28th ANNUAL EASTERN FISH HEALTH WORKSHOP April 21-25, 2003 Electroporation Of DNA Vaccines Into Guppies (Poecilia reticulata) And Channel Catfish (Ictalurus punctatus) Kenneth E. Nusbaum 1, Rebecca L. Sanchez 2, Bruce F. Smith3, and R. Curtis Bird1 1Department of Pathobiology, College of Veterinary Medicine; 2Undergraduate Research Fellows Program, 202 Spidle Hall, Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, AL 36849 The emergent technology of DNA vaccination of fish has been well established in salmonids and is emerging in channel catfish with a DNA vaccine against channel catfish virus (CCV).  The relatively low cost per fish of catfish and the young age and small size coincident with CCV disease argue against vaccination of individual fish, and highlight the need for a means of vaccinating fry or eggs before they leave the hatching shed.  Electroporation is a well-established means of introducing foreign or marker DNA into tissues and has been widely used in molecular biology.  Because of the brief availability of catfish fry or eggs, guppies, a species that reproduces readily in confinement were chosen as a model species for vaccination procedures when catfish were unavailable.  In addition, guppies are scaled fish, and may serve as a model for electroporation immunization of other fishes.  Optimization of electroporation conditions determined that, in the absence of handling injuries, 100% of electroporated fish survived treatment at 50 to 200 volts/cm using 2 pulses of  100 secs over a one second interval.  Vaccination was conducted with 6 to 24 mg of DNA in 600 ml of distilled water in an electroporation cuvette.  The vaccine was either CCV open reading frame (ORF) 8a or ORF 59.  Fish were removed from the cuvette and held from 24 to 72 hours in 500 ml beakers (betta tanks).  Fish were then harvested and DNA or RNA was extracted to determine the presence of the vaccine through PCR and the expression of the vaccine through rt-PCR.  Vaccine presence has been demonstrated repeatedly, but expression is still elusive. Successful use of electroporation for DNA vaccination will depend on the ability of the introduced plasmid to express itself and on the ability of the system to handle a much greater biomass than that studied here. Return to 28th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page