Source: ARIZONA STATE UNIVERSITY submitted to
FOOD SAFETY VACCINE OF ECONOMIC BENEFIT TO POULTRY PRODUCERS TO PREVENT SALMONELLA AND APEC INFECTIONS AND FOODBORNE TRANSMISSION TO HUMANS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0211303
Grant No.
2007-35201-18519
Project No.
ARZR-2007-02060
Proposal No.
2007-02060
Multistate No.
(N/A)
Program Code
32.0
Project Start Date
Aug 15, 2007
Project End Date
Aug 14, 2011
Grant Year
2007
Project Director
Curtiss, R.
Recipient Organization
ARIZONA STATE UNIVERSITY
(N/A)
TEMPE,AZ 85287
Performing Department
BOARD OF REGENTS
Non Technical Summary
Salmonella and Escherichia coli infections in poultry are a human health threat by transmission to humans via contaminated poultry products. Safe effective vaccines to prevent Salmonella infection of poultry exist but are not extensively used since there is no adverse economic consequence associated with Salmonella infection of chickens. On the other hand, avian pathogenic E. coli (APEC) infections cause morbidity, mortality and carcass condemnation and therefore have a negative economic impact. The difficulty of having an effective vaccine against APEC strains is related to the high level of diversity of these strains. Avirulent strains of Salmonella unable to cause disease will be engineered, these bacteria will possess many mutations to minimize induction of immune responses to serotype-specific antigens and maximize induction of cross-protective immunity to common related antigens of S. enterica strains of diverse serotypes; and these bacteria will be genetically modify to harbor plasmids from other enteric bacteria. Introduction of pAPEC-1 plasmid harboring common APEC antigens in these strains will enhance induction of cross-protective immunity to APEC serotypes. None of the bacterial vaccine cells are able to survive and thus exhibit complete biological containment. The objective is to develop and evaluate vaccines for chickens to prevent Salmonella and APEC infections and foodborne transmission to humans The purpose of this project is to develop safe and inexpensive vaccines for chickens to prevent Salmonella and APEC infections and foodborne transmission to humans
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123299109030%
7123299110050%
7123299116020%
Goals / Objectives
The objectives are to genetically engineer the S. Typhimurium vaccine strain to: (i) minimize induction of immune responses to serotype-specific antigens and maximize induction of cross-protective immunity to common related antigens of S. enterica strains of diverse serotypes; (ii) cure its virulence plasmid and insert the virulence plasmid spv operon into the chromosome; and (iii) express APEC antigens to enhance induction of cross-protective immunity to APEC serotypes. We will evaluate abilities of the different RASV strains to colonize lymphoid tissues in mice and chickens, exhibit biological containment attributes, induce mucosal, systemic and cellular immunities to different Salmonella and APEC serotypes and eliminate or reduce Salmonella colonization and APEC infection of poultry. Success will provide improved poultry health, an economic benefit to producers and enhanced food safety.
Project Methods
A safe efficacious vaccine for the prevention of Salmonella and APEC infections in poultry will be engineered by using a regulated delayed attenuation phenotype such that vaccine strains at the time of immunization exhibit nearly wild-type attributes for survival and colonization of lymphoid tissues but after five to ten cell divisions in the host become avirulent. The first strategy is constructing S. Typhimurium vaccine strains with the mutations Δpmi-2426 and Δ(gmd-fcl)-26. These mutations will turn off ability to synthesize LPS-O-antigen in vivo. The second strategy is to replace promoters for virulence genes needed to display with regulatable promoters (ΔPfur81::TT araC PBAD fur and ΔPcrp527::TT araC PBAD) that will be on for vaccine strain growth and initial colonization of the gastrointestinal tract and be turned off in vivo. Growth of such strains in the presence of arabinose leads to transcription of the fur and/or crp genes but expression ceases in vivo since there is no free arabinose. Attenuation develops as the products of these genes are diluted at each cell division. We also delay onset of attenuation by including ΔaraBAD23, which prevents use of arabinose retained in the cell cytoplasm at the time of oral immunization, and/or ΔaraE25 which enhances retention of arabinose. Additionally, we will include ΔfliC180 and ΔfljB180 mutations which have deletions in the antigenically variable serotype-specific domains of FliC and FljB to enhance the likelihood for repeat use of the host-vector system for other vaccines. The portion of the flagellar protein thatinteracts with TLR5 to recruit/stimulate innate immune responses represents the conserved N- and C-terminal regions of the flagellar proteins. We designed our Salmonella vaccine strains capable of carrying either cloning vectors engineered to express a recombinant protective antigen or wild-type plasmids specifying synthesis of different antigens of targeted pathogens. We first cured S. Typhimurium of its pSTV plasmid and then cloned the spv region essential for virulence and internal tissue growth into either the endA or cysG gene thus deleting these genes in the process. The Salmonella cured strain containing spv in its chromosome will be attenuated as described above and then used to contain the virulence plasmid pAPEC-1 of an APEC strain which encodes three Fur-regulated operons important for iron and manganese uptake. pAPEC-1 will then be engineered to be DadB+ and Tra- to establish a balanced-lethal vector-host system. We will then fully evaluate our recombinant attenuated Salmonella Typhimurium strain with pAPEC-1 as an anti Salmonella and E. coli vaccine for poultry that should enhance food safety by lessening the likelihood of transmission of Salmonella and APEC to humans via contaminated poultry products.

