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Diseases of Aquatic Organisms

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DAO 34:13-20 (1998)  -  doi:10.3354/dao034013

Rapid and sensitive reverse transcriptase-polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells

T. A. Miller1,2, J. Rapp1, U. Wastlhuber1, R. W. Hoffmann3, P.-J. Enzmann2,*

1Staatliches Tierärztliches Untersuchungsamt Aulendorf, Löwenbreitestr. 18/20, D-88326 Aulendorf, Germany
2Bundesforschungsanstalt für Viruskrankheiten der Tiere, Paul-Ehrlich-Str. 28, D-72076 Tübingen, Germany
3Institut für Zoologie, Fischerei und Fischkrankheiten, Kaulbachstraße 37, D-80539 München, Germany

ABSTRACT: A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed and applied to the detection and differentiation of viral haemorrhagic septicaemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) in organ samples and cultured cells, regardless of the serotype. This method was developed by selecting primer sets corresponding to highly conserved regions of the glycoprotein G-gene sequences of the 2 viruses. The very fast RNA extraction, reverse transcription and PCR permitted us to read the agarose gels within 7 to 9 h after samples, cultured cells and whole fish arrived, which is of great importance when there is reason to believe that VHSV or IHNV may be present. This is also the first report of a large-scale field trial comparing the RT-PCR assay in trout from 30 German fish farms (a total of 330 rainbow trout) with the usual virus isolation and identification method in order to evaluate the efficiency of the RT-PCR assay for general use in fish health management programs. RT-PCR followed by semi-nested PCR using RNA directly extracted from fish tissue turned out to be the most sensitive method. It recognized 9 fish farms as VHS-positive and 7 as ihn-positive. This is 3 VHS- and 4 ihn-farms more than detected by the traditional virus isolation method. By directly examining the tissue by means of a pcr test it was possible to detect viral RNA in acutely and subacutely to chronically diseased fish as well as in asymptomatic vhs/IHN-carrier fish. Therefore, this effective and powerful assay for detecting VHSV and IHNV by means of PCR has great advantages compared with the presently used procedures.

KEY WORDS: RT-PCR · VHSV/IHNV · differential diagnosis in organ samples

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