Nested PCR for the Non-Lethal Detection of Mycobacteriosis in Striped Bass (Morone saxatilis)
IAAAM Archive
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

Abstract

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.

References

1.  Bleiker TO, JE Bourke, DA Burns. 1996. Fish tank granuloma in a 4-year-old boy. Br. J. Dermatol. 135:863-864.

2.  Borradori L, et al. 1991. Granulomes a Mycobacterium marinum chez un aquariophile. Schwiez med Wschr 121:1340-1344.

3.  Bruckner-Tuderman L, AA Blank. 1985. Unusual cutaneous dissemination of a tropical fish tank granuloma. Cutis 36:405-408.

4.  Buck GE, LC O'Hara, JT Summersgill. 1992. Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J. Clin. Microbiol. 30:1331-1334.

5.  Chow SP, et al. 1983. Mycobacterium marinum infection of the hand involving deep structures. J. Hand. Surg. 8:568-573.

6.  Colorni A, et al. 1993. Detection of mycobacteriosis in fish using the polymerase chain reaction technique. Bull. Eur. Ass. Fish Pathol. 13:195-198.

7.  Davis LG, WM Kuehl, JF Battey. 1994. Basic Methods in Molecular Biology, 2 ed. Appleton and Lange, Norwalk, Connecticut, USA.

8.  Dieffenbach CW, GS Dveksler, eds. 1995. PCR Primer, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY, USA.

9.  Dulin MP. 1979. A review of tuberculosis (mycobacteriosis) in fish. Vet. Med. Small Anim. Clin. 74:731-735.

10. Herrington CS, JJ O'Leary, eds. 1998. PCR 3, PCR In Situ Hybridization, A Practical Approach. The Practical Approach Series, B.D. Hames ed., IRL Press at Oxford University Press, Oxford, UK.

11. Hoss M, S Paabo. 1993. DNA extraction from Pleistocene bones by a silica-based purification method. Nucl. Acids Res. 21:3913-3914.

12. Howe C. 1995. Gene Cloning and Manipulation. Cambridge University Press. Cambridge, UK.

13. Knibb W, et al. 1993. Detection and identification of a pathogenic marine Mycobacterium from the European seabass Dicentrarchus labrax using polymerase chain reaction and direct sequencing of 16S rDNA sequences. Mol. Mar. Biol. Biotechnol l2:225-32.

14. McCormick JI, MS Hughes, MF McLoughlin. 1995. Identification of Mycobacterium chelonae in a cichlid oscar, Astronotus ocellatus Cuvier, by direct cycle sequencing of polymerase chain reaction amplified 16s rRNA gene sequences. J. Fish Dis. 18:459-461.

15. Miller SA, DD Dykes, HF Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucl. Acids Res. 16:1215.

16. Noga EJ. 1996. Fish Disease: Diagnosis and Treatment. Mosby, St. Louis.

17. Noga EJ, JF Wright, L Pasarell. 1990. Some unusual features of mycobacteriosis in the cichlid fish Oreochromis mossambicus. J. Comp. Pathol. 102:335-344.

18. Pungkachonboon T, et al. 1990. Isolation and characterization of Mycobacterium sp. from Siamese fighting fish, Betta splendens Regan. In: Symposium on Diseases in Asian Aquaculture. Bali (Indonesia).

19. Ross AJ, HE Johnson. 1962. Studies of transmission of mycobacterial infections in Chinook salmon. The Progressive Fish-Culturist 24:147-149.

20. Ross JA, FP Brancato. 1959. Mycobacterium fortuitum Cruz from the tropical fish Hyphessobrycon innesi. J. Bacteriol. 78:392-395.

21. Shamsudin MN, et al. 1990. Characterization of the causative organism of ornamental fish mycobacteriosis in Malaysia. Fish Pathology 25:1-6.

22. Smith SA. 1995. Infectious and zoonotic implications of mycobacteriosis in spawning tropical fish. International Association For Aquatic Animal Medicine. Mystic, Connecticut, USA 26:41.

23. Talaat A, R Reimschuessel, M Trucksis. 1997. Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis. Vet. Microbiol. 58:229-237.

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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