Fluorescent <em>In Situ</em> Hybridization: A New Tool for the Direct Identification and Detection of <em>F. psychrophilum</em>

<div><p><em>F. psychrophilum</em> is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent <em>in situ</em> hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect <em>F. psychrophilum</em> in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of <em>Flavobacterium</em> species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of <em>F. psychrophilum</em> infections in fish. We constructed a generic probe for the genus <em>Flavobacterium</em> (“Pan-Flavo”) and two specific probes targeting <em>F. psychrophilum</em> based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different <em>Flavobacterium</em> and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the <em>F. psychrophilum</em> probe, respectively). FISH was able to detect <em>F. psychrophilum</em> in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of <em>Flavobacterium</em> spp. and <em>F. psychrophilum</em>.</p> </div

FISH assays of pure cultures.

<p>DAPI staining (A, B, C); Pan-Flavo probe (D, E, F); <i>F. psychrophilum</i> probes (G, H, I) (100x). <i>F. psychrophilum</i> (DSM3660) (A, D, G); <i>Flavobacterium</i> spp. (B, E, H); <i>Chryseobacterium</i> spp. (C, F, I).</p

Agreement between FISH and 16S rDNA sequencing (SEQ, used as gold standard) in the experiments carried out with the Pan-Flavo (Flavo285) probe and the combination of two <i>F. psychrophilum</i> (FlavoP77, FlavoP477) probes.

<p>SE: sensitivity; SP: specificity; PPV: positive predictive value, NPV: negative predictive value.</p

ROC curves for cell suspension of pure strains; area under the curve (AUC) for FISH: 0.89, for culture method: 0.79.

<p>(A). ROC curves for spiked spleens; AUC for FISH: 0.84, for culture method: 0.6 (B).</p

FISH assay on infected fish tissues.

<p>Pan-Flavo probe (A, B); <i>F. psychrophilum</i> probes (C, D). <i>F. psychrophilum</i> on skin (A, C) and <i>F. psychrophilum</i> in a spleen (B, D).</p

Probes used, target microorganisms and DNA target regions. [Cyanine dye (CY3); Carboxyfluorescein (FAM)].

*<p><i>E.coli</i>, GenBank sequence HM371196.</p