van Goor, FSHN, Iowa State University (5/2016-present) Graduate students Graham Redweik, PhD student (8/2016-present) Undergraduate students Seerat Marwaha (5/16-7/16); Ellen Swartz (5/16-4/17); Kyle Anderson (5/16-present); Caroline Treadwell (1/2017-present); David Couri (1/2017-present) Undergraduate students have been trained in basic laboratory techniques such as laboratory safety, sterilizing dishes, preparing solutions, and pipetting. They gained experience in laboratory techniques in the areas of microbiology, including culturing bacteria, PCR, and cell culture. They learned to devise and execute hypothesis driven experiments. All individuals involved in this project, had training in bacterial food safety, animal vaccination, and bacterial challenges. New research skills acquired from this project include techniques in animals (inoculations, bleeding, necropsy, processing organs), cloning, ELISA, protein purification and western blot. For post-docs, additional experience was gained in technical skills, experimental design and student mentoring. This project, enhanced post-doc training in food safety and allowed them to develop independent scientific programs, which is beneficial for obtaining future positions such as faculty, government, or industry scientist position. Zach Stromberg has been awarded a post-doc research fellowship from USDA-NIFA on food safety. Angelica has applied as co-investigator for two research grant proposals to the U.S. Poultry & Egg Association and to the Egg Industry Center. Funding has been approved for the Egg Industry Center research grant. For undergraduate students, Alyssa Stacy was awarded a fellowship to do research at the National Institutes of Health. Caroline Treadwell and David Couri applied for and received the First Year Honors Research Grant through ISU to pursue research in Dr. Mellata's lab. The two students presented their projects at the Symposium for Undergraduate Research and Creative Expression, and the Honors Poster Symposium in Spring 2017 at ISU. Caroline, David, Dr. Van Goor, and Dr. Mellata were all featured in a video on the First Year Honors Mentor Program at ISU (https://www.youtube.com/watch?v=AOFGTmcYwSg). Due to their significant contributions to research projects, some involved in this project are listed as authors on peer-reviewed published or in preparation manuscripts (Jacob Maddux, Natalie Mitchell, Kristen Morrow, Timothy Nam, Alyssa Stacy, Zach Stromberg, Angelica Van Goor, and Graham Redweik). Additionally, they presented research posters at meetings, including the General Meeting of the American Society for Microbiology in June, 2012 in San Francisco (Jacob Maddux, Alyssa Stacy) and in May, 2014 in Boston (Natalie Mitchell), and at the Conference of Research Workers in Animal Diseases (CRWAD) in December, 2016 in Chicago (Zach Stromberg and Angelica Van Goor). Zach Stromberg, Angelica Van Goor, and Graham Redweik each presented research posters at American Society for Microbiology, Microbes in June 2017 in New-Orleans. The training in this project has significantly influenced and fostered the careers of these young scientists, below are some examples: George Vo (Scientist at ThermoFisher); Jacob Maddux (an MD student 1ST year intern in internal medicine, Mayo Clinic in Arizona); Alyssa Stacy and Alec Zamarripa (MD student at University of Arizona); Brittany Chou (Dental student at University of California, San Francisco dental school); Jason Clark (Lab tech at Pima community school); Natalie Mitchell (Graduate student, ASU); Megan Mcfeely (Manager at EPic); Kristen Morrow (Graduate student in immunology at Emory University). In summary, this project provided students, researchers, and post-docs opportunities for extensive training in microbiology, immunology, and vaccine development. This impacted their perceptions of food safety issues and increased their understanding of basic science and molecular microbiology, which is essential to the ability of future generations to ensure a safe and quality product and improve animal health. Introducing food safety issues to young people increases their awareness and prepares them for successful careers in the field. This funding has provided students to acquire proficiency of laboratory skills, and increased scientific competencies. It