Progress 09/01/09 to 08/31/13
Outputs
Target Audience: 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
Impacts
What was accomplished under these goals?
In our research, we developed new genetic technologies, discovered new aspects of E. ictaluri pathogenesis and physiology, and discovered and created new vaccine strains. Our work included: (i) Developing methods to precisely delete genes in-frame from the E. ictaluri chromosome in the absence of antibiotic-resistance markers. This technology allows the construction and design of stable vaccines in the absence of antibiotic resistant genes, in contrast to the current antibiotic-resistant live vaccines for catfish. In addition to publishing these methods and discussing them at international, national and regional aquaculture meetings, we have facilitated transfer of our technology to other groups, including Dr. Attila Karsi and Dr. Mark Lawrence at Mississippi State University (Abdelhamed et al., Vet Microbiol (2012) 162:858-565). (ii) Developing a balanced-lethal plasmid-host system to specify synthesis of recombinant antigens in E. ictaluri in the absence of antibiotic-resistance markers. By using this technology, E. ictaluri becomes plasmid dependent in the absence of antibiotic-resistance genes. This plasmid is used to specify synthesis of heterologous antigens. (iii) Evaluating GAPDH from F. columnare (using codon optimized and non-codon optimized DNA sequences specifying its synthesis) as a possible protective antigen in catfish. This antigen was evaluated as an injectable protein and in a RAEV strain with no immune protection when injected and no synthesis by recombinant E. ictaluri and therefore no protection induced. (iv) Constructing a series of bath-oral E. ictaluri attenuated vaccines. Currently, we are collaborating with several groups to design vaccines against several catfish pathogens (although this effort will now cease). (v) Discovering new molecular mechanisms of E. ictaluri pathogenesis and physiology, including: an iron acquisition system in absence of siderophores; a heme-hemoglobin acquisition system; effects of cAMP receptor protein on E. ictaluri physiology; functionality of asd genes; mechanism of resistance to antimicrobial peptides; lipopolysaccharide synthesis and impact on catfish gut inflammation.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Guan, L., J. Santander, M. Mellata, Y. Zhang, and R. Curtiss 3rd. 2013. Identification of an Iron Acquisition Machinery of Flavobacterium columnare. Dis. Aqua. Org. (accepted).
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Santander, J. 2012. Edwardsiellosis, an Emerging Enteric Zoonotic of Aquatic Animals. Immunity and Diseases. 1(1):1-2.
- Type:
Book Chapters
Status:
Under Review
Year Published:
2014
Citation:
J. Santander. 2014. Zebrafish bacterial host model, In: Zebrafish: Development, Breeding and Implications for the Environment.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Diaz, M. I., R. Curtiss III and J. Santander. 2013. The Sticky Factor: A Novel Adhesin in Edwardsiella ictaluri. Annual Eastern Fish Heath Workshop. Gettysburg, PA, USA. Oral presentation
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Santander, J., T. Martin, A. Loh, C. Pohlez, G. M. Gatlin III and R. Curtiss III. 2013. Mechanism of Intrinsic Resistance to Antimicrobial Peptides of Edwardsiella ictaluri and its Influence on Fish Virulence and Gut Inflammation. Microbiology. 159:1471-1486. doi:10.1099/mic.0.066639-0
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Santander, J. 2013. Appling Bacterial Virulence Evolution To Edwardsiella ictaluri Vaccine Design for Multiple Channel Catfish Pathogens. Annual Eastern Fish Heath Workshop. Gettysburg, PA, USA. Oral presentation
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Santander, J., T. Martin and R. Curtiss III. 2013. Lipopolysaccharide Synthesis, Antimicrobial Peptide Resistance and Fish Virulence in Edwardsiella ictaluri. Annual Eastern Fish Heath Workshop. Gettysburg, PA, USA. Oral presentation
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Martin, T., R. Curtiss III and Santander, J. 2013. UDP-glucuronic Acid Transferase of Edwardsiella ictaluri and Virulence. American Association for the Advancement of Science, Boston, MA. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Loh, A. and Santander J. 2013. Phenotype, Virulence and Immunogenicity of Aeromonas salmonicida Cyclic Adenosine 3,5-Monophosphate Receptor Protein (Crp) Mutants in fish Host. American Association for the Advancement of Science, Boston, MA. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Santander, J., Martin T., Loh A. and R. Curtiss III. 2013. Intrinsic Resistance to Antimicrobial Peptides of Edwardsiella ictaluri and its Influence on Fish Gut Inflammation and Virulence. Aquaculture America Annual Meeting. Tennessee, Nashville, TN, USA. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2012
Citation:
Santander, J., Martin T., Loh A. and R. Curtiss III. 2012. Mechanisms of Intrinsic Resistance to Antimicrobial Peptides of Edwardsiella ictaluri and its Influence on Fish Gut Inflammation and Virulence. Conference of Research Workers in Animal Diseases, Chicago, Illinois, USA. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2012
Citation:
Loh, A., Martin T., R. Curtiss III and J. Santander. 2012. Zebrafish Larvae as Model to Evaluate Lipopolysaccharide Toxicity. Conference of Research Workers in Animal Diseases, Chicago, Illinois, USA. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2012
Citation:
Santander, J., T. Martin and R. Curtiss III. 2012. Edwardsiella ictaluri Sweeteners and Fish Pathogenesis. Western Fish Disease Workshop. Boise, Idaho, USA. Oral presentation
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: 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. PARTICIPANTS: 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.
