Detection Methods

Mycobacteria in Striped Bass

electrophoresis

Objective:To develop and evaluate methods for detecting mycobacteria in striped bass.

Scientists have traditionally used three main methods to study the occurrence of mycobacterial infections in striped bass: histological, cultural, and molecular. VIMS researchers are working to adapt and enhance these methodologies for detection of mycobacteria in fish tissues.

Histological assessment is based on detection of granulomatous lesions formed by the host in response to infecting bacteria. Thin slices of preserved fish tissue are examined for the presence of inflammatory cells that surround the mycobacteria in an attempt to "wall-off" the infection. Special staining techniques are used to detect the mycobacteria within these lesions.

Culturing mycobacteria requires growing mycobacteria on various artificial culture media. Scientists collect fish tissue under sterile conditions beneath a laminar flow hood, apply appropriate temperature and incubation periods to maximize mycobacterial growth, and analyze results using a quantitative approach. Although laborious and time-consuming, the culture method allows researchers to determine the density of mycobacteria in fish tissue and to recognize the types of mycobacteria present. Once mycobacteria are isolated, they can be characterized by biochemical tests, growth characteristics, and molecular methods (see articles in Emerging Infectious Diseases and International Journal of Systematic and Evolutionary Microbiology).

Molecular detection involves the use of polymerase chain reactions (PCR) and a further amplification technique called nested PCR. These techniques use oligonucleotide primers that detect and amplify a portion of the Mycobacterium genus-specific 16S rRNA gene. This gene is highly conserved amongst all Mycobacterium species and is thus a reliable indicator of mycobacterial infections. Molecular detection of mycobacteria can be accomplished within hours or days compared to the cultural approach, which requires weeks to months because of the comparatively slow growth rate of some Mycobacterium species. Additional molecular techniques such as gene sequencing and restriction fragment length polymorphism (RFLP) patterns are used to characterize isolates.

A recent VIMS investigation compared these methods for assessing mycobacteriosis prevalence. Cultural and molecular-based methods yielded significantly higher rates of detection than the histological method.

Principal Investigators: