Progress 02/01/10 to 01/31/15

Outputs
Target Audience: Aquatic animal health researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate and professional students are involved in the project. How have the results been disseminated to communities of interest? Results have been dissiminated through a journal article and presentations at aquatic workshops and a symposium. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Under goal I, objective I, proprietary vaccineresults have been obtained and are pending subsequent patent application.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Baumgartner, W., L. Dubytska, M. Rogge, P. J. Mottram, and R. L. Thune. 2014. Modulation of vacuolar pH is required for replication of Edwardsiella ictaluri in channel catfish macrophages. Infection and Immunity. 82(6):2329-2336.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Dubytska, L. and R. L. Thune. 2013. Putative Edwardsiella ictaluri type III secretion system effectors are translocated to the cytoplasm of host cells. Proceedings of the 54th Joint Fish Health Workshop & AFS Fish Health Section Meeting. Port Townsend, WA 6/18-20/2013. p. 34.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Thune, R. L. and L. Dubytska. 2014. Studies on the Edwardsiella ictaluri Type Three Secretion System Effectors. Proceedings of the Seventh International Symposium on Aquatic Animal Health. p. 57.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Dubytska, L. and R. L. Thune. 2014. Early intracellular trafficking of Edwardsiella ictaluri in channel catfish macrophages. Proceedings of the Seventh International Symposium on Aquatic Animal Health. p. 56.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate and post doctorate students have been involved with the project. How have the results been disseminated to communities of interest? Publication in refereed journals. What do you plan to do during the next reporting period to accomplish the goals? Goal I. To develop immunological procedures for studying, diagnosing and preventing aquatic animal diseases. Objective I. Evaluate defined mutants of E. ictaluri Type III secretion system effector as vaccine candidates by evaluating their persistance in catfish tissues following an immersion challenge. Objective II. Toevaluate thespecific effector\ mutants of the E. ictaluri T3SS vaccine candiates. Goal II. To examine the structure, biology, and pathology of aquatic animal disease organisms.

