Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? From 1987 to 1989, columnaris was the most frequently reported infectious disease in the U.S. catfish industry accounting for 58% of all bacteria cases. Currently, F. columnare is considered the second most important bacterial pathogen in commercial culture of channel catfish in Alabama, second only to Edwardsiella ictaluri (NAHMS, 1997). Although columnaris negatively impacts aquaculture production around the world, bibliographical references to this species are mostly related to taxonomical status rather than virulence properties. To date, the only F. columnare sequence information available in GenBank (http://www.ncbi.nlm. nih.gov/Genbank/) corresponds to ribosomal genes. One of the main objectives within this project was to increase our genetic knowledge on F. columnare. This new genetic
information would be applicable to the future development of new vaccines against columnaris. 2. List the milestones (indicators of progress) from your Project Plan. Milestone 1: Genotyping of Flavobacterium columnare isolates Milestone 2: Developing a new Polymerase Chain Reaction (PCR) detection protocol for F. columnare Milestone 3: Assessing the prevalence of Flavobacterium columnare and Edwarsiella ictaluri in pond raised catfish. Milestone 4: Comparing gene expression profiles between different F. columnare strains by using cDNA-AFLP analysis. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Genotyping and incorporation of new F. columnare isolates into the existing database. Milestone Fully Met 2. Adapting the newly developed PCR detection method into a real-time PCR protocol. Milestone Substantially Met 3. Performing regular samplings using both
cultural methods and PCR detection protocols, to evaluate the presence of F. columnare and E. ictaluri in a catfish farm environment. Milestone Substantially Met 4. Isolate and identify genes that are differentially expressed by two different F. columnare isolates with distinct virulence properties. Milestone Not Met Redirection of Research focus due to change in priorities 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This project has been terminated. The project number 6420-32000-019-06S (from 03/05 to 02/10) will continue the research started by this project. 4a What was the single most significant accomplishment this past year? The PCR-based detection of F. columnare in asymptomatic catfish during regular samplings carried out at the North Fisheries Station, Auburn University. For the first time, routine samplings were performed in pond-
raised catfish in a farm-like environment throughout the complete growing season. The presence of main catfish pathogens, E. ictaluri and F. columnare, was investigated using different techniques: PCR-based direct detection, classical culture-based techniques and indirect ELISA. The genetic-based approach (direct PCR) proved to be more sensitive than culture-based methods since a higher number of positive samples were found by PCR. Edwardsiella ictaluri could not be detected/isolated during the study. This correlates well with the absence of enteric septicemia in catfish (ESC) outbreaks. Flavobacterium columnare did cause two epizootic episodes during the growing season. 4b List other significant accomplishments, if any. A total of 15 new F. columnare (some isolated during the above mentioned samplings and some provide by the Fish Disease Lab at Auburn University) were fingerprinted and added to the existing database. A total of 45 isolates are currently typed by AFLP and ITS
sequencing. The 23S rDNA gene from F. columnare has been fully sequenced. This information is new to the scientific community and will be deposited into GenBank shortly. This newly generated information will be used as target for a real-time PCR-based detection method. Real time PCR has the advantage over the previously developed method of being quantitative. The adaptation of the current standard PCR protocol into a real-time PCR was not possible due to the length of the amplified fragment (the tested real- time PCR primers did not provide the expected sensitivity). The optimal length for real-time PCR fragment should be between 80 to 250 bp. Specific sequences for F. columnare within the very limited genetic information available up to date made it impossible to design specific primers for real-time PCR. A shotgun whole-genome sequencing project has been started. In order to complete this project additional funding is needed (Dr. Arias is seeking support from the NSF). To date,
approximately 3,000 clones containing 1,2 kb of F. columnare genetic information have been sequenced. These clones yielded around 800 contings (contiguous sequences) that have been Blasted in GenBank. Some putative virulence-related genes have been identified and these genes are: iron capitation mechanisms, adhesions, invasion related proteins, and lipopolisaccharide biosynthesis. Currently, we are responsible for more than 95% of the total F. columnare genome sequenced. 4d Progress report. This project has been terminated. The project number 6420-32000-019-06S (from 03/05 to 02/10) will continue the research started by this project. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The major accomplishment of this project was the developing of a rapid, sensitive and specific method for the detection of F. columnare by PCR. This new method allows the direct detection of as few as 100 cells/ml from fish and water samples.
