Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction

Abstract

We evaluated a polymerase chain reaction (PCR) method for detecting Yersinia ruckeri, the bacterial pathogen causing enteric redmouth disease (ERM), in blood of rainbow trout Oncorhynchus mykiss. Identification of the PCR product was confirmed by Southern blot hybridization with a 32 P-labeled oligonucleotide probe matching a sequence within the small subunit ribosomal RNA gene of Y. ruckeri. Following a 1 h immersion of rainbow trout in water with 4.5 × 10 6 colonyforming units of Y. ruckeri l -1 , the PCR was positive for all blood samples from 1 h (first sample) to 5 d and was negative from 9 to 30 d (last sample). Fish in this experiment did not show signs of disease, probably because they had been vaccinated against Y. ruckeri. To test this method with naturally infected fish, 42 rainbow trout from hatcheries were examined. Four of these fish had clinical signs of ERM and were infected with Y. ruckeri based on bacteriological culture. The PCR method detected Y. ruckeri in blood, intestine, liver, and trunk kidney from the 4 fish with ERM and from 5 additional rainbow trout that were bacteriologically negative for Y. ruckeri. Three of 5 rainbow trout from streams receiving effluent from hatcheries were positive for Y. ruckeri when tested with PCR, although there was no growth of Y. ruckeri on culture plates inoculated with the same samples. Samples were successfully stored for 1 wk in lysis buffer at 25°C. This study demonstrated that a nonlethal blood sample can be used with PCR to detect Y. ruckeri.

PCR detection and PFGE DNA macrorestriction analyses of clinical isolates of Pseudomonas anguilliseptica from winter disease outbreaks in sea bream Sparus aurata

“…Although the presence of host DNA or inhibitory substances in tissue extracts has been reported to reduce the sensitivity of the PCR assays (Coleman et al 1996, McIntosh et al 1996, the sensitivity of the PCR assay with tissue extracts was very similar to that obtained with cell suspensions. The absence of inhibitory effects may be due to the 10-fold dilution step of the tissues and/or the DNA extraction procedure used, which would reduce or eliminate the possible inhibitory substances present in the tissue extracts (Altinok et al 2001).…”

Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease

“…After 1 h of incubation at 37°C, 100 µl of 5 M NaCl were added and mixed thoroughly. Then 80 µl cetyltrimethylammonium bromide were mixed with the sample, which was incubated at 65°C for 10 min (Altinok et al 2001). The DNA was then purified as described above for tissue samples, resuspended in 100 µl TE buffer, quantified spectrophotometrically, and stored at 4°C until required.…”

“…The lysis buffer and TE buffer were prepared with DNase-and RNase-free ultra pure autoclaved water. Samples were incubated at 60°C for 16 h. After extraction of the DNA with a mixture of phenolchloroform-isoamyl alcohol (50:48:2; Altinok et al 2001), the DNA was precipitated at -20°C for 2 h by adding 0.6 ml of ice-cold 100% ethanol, then centrifuged at 21 000 × g for 20 min at 4°C and the ethanol was decanted. The DNA was centrifuged following addition of 70% ethanol at 21 000 × g for 20 min at 4°C and dried overnight.…”

Molecular diagnosis of francisellosis, a systemic granulomatous inflammatory disease in Atlantic cod, Gadus morhua L

“…Møre og Romsdal, Norway isolate (Ottem et al 2008); Lane 7: Aeromonas salmonicida NCMB 1102; Lane 8: Vibrio anguillarum NCMB 6; Lane 9: V. anguillarum NCMB 2133; Lane 10: V. anguillarum 02β VI-F-258-3; Lane 11: Yersinia ruckeri VI-3629; Lane 12: Y. ruckeri NCMB 2196; Lane 13: PCR negative control. PCR was done at an annealing temperature of 60°C and 40 cycles PCR techniques have excellent advantages over the other detection methods and these protocols have been developed for the diagnosis of many important bacterial diseases in fish (Altinok et al 2001;del Cerro et al 2002;Gonzalez et al 2003;Osorio and Toranzo 2002;Romalde and Toranzo 2002). Hsieh et al (2006Hsieh et al ( , 2007 used the primers for the PCR amplification of the 16S rDNA and an in situ hybridization (ISH) protocol to detect Francisella-like bacterium in ornamental cichlids, but did not test for specificity and sensitivity.…”