Progress 07/01/05 to 06/30/11
Outputs
OUTPUTS: This research is having a broad impact on bacterial pathogenesis research: plasmids constructed as a component of this project were made publicly available on AddGene (http://www.addgene.org/pgvec1). As a result, plasmids have been disseminated to researchers in the U.S. and other countries for use in multiple gram-negative species. Research results have disseminated in peer-reviewed journals and at aquatic animal health meetings (International Symposium on Aquatic Animal Health, Eastern Fish Health Workshop, Annual Meeting of the Fish Health Section of the American Fisheries Society). PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine is the project director. Dr. Attila Karsi at MSU-CVM is assisting in direction of this project. Research was conducted by a Ph.D. student under the direction of Dr. Lawrence, Simon Menanteau-Ledouble, and by research associate Michelle Banes. Joey Greene, a veterinary student at the MSU College of Veterinary Medicine, conducted some of the research for this project as a component of the MSU-CVM Summer Research Experience program. Dr. Nagihan Gulsoy, a visiting scientist from Turkey, also assisted in the project. TARGET AUDIENCES: Fundamental knowledge about the pathogenesis of enteric septicemia of catfish (caused by Edwardsiella ictaluri) from this project will benefit the catfish health research community. The basic knowledge gained should stimulate new research on pathogenesis of enteric septicemia of catfish (ESC), the immune response to E. ictaluri, and on vaccine development for catfish. This research ultimately targets catfish producers to provide new solutions to help control ESC, which is their most important disease problem. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have developed plasmids that can be used to constitutively or inducibly express luminescence or fluorescence in a broad range of gram-negative bacteria. We have developed methods for monitoring and quantifying E. ictaluri infection in living channel catfish, yielding experimental results with less variability and using fewer fish. This research demonstrates that bioluminescence is a sensitive, effective method for monitoring E. ictaluri infections in vivo. Fundamental knowledge on the pathogenesis of ESC has been obtained; in vivo imaging analysis confirmed previous findings that the anterior kidney, posterior kidney, and spleen are primary target organs, and it also showed that other organs (gill and heart) are sites with high E. ictaluri concentrations. Our studies also revealed a previously unreported aspect of E. ictaluri pathogenesis: E. ictaluri attaches on channel catfish skin abrasions, and skin abrasions can contribute to the development of septicemia. Identification of skin abrasions as a site for E. ictaluri invasion of the channel catfish host has potential implications for management of disease prevention. Genes necessary for E. ictaluri skin attachment and colonization were determined, including genes encoding regulatory proteins for adhesins and putative novel adhesins. This knowledge could lead to development of new strategies for blocking the pathogen's attachment to channel catfish.
Publications
- Menanteau-Ledouble, S. and M. L. Lawrence. 2009. Genes involved in skin colonization of channel catfish Ictalurus punctatus by Edwardsiella ictaluri as determined by in vivo screening of transposon mutants. Annual Meeting of the Fish Health Section of the American Fisheries Society, Park City, Utah.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: This research is having a broad impact on bacterial pathogenesis research: plasmids constructed as a component of this project were made publicly available on AddGene (http://www.addgene.org/pgvec1). As a result, plasmids have been disseminated to researchers in the U.S. and other countries for use in multiple gram-negative species. Research results have disseminated in peer-reviewed journals and at aquatic animal health meetings (International Symposium on Aquatic Animal Health, Eastern Fish Health Workshop, Annual Meeting of the Fish Health Section of the American Fisheries Society). PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine is the project director. Dr. Attila Karsi at MSU-CVM is assisting in direction of this project. Research was conducted by a Ph.D. student under the direction of Dr. Lawrence, Simon Menanteau-Ledouble, and by research associate Michelle Banes. Joey Greene, a veterinary student at the MSU College of Veterinary Medicine, conducted some of the research for this project as a component of the MSU-CVM Summer Research Experience program. Dr. Nagihan Gulsoy, a visiting scientist from Turkey, also assisted in the project. TARGET AUDIENCES: Fundamental knowledge about the pathogenesis of enteric septicemia of catfish (caused by Edwardsiella ictaluri) from this project will benefit the catfish health research community. The basic knowledge gained should stimulate new research on pathogenesis of enteric septicemia of catfish (ESC), the immune response to E. ictaluri, and on vaccine development for catfish. This research ultimately targets catfish producers to provide new solutions to help control ESC, which is their most important disease problem. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have developed plasmids that can be used to constitutively or inducibly express luminescence or fluorescence in a broad range of gram-negative bacteria. We have developed methods for monitoring and quantifying E. ictaluri infection in living channel catfish, yielding experimental results with less variability and using fewer fish. This research demonstrates that bioluminescence is a sensitive, effective method for monitoring E. ictaluri infections in vivo. Fundamental knowledge on the pathogenesis of ESC has been obtained; in vivo imaging analysis confirmed previous findings that the anterior kidney, posterior kidney, and spleen are primary target organs, and it also showed that other organs (gill and heart) are sites with high E. ictaluri concentrations. Our studies also revealed a previously unreported aspect of E. ictaluri pathogenesis: E. ictaluri attaches on channel catfish skin abrasions, and skin abrasions can contribute to the development of septicemia. Identification of skin abrasions as a site for E. ictaluri invasion of the channel catfish host has potential implications for management of disease prevention. Genes necessary for E. ictaluri skin attachment and colonization were determined, including genes encoding regulatory proteins for adhesins and putative novel adhesions. This knowledge could lead to development of new strategies for blocking the pathogen's attachment to channel catfish.
