Source: MISSISSIPPI STATE UNIV submitted to
DEVELOPMENT OF BROAD EFFICACIOUS MULTIVALENT LIVE VACCINES AGAINST CATFISH PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0228444
Grant No.
(N/A)
Project No.
MISV-371560
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2011
Project End Date
Jun 30, 2013
Grant Year
(N/A)
Project Director
Karsi, A.
Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762
Performing Department
College Of Veterinary Medicine
Non Technical Summary
Aquaculture, with a sales value of $1.2 billion, is one of the fastest growing agricultural commodities in the United States, and channel catfish continues to be the most important cultured fish, with an economic impact of $403 million in 2010. Enteric septicemia of catfish (ESC) caused by Edwardsiella ictaluri and columnaris disease caused by Flavobacterium columnare are recognized as the most important bacterial diseases impacting catfish aquaculture. Moreover, Aeromonas hydrophila is a recently reemerging fish pathogen causing motile aeromonad septicemia (MAS) in catfish. The rapid spread of these diseases and large number of mortalities suggest that these pathogens are well-adapted to channel catfish probably due to their abilities to quickly regulate their pathogenic mechanisms in the host environment. However, virulence factors causing these diseases are still unexplored. Lack of this knowledge is a problem because a complete understanding of the molecular basis of bacterial pathogenesis, and thus, development of useful strategies to prevent diseases are unlikely to be realized The overall objective of this study is to develop multivalent live attenuated vaccines against ESC, columnaris, and MAS.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31137101100100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3710 - Catfish;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Our long-term goal is to determine mechanisms of bacterial pathogenesis, with the ultimate applied goal of developing effective live vaccines against fish diseases. The overall objective of this study is to develop multivalent live attenuated vaccines against ESC, columnaris, and MAS. The central hypothesis of this proposal is that expressing surface-associated proteins of F. columnare and A. hydrophila in live attenuated E. ictaluri vaccine will produce multivalent vaccines with broad efficacy against multiple catfish pathogens. The rationale of the proposed research is that expression of surface associated proteins of F. columnare and A. hydrophila in attenuated live E. ictaluri vaccine is expected to lead to broad effective vaccines against multiple catfish pathogens, as well as new knowledge on bacterial pathogenesis will be obtained. We plan to accomplish our overall objective by pursuing the following specific aims: 1. Identify surface associated proteins of F. columnare and A. hydrophila 2. Express F. columnare and A. hydrophila surface-associated proteins in E. ictaluri vaccine. 3. Characterize efficacy of multivalent vaccines and catfish immune responses.
Project Methods
1. Identify surface associated proteins of F. columnare and A. hydrophila We will use the resources available at of the Vaccine Investigation and Online Information Network (VIOLIN). Violin is designed to be a vital source of vaccine information and will provide researchers in basic and clinical sciences with curated data and bioinformatics tools to facilitate understanding and development of vaccines to fight infectious diseases. Specifically, Vaxign pipeline for vaccine target prediction will be used: Vaxign includes a pipeline of software programs to predict possible vaccine targets based on various vaccine design criteria using microbial genomic and protein sequences as input data. The predicted features in the Vaxign pipeline include antigen sublocation, adhesion, epitope binding to MHC class I and class II, and sequence similarities to human, mouse and/or pig proteins. 2. Express F. columnare and A. hydrophila surface-associated proteins in E. ictaluri vaccines. Genes for vaccine candidates will be cloned into pBBRMCS4 plasmid. This plasmid was successfully used by our group to express green fluorescent protein and bacterial luciferase genes in E. ictaluri. 3. Characterize efficacy of multivalent vaccines and catfish immune responses. Efficacy of multivalent vaccines will be analyzed following the procedures developed in our lab. Briefly, four replicate tanks with 25 fish/tank will be used for vaccination with each multivalent vaccine. Fish will be vaccinated by immersion in water containing 1 x 107 CFU/ml for one hour. After 21 days, immunized fish will be infected with wild-type E. ictaluri, F. columnare and A. hydrophila (1 x 107 CFU/ml) as well as their combinations (E. ictaluri + F. columnare; E. ictaluri + A. hydrophila; F. columnare + A. hydrophila; E. ictaluri + F. columnare + A. hydrophila) by immersion in water with 2 x 107 - 3 x 107 CFU/ml for one hour. Mean percent mortalities for each group will be calculated and data will be analyzed.

Progress 07/01/11 to 06/30/13

Outputs
Target Audience: This project is directly related to the catfish health research community. Research outcomes should help gain more information on motile aeromonad septicemia in catfish and accelerate research on vaccine development for this disease. The targeted audiences also include fish health community, catfish producers, and microbiologists. Changes/Problems: Transposon end mapping in A. hydrophila mutants has not been as efficient as we were expecting, which slowed down the progress. What opportunities for training and professional development has the project provided? A MS student, Safak Kalindamar, has been trained as part of his MS research. How have the results been disseminated to communities of interest? Findings were presented in the American Society for Microbiology South Central Branch Meeting and departmental seminar. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Random transposon mutations have been introduced into Aeromonas hydrophila genome. Efforts are focused on identification of transposon insertion sites. In addition, potential vaccine targets on the A. hydrophila genome have been identified by submitting its genome to vaccine target prediction and analysis system.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2012 Citation: Kalindamar S, Fuqua K, Lu J, M, Lawrence ML, Karsi A. 2012. Identification of the Edwardsiella ictaluri genes causing impaired growth. American Society for Microbiology South Central Branch Meeting, Starkville, MS, USA.


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

Outputs
OUTPUTS: Random transposon mutations have been introduced into Aeromonas hydrophila genome. Effects of random transposon insertions are being analyzed to identify possible virulence related A. hydrophila genes. In addition, the A. hydrophila genome sequence has been submitted to vaccine target prediction and analysis system to predict potential vaccine targets. PARTICIPANTS: Dr. Attila Karsi is the project director and a MS student has been working on this project. TARGET AUDIENCES: This project is directly related to the catfish health research community. Research outcomes should help gain more information on motile aeromonad septicemia in catfish and accelerate research on vaccine development for this disease. The targeted audiences also include fish health community, catfish producers, and microbiologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We produced a random transposon mutant library from an epidemic strain of A. hydrophila. Functional characterization of interrupted genes is being conducted. Moreover, surface-exposed antigens with potential immunogenic properties have been identified, some of which will be expressed in a live attenuated Edwardsiella ictaluri strain.

Publications

  • No publications reported this period


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

Outputs
OUTPUTS: Because of the late release of funds, this project started recently. PARTICIPANTS: Dr. Attila Karsi is the project director and a MS student has been assigned to this project. TARGET AUDIENCES: This project is directly related to the catfish health research community. Research outcomes should help gain more information on motile aeromonad septicemia in catfish and accelerate research on vaccine development for this disease. The targeted audiences also include fish health community, catfish producers, and microbiologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have completed several conjugations to transfer plasmid carrying the bacterial lux genes into Aeromonas hydrophila. Although we were able to obtain bioluminescent A. hydrophila colonies, it appears plasmid stability should be improved. Research undertaken to date revealed that transposon mutagenesis works in A. hydrophila. Thus, we are in process of producing random transposon mutant library. Functional characterization of interrupted genes should lead to a better understanding of A. hydrophila pathogenesis. Moreover, surface-exposed antigens are being identified using bioinformatics tools.

Publications

  • No publications reported this period