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Comparison of fixatives and fixation time for PCR detection of Mycobacterium in zebrafish Danio rerio
Tracy S Peterson1, Michael L Kent, Jayde A Ferguson
1Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA. fishpathman@gmail.com
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.
Insights
A PCR assay effectively detected mycobacterial DNA in paraffin-embedded zebrafish, aiding in diagnosing mycobacteriosis. This method works regardless of fixative type, time, or mycobacterial species, improving fish disease management.
Area of Science:
- Veterinary Pathology
- Molecular Diagnostics
- Aquatic Animal Health
Background:
- Mycobacteriosis is a prevalent disease in laboratory zebrafish (Danio rerio), with varying impacts based on the specific Mycobacterium species.
- Accurate species-level identification is crucial for effective disease management in zebrafish.
- Traditional diagnostic methods like acid-fast staining offer genus-level identification, while culture and molecular methods are more definitive but can be time-consuming.
Purpose of the Study:
- To evaluate the efficacy of a PCR assay targeting the hsp gene for detecting mycobacterial DNA in paraffin-embedded zebrafish tissues.
- To determine the influence of fixative type, fixation duration, and mycobacterial species on PCR assay performance.
Main Methods:
- Zebrafish were experimentally infected with Mycobacterium chelonae or Mycobacterium marinum.
- Fish tissues were preserved in either 10% neutral buffered formalin or Dietrich's fixative for varying durations (3, 7, 21, 45 days).
- Histological and Fite's acid-fast staining were performed, alongside PCR analysis using the hsp gene primer set (HS5F/hsp667R).
Main Results:
- The hsp gene PCR assay successfully amplified mycobacterial DNA from 75% of M. chelonae-infected and 88% of M. marinum-infected zebrafish.
- No statistically significant relationship was found between the PCR assay's efficacy and the type of fixative used, time in fixative, or the specific mycobacterial species.
- PCR provided a viable method for detecting mycobacterial DNA even from long-term preserved samples.
Conclusions:
- The hsp gene PCR assay is a robust tool for detecting mycobacterial DNA in paraffin-embedded zebrafish, facilitating species identification.
- This molecular diagnostic approach offers advantages over traditional methods, especially when dealing with archived tissue samples.
- The assay's reliability across different fixation conditions supports its utility in routine diagnostics and research for zebrafish mycobacteriosis.