Publications
- Menanteau-Ledouble, S. and M. L. Lawrence. 2011 In press. Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples. Journal of Visualized Experiments.
- Menanteau-Ledouble, S., A. Karsi, and M. L. Lawrence. 2011. Importance of skin abrasion as a primary site of adhesion for Edwardsiella ictaluri and impact on invasion and systemic infection in channel catfish Ictalurus punctatus. Vet. Microbiol. 148(2-4):425-430.
- Menanteau-Ledouble, S. and M. L. Lawrence. 2010. Construction and screening of a library of attenuated mutants in Edwardsiella ictaluri, identification of genes involved in bacterial invasion and potential as vaccines. International Symposium on Aquatic Animal Health, Tampa, Florida.
- Karsi, A. and M. L. Lawrence. 2007. Broad host range fluorescence and bioluminescence expression vectors for gram negative bacteria. Plasmid.
- Karsi, A., S. Menanteau-Ledouble, and M. L. Lawrence. 2006. Development of bioluminescent Edwardsiella ictaluri for non-invasive disease monitoring. FEMS Microbiol. Lett. 260:216-223.
- Menanteau-Ledouble, S., M. Leroux, A. Karsi, and M. L. Lawrence. 2008. Use of green fluorescent protein-labeled Edwardsiella ictaluri to investigate the pathogenesis of enteric septicemia in channel catfish. 33rd Annual Eastern Fish Health Workshop, Atlantic Beach, North Carolina.
- Menanteau-Ledouble, S., A. Karsi, and M. L. Lawrence. 2006. Importance of skin abrasion as a primary site of adhesion for Edwardsiella ictaluri and in the subsequent development of enteric septicemia in channel catfish Ictalurus punctatus. 6th International Symposium on Aquatic Animal Health, San Francisco, California.