will advance students to graduate education positions, and retention of students and young scientists in agricultural related fields. How have the results been disseminated to communities of interest?Oral and poster presentations at national meetings, manuscripts, press releases Through news coverage from College, University, local or national news services, faculty and departmental website content. Direct communications, website content, presentations in webinars or at scientific/professional meetings. Efforts to reach academic personnel included direct personal interactions, training and development of new research techniques, publication of peer reviewed research papers, oral or poster presentations at scientific meetings and recruitment materials (presentations, flyers, website content, etc.). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACT: Extraintestinal pathogenic Escherichia coli (ExPEC) constitutes ongoing health concerns for women, newborns, elderly, and immunocompromised individuals due to increased numbers of urinary tract infections (UTIs), newborn meningitis, abdominal sepsis, and septicemia. E. coli remains the leading cause of UTIs, with recent investigations reporting the emergence of E. coli as the predominant cause of nosocomial and neonatal sepsis infections. Our project was aimed at increasing our understanding the risk to humans of E. coli from chicken-derived food products, such as raw meat and eggs, and determining the effectiveness of a vaccine to control these contaminants in poultry production. During the project, retail chicken meat and eggs, and the feces of healthy commercial chickens were sampled to assess whether these food products and animals contained virulent E. coli isolates. Microorganisms from these sources were explored genetically to determine if they were similar to those that cause disease in humans and animals. During the project, vaccines were developed and studied for their efficacy in reducing extraintestinal E. coli infections in chickens. In this project, we confirmed the presence of E. coli in chicken meat and feces and showed for the first time their presence on egg shells. We found that some chicken-source E. coli isolates can cause one and even multiple diseases (sepsis, UTI, and meningitis) when tested in rodent infection models that mimic human disease and in chickens (colibacillosis). During the project, vaccines were developed to reduce extraintestinal E. coli infections in chickens. After testing, we showed that their use in the poultry industry will protect chickens against diseases caused by these bacteria and prevent their transmission to humans through contaminated food. Benefits of this work included a deeper understanding of virulent microorganisms within the U.S. food supply, development of therapeutics to decrease disease incidence from E. coli in chickens, enhancement of food safety, and reduction in cost of livestock production. Technology transfer of knowledge generated in this project is crucial to the successful implementation of screening and therapeutics to improve animal and consumer health. Project outputs were disseminated through numerous avenues including media coverage, presentations at scientific conferences, peer-reviewed journals, patenting new technologies, presentations at university symposium, and through training of young scientists. The results of this project will lead to improved animal health and reduced risks to consumers for contraction of food borne pathogens. Objective (i) identify the presence of different sub-pathotypes of ExPEC from healthy chicken intestine (a potential reservoir of ExPEC) and poultry meat and eggs from retail markets (potential vehicles of ExPEC to humans). During the project, retail chicken meat and eggs, and the feces of healthy commercial chickens were sampled to assess whether these food products and animals contained virulent E. coli isolates. Microorganisms from these sources were explored genetically to determine if they were similar to those that cause disease in humans and animals. The results showed that retail meat and eggs as well as chicken feces contained potentially virulent ExPEC based on genetic and phenotypic testing methods and are possible sources for transmission of ExPEC to humans. In addition, this