Detection of fish pathogens with PCR-DGGE in non-lethal mucus samples and molecular typing of Aeromonas salmonicida using the 16s-23s internal transcribed spacers

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Quinn, Robert Andrew
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University of Guelph

This thesis is an investigation of the detection of 'Aeromonas salmonicida' using nested PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) in non-lethal fish mucus samples. The technique was compared to conventional non-lethal and lethal methods. 'A. salmonicida' was detected by all techniques. Detection of 'A. salmonicida' by DGGE was based on co-migrating bands and was verified by sequencing. The mean level of detection was highest with culture of mucus on Coomassie brilliant blue agar in the Chinook (71.67% ± 7.64) and PCR-DGGE (69.10 ± 1.29) for the coho salmon. Mucus based techniques had a statistically significant agreement for detection of 'A. salmonicida. Flavobacterium psychrophilum, F. branchiophilun' and 'Yersinia ruckeri' were not detected with PCR-DGGE. Bands often co-migrated to these pathogens, but their nucleotide sequences belonged to other bacteria. Seeding 'Y. ruckeri ' in mucus showed that PCR-DGGE could detect the pathogen. In an attempt to detect strain differences, the 16S-23S rRNA internal transcribed spacers of 'A. salmonicida' were fingerprinted using RFLP and DGGE. These techniques verify the clonal nature of the pathogen, but do show the capacity to differentiate various 'Aeromonas' spp.

fish pathogens, non-lethal fish mucus, PCR-DGGE, Aeromonas slamonicida, molecular typing, 16s-23s internal transcribed spacers