- Karsi, A. and M. L. Lawrence. 2006. Development of fluorescent Edwardsiella ictaluri and visualization of the pathogen in channel catfish neutrophils. 106th General Meeting of the American Society for Microbiology, Orlando, Florida.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This research is having a broad impact on bacterial pathogenesis research: plasmids constructed as a component of this project were made publicly available on AddGene (http://www.addgene.org/pgvec1). As a result, plasmids have been disseminated to researchers in the U.S. and other countries for use in multiple gram-negative species. Research results have disseminated in peer-reviewed journals and at aquatic animal health meetings (International Symposium on Aquatic Animal Health, Eastern Fish Health Workshop, Annual Meeting of the Fish Health Section of the American Fisheries Society). PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine is the project director. Dr. Attila Karsi at MSU-CVM is assisting in direction of this project. Research is being conducted by a Ph.D. student under the direction of Dr. Lawrence, Simon Menanteau, and by Michelle Banes. Joey Greene, a veterinary student at the MSU College of Veterinary Medicine, conducted some of the research for this project as a component of the MSU-CVM Summer Research Experience program. Dr. Nagihan Gulsoy, a visiting scientist from Turkey, also assisted in the project. TARGET AUDIENCES: The fundamental knowledge about the pathogenesis of enteric septicemia of catfish (caused by Edwardsiella ictaluri) from this project will benefit the catfish health research community. The basic knowledge gained should stimulate new research on pathogenesis of enteric septicemia of catfish (ESC), the immune response to E. ictaluri, and on vaccine development for catfish. This research ultimately targets catfish producers to provide new solutions to help control ESC, which is their most important disease problem. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have developed plasmids that can be used to constitutively or inducibly express luminescence or fluorescence in a broad range of gram-negative bacteria. We have developed methods for monitoring and quantifying E. ictaluri infection in living channel catfish, yielding experimental results with less variability and using fewer fish. This research demonstrates that bioluminescence is a sensitive, effective method for monitoring E. ictaluri infections in vivo. Fundamental knowledge on the pathogenesis of ESC has been obtained; in vivo imaging analysis confirmed previous findings that the anterior kidney, posterior kidney, and spleen are primary target organs, and it also showed that other organs (gill and heart) are sites with high E. ictaluri concentrations. Our studies also revealed a previously unreported aspect of E. ictaluri pathogenesis: E. ictaluri attaches on channel catfish skin abrasions, and skin abrasions can contribute to the development of septicemia. Identification of skin abrasions as a site for E. ictaluri invasion of the channel catfish host has potential implications for management of disease prevention. Genes necessary for E. ictaluri skin attachment and invasion were determined; this knowledge could lead to development of new strategies for blocking the pathogen's attachment to channel catfish .
Publications
- Menanteau-Ledouble, S. and M. L. Lawrence. 2009. Genes involved in skin colonization of channel catfish Ictalurus punctatus by Edwardsiella ictaluri as determined by in vivo screening of transposon mutants. Annual Meeting of the Fish Health Section of the American Fisheries Society, Park City, Utah.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This research is having a broad impact on bacterial pathogenesis research: plasmids constructed as a component of this project were made publicly available on AddGene (http://www.addgene.org/pgvec1). As a result, plasmids have been disseminated to researchers in the U.S. and other countries for use in multiple gram-negative species. Research results have disseminated in peer-reviewed journals and at aquatic animal health meetings (International Symposium on Aquatic Animal Health, Eastern Fish Health Workshop). PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine is the project director. Dr. Attila Karsi at MSU-CVM is assisting in direction of this project. Research is being conducted by a Ph.D. student under the direction of Dr. Lawrence, Simon Menanteau, and by Michelle Banes. Joey Greene, a veterinary student at the MSU College of Veterinary Medicine, conducted some of the research for this project as a component of the MSU-CVM Summer Research Experience program. Dr. Nagihan Gulsoy, a visiting scientist from Turkey, also assisted in the project. TARGET AUDIENCES: The fundamental knowledge about the pathogenesis of enteric septicemia of catfish (caused by Edwardsiella ictaluri) from this project will benefit the catfish health research community. The basic knowledge gained should stimulate new research on pathogenesis of enteric septicemia of catfish (ESC), the immune response to E. ictaluri, and on vaccine development for catfish. This research ultimately targets catfish producers to provide new solutions to help control ESC, which is their most important disease problem. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have developed plasmids that can be used to constitutively or inducibly express luminescence or fluorescence in a broad range of gram-negative bacteria. We have developed methods for monitoring and quantifying E. ictaluri infection in living channel catfish, yielding experimental results with less variability and using fewer fish. This research demonstrates that bioluminescence is a sensitive, effective method for monitoring E. ictaluri infections in vivo. Fundamental knowledge on the pathogenesis of ESC has been obtained; in vivo imaging analysis confirmed previous findings that the anterior kidney, posterior kidney, and spleen are primary target organs, and it also showed that other organs (gill and heart) are sites with high E. ictaluri concentrations. Our studies also revealed a previously unreported aspect of E. ictaluri pathogenesis: E. ictaluri attaches on channel catfish skin abrasions, and skin abrasions can contribute to the development of septicemia. Identification of skin abrasions as a site for E. ictaluri invasion of the channel catfish host has potential implications for management of disease prevention.
Publications
- Menanteau-Ledouble, S., M. Leroux, A. Karsi, and M. L. Lawrence. 2008. Use of green fluorescent protein-labeled Edwardsiella ictaluri to investigate the pathogenesis of enteric septicemia in channel catfish. 33rd Annual Eastern Fish Health Workshop, Atlantic Beach, North Carolina.