project was the first to screen and find ExPEC in chicken eggs. Identification of these microorganisms in chicken's feces, meat, and eggs advances our understanding of the sources of foodborne illness and increases the need for effective microbial interventions of these food products to eliminate foodborne illnesses and risk to consumers. Objective (ii) ascertain if subset(s) of these ExPEC are able to cause different human ExPEC infections in animal models We selected E. coli isolates from chicken meat, eggs and healthy chickens based on genetic and phenotypic virulence characteristics to assess their virulence in rodent models of human extraintestinal diseases (UTI, sepsis, meningitis) and chicken airsacculitis. The results showed that some E. coli from chicken meat, eggs, and healthy chickens could cause disease in animal models for human and avian extraintestinal diseases. The impact of this finding ultimately demonstrates that healthy chickens as well as chicken products contain microorganisms that have the potential to cause extraintestinal disease in humans and may pose a threat to consumers and those that are in direct contact with chickens such as workers on poultry farms. Objective (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. During the project, vaccines were developed to reduce extraintestinal E. coli infections in chickens and improve animal health and food safety. We first evaluated 8 E. coli proteins (EcpA, EcpD, IutA, IroN, TraT, EtsC, OmpT, and OmpA) for their vaccine potential against ExPEC using active immunization in mice and chickens. The results showed that the E. coli proteins used elicited a strong immune response in vaccinated animals. In addition, broad protection against enteric bacteria was found using in vitro analyses and in avian pathogenic E. coli challenge of chickens. These experiments were a first step towards developing a vaccine that could significantly reduce disease caused by E. coli in poultry and provide improved human and poultry health, an economic benefit to poultry producers, and enhanced food safety. To further evaluate vaccines for the reduction of ExPEC in chickens, we developed two recombinant attenuated Salmonella vaccines chi9373 and chi8025 that synthesized a conserved E. coli antigen (ECP). We evaluated the vaccine construct in vivo by oral vaccination of day-old chickens. The protection ability of vaccines against multiple avian pathogenic E. coli serogroups were evaluated using air sac challenges. The results showed that these vaccines elicited immune responses against E. coli antigens. The vaccine reduced the level of E. coli and lesions caused by E. coli in an animal challenge model. The results also showed that based on in vitro analyses that these vaccines have the potential to protect against multiple avian pathogenic E. coli serogroups. Overall, a significant effort was made to develop broadly protective vaccines against extraintestinal E. coli infections to decrease disease incidence of E. coli in chickens, enhance food safety, and reduce the cost of poultry production.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Melha Mellata, James Johnson, and Roy Curtiss. 2017. Escherichia coli Isolates from Commercial Chicken Meat and Eggs Cause Sepsis, Meningitis, and Urinary Tract Infection in Rodent Models of Human Infections. Zoonoses Public Health (under review).
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Jacob T. Maddux, Zachary R. Stromberg, Roy Curtiss 3rd, Melha Mellata. 2017. Recombinant Attenuated Salmonella Vaccine Strain Delivering Escherichia coli Common Pilus has Broad Protection Potential Against Extraintestinal Pathogenic E. coli and Salmonella enterica. Front Immunol (under-review).
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Zachary R. Stromberg, James R. Johnson, John M. Fairbrother, Angelica Van Goor, Roy Curtiss 3rd, Melha Mellata. 2017. Evaluation of Escherichia coli isolates from healthy chickens to determine their potential risk to poultry and human health. PlosOne (under-review).
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Angelica Van Goor, Zachary R. Stromberg, and Melha Mellata. 2017. A Recombinant Multi-Antigen Vaccine With Broad Protection Potential Against Avian Pathogenic Escherichia coli. Clin Vaccine Immunol (under-review).
