Bath/Oral Live Recombinant Edwardsiella Vaccine for the Aquaculture Industry

<p>NON-TECHNICAL SUMMARY: <br/>The largest aquaculture industry in the United States is the production of channel catfish (Ictalurus punctatus). The most serious bacterial pathogens affecting this industry are Edwardsiella ictaluri and Flavobacterium columnare. Losses due to these bacterial pathogens are estimated to be $50-80 million, annually. Our objective is to develop a non-virulent and antibiotic-sensitive E. ictaluri strain that will prevent E. ictaluri and F. columnare infections in channel catfish (and other fish, such as blue catfish and hybrids) in a single safe bath/oral vaccine introducing an economical boosting immunization through food. The E. ictaluri vaccine vector can have later applications for virus/parasite antigen delivery or DNA vaccine delivery. The best way to enhance production by vaccination is generating a safe, efficient oral/bath E. ictaluri vaccine vector that can be applied in any age of fish, from egg to adults, and introducing a boost immunization through food. The E. ictaluri vaccine vector will pose no threat for the environment, since it is programmed to self-lyse after five to ten cell divisions and after induction of the immune response. We believe that development of this vaccine will enhance catfish production to result in widespread use of E. ictaluri vaccines against other important pathogens of the aquaculture industries.
<p>APPROACH:<br/> Live recombinant vaccines, which protect against several diseases at low cost, have not yet been designed for the aquaculture industry. E. ictaluri, the causative agent of catfish enteric septicemia, is an invasive intracellular pathogen; an excellent candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the aquaculture industry. We propose to develop an antibiotic-sensitive bath/oral live RAEV to prevent Edwardsiella and F. columnare infections in catfish. RAEV will be engineered by using regulated delayed attenuation, delayed antigen synthesis, and delayed lysis systems. The RAEV strain at the time of immunization will exhibit nearly wild-type attributes for survival and colonization of lymphoid tissues but after five to ten cell divisions in the host become avirulent and ultimately lyse in the absence of arabinose. The strategy is to construct an E. ictaluri strain that gradually eliminates virulence in vivo and increases synthesis of the heterologous protective antigen before finally lysing. The delayed attenuation will be achieved by deletion of the promoter region for the crp, insA and fur genes and substituting the araC PBAD cassette to yield E. ictaluri strains with the DELTAPcrp11::TT araC PBAD crp, DELTAPinsA31::TT araC PBAD insA, and DELTAPfur70::TT araC PBAD fur deletion-insertion mutations. Growth of such strains in the presence of arabinose leads to transcription of the crp, fur and LPS O-antigen genes, but expression ceases in vivo since there is no free arabinose in fish tissues. To eliminate use of plasmid vectors with drug resistance genes and to stabilize plasmid vectors in RAEVs in vivo, the asdA balanced-lethal host-vector system with an Asd+ plasmid vector will be utilized. Delayed antigen synthesis will be constructed by replacement of the relA gene with a TT araC PBAD lacI cassette. The LacI repressor is synthesized in the presence of arabinose. LacI binds to the Ptrc promoter, which controls transcription of the cross-protective antigen glyceraldehyde-3-phosphate dehydrogenase. The regulated programmed cell lysis is achieved by using a strain with the DELTAasdA02::TT araC PBAD c2 and DELTAPmurA60::TT araC PBAD murA mutations and complementing the two mutations by a plasmid vector that possesses the wild-type asdA and murA genes under control of araC PBAD. The plasmid vector contains P22 PR, a C2-regulated promoter, with opposite polarity at the 3' end of the asdA and murA genes. P22 PR directs synthesis of antisense mRNA to block translation of mRNA transcribed from asdA and murA genes during programmed lysis when arabinose is absent. The bath/oral RAEV will consists of 5 to 7 genetic modifications: DELTAPcrp11::TT araC PBAD crp, DELTAPfur70::TT araC PBAD fur; DELTAPinsA40::TT araC PBAD insA or DELTAgne-25; DELTAesrB80; DELTArelA50::araC PBAD lacI TT; DELTAasdA02::TT araC PBAD c2; and DELTAPmurA60::TT araC PBAD murA. We will fully evaluate the abilities of the RAEV to colonize lymphoid tissues in catfish, induce cross immune-protection, and exhibit in vivo lysis. Success will provide improved fish health, economic benefit, and enhanced food safety.