- Karsi, A. and M. L. Lawrence. 2007. Broad host range fluorescence and bioluminescence expression vectors for gram negative bacteria. Plasmid.
- Karsi, A., S. Menanteau-Ledouble, and M. L. Lawrence. 2006. Development of bioluminescent Edwardsiella ictaluri for non-invasive disease monitoring. FEMS Microbiol. Lett. 260:216-223.
- Menanteau-Ledouble, S., A. Karsi, and M. L. Lawrence. 2006. Importance of skin abrasion as a primary site of adhesion for Edwardsiella ictaluri and in the subsequent development of enteric septicemia in channel catfish Ictalurus punctatus. 6th International Symposium on Aquatic Animal Health, San Francisco, California.
- Karsi, A. and M. L. Lawrence. 2006. Development of fluorescent Edwardsiella ictaluri and visualization of the pathogen in channel catfish neutrophils. 106th General Meeting of the American Society for Microbiology, Orlando, Florida.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We are experimentally infecting channel catfish with virulent E. ictaluri strain 93-146 that constitutively expresses dual reporter genes, bacterial luciferase and green fluorescent protein. The progress of infection is monitored and quantified using an imaging system equipped with a CCD camera. Bioluminescence is being used to confirm colonized tissues, and luminescent tissues will be collected for subsequent microscopic examination for cellular and subcellular localization using fluorescence. We will construct a recombinant E. coli strain expressing E. ictaluri fimbriae and an isogenic E. ictaluri fimbrial mutant strain. The E. ictaluri mutant strain will be used to determine if fimbriae are required for host invasion and virulence, and the recombinant E. coli strain will be used to determine if E. ictaluri fimbriae are sufficient for mediating host attachment and invasion. PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine is the project director. Dr. Attila Karsi at MSU-CVM is assisting in direction of this project. Research is being conducted by a Ph.D. student under the direction of Dr. Lawrence, Simon Menanteau, and by Michelle Banes. Joey Greene, a veterinary student at the MSU College of Veterinary Medicine, conducted some of the research for this project as a component of the MSU-CVM Summer Research Experience program. Dr. Nagihan Gulsoy, a visiting scientist from Turkey, also assisted in the project. TARGET AUDIENCES: The fundamental knowledge about the pathogenesis of enteric septicemia of catfish (caused by Edwardsiella ictaluri) from this project will benefit the catfish health research community. The basic knowledge gained should stimulate new research on pathogenesis of ESC, the immune response to E. ictaluri, and on vaccine development for catfish. This research ultimately targets catfish producers to provide new solutions to help control ESC, which is their most important disease problem. PROJECT MODIFICATIONS: No Project Modifications information reported.
Impacts
Plasmids for expression of both bacterial luciferase and green fluorescent protein in E. ictaluri have been constructed and used to create E. ictaluri strains with bioluminescence and fluorescence. Using bioluminescent E. ictaluri, experimental infections of channel catfish have been monitored and quantified in the living host using a photon-counting intensified-charge-coupled device (ICCD) camera. In vivo imaging analysis confirmed previous findings that the anterior kidney, posterior kidney, and spleen are primary target organs, and it also showed that other organs (gill and heart) are sites with high E. ictaluri concentrations. In addition, these studies revealed that E. ictaluri attaches on channel catfish skin abrasions and that skin abrasions can contribute to the development of septicemia. Work on objective 2 using fluorescent E. ictaluri in experimental infections of channel catfish is currently underway to enable investigation of cellular and subcellular localization of E. ictaluri. Our research demonstrates that bioluminescence is a sensitive, effective method for monitoring E. ictaluri infections in vivo. Because fish can be anesthetized, imaged, and recovered, infection can be tracked in the same fish over multiple time points. As a result, fewer fish are required to conduct experimental infections with this method than when conventional methodologies are used, and the amount of fish to fish variation is reduced. In particular, identification of skin abrasions as a site for E. ictaluri invasion of the channel catfish host has potential implications for management of disease prevention. The bioluminescence and fluorescence plasmids constructed for this study have a broad host range; we have sent them to multiple investigators who have requested them for use in several different bacterial species. Therefore, this research is having a broad impact on bacterial pathogenesis research.
