TWENTY-FOURTH ANNUAL EASTERN FISH HEALTH WORKSHOP Royal Pavilion Resort, Atlantic Beach, NC MARCH 9-11, 1999 Nested PCR For The Non-lethal Detection Of Mycobacteriosis In Striped Bass (Morone saxatilis) Jeffrey C. Wolf1, Stephen A. Smith1, Brent R. Whitaker2 1Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg VA 21046; 2The National Aquarium in Baltimore, Baltimore, MD Piscine mycobacteriosis is a chronic debilitating disease of freshwater, brackish, and marine fishes throughout the world. It has been suggested that virtually all fish species are susceptible to infection. Mycobacteriosis has been documented in fishes raised for food, sportfishing, and ornamental display. Fish-borne mycobacteria are capable of causing serious wound-related infections in humans. Thus, infected captive fishes represent a public health hazard. Clinical signs of piscine mycobacteriosis are non-specific and there currently are no established methods for the antemortem diagnosis of this disease. At present, control of mycobacteriosis in affected fish populations is based upon a principle of "cull and test". This method is undesirable, especially when working with fishes that are expensive or difficult to replace, such as valuable broodstock, research animals, or rare and exotic aquarium specimens. Polymerase chain reaction (PCR) is a rapid, dependable, and relatively economical method for the diagnosis of mycobacteriosis from blood or tissues. We are currently evaluating a nested PCR procedure using a variety of hematologic specimens from experimentally and spontaneously infected striped bass including whole blood, buffy coat, plasma, and red cell preparations. The three basic stages of this assay are: 1) the liberation of mycobacterial DNA by the disruption of host and bacterial cell membranes; 2) the amplification of a specific mycobacterial 16S rRNA genomic sequence using two polymerase chain reactions (PCRs) run serially; 3) the sizing and detection of mycobacterial DNA amplicons bound to ethidium bromide, by agarose gel electrophoresis and ultraviolet light. Results indicate that nested PCR is a highly sensitive and specific method for detecting mycobacterial DNA in fish blood, and is potentially valuable in the antemortem diagnosis of mycobacteriosis in fish. Return to 24th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page