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

• 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
• 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.

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.