Impacts
What was accomplished under these goals? Goal II. To examine the structure, biology, and pathology of aquatic animal disease organisms. Enteric septicemia of catfish (ESC) caused by Edwardsiella ictaluri is becoming an increasing problem in aquaculture and has been reported extensively from catfish species in Asia. In order to evaluate relative risk of the Asian isolates to the US catfish industry, isolates from Asia were compared to US isolates of E. ictaluri. Comparison to isolates from Vietnam using rep-PCR fingerprinting demonstrated no significant differences between the isolates, but plasmid analysis indicated that the Vietnamese isolates grouped into four plasmid profiles, each different from the typical pEI1 and pEI2 plasmid profile found in the US isolates. Sequencing plasmids representative of the four profiles indicated that all contained derivatives of the E. ictaluri plasmid pEI1, whereas only one contained a plasmid derivative of the E. ictaluri plasmid pEI2. The pEI2 encoded type three secretion effector EseI and its chaperone EscD were found to be present on the chromosome in isolates lacking a pEI2 derivative. In addition, one isolate carried a 5023 bp plasmid that does not have homology to either pEI1 or pEI2. Furthermore, Vietnamese isolates were PCR positive for the type III and type VI secretion system genes esrC and evpC, respectively, and the urease enzyme, but were PCR-negative for the putative type IV secretion system gene virD4. A monoclonal antibody against the LPS of E. ictaluri ATCC 33202 did not react with the Asian isolates or with the more recent US isolates. Antibiotic resistance patterns were variable and did not correlate to the presence of any particular plasmid profile. Finally, the Vietnamese isolates were avirulent and had a significantly reduced capacity for intracellular replication within head-kidney-derived channel catfish macrophages. Bacterial isolates from Thailand were also identified as E. ictaluri by conventional and rapid identification systems, as well as by genetic and phylogenetic characterization. Analysis of 16s rRNA indicated 100% homology to the 16s rRNA sequence of several E. ictaluri strains in GenBank. Plasmid profiles demonstrated 4.0 and 5.6 kb plasmids, compared to the 4.8 and 5.6 kb plasmids in the US isolates; and representative genes of 3 of the 4 known pathogenicity islands of US isolates were present. Serologically, lipopolysaccharide (LPS) purified from the Thai isolates was not recognized by a monoclonal antibody against the LPS of US isolates. Fish experimentally infected with E. ictaluri showed 23-100% mortality within 14 days with a 168-h LD50 of 7.946 x 107 CFU mL-1 by immersion and a 96-h LD50 of 2.35 x 106 CFU fish-1 by intra-peritoneal injection. Examination of tissue sections obtained from both naturally and experimentally infected fish indicated that infection of hybrid catfish with E. ictaluri produced lesions in several organs including liver, kidney, spleen, heart and brain. Histopathology findings included cellular necrosis, focal hemorrhage, infiltration of lymphocytes and multifocal granulomatous inflammation in the infected organs.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Suanyuk, N., Rogge, M., Thune, R., Watthanaphiromsakul, M., Jampat, N., Wianagkum, W. 2013. Mortality and pathology of hybrid catfish, Clarius macrocephalus x Clarius gariepinus, associated with Edwardsiella ictaluri infection in southern Thailand. Journal of Fish Diseases. DOI: 10.1111/jfd.12127.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Rogge, M., L Dubytska, T. S. Jung, J. Wiles, A. A. Elkamel, A. Rennhoff, and R. L Thune. 2013. Comparison of Vietnamese and United States Isolates of Edwardsiella ictaluri. Diseases of Aquatic Organisms. 106(1):17-29.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Completion of the Francisella genome allows genetic analysis, Francisella pathogenesis, and the possible developement of new treatment and preventative modalities. Work on the E. ictaluri isolates from Asia indicated that they are different from US isolates and appear to be relatively avirulent in channel catfish. Results were disseminated to fish health professionals at the Fish Health Section of the American Fisheries Society and to the aquaculture community at the World Aquaculture Society meeting, Aquaculture 2012. One journal article, one symposium presentation, five national meeting abstracts, and two international meeting abstracts resulted from this project. PARTICIPANTS: RL Thune, (PI), LSU AgCenter; Lidiya Dubytska, LSU SVM. TARGET AUDIENCES: Aquaculturists, fish veterinarians and diagnosticians, and fish infectious disease researchers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Progress Goal II, Objective II. Sequencing of the Francisella asiatica genome was completed using optical mapping and gap closure. The genome totals 1,858,987 bp, is 32.3% GC and encodes a putative 2284 open reading frames. Manual annotation is ongoing. In other work, E. ictaluri isolated from striped catfish in Vietnam and hybrid walking catfish in Thailand were characterized. Plasmid analysis of 22 Asian E. ictaluri isolates grouped into four different profiles. The Asian E. ictaluri isolates were positive for indicator genes of the urease enzyme, the type III secretion system, and the type VI secretion system that are essential for virulence in U. S. isolates. The virD4 gene of the type IV secretion system was not amplified from the Asian E. ictaluri isolates. None of the Asian isolates were detected by Ed9, a monoclonal antibody directed against the LPS of the ATCC E. ictaluri strain, 33202. Sequencing of the plasmids from representative isolates revealed that a 4 kb plasmid was pEI1 that lost the ISEI1 insertion element from pEI1 and that a 5.6 kb plasmid that was present only in one strain had the ISEI1 insertion element replaced by a TnpAIS26 transposase. A 9 kb plasmid had repA and mob genes added to the backbone of pEI2, which was the only Asian isolate that retained pEI2. The apparently 5.6 kb plasmid that was present in the seven strains along with the 4.0 kb plasmid encoded a methyl accepting chemotaxis protein, a hypothetical integrase protein, a trimethoprim resistance protein, and two transposases, and was not related to either pEI1 or pEI2. In HKDM, the channel catfish isolate of E. ictaluri replicated greater than 10-fold after 10 hrs post-infection, while the Asian isolates did not replicate more than 4-fold in the same period. The Vietnamese E. ictaluri isolates were avirulent in channel catfish. Asian isolates were resistant to a number of antibiotics, but resistance did not correlate to plasmid profiles. In addition, analysis of the E. ictaluri genome identified a 16-gene pathogenicity island putatively encoding a T6SS that is required for virulence. Additional results demonstrated that T6SS gene expression is induced during culture in acidic minimal media, but not during culture in BHI broth. Two proteins not previously described in the literature were found to be secreted into the culture media in a T6SS dependent fashion. Furthermore, mutation of the genes encoding these proteins significantly reduced the virulence of E. ictaluri following immersion-infection of channel catfish. These results demonstrate that the T6SS encoded by E. ictaluri is active and required for virulence. Furthermore, two proteins encoded by the T6SS pathogenicity island were identified as potential secreted effector proteins. Further work is required to determine if these proteins are secreted into the extracellular milieu or translocated directly into host cells and to determine the role of these secreted proteins in E. ictaluri pathogenesis.

