Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum.
OBJECTIVE
To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples.


RESULTS
The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. psychrophilum. The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated.


CONCLUSIONS
The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. psychrophilum in the ovarian fluid.


CONCLUSIONS
The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.
Baliarda
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Laboratoire de Microbiologie, ENITA Bordeaux, 1 cr. G. de Gaulle, 33175 Gradignan, France.
Faure
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Urdaci
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D000818:Animals; D017931:DNA Primers; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D005260:Female; D005393:Fish Diseases / Q000382:microbiology*; D005417:Flavobacterium / Q000235:genetics / Q000302:isolation & purification*; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology / Q000662:veterinary*; D017684:Oncorhynchus kisutch / Q000382:microbiology*; D017686:Oncorhynchus mykiss / Q000382:microbiology*; D010053:Ovary / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012680:Sensitivity and Specificity; D013154:Spleen / Q000382:microbiology
D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S