Use of the polymerase chain reaction (PCR) to detect DNA from Renibacterium salmoninarum within individual salmonid eggs

Abstract

Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD) in salrnonids, 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. sahoninarum 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 lusutch and 0 . 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 ('p577 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. salrnoninarum 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 heatdenatured, double-stranded amplification product was carried out by dideoxy termination using T7 DNA polymerase and 35S 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. s a l m o n i n a m .

Production of the 57 kDa major surface antigen by a non-agglutinating strain of the fish pathogen Renibacterium salmoninarum

“…The polymerase chain reaction (PCR) was a variation of the protocol described by Brown et al (1994). Renibactedum salmoninarum was suspended to an ODGOOnm of 1.0 in sterile buffer (10 mM Tris-HC1, 1 mM sodium-EDTA, 0.35 M sucrose) and a 200 pl volume was incubated for 30 min at 25'C in the presence of 2% SDS.…”

“…The primers Rsl and Rs2, which amplify a 501 basepair (bp) region of the gene encoding p57, were described by Brown et al (1994) and were synthesized by the Laboratory Services Division, University of Guelph. The PCR assay was conducted under the following conditions: 1 mM Tris-HC1,5 mM KC1,O.…”

Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

“…In previous work, it was demonstrated that 16s rRNA gene primers can be used to detect as little as 1 pg to 10 fg of Photobacterium damselae purified DNA (Osorio et al 1999). Nevertheless, the sensitivity of the multiplex PCR should not be affected by the difference in gene copy numbers, since very good results and low detection levels are usually obtained for other fish pathogens when a single-copy gene is used as a target for PCR (Brown et al 1994).…”

Immunosuppression in progeny of chinook salmon infected with Renibacterium salmoninarum: re-analysis of a brood stock segregation experiment

“…Other studies found even lower levels of vertical transmission (1 to 6%) (Evelyn et al 1986, Lee & Evelyn 1989. While a more recent study has suggested that 31 Dis Aquat Org 65: [29][30][31][32][33][34][35][36][37][38][39][40][41] 2005 these numbers might underestimate actual vertical infection rates due to incomplete detection (Brown et al 1994), vertical transmission alone, without evidence of increased horizontal transmission, immunosuppression, or both, is unlikely to account for large increases in infection or decreases in survivorship of progeny of infected females, especially if the majority of these spawners have only low to moderate infection levels, as was the case in the 1988 Dworshak brood stock segregation study.It may be that the p57 antigen in soluble form is taken up by eggs from the surrounding ovarian fluid or introduced directly from the bloodstream during oogenesis. Alternatively, vertical infection and subsequent antigen secretion could result in high antigen concentrations within the egg.…”