This field efficiency of this protocol was tested during a number of routine catfish samplings. This protocol yielded a higher sensitivity for the detection of F. columnare than culture-based methods. Results can be obtained in less than 8 h. This protocol might be developed into a commercial kit although its cost- effectiveness should be improved for catfish farm surveillance studies and routine diagnosis. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The above mentioned specific PCR protocol has been published and other scientists might use it for a rapid detection of F. columnare. This protocol is being currently used at the Fish Diagnostics Laboratory, Department of Fisheries and Allied Aquacultures, Auburn University, as confirmatory
identification (as alternative to cumbersome classical microbial identification) when atypical Flavobacterium colonies are isolated. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Arias, C. R., Shoemaker, C., Welker, T., Klesius, P. Identification and genetic characterization of Flavobacterium columnare isolates. 21st Annual Meeting of the Alabama Fisheries Association. February 16-18, 2004. Gulfshores, Alabama, USA. Arias, C., Welker, T., Shoemaker, C., Klesius, P. Genetic fingerprinting of Flavobacterium columnare isolates from culture fish. VII International Meeting on Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases. July 18-23, 2004. Valencia, Spain. Arias, C., Shoemaker, C., Welker, T., Klesius, P. Intraspecific diversity of Flavobacterium columnare. Aquaculture 2004. March 1-5, 2004. Honolulu, Hawaii, USA.
Impacts
(N/A)
Publications
- Klesius, P.H., Lovy, J., Evans, J.J., Washuta, E., Arias, C. 2004. Isolation of Edwardsiella Ictaluri from tadpole madtom, Noturus gyrinus in a southwestern New Jersey River. Journal of Aquatic Animal Health. 15(14) :295-301.
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Progress 04/28/00 to 02/28/05
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Aquatic animal farmers in the U.S. continue to identify disease as a major problem in their industry(ies). Several areas of inadequate research are: rapid identification of pathogens, epidemiology of infectious diseases, pathogenesis (i.e. mechanism of disease) of aquatic animal pathogens, and vaccine effectiveness in field studies. The overall objective for this project was to increase our knowledge on bacterial catfish pathogens and to provide fish health specialist with better tools for catfish diseases diagnosis and better management practices. 2. List by year the currently approved milestones (indicators of research progress) Objective 1: Develop and apply rapid methods to detect and diagnose bacterial catfish pathogens. Objective 2: Conduct epidemiological studies using a
multidisciplinary approach (i.e., microbiology, immunology, etc.) to determine and/or assess the risk factors associated with disease and conduct research to determine the disease prevalence, incidence, sources and origin of economically important aquatic animal pathogens, especially, Edwardsiella ictaluri, and Flavobacterium columnare. Objective 3: Isolation and characterization of virulence genes from Flavobacterium columnare. 4a List the single most significant research accomplishment during FY 2006. First time gene expression has been evaluated in F. columnare. 4b List other significant research accomplishment(s), if any. - Development of a rapid, sensitive and specific PCR method for the detection of F. columnare in fish samples - Instrapecies characterization of F. columnare and E. ictaluri using a polyphasic approach - Characterization of the avirulent mutants used as commercial vaccines - Cloning and sequencing approximately 3,000 kb from F. columnare genome 5. Describe the
major accomplishments to date and their predicted or actual impact. Intraspecific characterization of F. columnare and E. ictaluri using a polyphasic approach has resulted in the creation of a data base with 100+ characterized isolates. The generated information has been compiled in a computerized data base that will be the basis for epidemiological studies in the future. This data base will allow isolate tracking (i.e. geographic origin/farm to farm transfer) in the aquaculture environment. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The molecular biology and epidemiology research accomplishments have been disseminated to other scientists and industry through scientific presentations and publications. The immediate benefits of the research will
be realized by other scientists, nationally and internationally. These studies complement the molecular biological and vaccine studies being conducted at ARS, Aquatic Animal Health Research Unit at Auburn, AL, and health research programs at the Catfish Genetics Unit in Stoneville, MS.