  • Type: Journal Articles Status: Other Year Published: 2017 Citation: Zachary R. Stromberg*, Angelica Van Goor*, and Melha Mellata. 2017. Host response and prevention of avian pathogenic Escherichia coli following vaccination with an attenuated Salmonella expressing E. coli common pilus in chickens. Front Vet Sci. (in preperation). *co-first authors.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Roy Curtiss 2015. Recombinant attenuated Salmonella vaccines to enhance food safety. Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida. - discussed induction of cross protective immunity to enteric bacterial pathogens including Salmonella, E. coli and C. perfringens 9-8-15
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Roy Curtiss 2015. New recombinant attenuated Salmonella vaccine platforms. Department of Molecular Genetics and Microbiology, College of Medicine, UF 9-29-15 and 10-14-15Department of Microbiology, University of Virginia  talked about protection against Salmonella and avian influenza
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Roy Curtiss 2015. New technologies in developing recombinant attenuated Salmonella vaccines. Department of Animal Sciences, College of Agriculture, University of Florida  discussed RASVs against APEC, C. perfringens, Eimeria and influenza in poultry11-20-15
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Roy Curtiss 2016. Salmonella vectored recombinant vaccines: new platform technologies. Center of Excellence for Emerging and Zoonotic Animal Diseases, Nebraska City, Nebraska  discussed Salmonella, C. perfringens and Eimeria10-31-16
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: M Mellata, J Johnson, and R Curtiss 3ed 2016 Escherichia coli from Poultry Meat and Eggs Cause Sepsis, Meningitis, and Urinary Tract Infections in rodent models for Human Infections. CRWAD, Chicago, Dec 3-6th.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: D. Couri, A. Van Goor, M. Mellata. Evaluation of Immune Protection Elicited by Recombinant Antigen EtsC. 2017 Symposium for Undergraduate Research and Creative Expression (SURCE), Iowa State University, April 11.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: C. Treadwell, A. Van Goor, M. Mellata. Evaluate broad protection of the Escherichia coli Outer Membrane Protein A (Omp A) against Avian Pathogenic E. coli infections in chickens. 2017 Symposium for Undergraduate Research and Creative Expression (SURCE), Iowa State University, April 11.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: A Van Goor, ZR. Stromberg, JR. Johnson, JM. Fairbrother, R Curtiss 3rd, and M Mellata. 2016 Escherichia coli isolates from healthy poultry feces: their potential risk to poultry and human health. CRWAD, Chicago, Dec 3-6th.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: ZR. Stromberg, A Van Goor, R Curtiss 3rd, and M Mellata. 2016 Recombinant attenuated Salmonella vaccine strains with broad protection of colibacillosis in chickens. CRWAD, Chicago, Dec 3-6th. (Poster presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Angelica Van Goor, Zachary R. Stromberg, Graham A. Redweik, and Melha Mellata. Novel Recombinant Antigen Vaccine with Broad Protection Potential Against Avian Pathogenic Escherichia coli and Other Enterobacteria. #5390. ASM Microbe. June, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Graham A. Redweik, Zachary R. Stromberg, and Melha Mellata. Vaccination and Probiotics Treatment Provide Enhanced Protection against Bacterial Infections in Chickens. #4731. ASM Microbe. June, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: D. Couri, A. Van Goor, M. Mellata. Evaluation of Immune Protection Elicited by Recombinant Antigen EtsC in Chickens. 2017 Honors Poster Symposium, Iowa State University, April 26.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: C. Treadwell, A. Van Goor, M. Mellata. Evaluation of Outer Membrane Protein A (OmpA) against Avian Pathogenic Escherichia coli infections in chickens. 2017 Honors Poster Symposium, Iowa State University, April 26.