Progress 08/15/07 to 08/14/11

Outputs
OUTPUTS: During the time-period of the project, we were able to cover the three objectives of the proposal: 1) minimize induction of immune responses to serotype-specific antigens and maximize induction of cross-protective immunity to common related antigens of S. enterica strains of diverse serotypes; 2) cure its virulence plasmid and insert the virulence plasmid spv operon into the chromosome; and 3) express APEC antigens to enhance induction of cross-protective immunity to APEC serotypes. Although we have made a lot of progress in improving our developed means to permit a regulated delayed-attenuation phenotype so that vaccine strains, at the time of immunization, exhibit nearly wild-type attributes for survival and colonization of lymphoid tissues and after five to ten cell divisions become avirulent due to an inability to synthesize the LPS O-antigen and outer core in vivo, further studies are still needed to determine the best combination of mutations to limit LPS O-antigen synthesis in vivo to induce the highest level of cross-protective immunity against all Salmonella serotypes. We have also made a lot of progress in developing genetic attenuations of S. Typhimurium that upregulate genes for the iron regulated outer membrane proteins (IROMPs) and for manganese uptake in vivo to induce cross-protective immunity to diverse pathogens; many mutations have been generated for this purpose and studies under way will indicate the best one to use in our vaccine strains. We have genetically modified Salmonella vaccine strains to carry plasmids from other enteric bacteria by curing the S. Typhimurium UK-1 strain of its large virulence plasmid pSTUK-100 and inserted the virulence-enhancing spvABCD operon into the Salmonella deltacysG chromosome deletion-mutation. The promoter of the spvABCD operon was modified to enhance the expression of the inserted spvABCD operon. The genetically engineered strains were fully characterized. The virulence of this strain increased as tested in mice and it colonized the internal organs of infected mice and chicks as well as the wild-type. The insertion of spvABCD in the cysG deletion with improved promoter was then moved into different attenuated Salmonella strains cured of their pSTUK-100 plasmid. We have fully sequenced and characterized a virulence plasmid pAPEC-1 of APEC strain chi7122 that encodes for common virulence of APEC strains, including four iron acquisition systems. We have successfully moved this plasmid into the generated strains and have shown that pAPEC-1 genes, including iron-uptake systems, are expressed in Salmonella. We have evaluated our vaccine strains in chickens for protection against APEC; our preliminary data are very encouraging and warrant further studies to evaluate the best combination of mutations and conditions that will provide maximum efficiency of the vaccines. Some results of our research have been published or reported in scientific meetings. Some of our products have already been shared; after request, we have provided our large plasmid DNA pAPEC-1 to scientists from other institutions. Our project was published on the CSREES Web site and reported by our University's media outlet. PARTICIPANTS: Participants: Roy Curtiss, Principal Investigator-Experimental design and supervised; Melha Mellata, Assistant Research Professor-designed and performed experiments, supervised students, prepared manuscripts, and presented data in international meetings. Jacob Maddux, and Timothy Nam, undergraduate researchers-assisted in strains construction, animal experiments, and preparation of media and solutions; Hua Mo, Assistant Research Technologist-conducted animal experiments; Tina Hartig, Lab Coordinator-provided sterile materials and managed dishwashing facility. TARGET AUDIENCES: Poultry veterinarians PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The U.S. poultry industry is the world's largest producer. It is also the second largest exporter of poultry meat. Salmonella and Escherichia coli infections in poultry are a human health threat and can cost billions of dollars to the industry. Poultry products are often associated with human food-borne disease involving Salmonella. Moreover, avian pathogenic E. coli (APEC) infections cause significant morbidity, mortality and carcass condemnation and therefore have a negative economic impact. With funding from this grant, we are in the process of making a unique vaccine against both Salmonella and E. coli that will provide economical benefit to producers and enhanced food safety. We were also able to fully sequence and characterize a large virulence plasmid (103kb), pAPEC-1 from an APEC strain as a potential vector for Salmonella vaccines against APEC/ExPEC infections. The analysis of the plasmid sequence revealed the presence of most common virulence genes among APEC strains, mostly different iron acquisition systems, adhesins and other ABC transport systems that have been proved to be very immunogenic.