Impacts
(N/A)
Publications
- Klesius, P.H., Lovy, J., Evans, J.J., Washuta, E., Arias, C. 2004. Isolation of Edwardsiella Ictaluri from tadpole madtom, Noturus gyrinus in a southwestern New Jersey River. Journal of Aquatic Animal Health. 15(14) :295-301.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Auburn University. Additional details of research can be found in the report for parent project 6420- 32000-012-00D, "Integrated Management of Fish Health by Multi- Disciplinary Approaches." Infectious diseases are the cause of significant economic losses in channel catfish production in the U.S., especially the southeastern states. Edwardsiella ictaluri and Flavobacterium columnare are major bacterial pathogens and results in greater than $80 million per year in losses to U.S. farmers. No effective treatments are available. Development of methods to detect and prevent diseases of farmed fish will decrease the use of drugs and chemicals in the aquatic environment and food chain. Development of
effective vaccines in channel catfish production systems is needed. Little is known about virulence factors of these pathogens at the molecular level. The development of new types of vaccines against these pathogens depends on the understanding of virulence factors at the molecular level (i.e. genomics). 2. List the milestones (indicators of progress) from your Project Plan. 1. Genotyping of Flavobacterium columnare isolates. 2. Developing a new PCR detection protocol for F. columnare. 3. Assessing the prevalence of F. columnare and Edwardsiella ictaluri in pond raised catfish. 4. Comparing gene expression profiles between different F. columnare strains by using cDNA-AFLP analysis. 3. Milestones: Accomplishments during FY 2004: Milestone 1: A total of 30 F. columnare strains were fully characterized by genotypic methods. All data was incorporated into a computerized database to be used in further epidemiological studies. Milestone 2: New specific primers against the
intergenic spacer region in F. columnare were designed. This new protocol was sensitive and specific to detect F. columnare from fish and water samples. Milestone 3: Several classical and nucleic acid-based methodologies were compared to isolate and/or detect F. columnare and E. ictaluri from catfish. Milestone 4: Total and messenger RNA extraction, and double strand cDNA construction protocols were optimized for F. columnare. Expected accomplishments for FY 2005: Milestone 1: Genotyping and incorporation of new F. columnare isolates into the existing database. Milestone 2: Adapting the newly developed PCR detection method into a real- time PCR protocol. Milestone 3: Performing regular samplings using both standard culture methods and PCR detection protocols, to evaluate the presence of F. columnare and E. ictaluri in a farm environment. Milestone 4: Isolate and identify genes that are differentially expressed by two different F. columnare isolates with distinct virulence
properties. 4. What were the most significant accomplishments this past year? The instraspecific variability of Flavobacterium columnare isolates from cultured fish was analyzed. Genetic diversity of this fish pathogen was evaluated using three different fingerprinting methods: restriction fragment length polymorphism (RFLP) of the 16S ribosomic gene, intergenic spacer region (ISR) sequence, and amplified fragment length polymorphism (AFLP). Thirty F. columnare strains from different sources were analyzed using these three methods. The results confirmed the polyphyletic nature of F. columnare isolates from fish. The results also showed that genomovar I and II (previously descibed) are present in the channel catfish from the USA. A unique group within the species formed by isolates from Brazil was described. The three methods were able to find genetic differences between isolates, however, AFLP was the technique with the highest resolution level. 5. Describe the major
accomplishments over the life of the project, including their predicted or actual impact. Major accomplishments over the life of the project includes the development of genomic technology that can be applied to bacterial pathogens of catfish. The research is expected to identify virulence genes of Edwardsiella ictaluri and Flavobacterium columnare. This information will be important in the development of molecular vaccines against these bacterial pathogens. The application of these vaccines is expected to prevent the economic losses due to infection that is estimated on the identification of pathogens and mechanisms of pathogenesis expressed by these bacterial pathogens in fish. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? New tools to detect
and prevent disease of farmed fish are expected. The development of vaccines against major bacterial disease is expected. Research on improving the delivery and efficacy of fish vaccines is expected to lead to new technology and knowledge. The genomic research was initiated on Flavobcterium columnare and the results on identification of isolates and expression of virulence are expected within the next year. Information on genomic disease research will be transferred to scientists presently in the form of peer reviewed journals and national and international workshops and congresses. Presentations and proceedings will be published which will reach industry representatives. The major constrainsts on this technology are the modification and development of new genomic methods and procedures suitable for research on bacterial pathogens of fish.