Publications
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Progress 01/01/06 to 12/31/06
Outputs
Objective 1 has been met, with additional work still ongoing. Plasmids for expression of both bacterial luciferase and green fluorescent protein in E. ictaluri have been constructed and used to create E. ictaluri strains with bioluminescence and fluorescence. Using bioluminescent E. ictaluri, experimental infections of channel catfish have been monitored and quantified in the living host using a photon-counting intensified-charge-coupled device (ICCD) camera. In vivo imaging analysis confirmed previous findings that the anterior kidney, posterior kidney, and spleen are primary target organs, and it also showed that other organs (gill and heart) are sites with high E. ictaluri concentrations. In addition, these studies revealed that E. ictaluri attaches on channel catfish skin abrasions and that skin abrasions can contribute to the development of septicemia. Work on objective 2 using fluorescent E. ictaluri in experimental infections of channel catfish is currently
underway to enable investigation of cellular and subcellular localization of E. ictaluri.
Impacts
Our research demonstrates that bioluminescence is a sensitive, effective method for monitoring E. ictaluri infections in vivo. Because fish can be anesthetized, imaged, and recovered, infection can be tracked in the same fish over multiple time points. As a result, fewer fish are required to conduct experimental infections with this method than when conventional methodologies are used, and the amount of fish to fish variation is reduced. In particular, identification of skin abrasions as a site for E. ictaluri invasion of the channel catfish host has potential implications for management of disease prevention. The bioluminescence and fluorescence plasmids constructed for this study have a broad host range; we have sent them to multiple investigators who have requested them for use in several different bacterial species. Therefore, this research is having a broad impact on bacterial pathogenesis research.
Publications
- Karsi, A. and M. L. Lawrence. 2007. Broad host range fluorescence and bioluminescence expression vectors for gram negative bacteria. Plasmid.
- Karsi, A., S. Menanteau-Ledouble, and M. L. Lawrence. 2006. Development of bioluminescent Edwardsiella ictaluri for non-invasive disease monitoring. FEMS Microbiol. Lett. 260:216-223.
- Menanteau-Ledouble, S., A. Karsi, and M. L. Lawrence. 2006. Importance of skin abrasion as a primary site of adhesion for Edwardsiella ictaluri and in the subsequent development of enteric septicemia in channel catfish Ictalurus punctatus. 6th International Symposium on Aquatic Animal Health, San Francisco, California.
- Karsi, A. and M. L. Lawrence. 2006. Development of fluorescent Edwardsiella ictaluri and visualization of the pathogen in channel catfish neutrophils. 106th General Meeting of the American Society for Microbiology, Orlando, Florida.
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Progress 01/01/05 to 12/31/05
Outputs
Plasmids for expression of both bacterial luciferase and green fluorescent protein in E. ictaluri have been constructed. The plasmids are stable in E. ictaluri in the absence of antibiotic selection. The expression of bacterial luciferase in E. ictaluri causes bioluminescence, which allows detection of the pathogen by a photon-counting intensified-charge-coupled device (ICCD) camera. The relationship between E. ictaluri density in suspension as measured by luminescence and by plate counts was linear over five orders of magnitude. Experimental infections of juvenile channel catfish with luminescent E. ictaluri have been conducted by both intraperitoneal injection and by bath immersion, and methods were developed for anesthetizing and imaging E. ictaluri in living catfish. In vivo imaging analysis clearly showed that the anterior and posterior kidneys and spleen are the primary target organs.
Impacts
Our research demonstrates that bioluminescence is a sensitive, effective method for monitoring E. ictaluri infections in vivo. Because fish can be anesthetized, imaged, and recovered, infection can be tracked in the same fish over multiple time points. As a result, fewer fish are required to conduct experimental infections with this method than when conventional methodologies are used, and the amount of fish to fish variation is reduced.
Publications
- Lawrence, M. L. and M. M. Banes. 2005. Tissue persistence and vaccine efficacy of an O polysaccharide mutant strain of Edwardsiella ictaluri. J. Aquat. Anim. Health 17:228-232.
- Williams, M. L. and M. L. Lawrence. 2005. Identification and characterization of a two-component hemolysin from Edwardsiella ictaluri. Vet. Microbiol. 108:281-289.
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