Nested PCR for the Non-Lethal Detection of Mycobacteriosis in Striped Bass (Morone saxatilis)
IAAAM 1999
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, USA; 2The National Aquarium in Baltimore, Baltimore, MD, USA


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.9 Mycobacteriosis has been documented in fishes raised for food17, sportfishing19, and ornamental display.14,18,20-22 Fish-borne mycobacteria are capable of causing serious wound-related infections in humans.1-3,5 Thus, infected captive fishes represent a public health hazard. Clinical signs of piscine mycobacteriosis are non-specific, and there are currently no established methods for the antemortem diagnosis of this disease.16 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.

We have developed a rapid, dependable, non-lethal, and relatively economical assay to detect mycobacterial DNA from frozen heparinized fish blood. The three basic stages of this assay are: 1) liberation of mycobacterial DNA from fish buffy coat cells by sonication; 2) amplification of a specific mycobacterial 16S rRNA genomic sequence using two polymerase chain reactions (PCRs) run serially; and 3) sizing and detection of ethidium bromide-bound mycobacterial DNA amplicons by agarose gel electrophoresis and ultraviolet light.

Several methods have been used to isolate DNA from intact cells and tissues, including phenol/chloroform extraction7, salting out15, and silica-based purification.11 Sonication, the use of ultrasonic energy to disrupt cell membranes, is a rapid, simple, inexpensive, and safe alternative for DNA isolation.4 Sonication usually involves the insertion of a cylindrical metal probe directly into fluid containing suspended cells. The technique used in this study, adapted from Buck et al.4, differs in that the sonicator probe is placed in a container of water adjacent to a floating rack of capped sample tubes. This simple modification minimizes both sample cross-contamination and DNA contamination of the laboratory environment.

The polymerase chain reaction is "...the repetitive bidirectional DNA synthesis via primer extension of a region of nucleic acid..."8 Useful information on PCR theory and application is available.7,8,10,12 In the nested polymerase chain reaction (nested PCR), a small sample of amplified DNA from the primary reaction is re-amplified using a second set of oligonucleotide (18-24 base pair) primers positioned internal to the first set. This double amplification generates large amounts of DNA product and is thus highly sensitive to minute amounts of template DNA. The genus-specific oligonucleotide primers used in the primary reaction of our assay were originally designed by Talaat et al. to amplify a strongly conserved segment of the mycobacterial 16S rRNA gene.23 The secondary (nested) primers that we designed are also Mycobacterium species specific. When the product of the secondary reaction is placed within 2% agarose gel containing ethidium bromide, electrophoresis results in a predicted-length 349 base pair (bp) band that is readily visualized under ultraviolet light.

Although not widely utilized for the diagnosis of piscine mycobacteriosis, PCR has been used experimentally to detect and identify fish-borne mycobacteria from culture6,13,23 and from the blood6 and tissues6,23 of infected fish. We are currently evaluating our procedure using a variety of specimens from experimentally and spontaneously infected striped bass including whole blood, buffy coat, plasma, and red cell preparations. Results indicate that nested PCR is a highly sensitive and specific method for detecting mycobacterial DNA in fish blood and is potentially valuable for the antemortem diagnosis of mycobacterioses in fishes.


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Speaker Information
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Jeffrey C. Wolf, DVM
Department of Biomedical Sciences and Pathobiology
Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute and State University
Blacksburg, VA, USA

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