Impacts
(N/A)
Publications
- Arias, C.R., Welker, T.L., Shoemaker, C.A., Abernathy, J.W., Klesius, P.H. 2004. Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish. Journal of Applied Microbiology. 97:421-428.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Infectious diseases are the cause of significant economic losses in channel catfish production in the U.S., especially the southeastern states. Edwardsiella ictaluri and Flavobacterium columnare are major bacterial pathogens and results in more than $80 million per year. No effective treatments are available. Development of effective vaccines in channel catfish production systems is needed. Little is known about virulence factors of these pathogens at the molecular level. The development of new types of vaccines against these pathogens depends on the understanding of virulence factors at the molecular level (i.e. genomics). 2. How serious is the problem? Why does it matter? Diseases are a major threat to the sustainability of fish farming and losses of channel catfish due to disease are estimated at $80 to $100 million per year. In the catfish industry alone, two bacterial diseases
(enteric septicemia and columnaris) are responsible for 70% of the cases. Presently, only two antibiotics and a few chemicals are approved for food fish. Development of methods to detect and prevent diseases of farmed fish will decrease the use of drugs and chemicals in the aquatic environment and food chain. Development of effective vaccines will limit the use of chemicals or drugs and result in less use and thus a healthier environment and safer products. It will also help reduce the development of antibiotic resistant microbes caused by antibiotic overuse. Information gained from this project will aid in the development of vaccines to control and prevent these diseases in the farmed catfish industry. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? The research is directly related to the National Aquaculture Program - 106. 4. What were the most significant accomplishments this past year? This report documents
research conducted in collaboration with the Department of Fisheries and Allied Aquacultures of Auburn University. This research has been conducted at the ARS Aquatic Animal Health Research Lab in collaboration with Dr. Cova Arias. We compared the biological properties of Edwardsiella ictaluri RE-33 rifampicin-mutant and its parent strain EILO. RE-33 is an avirulent isolate used as a modified live vaccine against enteric septicemia of catfish (ESC). Both strains were fully characterized by biochemical, phenotypic, serological, and genotypic methods. Main differences between both strains relied on the lipopolisaccharides (LPS) composition. Additional characterization of LPS by immunoblotting revealed main differences in LPS composition. RE-33 isolate lacks the high molecular weight bands of the LPS (HMW-LPS). Seven genes related to lipopolysaccharides (LPS) biosynthesis were cloned and sequenced from both strains; however, sequence homology was 100% between the avirulent mutant
and the parent strain. Genotyping using PCR-based methods (such as ERIC-PCR, Box-PCR, and AFLP) did not provide enough resolution to discriminate between the strains. On the other hand, phenotypic-based methodologies such as Biolog and FAME (fatty acid methyl esters) were able to differentiate the mutant strain RE-33 from its parent EILO. Both methods can be used to identify the strain used as vaccine from the wild type E. ictaluri in less than 24 h. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Major accomplishments over the life of the project includes the development of genomic technology that can be applied to bacterial pathogens of catfish. The research is expected to identify virulence genes of Edwardsiella ictaluri and Flavobacterium columnare. This information will be important in the development of molecular vaccines against these bacterial pathogens. The application of these vaccines is expected to
prevent the economic losses due to infection that is estimated at $100 to $150 million per year. In addition, new information is expected on the identification of pathogens and mechanisms of pathogenesis expressed by these bacterial pathogens in fish. 6. What do you expect to accomplish, year by year, over the next 3 years? New tools to detect and prevent disease of farmed fish are expected. the development of vaccines against major bacterial disease is expected. Research on improving the delivery and efficacy of fish vaccines is expected to lead to new technology and knowledge. The genomic research was initiated on Flavobacterium columnare and the results on identification of isolates and expression of virulence are expected within the next year. A project that will compare the lipopolysaccarides between our vaccine avirulent mutant (AquaVac-ESC) and the virulent parent ELIO Edwardsiella ictaluri with other genomic and phenotypic characteristics was initiated and results are
expected next year. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Information on genomic disease research will be transferred to scientists presently in the form of peer reviewed journals and national and international workshops and congresses. Presentations and proceedings will be published which will reach industry representatives. The major constraints on this technology are the modification and development of new genomic methods and procedures suitable for research on bacterial pathogens of fish.
Impacts
(N/A)
Publications
- Arias, C., Shoemaker C., and Klesius P. A comparative study of Edwardsiella ictaluri parent (EILO) and Edwardsiella ictaluri rifampicin- mutant (RE33) isolates using lipopolysaccharides, outer membrane proteins, fatty acids, Biolog, API 20E, and genomic analyses. Journal of Fish Diseases. 2003. v. 26. p. 415-421.
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