USE OF THE POLYMERASE CHAIN-REACTION (PCR) TO DETECT DNA FROM RENIBACTERIUM-SALMONINARUM WITHIN INDIVIDUAL SALMONID EGGS

Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD) in salmonids, can be transmitted vertically, i.e. from parent to progeny via the egg. The usual procedure to determine the likelihood of egg infection has been to examine non-egg tissues from mature female fish for evidence of R. salmoninarum cells or antigens. In the present study we examined the feasibility of detecting the pathogen directly within individual salmonid eggs by use of the polymerase chain reaction (PCR) to amplify segments of its DNA. Total DNA was extracted from the contents of eggs taken from several coho and chinook salmon (Oncorhynchus kisutch and O. tshawytscha), including coho eggs that had been injected with numbers of R. salmoninarum cells ranging from 2 to 2000 cells per egg. We also isolated DNA from kidney samples taken from these fish. Utilizing the PCR, two 24-base oligonucleotide primers were used to amplify a 501 base-pair region of the gene encoding a 57 kDa soluble protein ('p57') produced by R. salmoninarum. A segment of DNA of the appropriate size (501 base-pairs) from R. salmoninarum was detected by the PCR in all of the injected eggs. R. salmoninarum DNA was also detected in some of the eggs taken from the coho and chinook samples that had previously been diagnosed as 'high-BKD' or 'negative-BKD' fish. Direct sequencing of the heat-denatured, double-stranded amplification product was carried out by dideoxy termination using T7 DNA polymerase and S-35 labelling. There was 100% homology between the partial sequence obtained from the amplified DNA fragment and the sequence of the p57 gene. The DNA extracted from other bacterial fish pathogens did not yield a positive result. These results indicate that the PCR is able to detect as few as 2 R. salmoninarum cells per egg and that it may be feasible as a broodstock screening technique specific for R. salmoninarum.