PUBLICATION

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

Authors
Peterson, T.S., Kent, M.L., Ferguson, J.A., Watral, V.G., and Whipps, C.M.
ID
ZDB-PUB-130708-1
Date
2013
Source
Diseases of aquatic organisms   104(2): 113-120 (Journal)
Registered Authors
Kent, Michael
Keywords
none
MeSH Terms
  • Animals
  • Bacteriological Techniques
  • Fish Diseases/diagnosis
  • Fish Diseases/microbiology*
  • Fixatives/analysis*
  • Mycobacterium/isolation & purification*
  • Mycobacterium Infections/microbiology
  • Mycobacterium Infections/veterinary*
  • Polymerase Chain Reaction/veterinary*
  • Time Factors
  • Zebrafish*
PubMed
23709464 Full text @ Dis. Aquat. Organ.
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.

Genes / Markers
Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes