Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

Abstract

Rainbow trout fry syndrome and cold‐water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex® 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0·4 cfu in the PCR tube, corresponding to 17 cfu mg−1 brain tissue. The PCR‐assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non‐sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR‐assay was 1·7 cfu in the PCR tube, corresponding to 110 cfu ml−1 water. The PCR‐assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum.

Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss

“…For specific detection of F. psychrophilum in fish tissue and aquarium water using epifluorescence microscopy, rabbit polyclonal antiserum against F. psychrophilum strain T1-1 was prepared according to the method of Lancefield et al (1975) modified by Lorenzen & Karas (1992). In order to eliminate a slight cross-reaction of the anti-T1-1 serum observed with yellow-pigmented, non-characterised bacterial isolates biochemically different from F. psychrophilum (Wiklund et al 2000), absorption of the antiserum was done by the following method: Two cross-reactive isolates were cultivated in separate tubes containing TYES broth for 3 d at 15°C with gentle shaking. The cultures were centrifuged at 2500 × g for 15 min at 15°C and the bacteria were washed twice with PBS (pH 7.2).…”

Phase variation in Flavobacterium psychrophilum: characterization of two distinct colony phenotypes

“…1B) colony phenotypic variants that were separated for the study and thereafter named after the corresponding colony type as P13-4S/96, P13-4R/96, P6-1S/07, P6-1R/07, P6-3S/07, P6-3R/07, P6-8S/07 and P6-8R/07. All variants were identified as F. psychrophilum by polymerase chain reaction (PCR) (Wiklund et al 2000). The F. psychrophilum type strain NCIMB 1947 T and the wild type (WT) isolate P13-4/96 were also included in the study for comparison.…”

Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule?

“…The isolates were all characterized as members of the 87 Flavobacteriaceae, based on morphological and biochemical criteria. Isolates were also tested by a 88 16S rRNA nested PCR method (Wiklund et al 2000) to determine whether they were Flavobacterium 89 psychrophilum or other species of Flavobacteriaceae. A subset of these other Flavobacteriaceae 90 were further identified on the basis of partial 16S rRNA gene sequencing and analysis using the 91 online tool RDP SEQMATCH (Cole et al 2005).…”