Microbiology

The Fish Health/Microbiology Team has developed several molecular-based methods that can be used to identify bacterial pathogens in fish tissue, without the need for culturing the microorganisms. Additionally, methods are being developed to differentiate strains of individual bacterial species for epidemiological/epizootiological studies.

Sensitive and specific diagnostic tests
RT-PCR: We have developed a diagnostic application using the techniques of reverse transcription (RT) and polymerase chain reaction (PCR) to specifically detect the presence of Renibacterium salmoninarum in a variety of fish tissues, including blood ( Abstract). This technique is capable of identifying the presence of as few as 1-10 bacteria per mg of tissue. It involves targeting the 16S ribosomal RNA of the bacterium with a specific oligonucleotide primer followed by RT. The resulting product is then amplified via PCR. We routinely use the assay to screen eggs or salmon fry for the presence of R. salmoninarum prior to using the fish in virulence, therapeutic, or vaccine trials.

Running a 310 Genetic Analyzer

T-RFLP: We have extended a nucleic acid-based assay to detect and identify bacterial pathogens in infected tissue without the need for bacterial culture ( Abstract). The technique, known as terminal restriction fragment length polymorphism (T-RFLP), uses a pair of fluorescent-labeled PCR primers that target two highly conserved regions in the gene for eubacterial 16S ribosomal RNA. The resulting amplicon (with labeled ends) is then digested with restriction endonucleases and the size of the terminal fragments determined by capillary electrophoresis with laser-induced fluorescence detection. The size of these fragments is unique and specific for bacterial genera and species. Application of the test results in highly specific identification of infecting bacteria in fish kidney, and is particularly useful in detection of those bacteria that do not grow well on standard laboratory media.

Strain differentiation
The potential for horizontal transmission of Bacterial Kidney Disease (BKD) from hatchery-reared salmonids to wild stocks is a management concern. A barrier to testing this possibility has been the inability to genetically or phenotypically distinguish isolates of R. salmoninarum, the causative agent of BKD. Recent identification of genetic variation among R. salmoninarum isolates has introduced the possibility of applying methods that can determine and monitor horizontal transmission of BKD between captive and feral salmon populations.

IS994 and other genetic markers: We have characterized an insertion sequence (IS) element designated IS994 present in multiple copies in the chromosome of R. salmoninarum ( Abstract). IS elements are mobile genetic elements present in the DNA of many bacterial species. Because of their ability to often create multiple and random insertions in the chromosome, their location and copy number can often be used to differentiate strains of bacteria from widely different geographic areas by probing restriction endonuclease digested DNA with an IS994 probe. We are working on combining IS994 chromosomal patterns with two PCR-based methods (RAPD and tDNA-ILP) to develop molecular fingerprints of R. salmoninarum strains isolated from different geographical areas.