Project Number: 6420-32000-020-00
Project Type: Appropriated

Start Date: Dec 14, 2004
End Date: Dec 13, 2009

Objective:
Objective: A multiplex PCR for simultaneous detection of three fish-pathogenic bacteria, Edwardsiella ictaluri, Flavobacterium columnare and Aeromonas hydrophila. The current project will be expanded to include development of molecular-based vaccines for use in aquaculture to prevent fish diseases.

Approach:
Traditional methods of diagnosing infection using culture techniques are laborious, time consuming, often unreliable and require several days to arrive at a definitive diagnosis; thereby increasing the potential of spreading the disease and delaying implementation of disease control strategies. When multiple bacterial pathogens are likely to occur as in aquatic environment, amplification of multiple target genes in a single reaction mixture is possible with the multiplex-PCR technique. We will develop three specific primer sets, each targeting a unique gene segment of the homologous bacterium. The specificity and sensitivity of the primer pairs will be rigorously tested and validated for rapid/specific detection of the infecting organism with a minimum of time, cost and effort. Impact: Warmwater aquaculture is slated to be the fastest growing sector in the U.S. agro-business. Notwithstanding, bacterial disease has been spot-lighted as the preeminent cause of monitory loss, accounting for millions of dollars to the industry. Thus, the development of a multiplex-PCR for rapid/accurate detection of multiple pathogens simultaneously is imperative for making prompt management decisions as well as for bio-security reasons to negate the spread of disease and to cut down the monitory losses sustained by aquaculture farmers and industry. Bacteria and viruses (channel catfish virus and channel catfish reovirus) are selectively tropic and attach to specific receptors on specific fish-host tissues. Thus, in this research, primary cell cultures (gill, kidney and intestine) from two to three week old channel catfish will be harvested aseptically and maintained in Leibowitz L 15 medium supplemented with antibiotics, 10% fetal bovine serum and growth hormones. Primary cells will be transfected with the pSV2-neo plasmid carrying the SV40 T-antigen gene by lipofectamin transfection procedure to establish continuously dividing immortalized cells. Transformed cells will be selected by their growth resistance in antibiotic geneticin (G418) containing medium. Cells will be tested for the presence of the SV40 T-antigen with the monoclonal antibody (TIB-115) against the T-antigen by immunofluorescence staining and epifluorescence microscopy with appropriate controls. Immortalized cells will be karyotyped and the established permanent cell lines banked in the ATCC repository. The permanent epithelial cell lines will be used for the growth of channel catfish virus (CCV) for large scale propagation of the virus for development of an efficient vaccine. The immediate benefits of this research will be the development of vaccines against viral pathogens of catfish.