Progress 09/15/15 to 09/14/16

Outputs
Target Audience:Poultry veterinarians; Poultry product consumers; Physicians; Consumers Changes/Problems: Dr Mellata (PD) relocated her lab from Arizona State University (ASU) to Iowa State University (ISU), there was a long delay in transferring the grant to her new institution. There was also a delay in starting the project at ISU because Dr Mellata's new lab at ISU required some renovation, new personal had to be hired and trained, and lab protocols have to be approved by ISU IBC and IACUC We will include high throughput sequencing approach (RNA-sequencing) to evaluate the immune response to live Salmonella vaccine in chickens. The spleen samples will be sequenced on the newest platform (Illumina HiSeq 3000) at the ISU sequencing facility. This project will result in a stand-alone manuscript that will give insight into the immune response to vaccination. We chose to use this method for evaluation because Dr. Van Goor (post-doc) has experience with transcriptome data. What opportunities for training and professional development has the project provided?Post-doctoral research positions at Iowa State University Post-doc 1 (02/2016-present) Post-doc 2 ( 05/2016-present) Graduate student Graduate Student 1 (08/2016-present) Undergraduate students trained at Iowa State University Student 1 (5/2016-present); Student 2 (5/2016-present); Student 3 (5/2016-7/2016) They all had training in bacterial food safety, animal vaccination and challenges. Post-doc 1 has been awarded a Post-doc research fellowship from USDA-NIFA on food safety. Post-doc 2 has applied a research grant pre-proposal to the US Poultry & eggs association and is planning on applying to a Post-doctoral fellowship to both NIH and USDA. How have the results been disseminated to communities of interest?Webinar Mellata, M: "Can E. coli from contaminated poultry food products cause human extra-intestinal infections? Development and evaluation of a vaccine to improve poultry health and enhance food safety" Institute of Food Safety and Nutrition (IFSN)Seminar: Webinar. 12/11/2015 Oral presentation Mellata, M: presented the USDA project to "Food Science and Human Nutrition (FSHN) External Advisory Council Spring 2016 Meeting April 15, 2016 Mellata, M. "The Unrecognized Foodborne E. coli : their Prevalence in Chicken Products and Virulence potential" USDA NIFA Food Safety Project Directors' Meeting, Saint Louis, MO July 30th 2016 Press and news releases New Iowa State molecular microbiologist brings expertise in E. coli, vaccine development, by Shannon Stump 10/26/2015 : http://www.hs.iastate.edu/news/2015/10/26/e-coli-vaccine/ ISU prof researches E. coli, vaccine development, by Julie Ferrell10/31/2015, in Ames Tribune, http://amestrib.com/news/isu-prof-researches-e-coli-vaccine-development What do you plan to do during the next reporting period to accomplish the goals?Aim 1: All parts of the Aim 1 have now been completed. Egg, meat, and intestine isolates have been tested. Data of egg and meat isolates are already published. The data of intestine isolates are now analyzed and will be included in a second manuscript (in preparation). Aim 2: All parts of the Aim 2 have now been completed. Data of egg and meat isolates are included in a manuscript submitted to AEM journal (under revision). Data of intestine isolates are now analyzed and will be included in a second manuscript (in preparation). Aim 3: Regarding the experiment on vaccination of chickens using attenuated Salmonella expressing ECP, we will collect sera and intestinal washes from vaccinated and non-vaccinated chickens in the next two weeks. Samples will be analyzed for IgY and IgA responses using ELISA. To evaluate vaccine protection, we will challenge vaccinated and non-vaccinated chickens with three serotypes (O1, O2, and O78), respectively. We will complete a transcriptome evaluation using high throughput RNA-sequencing on the spleen of chickens in response to the live Salmonella vaccination. The transcriptome data will be analyzed and published as a stand-alone manuscript to determine an in depth understanding of immune response to vaccination. Additionally, we will collect other relevant immune tissues (bursa, thymus, and PBMCs) in response to vaccination and challenge with APEC. Regarding the experiment on vaccination of chickens using recombinant antigens (EtsT, TraT, OmpT, and OmpA), we will start the vaccination schedule in one week, we will vaccinate chickens with antigens individually or in combination with 4 antigens. We will evaluate IgY responses in sera and protection using E. coli challenge.