Publications

  • Mellata, M., J. Maddux, N. Thomson, H. Hauser, M. P. Stevens, S. Mukhopadhyay, S. Sarker, A. Crabbe, C. Nickerson, and R. Curtiss III. 2011. New Insights into the Fitness-associated Mechanisms of ExPECs Revealed by the Genotypic and Phenotypic Characterization of Large Plasmids of APEC χ7122 (O78:K80:H9). Submitted to PlosOne.
  • Mellata M., Ameiss K., Mo H., and Curtiss R. III. 2010. Characterization of the Contribution to Virulence of Three Large Plasmids of Avian Pathogenic E. coli χ7122 (O78:K80:H9). Infect. Immun. 78(4):1528-41.


Progress 08/15/08 to 08/14/09

Outputs
OUTPUTS: During this reporting period, we have continued working on the second objective of our research proposal, improving our modified Salmonella vaccine strain to carry plasmids from other enteric bacteria, Salmonella that has been cured from its virulence plasmid pSTUK-100 and inserted the virulence enhancing spvABCD operon into the Salmonella deltacysG chromosome deletion mutation. We have modified the promoter of the spvABCD operon to enhance the expression of the inserted spvABCD operon. The genetically engineered strains were fully characterized in vitro and in vivo. The virulence of this strain increased as tested in mice, and it colonized the internal organs of infected mice as well as the wild type. The insertion of spvABCD in the cysG deletion with improved promoter was then moved into different attenuated Salmonella strains cured of their pSTUK-100 virulence plasmid. We have start working on the third objective of our proposal. We have successfully moved the plasmid pAPEC-1 of APEC into the new generated vaccine strains and we are now in the process of fully characterizing theses strains. We will very soon start the last objective of our proposal which consist in evaluating the protection of our vaccines against both Salmonella and APEC in chickens. Some results of our researches have been published or reported in scientific meetings. Some of our products have been already shared; after request, we have provided our large plasmid DNA pAPEC-1 to scientists from other institutions. Our project was published on the CSREES Web site and reported by our University's media outlet. PARTICIPANTS: Roy Curtiss, Principal Investigator--experimental design, supervising Melha Mellata, Assistant Research Scientist--experimental design, animal experiments, strain construction, supervising students and assistants, MS preparation Jacob Maddox, Undergraduate Researcher--strain construction, preparing media, and animal experiments George Vo, Undergraduate Researcher--strain construction, preparing media, and animal experiments (unpaid undergraduate) Hua Mo, Assistant Research Technologist--animal experiments Tina Hartig, Lab Coordinator--provides sterile materials and manages dishwashing facility Two ASU undergraduates have been involved in the project. They learned different methods of genetic engineering of salmonella vaccine strains including mutation, and analyzing large virulence plasmids of Salmonella and APEC , such as purification. They also learned different techniques used in animal experimentation, such as necropsy, inoculation, and processing organs. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The U.S. poultry industry is the world's largest producer. It is also the second largest exporter of poultry meat. Salmonella and Escherichia coli infections in poultry are a human health threat and can cost a billion dollars to the industry. Poultry products are often associated with human food-borne disease involving Salmonella and, on the other hand, avian pathogenic E. coli (APEC) infections cause significant morbidity, mortality and carcass condemnation and therefore have a negative economic impact. With funding from this grant, we are in the process of making a unique vaccine against both Salmonella and E. coli that will provide economical benefit to producers and enhanced food safety. We were also able to fully sequence and characterize a large virulence plasmid (103kb), pAPEC-1 from an APEC strain as a potential vector for Salmonella vaccines against APEC/ExPEC infections, the analysis of the plasmid sequence revealed the presence of most common virulence genes among APEC strains, mostly different iron acquisition systems, adhesins and other ABC transport systems that have been proved to be very immunogenic.

