Progress 10/01/04 to 09/30/09
Outputs
OUTPUTS: Identification of Edwardsiella ictaluri virulence factors using signature tagged mutagenesis (STM) resulted in the elicitation of a model for pathogenesis. A proposal submitted to the USDA National Research Initiative Competitive Grants Program for validation of this model using transcriptional and translational analysis in catfish macrophages was funded, and the work was initiated in 2009. In addition, the development of the pathogenesis model, combined with the completion of our E. ictaluri genome sequencing project, helped to identify mutations that might be useful in the development of a live attenuated vaccine. The results of this research have been disseminated through publication in scientific journals, through presentations at several Fish Health Section of the American Fisheries Society annual meetings, and through additional presentations to the Catfish Farmers of America in their Catfish Research Symposiums. PARTICIPANTS: Ms. Chris Darnell conducted the oral vaccination studies as part of an MS program. Ms. Ildiko Polyack completed an MS thesis working on an E. ictaluri adhesion protein identified by STM. Ms. Natha Booth completed a PhD working on the role of the urease enzyme in pathogenesis. Mr. Matt Rogge completed a doctoral degree concerning the regulation of gene expression of the E. ictaluri TTSS, and is currently working on the USDA grant as a post-doctoral researcher. Dr. Wes Baumgartner is working on the USDA project as PhD graduate student. Ms. Denise Fernandez and Ms. Judith Beekman worked on several of the projects as research associates. Ms. Meridith Mooney completed a research project supported by the LSU School of Veterinary Medicine's Summer Research Scholars program. TARGET AUDIENCES: The target audience is other researchers in the field of bacterial pathogenesis, especially concerning E. ictaluri, and people associated with the commercial production of channel catfish. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Analysis of the putative biological activities of virulence associated genes of E. ictaluri enabled the development of a unique preliminary model to explain the mechanisms associated with intracellular survival and replication of E. ictaluri. A full knowledge of the pathogenesis of E. ictaluri will enable the development of unique prevention and control measures. Several of these mutants are currently in laboratory trials to evaluate efficacy following both oral and immersion delivery. Comparing the use of vaccine coated feed and oral delivery of a vaccine filled capsule to a typical immersion dosage, oral uptake is significantly lower than via immersion. Possible explanations include limited uptake of bacteria in the digestive tract versus the gills, either due to limited bacterial adhesion, limited phagocytosis, or differences in the surface area of the two organs. Edwardsiella ictaluri encodes a type III secretion system (TTSS) similar to the Salmonella pathogenicity island 2 (SPI2) TTSS, which is involved in intracellular replication. TTSS inject effector proteins directly into the host cell cytoplasm and have a variety of effects on host cells, including cytoskeletal manipulation, inducing or preventing apoptosis, and manipulation of the host immune response. In order to evaluate the role of the E. ictaluri TTSS effectors, it is important to know how their expression is regulated. Data indicates that the E. ictaluri TTSS promoters are active in acidic and phosphate limiting conditions as well as at pH 7 in the presence of phosphate. Further studies are required to define regulation of expression of the E. ictaluri TTSS. Further work on other virulence genes identified by STM indicated that an acid inducible urease enzyme is important in intracellular survival and replication. The concept of DNA vaccination against channel catfish virus was found to be unsuccessful in channel catfish. Finally, the iglABCD operon of Francisella asiatica was found to be important in intracellular replication in tilapia macrophages.
Publications
- Booth, N.A. and R. L. Thune. 2009. Edwardsiella ictaluri encodes an acid activated urease that is required for intracellular replication in channel catfish (Ictaluris punctatus) macrophages. Applied and Environmental Microbiology. 75(21):6712-6720.
- Rogge, M. L. and R. L. Thune. 2009. Regulatory proteins encoded by the Edwardsiella ictaluri type III secretion system are required for virulence in channel catfish. Proceedings of the 2009 AFS Fish Health Section Annual Meeting, Park City, Utah. p. 13.