Publications

• Williams, M.L, Gillaspy, A.F, Dyer, D.W., Thune, R.L., Waldbieser, G.C., Schuster, S.C., Gipson, J., Zaitshik, J., Landry, C., Banes, M.M., Lawrence, M.L. 2012. Genome sequence of Edwardsiella ictaluri 93-146, a strain associated with a natural channel catfish outbreak of enteric septicemia of catfish. Journal of Bacteriology. 194(3):740-741
• Thune, RL, M. Rogge, N. Booth, W. Baumgartner and J. Beekman. 2012. A Developing Model for E. ictaluri Pathogenesis. Catfish Farmers of America Research Symposium. Savannah, GA. 02/16/2012-02/18/2012.
• Rogge M. L., T. S. Jung, A. Elkamel, A. Rennhoff, and R. L. Thune. 2012. Comparison of Vietnam and United States Isolates of Edwardsiella ictaluri. Aquaculture America 2012. Las Vegas, NV. 02/29/2012-03/02/2012.
• Thune, RL, M. Rogge, N. Booth, and W. Baumgartner. 2012. Towards the Intelligent Design of a Vaccine Against Edwardsiella ictaluri. World Congress of Vaccine 2012. Beijing, China. 03/26/2012-03/28/2012.
• M. L. Rogge and R. L. Thune. 2012. Mutagenic Analysis of the Edwardsiella ictaluri Type VI Secretion System. Fish Health Section of the American Fisheries Society Annual Meeting. LaCrosse, WI. 07/30/2012-08/02/2012. p. 29
• M. L. Rogge, T. S. Jung, A. ElKamel, A. Rennhoff, R. L. Thune. 2012. Genotypic Characterization of Edwardsiella ictaluri Isolated from Asian Catfish. Fish Health Section of the American Fisheries Society Annual Meeting. LaCrosse, WI. 07/30/2012-08/02/2012. p. 28.
• Thune R. L.and L. P. Dubytska. 2012. Modulation of vacuolar pH is required for replication of Edwardsiella ictaluri in catfish macrophages. Fish Health Section of the American Fisheries Society Annual Meeting. LaCrosse, WI. 07/30/2012-08/02/2012. p. 31.
• Dubytska, L. P. and R. L. Thune. 2012. Characterization of Atypical Plasmids of Edwardsiella ictaluri. Fish Health Section of the American Fisheries Society Annual Meeting. LaCrosse, WI. 07/30/2012-08/02/2012. p. 31.
• M. L. Rogge, Dubytska L., Jung T. S., Elkamel A., Rennhoff A., Suanyuk N., and Thune R. L. 2012. Atypical strains of Edwardsiella ictaluri from Asia. AQUA 2012, Prague, Czech Republic. 09/01/2012-09/05/12.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Project results were presented to producer groups, local and national scientific community, and aquaculture researchers through oral communications and one poster. Dr. Matt Rogge was awarded a competetive grant by the USDA National Institute of Food and Agricultures (NIFA) Agriculture and Food Research Initiative (AFRI)for his project entitled "The role of Type VI secretion in Edwardsiella virulence". PARTICIPANTS: Matt Rogge, post-doctoral researcher, and Lidiya Dubytska, post-doctoral researcher. TARGET AUDIENCES: Local and national scientific communities. PROJECT MODIFICATIONS: Because of the complexity of the P. damselae peliminary genome, completion of the genome sequence has been suspended pending additional funding.