<p>PROGRESS: 2009/09 TO 2013/08 <p>Target Audience: <br/>Culture of channel catfish (Ictalurus punctatus) is the largest aquaculture in the U.S. Also this aquaculture is significantly growing in Brazil and African countries. The most serious bacterial pathogen affecting this industry is Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). The current vaccine against ESC is a live E. ictaluri rifampicin-resistant strain attenuated by unknown genetic modifications. Although USDA has licensed this vaccine, it has marginal safety in terms of animal health and environmental release and is also not very efficacious. Live recombinant vaccines, which protect against several diseases at low cost, have not yet been developed for the aquaculture industry. E. ictaluri is an invasive intracellular pathogen; an excellent candidate to develop a bath-oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the aquaculture industry. The objective of this proposal was to develop an antibiotic-sensitive bath/oral live RAEV to prevent Edwardsiella and F. columnare infections in catfish. In addition, this vaccine vector could be modified for use against other fish pathogens. The specific objectives of this proposal were to genetically engineer E. ictaluri to: (i) develop a balance between attenuation and immune protection; (ii) develop a balanced-lethal plasmid vector-bacterial host system; and (iii) synthesize recombinant antigens in attenuated E. ictaluri vaccine strains. In terms of dissemination of information learned,we have published 9 manuscripts, are finalizing one addition article for a peer-reviewed journal and a review article. We have given 27 oral and poster presentations at 20 regional, national and international meetings in the fields of aquaculture, animal health and microbiology. We established collaborations with aquaculture researchers at Louisiana State University, Mississippi State University, Auburn University and the University of Georgia. In regard to teaching and mentoring, this project was written and developed by Javier Santander for his Ph.D. research. During this period, Javier Santander and Dr. Curtiss mentored two Ph.D. students from the East Chinese University of Science and Technology in Shanghai and three undergraduate students from Arizona State University, School of Life Sciences. Changes/Problems: The major problem encountered was our inability to construct a RAEV with regulated delayed in vivo attenuation. This was because the araC PBAD arabinose-dependent regulatory cassette derived from E. coli is not functional in E. ictaluri, even in the presence of AraE, one of the main systems for arabinose import. Another significant problem was the poor null protection triggered by the F. columnare GAPDH antigen. Research on fish pathogens and development of vaccines to prevent diseases of fish has ceased at ASU due to lack of funding necessitating Dr. Santander�s relocation to Chile where he will hopefully be able to continue this research. What opportunities for training and professional development has the project provided? Dr. Roy Curtiss III was the Principal Director in this proposal. Dr. Curtiss provided his expertise on the field of live attenuated recombinant vaccines and laboratory facilities. Dr. Javier Santander participated as Research Scientist in this proposal. Dr. Santander designed, wrote and developed this proposal to fund his Ph.D. research. He developed the research, published eight articles and gave twenty-seven conference presentations in different scientific societies regarding this research. Also Dr. Santander become elected member of CRWAD. Currently, Dr. Santander accepted an Assistant Professor position at Universidad Mayor, Chile and I am sponsoring him for an Adjunct Faculty position in the School of Life Sciences, Arizona State University. During this period, Dr. Santander and Dr. Roy Curtiss III have mentored two Ph.D. students from the East Chinese University of Science and Technology in Shanghai. Dr. Santander mentored two undergraduates students, Taylor Martin (School of life Sciences, Microbiology major) and Amanda Loh (School of Life Sciences, Biology major). Also, Dr. Curtiss and Dr. Santander contributed to Ignacia Diaz (MSc Nanonosciences, Department of Physics) program. How have the results been disseminated to communities of interest? Collaborations were established with aquaculture researchers at Louisiana State University, Mississippi State University, Auburn University and the University of Georgia. Dr. Santander has interacted with several catfish aquaculture farms. Also we are in contact with Merial and Merck relative to vaccine licensing. Dr. Santander has given oral presentations in the Eastern Fish Health Workshop and in the Western Fish Health Workshop and talks and poster presentations at 18 other regional, national and international meetings in the fields of aquaculture, animal health and microbiology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported
<p>PROGRESS: 2009/09/01 TO 2010/08/31 <p>OUTPUTS: <br/>Culture of channel catfish (Ictalurus punctatus) is the largest aquaculture in the U.S. The most serious bacterial pathogen affecting this industry is Edwardsiella ictaluri. The current live vaccine against E. ictaluri is a rifampicin-resistant strain attenuated by unknown genetic modifications. Although USDA has licensed this vaccine, it has marginal safety in terms of animal health and environmental release. Live recombinant vaccines, which protect against several diseases at low cost, have not yet been developed for the aquaculture industry. E. ictaluri, the causative agent of catfish enteric septicemia, is an invasive intracellular pathogen; an excellent candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the aquaculture industry. The objective of this proposal is to develop an antibiotic-sensitive bath/oral live RAEV to prevent Edwardsiella and F. columnare infections in catfish. In addition, this vaccine vector can be modified to be used against other types of pathogens. The specific objectives of this proposal are to genetically engineer E. ictaluri for: (i) balance attenuation and immune protection; (ii) develop a balanced-lethal system; and (iii) synthesize recombinant antigens in E. ictaluri vaccine. We have developed several technologies, discover new aspects of E. ictaluri pathogenesis and contribute to education, including: (i) Methodology to delete genes in-frame from the E. ictaluri chromosome in absence of antibiotic markers. This technology allows the construction and design of stable vaccines in absence of antibiotic resistant genes, in contrast to the current vaccines for catfish. We have facilitated our technology to other groups, including Dr. Attila Karsi from Mississippi State University. (ii) Balanced-lethal system to synthesize recombinant antigens in E. ictaluri in absence of antibiotic markers. By using this technology, E. ictaluri becomes plasmid dependent in absence of antibiotic resistant genes. This plasmid can be used to synthesize heterologous antigens. (iii) We evaluated GAPDH from F. columnare as possible protective antigen in catfish. This antigen was evaluated as injectable protein and in a RAEV strain with no immune protection. (iv) We have constructed a series of bath/oral E. ictaluri attenuated vaccines. Currently, are collaborating with several groups to design vaccines against several catfish pathogens. (v) We have discovered new molecular mechanisms of E. ictaluri pathogenesis and physiology, including: iron acquisition system in absence of siderophores; heme-hemoglobin acquisition system; effects of cAMP
receptor protein in the E. ictaluri physiology; functionality of asd genes; mechanism of resistance to antimicrobial peptides. (vi) Teaching and mentoring. This project was written and developed by Dr. Javier Santander as part of his Ph.D. thesis. During this period, Dr. Santander and Dr. Curtiss III mentored two Ph.D. students from the East Chinese University of Science and Technology. Currently three undergraduates students are participating in this project under Dr. Santander direction.<p> PARTICIPANTS: <br/>Dr. Roy Curtiss III is participating as Principal Director in this proposal. Dr. Curtiss has provided with his expertise on the field of live attenuated recombinant vaccines and laboratory facilities. Dr. Javier Santander is participating as Research Scientist in this proposal. Dr. Santander designed, wrote and developed this proposal as part of his Ph.D. thesis. He has developed most of the research, published four articles and given twenty-seven conference presentations in different scientific societies regarding this research. During this period, Dr. Santander and Dr. Roy Curtiss III have mentored two Ph.D. students from the East Chinese University of Science and Technology. Currently, three undergraduates students, Taylor Martin (School of life Sciences, Microbiology major), Amanda Loh (School of life Sciences, Biology major) and Ignacia Diaz (Department of Chemistry), are participating in this project under Dr. Santander direction. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS:Not relevant to this project.