- Soto, E., D. Fernandez, R. L. Thune, and J. P. Hawke. 2009. Attenuation of the fish pathogen Francisella sp. by mutation of the iglC gene. Proceedings of the 2009 AFS Fish Health Section Annual Meeting, Park City, Utah. p. 6.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The long term objective is to develop preventative methods for bacterial diseases of fish. In the past year, effort was concentrated on Edwardsiella ictaluri in the channel catfish. Identification of E. ictaluri virulence factors using signature tagged mutagenesis has resulted in the elicitation of a model for pathogenesis. A proposal submitted to the USDA National Research Initiative Competitive Grants Program for validation of this model using transcriptional and translational analysis in catfish macrophages was funded, and the work will be initiated in 2009. In addition, the pathogenesis model helped to identify mutations that might be useful in the development of a live attenuated vaccine. Several of these mutants are currently in laboratory trials to evaluate efficacy following both oral and immersion delivery. The targeted community of interest, the catfish farmer, will have little interest until the final product is developed. In the interim, publications in the scientific community serve to inform other scientists working in the field. Specifically, one abstract was published in the Proceedings of the 2008 AFS Fish Health Section Annual Meeting. PARTICIPANTS: The primary participants in the project in the past year other than the principal investigator were Mr. Matt Rogge and Ms. Judith Beekamn. Mr. Rogge is a PhD candidate that should complete his program in 2009. Ms. Beekman is a research associate who has been working to improve our proceedures for making mutants in E. ictaluri. TARGET AUDIENCES: Catfish producers and the research community. PROJECT MODIFICATIONS: A research proposal to the USDA NRI/CGP was funded that has significant objectives to validate the pathogenesis model that was developed based on previous data.
Impacts
The gram-negative enteric bacterium Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farmed-raised channel catfish, Ictalurus punctatus. Commercial catfish production accounts for 85-90% of the total fin fish aquaculture production in the United States, with almost 300,000 tons produced annually. Significant losses due to ESC were reported on over 60% of all farms in operation. Work completed includes partial completion of the E. ictaluri genome sequence and the identification of 34 genes that encode proteins with putative roles in the pathogenesis of E. ictaluri, including 10 proteins with known functions in other pathogens, 11 hypothetical proteins with known functions from other pathogens, and 13 proteins with no matches in the bacterial databases. Using bioinformatics and the E. ictaluri genomic database, all of those putative virulence factors were analyzed; and several were identified as having a strong likelihood of involvement in intracellular replication. Phenotypic characterization of strains carrying mutations in those genes confirmed that each are required for virulence in channel catfish and for successful survival and replication within channel catfish head-kidney derived macrophages (CC-HKDM). Analysis of the putative biological activities of the associated genes enabled the development of a unique preliminary model to explain the mechanisms associated with intracellular survival and replication of E. ictaluri. A full knowledge of the pathogenesis of E. ictaluri will enable the development of unique prevention and control measures.
Publications
- Rogge, M. L. and R. L. Thune. 2008. Expression of Edwardsiella ictaluri type III secretion effectors in vitro. Proceedings of the 2008 AFS Fish Health Section Annual Meeting, Charlottetown, Prince Edward Island, Canada. p. 110.
- Plant, K. P. and R. L. Thune. 2008. Genomic organization of the channel catfish Mx1 gene and characterization of multiple channel catfish Mx gene promoters. Fish and Shellfish Immunology. 24:575-583.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Support for this project resulted in four presentations to fish health scientists at the 2007 Annual Meeting of the Fish Health Section of the American Fisheries Society, one high impact journal publication, and one MS Thesis. Available data were integrated into a model for the pathogenesis of Edwardsiella ictaluri that was the basis of a USDA NRI-CGP grant application that is still pending.
PARTICIPANTS: Ms. Chris Darnell who is working on completing an MS degree while in Veterinary School conducted the oral vaccination studies. Ms. Ildiko Polyack completed an MS thesis working on the E. ictaluri adhesion protein. Mr. Matt Rogge is working on a doctoral degree concerning the regulation of gene expression of the E. ictaluri TTSS.
TARGET AUDIENCES: The target audience is other researchers in the field of bacterial pathogenesis, especially concerning E. ictaluri.