Impacts
What was accomplished under these goals? Overall impact statement: A group of E. coli named extra-intestinal pathogenic E. coli (ExPEC) cause diseases in humans and animals, especially chickens. Chicken-derived food products, such as raw meat and eggs can be contaminated with some E. coli bacteria that are similar to those that cause ExPEC human diseases. We have successfully developed and evaluated a Salmonella-based vaccine expressing ExPEC antigens to protect against ExPEC infections in chickens. Protection against bacterial colonization will help eradicate the presence of ExPEC in chicken products and eliminate their transmission through the food chain to humans. We are testing multiple new ExPEC proteins in chickens to evaluate their vaccine potential to provide broad protection against multiple ExPEC serotypes. Success will provide improved human and poultry health, an economic benefit to poultry producers, and will enhance food safety. This project provided students and post-docs opportunities for extensive training in microbiology, immunology, and vaccine development. This impacted their perceptions of food safety issues and increased their understanding of basic science and molecular microbiology, which is essential to the ability of future generations to ensure a safe and quality product and improve animal health. Introducing food safety issues to young people increases their awareness and prepares them for successful careers in the field. This funding has provided students to acquire proficiency of laboratory skills, and increased scientific competencies. It will advance students to graduate education positions, and retention of students in agricultural related fields Objective 1... Identify the presence of different sub-pathotypes of ExPEC from healthy chicken intestine (a potential reservoir of ExPEC) and poultry meat and eggs from retail markets (potential vehicles of ExPEC to humans) Completed Objective 2... Ascertain if subset(s) of these ExPEC are able to cause different human ExPEC infections in animal models Experiments were completed. The data generated were included in a manuscript entitled "Escherichia coli Isolates from Commercial Chicken Meat and Eggs Cause Sepsis, Meningitis, and Urinary Tract Infection in Rodent Models of Human Infections" that was submitted to AEM journal (reviewed, under revision). Objective 3... 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. We repeated the chicken oral vaccination experiment using Salmonella attenuated vaccine c8025 (Dcya-27 Dcrp-27 DasdA16) with empty Asd-plasmid pYA3337 or with Asd-plasmid with E. coli common pilus operon, ecpABCD, pYA4428 using a lower dose 108 CFU instead of 109 CFU, the latter was slightly virulent. Both mucosal (IgA) and circulatory (IgY) immune responses were measured in vaccinated and non-vaccinated chickens. The protection ability of vaccines against avian pathogenic E. coli (APEC) (O1, O2, and O78) were evaluated using Intra-tracheal challenge. Based on our preliminary data, we determined that a vaccine containing c8025 (pYA3337) + c8025 (pYA4428) could provide more broad protection. This new vaccine formula is now being evaluated in a new chicken trial which is occurring. We have prepared 4 more E. coli His-tag proteins (TraT, EtsC, OmpT, and OmpA) that will be tested for their vaccine potential using active immunization in chickens and then evaluation of challenge with (APEC) (O1, O2, and O78) to determine broad protection.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: M. Mellata, NM. Mitchell, F. Sch�del, R. Curtiss 3rd, GB. Pier. 2016. Novel vaccine antigen combinations elicit protective immune responses against Escherichia coli sepsis. Vaccine 34 (5): 656-662.
  • Type: Other Status: Published Year Published: 2015 Citation: Mellata, M. "Can E. coli from contaminated poultry food products cause human extra-intestinal infections? Development and evaluation of a vaccine to improve poultry health and enhance food safety Institute of Food Safety and Nutrition (IFSN) Seminar: Webinar. 12/11/2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mellata, M. The Unrecognized Foodborne E. coli : their Prevalence in Chicken Products and Virulence potential USDA NIFA Food Safety Project Directors Meeting, Saint Louis, MO July 30th 2016
  • Type: Other Status: Published Year Published: 2015 Citation: New Iowa State molecular microbiologist brings expertise in E. coli, vaccine development, by Shannon Stump 10/26/2015 : http://www.hs.iastate.edu/news/2015/10/26/e-coli-vaccine/
  • Type: Other Status: Published Year Published: 2015 Citation: ISU prof researches E. coli, vaccine development, by Julie Ferrell10/31/2015, in Ames Tribune, http://amestrib.com/news/isu-prof-researches-e-coli-vaccine-development
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Mellata, M. Extraintestinal Pathogenic Escherichia coli : From Understanding Virulence and Zoonotic Risk to Vaccine Development Presented at ISU Vet Med
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Curtiss III, R. Enhancing food safety for human consumers by eliminating food-borne enteric pathogens. CRAWD Chicago 12/7/2015