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INVESTIGATOR: Karsi, A.

PERFORMING INSTITUTION:
MISSISSIPPI STATE UNIV
MISSISSIPPI STATE, MISSISSIPPI 39762

PREVENTION OF ENTERIC SEPTICEMIA OF CATFISH BY NOVEL LIVE ATTENUATED VACCINES

NON-TECHNICAL SUMMARY: Channel catfish farming is the largest aquaculture industry in the U.S., and Edwardsiella ictaluri is recognized as the most economically important bacterial pathogen of catfish. The type six secretion system (T6SS) and universal stress proteins (USP) are essential for E. ictaluri virulence in catfish. However, critical work needs to be conducted to develop safe and efficacious live attenuated vaccines (LAV) against E. ictaluri. Our long-term goal is to characterize mechanisms of E. ictaluri pathogenesis in order to develop effective control strategies for the disease. The immediate objective of this study is to develop novel LAVs by mutating T6SS and USP genes and by characterizing mutants' safety and efficacy in catfish. The rationale of the proposed research is that T6SS and USPs are essential for E. ictaluri virulence in catfish and mutation of genes in these systems is expected to yield safe and efficacious vaccines for catfish. This study is significant because of the large economic impact enteric septicemia has on the catfish industry. Current disease treatments are often ineffective, costly, and can result in drug resistance or environmental contamination. Therefore, vaccine-based strategies for control of diseases are essential to support the development of an environmentally and economically sustainable aquaculture industry in the U.S.

OBJECTIVES: Construct T6SS and USP mutantsCharacterize T6SS and USP mutants

APPROACH: -EffortsThe type six secretion system (T6SS) and universal stress protein (USP) genes will be deleted in-frame. Then, attenuation and efficacy of mutants will be characterized in specific pathogen free catfish by immersion exposure. Briefly, catfish will be vaccinated by immersion, and three weeks following vaccination, fish will be immersion exposed to virulent parent strain. Percent mortalities will be calculated after two weeks to evaluate protection levels.-EvaluationPercent mortalities will be arcsine-transformed and compared by ANOVA. If significant differences among vaccine candidates are found at α = 0.05, means will be separated using the least-significant-difference test.

PROGRESS: 2016/02 TO 2020/01
Target Audience:-Target Audiences Microbiologists Immunologists Veterinarians Fish health researchers Catfish producers -Efforts Science-based knowledge has been delivered in laboratory teaching, fish health-related courses, and workshops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided training and professional development opportunities for one undergraduate student, one graduate student, and one post-doctoral scientist. How have the results been disseminated to communities of interest? Three peer-reviewed journal articles were published. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2016/02 TO 2020/01
What was accomplished under these goals? -Goal one: Construct T6SS and USP mutants. Nothing to report. -Goal two: Characterize T6SS and USP mutants Phagocytic properties of macrophages against selected mutants were determined Bactericidal properties of macrophages against selected mutants were determined Apoptotic changes in macrophages exposed to selected mutants was conducted Innate immune responses against selected mutants were determined by real-time qPCR Adaptive immune responses against selected mutants were determined by real-time qPCR IgM titers in catfish sera exposed to selected mutants were determined. 2) Specific objectives met -Goal one: Construct T6SS and USP mutants. Nothing to report. -Goal two: Characterize T6SS and USP mutants Innate and adaptive immune responses against selected mutants were determined. 3) Significant Results Achieved Safe and efficacious mutants were determined Vaccination increased the readiness of innate immunity Vaccination decreased wild-type replication in catfish Vaccination decreased the pathological lesions Vaccination induced protective immune responses Vaccination reduced inflammation caused by wild-type E. ictaluri Anterior kidney macrophages engulfed mutants more efficiently Anterior kidney macrophages destroyed internalized mutants more efficiently. Percentage of early and late apoptotic macrophages was increased after exposure to wild-type bacteria opsonized with serum from fish exposed to the wild-type strain. Gene expression analyses indicated that mutants could activate adaptive immune responses without causing inflammation. IgM titers indicated that mutants promote the development of primary immune responses and memory B cell formation. ?4) Key outcomes or other accomplishments realized Safe and efficacious mutant strains were identified Attenuated mutants increased the effectiveness of innate and adaptive immunity