Impacts
The current technology to develop mutant strains or attenuated vaccine strains in E. ictaluri is mediated random mutagenesis and/or directed transposon mutagenesis. These techniques have serious issues concerning vaccine safety and stability. We have adapted the suicide vector technology to E. ictaluri. This particular technology allows specific deletion of the gene fragments. This is a powerful tool to develop stable live attenuated vaccines without reversion to the virulent wild type phenotype. The key to develop a recombinant vaccine in absence of antibiotic marker is to create a balanced lethal system. We developed a balanced lethal system in E. ictaluri by deleting the asdA gene. asdA mutants have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Introduction of an Asd+ plasmid into a ΔasdA mutant makes the bacterial strain plasmid dependent. This dependence on the Asd+ plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. This recombinant plasmid can be used to synthesize antigens against a diversity of pathogens. One of the antigens evaluated was GAPDH from F. columnare. Although, this well conserved antigen induces protection against several bacterial fish pathogens, it does not induce immune protection against F. columnare. Currently, we are searching for new F. columnare antigens to be expressed in our E. ictaluri attenuated vaccines. In the context of basic research, we determined that deletion of conserved genes that control virulence factors could be used to develop universal live attenuated vaccines. Although, crp and fur control different virulence factors in Edwardsiella, in comparison to other enterics, individual deletion of these genes triggered protective immune response at the systemic and mucosal level of the fish. We suggest that crp and fur could be universally used to develop live attenuate recombinant vaccines for different hosts. Also, we have determined the mechanisms of intrinsic resistance to antimicrobial peptides of E. ictaluri. Intrinsic resistance to the cationic antimicrobial peptides is a specific property of the genus Edwardsiella. Particularly, E. ictaluri is highly resistant to cationic antimicrobial peptides (CAMP). We hypothesized that the lipopolysaccharide (LPS) of E. ictaluri plays role in resistance to antimicrobial peptides.
Publications
- Santander, J. and R. Curtiss III. 2010. Intrinsic Resistance of Edwardsiella ictaluri to Cationic Antimicrobial Peptides is Mediated by UDP-Glc Dehydrogenase, an Enzyme Required for the O-polysaccharide Synthesis. The 6th International Symposium on Aquatic Animal Health, Tampa, Florida, USA.
- Santander, J. G. Golden, Soo-Young Wanda, and R. Curtiss III. 2012. The Fur Regulated Iron Uptake System of Edwardsiella ictaluri and its Influence on Pathogenesis and Immunogenicity in the Catfish Host. Infection and Immunity 80(8): 2689-2703.
- Santander, J. 2012. Developing Live attenuated vaccines by deleting genes for iron metabolism in pathogenic bacteria. World Aquaculture. 43(3): 28-31
- Santander, J, A. Mitra, and R. Curtiss III. 2011. Phenotype, Virulence, and Immunogenicity of Edwardsiella ictaluri Cyclic Adenosine 3,5-Monophosphate Receptor Protein (Crp) Mutants in Catfish Host. Fish and Shellfish Immunology. 31:1142-1153.
- Santander, J., W. Xin, Z. Yang, and R. Curtiss III. 2010. The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology. PloS One. 5(12):e15944.