Publications

  • NEWSLETTERS Caspermeyer, J. (2008). Solving an avian scourge could also provide benefits to human health. Biodesign Institute at Arizona State University, News. http://biodesign.asu.edu/news/solving-an-avian-scourge-could-also-pro vide-benefits-to-human-health
  • Kish, S. (2008). Poultry Vaccines May also Improve Human Health. USDA, CSREES, NewsRoom. http://www.csrees.usda.gov/newsroom/impact/2008/nri/pdf/poultry_vacci ne.pdf
  • Caspermeyer, J. (2009). New insights into a leading poultry disease and its risks to human health. Biodesign Institute at Arizona State University, News. http://www.biodesign.org/news/new-insights-into-a-leading-poultry-dis ease-and-its-risks-to-human-health
  • Patent application (2008) Curtiss, R. III, M. Mellata, B. Zekarias, Z. Shi and C. Branger. Recombinant bacterium capable of eliciting an immune response against enteric pathogens. Salmonella against bacterial enteric pathogens. U.S. Provisional Application No. 60/978,084, filed 10/5/2007. PCT/US08/78991, filed 10/6/2008.
  • Mellata, M., Touchman, J.W., and Curtiss III, R. (2009). Full Sequence and Comparative Analysis of the Plasmid pAPEC-1 of Avian Pathogenic E. coli x7122 (O78:K80:H9). PlosOne 4(1):e4232.
  • Mellata, M., Ameiss, K., Mo, H., and Curtiss III, R. (2009). Characterization of the Contribution to Virulence of Three Large plasmids of Avian Pathogenic E. coli x7122 (O78:K80:H9), submitted


Progress 08/15/07 to 08/14/08

Outputs
OUTPUTS: We already start getting very promising results; based on our long experiment in genetically manipulating Salmonella, we have developed means that permit a regulated delayed attenuation phenotype so that vaccine strains at the time of immunization exhibit nearly wild-type attributes for survival and colonization of lymphoid tissues and after five to ten cell divisions become avirulent. We were able to modify our Salmonella vaccine strain to carry plasmids from other enteric bacteria. We have cured the S. Typhimurium virulence plasmid pSTUK-100 and inserted the virulence enhancing spvABCD operon into the Salmonella cysG chromosome. We have modified the promoter of the spvABCD operon to enhance the expression of the inserted spvABCD operon. The virulence of this strain increased as tested in mice model. The insertion-deletion of spvABCD in cysG with improved promoter was moved into a suicide vector and is now ready to move to any attenuated Salmonella strains. We were also able to fully sequence and characterize a large virulence plasmid (103kb) from an APEC strain as a potential vector for Salmonella vaccines against APEC infections, the analysis of the plasmid sequence revealed the presence of most common virulence genes among APEC strains, mostly different iron acquisition systems, adhesins and other ABC transport systems that have been proved to be very immunogenic. PARTICIPANTS: Roy Curtiss III, PD; Melha Mellata, Asst Res Sci; Hua Mo, Asst Res Technologist; Tina Hartig, Lab Coordinator TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The US is the largest poultry-producing nation in the world, with annual revenues near 50 billion US dollars. Salmonella and Escherichia coli infections in poultry are a human health threat by transmission to humans via contaminated poultry products. Approximately 1.4 million humans are infected with Salmonella enterica serotypes each year in the U. S. primarily causing gastroenteritis and leading to more severe infections and even death in individuals with an immunocompromising condition. On the other hand, avian pathogenic E. coli (APEC) infections cause significant morbidity, mortality and carcass condemnation and therefore have a negative economic impact. Our goals have been to genetically engineer the S. Typhimurium vaccine strain to (1) minimize induction of immune responses to serotype-specific antigens and maximize induction of cross-protective immunity to common related antigens of S. enterica strains of diverse serotypes, and to (2) carry plasmid from APEC strain by curing its virulence plasmid and inserting the virulence plasmid spv operon into the chromosome; to enhance induction of cross-protective immunity to APEC serotypes.

Publications

  • Melha Mellata, Jeffrey W. Touchman, and Roy Curtiss III. 2008. Full Sequence and Comparative Analysis of the Plasmid pAPEC-1 of Avian Pathogenic E. coli χ7122 (O78:K80:H9). Submitted
  • Curtiss, R. III, M. Mellata, B. Zekarias, Z. Shi, and C. Branger. 2008. Recombinant bacterium capable of eliciting an immune response against enteric pathogens. Salmonella vaccine against bacterial enteric pathogens. U.S. Provisional Application No. 60/978,084, filed 10/5/2007. PCT/US08/78991, filed 10/6/2008.