PUBLICATIONS (not previously reported): 2016/02 TO 2020/01
1. Type: Journal Articles Status: Other Year Published: 2020 Citation: Ibrahim I, Abdelhamed H, Baumgartner W, Wills RW, Awadin W, Elbegawy M, Lawrence ML, Karsi A. Pathological and ultrastructural characterization of an Edwardsiella ictaluri hemR mutant (Ei?hemR?frdA?sdhC). In preparation.
2. Type: Journal Articles Status: Other Year Published: 2020 Citation: Kalindamar S, Abdelhamed H, Kordon A, Pinchuk L, Karsi A. The effect of Hcp family proteins of type VI secretion system in Edwardsiella ictaluri virulence. In preparation.
3. Type: Journal Articles Status: Published Year Published: 2020 Citation: Kordon A, Kalindamar S, Majors K, Abdelhamed H, Tan W, Karsi A, Pinchuk L. 2020. Live attenuated Edwardsiella ictaluri vaccines enhance the protective innate immune responses of channel catfish B cells. Developmental and Comparative Immunology. 2020 Aug;109:103711. doi: 10.1016/j.dci.2020.103711. Epub 2020 Apr 18. PMID: 32311387.
4. Type: Journal Articles Status: Published Year Published: 2019 Citation: Kalindamar S, Kordon A, Abdelhamed H, Tan W, Pinchuk L, Karsi A. 2019. Edwardsiella ictaluri evpP is required for colonization of channel catfish ovary cells and necrosis in anterior kidney macrophages. Cellular Microbiology. 2019 Nov 19:e13135. doi: 10.1111/cmi.13135. [Epub ahead of print]. PMID: 31742869.
5. Type: Journal Articles Status: Published Year Published: 2019 Citation: Kordon A, Kalindamar S, Majors K, Abdelhamed H, Tan W, Karsi A, Pinchuk L. 2019. Effects of live attenuated vaccine and wild type strains of Edwardsiella ictaluri on phagocytosis, bacterial killing, and survival of catfish B cells. Frontiers in Immunology. 2019 Oct 9;10:2383. doi: 10.3389/fimmu.2019.02383. eCollection 2019. PMID: 31649682.

PROGRESS: 2017/02/01 TO 2018/01/31
Target Audience:Target Audiences Microbiologists Veterinarians Fish health research community Catfish producers Efforts Science-based knowledge has been delivered in laboratory teaching, fish health-related courses, and workshops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?- This project has provided training and professional development opportunities for two postdocs (Hossam Abdelhamed and Jingjun Lu) and four doctoral students (Ali Akgul, Safak Kalindamar, Adef Kordon, and Iman Ibrahim). - Hossam Abdelhamed accepted a faculty position in the College of Veterinary Medicine at Mississippi State University. - Jingjun Lu accepted a position in Saint Jude Children's Hospital. - Ali Akgul accepted a post-doctoral position in the College of Veterinary Medicine at the University of Florida. - Safak Kalindamar accepted a faculty position in a university in Turkey. How have the results been disseminated to communities of interest?- Five presentations were given at scientific conferences. - One manuscript was published in a peer-reviewed journal, Frontiers in Microbiology (https://doi.org/10.3389/fmicb.2017.02638). - One manuscript was submitted to peer-reviewed journals, PLOS One. What do you plan to do during the next reporting period to accomplish the goals?Goal one: Construct T6SS and USP mutants. - None. This goal is completed. Goal two: Characterize T6SS and USP mutants - Verify safety and efficacy of promising mutants in catfish fry - Assess vaccine-induced innate and adaptive responses.

IMPACT: 2017/02/01 TO 2018/01/31
What was accomplished under these goals? 1) Major activities completed Goal one: Construct T6SS and USP mutants.? - All T6SS and USP mutants were constructed. Goal two: Characterize T6SS and USP mutants. - Attenuation of mutants was determined in catfish fry - Efficacy of mutants was determined in catfish fry. 2) Specific objectives met Goal one: Construct T6SS and USP mutants. - Construction of all T6SS and USP mutants were completed. Goal two: Characterize T6SS and USP mutants - Initial screening of mutants' virulence and efficacy in catfish fry was completed. 3) Significant Results Achieved Initial characterization indicated the presence of attenuated and protective mutants in catfish fry. 4) Key outcomes or other accomplishments realized Several USP and T6SS genes contribute to E. ictaluri virulence in catfish Phagocytosis of attenuated mutants by macrophages was higher compared to wild-type. Uptake of E. ictaluri opsonized with sera from vaccinated catfish was more efficient.