- Santander, J., T. Martin and R. Curtiss III. 2012. Edwardsiella ictaluri Sweeteners and Fish Pathogenesis. Western Fish Disease Workshop. Boise, Idaho, USA.
- Santander, J. and R. Curtiss III. 2012. Iron uptake and virulence in Edwardsiella ictaluri. Annual Eastern Fish Heath Workshop. Lake Placid, NY, USA.
- Santander, J, and R. Curtiss III. 2012. The Fur Regulated Iron Uptake System of Edwardsiella ictaluri and its Influence on Pathogenesis. World Aquaculture Society, Aquaculture America annual meeting. Las Vegas, NV, USA.
- Santander, J, and R. Curtiss III. 2011. Edwardsiella ictaluri Cyclic Adenosine 3, 5-Monophosphate Receptor Protein (Crp) Mutant Vaccine and Boost Immunization in Catfish (Ictalurus punctatus). Conference of Research Workers in Animal Diseases, Chicago, Illinois, USA.
- Santander, J., L. Guan, M. Mellata and R. Curtiss III. 2011. Identification of iron acquisition machinery of Flavobacterium columnare. Conference of Research Workers in Animal Diseases, Chicago, Illinois, USA.
- Santander, J. and R. Curtiss III. 2011. Regulation of iron uptake in Edwardsiella ictaluri as a vaccine for fish. Conferencia Encuentros, connecting Chilean minds. University of Berkley, San Francisco, CA.
- Santander, J. and R. Curtiss III. 2011. Natural Resistance to Antimicrobial Peptides and its Influence on Pathogenesis in Edwardsiella ictaluri. Nexos USA-Chile, Creating networks for the scientific future of Chile, Massachusetts Institute of Technology, Boston, MA, USA.
- Santander, J, and R. Curtiss III. 2011. Natural Resistance of Edwardsiella ictaluri to Cationic Antimicrobial Peptides and its Influence on Catfish Pathogenesis. The 8th Cold Spring Harbor Laboratories meeting on Microbial Pathogenesis and Host Response. Cold Spring Harbor, NY, USA.
- Santander, J. and R. Curtiss III. 2011. Attenuation and Immunogenicity of Edwardsiella ictaluri mutants in catfish (Ictalurus punctatus). Animal Health-USDA annual meeting, Washington, DC, USA.
- Santander, J., Z. Yang, and R. Curtiss III. 2010. Aspartate-semialdehyde dehydrogenase (asdA) gene of Edwardsiella ictaluri and its use to develop a balanced-lethal vector-host system for construction of antibiotic-sensitive live attenuated recombinant vaccines for the aquaculture industry. World Aquaculture Society, Aquaculture meeting, San Diego, California, USA.
- Santander, J., Z. Yang and R. Curtiss III. 2009. Balanced-lethal Vector-Host System in Edwardsiella ictaluri as First Step to Develop Antibiotic-Sensitive Live Recombinant Attenuated Edwardsiella Vaccines for the Catfish Aquaculture Industry. Conference of research workers in animal diseases-Animal health-USDA annual meeting, Chicago, Illinois, USA.
- Curtiss, R. III, and J. Santander Morales. 2009. Recombinant Edwardsiella Bacterium. U.S. Provisional Application No. 61/182,569, filed 05/29/09. U.S. Patent Application 12/789,869, filed 5/28/10. PCT/US12/789,869, filed 05/28/10. (Internal Tech ID M9-119L). http://www.faqs.org/patents/app/20100303863
- Santander, J., A. Mitra and R. Curtiss III. 2011. Effects of cAMP receptor protein (Crp) gene in the fish pathogen Edwardsiella ictaluri, a potential strategy to develop live attenuated vaccines for the aquaculture industry. World Aquaculture Society, Aquaculture America annual meeting. Advances in Aquatic Veterinary Medicine, New Orleans, Louisiana, USA.
- Santander, J. and R. Curtiss III. 2010. Ferric Uptake Regulator (Fur) and Iron Uptake of Edwardsiella ictaluri, a potential Strategy for Catfish Vaccinology. Conference of Research Workers in Animal Diseases, Chicago, Illinois, USA.
- Santander, J. and R. Curtiss III. 2010. New genetic design of oral live recombinant vaccines for the aquaculture industry. Nexos USA-Chile, Creating networks for the scientific future of Chile, Washington, DC, USA.
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