Impacts
The utility of knowledge concerning bacterial pathogenesis was demonstrated by the selection of the genes for mutation and vaccine testing for Edwardsiella ictaluri. The use of optical mapping to resolve genome sequence confusion was a strong contributor to near completion of the F. asiatica genome. Markerless deletion mutants were constructed in genes encoding a Type III secretion system (T3SS) regulatory protein, a T3SS effector protein, and a Type VI secretion system translocon protein to produce three ictaluri strains for evaluation as live attenuated vaccines. Mutations in all three strains were stable after 30 in vitro passages, did not loose the ability to replicate in channel cafish cells, and invaded channel catish in an immersion invasion in numbers that were not significantly different from the wild-type E. ictaluri. One mutant persited for 9 days in the head kidney, while two of the mutants persisted for more than 2 weeks, although low level mortalities occurred in the early stages of infection. Fish challenged with wild type E. ictaluri 28 days following immersion vaccination with the three mutants experienced 95%, 100%, and 100% survival, while sham-vaccinated fish only had 11% survival. Because 454 sequencing resulted in a preliminary genome comprised of 624 contigs for Photobacterium damselae, and the Francisella asiatic preliminary gemome was only 19 contigs, efforts focused on the F. asiatica genome. After reducing the genome to three contigs it became difficult to complete, so DNA subjected to optical mapping, allowed the assembly of three contigs, with 8-9 kb gaps.

Publications

• Baumgartner, W., and Thune, R. Arginine metabolism in the Edwardsiella ictaluri-channel catfish macrophage dynamic. 2011. LSU SVM Phi Zeta Research Emphasis Day. Sept 28, 2011, Baton Rouge, LA.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: It was demonstrated that secretion of a Type VI secretion system [(T6SS protein, EvpC, is dependent on a Type III secretions system )(T3SS)] encoded regulatory proteins, suggesting the coordinated regulation of two separate secretion systems. Presented data at the 6th International Symposium on Aquatic Animal Health, Tampa, FL. September 5-9, 2010. PARTICIPANTS: RL Thune - principal investigator. Alison Graff, Veterinary Summer Scholars program. Matt Rogge, post-doctoral researcher. TARGET AUDIENCES: Information shared with fellow aquatic health researchers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A new aspect of E. ictaluri pathogenesis was discovered that is helping us further define the model of E. ictaluri pathogenesis we have developed, leading to an in-depth knowledge of E. ictaluri pathogenesis. This knowledge will allow identification of a specific gene or genes that will lead to the rational construction of an efficacious, live-attenuated vaccine. An E. ictaluri pathogenicity island encoding genes with homology to a Type IV secretion system (T4SS) also was described, but it was determined that there were no defects in invasion or intracellular replication, and that the mutant strain colonized the head kidney, persisted as long as the wild-type strain, and was virulent. These results demonstrate that virulence of E. ictaluri may not be dependent on the T4SS. Further characterization is needed to determine if the T4SS components are functional, under what environmental conditions they are expressed, and how they may be involved in virulence.

Publications

• Graff, AA, ML Rogge and RL Thune. 2010. Evaluation of the Edwardsiella ictaluri Type IV Secretion System Role in Virulence. Proceedings of the 6th International Symposium on Aquatic Animal Health, Tampa, FL. September 5-9, 2010. p. 80.