DAO 104:113-120 (2013)  -  DOI: https://doi.org/10.3354/dao02585

Comparison of fixatives and fixation time for PCR detection of Mycobacterium in zebrafish Danio rerio

Tracy S. Peterson1,*, Michael L. Kent1,2, Jayde A. Ferguson4, Virginia G. Watral1, Christopher M. Whipps

1Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA
2Department of Biomedical Sciences, Oregon State University, Corvallis, Oregon 97331, USA
3Department of Environmental Science and Forestry, State University of New York College of Environmental Science and Forestry, SUNY-ESF, 1 Forestry Drive, Syracuse, New York 13210, USA
4Alaska Department of Fish and Game, Commercial Fisheries Division, Pathology Laboratory, Anchorage, Alaska 99518, USA

ABSTRACT: Mycobacteriosis is a common disease of laboratory zebrafish Danio rerio. Different infection patterns occur in zebrafish depending on mycobacterial species. Mycobacterium marinum and M. haemophilum produce virulent infections associated with high mortality, whereas M. chelonae is more widespread and is not associated with high mortality. Identification of mycobacterial infections to the species level provides important information for making management decisions. Observation of acid-fast bacilli in histological sections or tissue imprints is the most common diagnostic method for mycobacteriosis in fish, but only allows for diagnosis to the genus level. Mycobacterial culture followed by molecular or biochemical identification is the traditional approach, but DNA of diagnostic value can also be retrieved from paraffin blocks. Here we investigated the type of fixative, time in fixative before processing, species of mycobacteria, and severity of infection as parameters to determine whether the hsp gene PCR assay (primer set HS5F/hsp667R) could detect and amplify mycobacterial DNA from paraffin-embedded zebrafish. Whole zebrafish were experimentally infected with either M. chelonae or M. marinum, and then preserved in 10% neutral buffered formalin or Dietrich's fixative for 3, 7, 21, and 45 d. Subsequently, fish were evaluated by hematoxylin and eosin and Fite's acid-fast stains to detect mycobacteria within granulomatous lesions. The PCR assay was quite effective and obtained PCR product from 75 and 88% of the M. chelonae- and M. marinum-infected fish, respectively. Fixative type, time in fixative, and mycobacterial species showed no statistical relationship with the efficacy of the PCR test.


KEY WORDS: PCR · Mycobacterium marinum · Mycobacterium haemophilum · 10% NBF · Formalin · Dietrich's · Zebrafish · Infection severity · Diagnostics


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Cite this article as: Peterson TS, Kent ML, Ferguson JA, Watral VG, Whipps CM (2013) Comparison of fixatives and fixation time for PCR detection of Mycobacterium in zebrafish Danio rerio. Dis Aquat Org 104:113-120. https://doi.org/10.3354/dao02585

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