Impacts
Oral delivery of vaccines via feed is less labor intensive and less stressful to fish than other delivery methods. The use of a live, attenuated Edwardsiella ictaluri mutant in an oral vaccine would give the channel catfish aquaculture industry an economical means of preventing enteric septicemia of catfish and would reduce the input of antibiotics into the environment. Comparing the use of vaccine coated feed and oral delivery of a vaccine filled capsule to a typical immersion dosage, oral uptake is significantly lower than via immersion. Possible explanations include limited uptake of bacteria in the digestive tract versus the gills, either due to limited bacterial adhesion, limited phagocytosis, or differences in the surface area of the two organs. In order to optimize bacterial vaccine uptake, a knowledge of the virulence factors involved in uptake is critical. Characterization of E. ictaluri pathogenesis led to discovery of a hypothetical protein that has
similarity to adhesion and hemolysin proteins in E. coli. Genes surrounding the mutation are very similar to a genomic island found in E. coli and Y. pestis. In order to evaluate its role in virulence, a mutant strain was created by inserting a kanamicin resistance cassette into the gene encoding the hypothetical protein. The mutation was introduced into E. ictaluri through homologous recombination and verified by PCR, sequencing, and southern blot. Using an in vivo catfish immersion assay, the mutated strain and the STM mutant strain were found to be attenuated in comparison to the E. ictaluri wild-type strain. In vitro studies in catfish head kidney derived macrophages and a channel catfish ovary cell line using a gentamicin exclusion assay demonstrated that survival and replication of the mutant strains was not statistically different from the wild type strain. Combined results of the in vivo and in vitro studies indicate that the hypothetical protein does have a role in host
infection but not specifically in intracellular invasion and replication. Edwardsiella ictaluri encodes a type III secretion system (TTSS) similar to the Salmonella pathogenicity island 2 (SPI2) TTSS, which is involved in intracellular replication. TTSS inject effector proteins directly into the host cell cytoplasm and have a variety of effects on host cells, including cytoskeletal manipulation, inducing or preventing apoptosis, and manipulation of the host immune response. In order to evaluate the role of the E. ictaluri TTSS effectors, it is important to know how their expression is regulated. Consequently, putative promoter regions of the E. ictaluri TTSS pathogenicity island were fused to GFP and inserted in the E. ictaluri genome to provide a method to detect expression. Data indicates that the E. ictaluri TTSS promoters are active in acidic and PO43--limiting conditions as well as at pH 7 in the presence of PO43-. Further studies are required to define regulation of expression
of the E. ictaluri TTSS.
Publications
- Thune, R. L., Fernandez, D.H., Benoit, J.L., Smith, M.K., Rogge, M.L., Booth, N.J., and Bologna, R. 2007. Signature-tagged mutagenesis of Edwardsiella ictaluri identifies virulence-related genes, including a Salmonella pathogenicity island 2 class of Type III secretion system. Applied and Environmental Microbiology. 73(24):7934-7946.
- Polyack, Ildiko. 2007. Characterization of a hypothetical protein of Edwardsiella icatluri. MS Thesis, PBS Department, Louisiana State University.
- Darnall, C. A. , R. L. Thune1, J. P. Hawke1, and R. C. Reigh. 2007. Development of oral vaccination methods for channel catfish (Ictalurus punctatus) using live Edwardsiella ictaluri. Proceedings of the 48th Western Fish Health Workshop and the 2007 AFS Fish Health Section Annual Meeting, Jackson Hole, Wyoming. p. 22.
- Booth, N. J., J. Beekman, and R. L. Thune. 2007. Urease activity and virulence in Edwardsiella ictaluri. Proceedings of the 48th Western Fish Health Workshop and the 2007 AFS Fish Health Section Annual Meeting, Jackson Hole, Wyoming. p. 14.
- Polyak, I. K., D, H. Fernandez, and R. L. Thune. 2007. A virulence related hypothetical protein in Edwardsiella ictaluri. Proceedings of the 48th Western Fish Health Workshop and the 2007 AFS Fish Health Section Annual Meeting, Jackson Hole, Wyoming. p. 15.
- Rogge, M. L., D. H. Fernandez, C. Landry, and R. L. Thune. 2007. Edwardsiella ictaluri Type III secretory system gene expression measured using promoter fusions to GFP. Proceedings of the 48th Western Fish Health Workshop and the 2007 AFS Fish Health Section Annual Meeting, Jackson Hole, Wyoming. p. 13.