PUBLICATIONS: 2017/02/01 TO 2018/01/31
1. Type: Journal Articles Status: Published Year Published: 2017 Citation: Kordon A, Abdelhamed H, Ahmed H, Park J, Karsi A, Pinchuk L. 2017. Phagocytic and bactericidal properties of channel catfish peritoneal macrophages exposed to Edwardsiella ictaluri live attenuated vaccine and wild-type strains. Frontiers in Microbiology (https://doi.org/10.3389/fmicb.2017.02638).
2. Type: Theses/Dissertations Status: Submitted Year Published: 2017 Citation: Akgul A. The role of universal stress proteins in Edwardsiella ictaluri virulence. Mississippi State University.
3. Type: Theses/Dissertations Status: Submitted Year Published: 2017 Citation: Kalindamar S. Characterization of type VI secretion system in Edwardsiella ictaluri. Mississippi State University.
4. Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Kalindamar S, Abdelhamed H, Kordon A, Lu J, Kumru S, Tekedar H, Pinchuk L, Karsi A. 2017. Characterization of type VI secretion system in Edwardsiella ictaluri. American Society for Microbiology South Central Branch Meeting, Little Rock, AR, USA.
5. Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Ibrahim I, Abdelhamed H, Baumgartner W, Lawrence M, Karsi A. 2017. Assessment of portals of entry for attenuated and wild-type Edwardsiella ictaluri. American Fisheries Society Fish Health Section Annual Meeting, East Lansing, MI, USA.
6. Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Ibrahim I, Abdelhamed H, Baumgartner W, Awadin W, Lawrence M, Karsi A. 2017. Pathological and ultrastructural effects of live attenuated Edwardsiella ictaluri vaccines. American Fisheries Society Fish Health Section Annual Meeting, East Lansing, MI, USA.
7. Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Abdelhamed H, Lawrence ML, Karsi A. 2017. A novel vaccine candidate protects catfish against enteric septicemia. American Fisheries Society Fish Health Section Annual Meeting, East Lansing, MI, USA.
8. Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Kalindamar S, Abdelhamed H, Kordon A, Al-Janabi N, Lu J, Tekedar H, Karsi A. 2017. The effect of Hcp family proteins of type VI secretion system in Edwardsiella ictaluri virulence. American Society for Microbiology ASM Microbe 2017 Meeting, New Orleans, LA, USA.

PROGRESS: 2016/02/01 TO 2017/01/31
Target Audience:The target audiences include: Fish health research community Veterinarians Catfish producers Microbiologists Efforts Science-based knowledge has been delivered in laboratory teaching, fish health related courses, and workshops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training and professional development opportunities for two postdocs (Hossam Abdelhamed and Jingjun Lu), three doctoral students (Ali Akgul, Safak Kalindamar, and Adef Kordon), and a visiting scholar (Iman Ibrahim). How have the results been disseminated to communities of interest?Results have been presented at scientific conferences and will be published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Finalize construction of all T6SS and USP mutants. Determine safety and efficacy of mutants in catfish fingerlings. Confirm safety and efficacy of potential vaccine candidates in catfish fry. Start testing tissue persistence and immune responses of vaccine candidates.

IMPACT: 2016/02/01 TO 2017/01/31
What was accomplished under these goals? 1) Major activities completed -Goal one: Construct T6SS and USP mutants Construction of T6SS mutants was completed. Construction of USP mutants was completed. -Goal two: Characterize T6SS and USP mutants Attenuation of mutants was determined in catfish fingerlings. Efficacy of mutants was determined in catfish fingerlings. 2) Specific objectives met -Goal one Construction of T6SS and USP mutants was completed. -Goal two Initial screening of mutants' virulence and efficacy was completed. 3) Significant Results Achieved We were able to construct all in-frame deletion mutants. Initial characterization indicated the presence of attenuated and protective mutants. 4) Key outcomes or other accomplishments realized Several USP and T6SS genes contribute to E. ictaluri virulence in catfish.

PUBLICATIONS: 2016/02/01 TO 2017/01/31
1. Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Akgul A, Akgul A, Lawrence ML, Karsi A. 2016. Expression of Edwardsiella ictaluri stress-related genes under various stresses. American Society for Microbiology South Central Branch Meeting, Lafayette, LA, USA.
2. Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Kalindamar S, Abdelhamed H, Tekedar H, Karsi A. 2016. Construction and evaluation of an Edwardsiella ictaluri virulence protein F mutant. 2nd International Conference of Fish and Shellfish Immunology. Portland, MA, USA.
3. Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Akgul A, Akgul A, Tekedar H, Lawrence M, Karsi A. 2016. Expression analysis of universal stress proteins in Edwardsiella ictaluri. American Society for Microbiology ASM Microbe 2016 Meeting, Boston, MA, USA.