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

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

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

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

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

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

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

Median-joining network based on the complete <i>G</i> gene sequence from 294 IHNV isolates (transition/transversion bias = 4).

<p>The country is indicated using a color code. The black triangles with the number 1 and 2 represent polytomy nodes (node 1 and 2). In accordance with a largely phylotemporal structure in the network, the black triangle with the number 1 (node 1) is surrounded by the oldest French isolate (X89213 from 1987, marked by an asterisk), Italian isolate (FJ711518 from 1987, marked by an asterisk), and the second eldest German isolate (LN897500 from 1993, marked by an asterisk). The <i>E</i> haplotypes are divided into two clades (the <i>E1</i> and <i>E2</i> clade). Isolate <i>M</i> is the outgroup in the network.</p

Consensus Maximum Likelihood tree and Median-joining network (transition/transversion bias = 4) of the <i>E</i> genogroup.

<p>The upper and the lower phylogeny show the phylogenetic relationship between <i>E</i> haplotypes of IHNV, based on the complete <i>G</i> gene sequence and were generated using 294 <i>E</i> isolates. The upper phylogeny is illustrated as a Consensus Maximum Likelihood tree (conflicting branching patterns are resolved by selecting the pattern seen in more than 50% of the trees). Numbers to the right of the branches represent the bootstrap support values obtained from 250 replicates. The lower phylogeny is illustrated as a Median-joining network. The subordinated lineages of clade <i>E</i>–<i>1</i> (<i>E–1–a</i> to <i>E–1–a–k</i>) and <i>E</i>–<i>2</i> (<i>E–2–a</i> to <i>E–1–c</i>) are indicated using a color code. Black triangles marked “1”or “2” represent nodes (node 1 and node 2) that correspond in the Median-joining network with a polytomy. Isolate <i>M</i> is the outgroup in each diagram.</p

Additional file 1: of An epidemiological model for proliferative kidney disease in salmonid populations

Linear stability analysis of the within-season system. (PDF 1644 kb

Mismatch distributions and Bayesian skyline plot of 5<i>i</i> sequences.

<p>The mismatch distribution is based on the complete <i>G</i> gene sequence and is calculated separately for the following: 152 <i>5i</i> sequences from Denmark, Germany, and the United Kingdom, collected between 2000 and 2011; and 137 Danish <i>5i</i> sequences from 2000 to 2009. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164475#pone.0164475.t001" target="_blank">Table 1</a> lists the respective values of the raggedness index (r), nucleotide diversity (PI), Tajima’s D, and Fu’s Fs of each dataset. The Bayesian skyline plot shows changes of the 5<i>i</i> population size between 2000 and 2009. The plot was generated using 137 Danish 5<i>i</i> sequences (complete <i>G</i> gene sequence). X axis: time in years, Y axis: population size. The middle solid line is the median estimate, and the area between the blue lines shows the 95% highest probability density (HPD).</p