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Progress 01/01/06 to 12/31/06
Outputs
Interferon (IFN) is the first line of defense against viral infection and induces a variety of proteins, including PKR and Mx. We have previously shown that poly I:C treatment transiently protects channel catfish against channel catfish virus (CCV) infection. This protection coincided with high levels of Mx transcription. It is unknown which mechanism is responsible for the protection and consequently in vitro studies were initiated to attempt to resolve this. A variety of 52 base pair oligonucleotide sequences of Mx1 were cloned into psiRNA. These fragments were designed in such a way that after transcription short hairpin (sh) RNAs are generated. Each shRNA was screened for effective knockdown of Mx1 using an alkaline phosphatase reporter vector. Three shRNA were chosen to create stably transfected cell lines using channel catfish ovary (CCO) cells. A control cell line was also created using psiRNA encoding a scramble sequence. Mx1 knockdown was confirmed by
quantitative RT-PCR. The stably transfected cell lines are being evaluated to study the role of Mx1 in cellular resistance to CCV.
Impacts
The establishment of RNA interference assays will allow for specific gene knockouts in catfish and catfish cell lines and facilitate pathogenesis studies with a variety of catfish pathogens.
Publications
- Plant, K.P. and R.L. Thune. 2006. RNA interference to elucidate the role of Mx in channel catfish virus infection in vitro. Proceedings of the International Symposium on Aquatic Animal Health. San Francisco, CA. September. p. 216.
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Progress 01/01/05 to 12/31/05
Outputs
In vivo studies were carried out to investigate the protective effect of the interferon inducer poly I:C against channel catfish virus (CCV). Channel catfish were stimulated by intraperitoneal injection of 50 mg of poly I:C or PBS at various days prior to immersion challenge with CCV. Mortality in the poly I:C group was significantly reduced from 70% to 3% at day 1 compared to the PBS controls. Mortality increased at day 3 but was still significantly different from the PBS controls. Mx1 transcription was significantly higher only at day 1. In an additional study Mx1 transcription was monitored in the liver, kidney, gills, spleen, and intestine at various time points post-stimulation with either poly I:C or CCV. Mx1 mRNA was significantly elevated in all organs only at day 1 post-injection with poly I:C. In response to CCV, Mx1 transcription was not significantly elevated until day 3 post-challenge, but remained elevated in certain organs until day 7. Invasion of
Edwardsiella ictaluri was evaluated in the catfish intestinal tract. After initial problems with contamination of the tissues in an intestinal surgical assay indicated that uptake from the infections was rapid, our latest studies indicate that E. ictaluri is not found in the head kidney as late as 30 min post-ex posure, although cells can be located in intestinal tissues by confocal microscopy after a 15 min exposure. Further work is planned to evaluate uptake over longer periods of time. As an alternative, a feed delivered challenge was developed to provide additional information. After comparing adherence, recovery and loss of bacteria in the water for feeds in which E. ictaluri culture was incorporated into the feed pellet or sprayed on the surface, surface coating resulted in a more stable pellet and less loss of the infectious dose to the water. The next step is to coat feed at culture to dry weight ratios of 10, 20, 40, and 60% to conduct a dose dependancy trial in fish. In a
preliminary trial at a 50% rate there was 100% mortality by day 10.
Impacts
The concept of DNA vaccination against channel catfish virus was found to be unsuccessful in channal catfish. Poly-IC induction, however, induced a protective response against CCV which was correlated to upregulation of transcription of the catfish Mx1 gene. Evaluation of intestinal invasion of E. ictaluri will provide important information for the prevention and control of infection.
Publications
- Plant K. P. , H. Harbottle, R. L. Thune. 2005. Poly-IC induces an antiviral state against Ictalurid herpesvirus 1 and Mx transcription in the channel catfish (Ictalurus punctatus). Developmental and Comparative Immunology. 29(7):627-635.
- Harbottle, H., K. P. Plant, R. L. Thune. 2005. DNA vaccination against channel catfish virus (CCV) is not efficacious although immune responses are elicited. Journal of Aquatic Animal Health. 17(3):251-262.
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