Dataset Information

Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum.

ABSTRACT: F. psychrophilum 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 in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium 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 F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium ("Pan-Flavo") and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium 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 F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum 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 Flavobacterium spp. and F. psychrophilum.

SUBMITTER: Strepparava N

PROVIDER: S-EPMC3494677 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum.

Strepparava Nicole N Wahli Thomas T Segner Helmut H Polli Bruno B Petrini Orlando O

PloS one 20121109 11

F. psychrophilum 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 in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum i ...[more]

PMID: 23152887

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Project description:BackgroundWhole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round.ResultsWe show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated.ConclusionSince POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

Project description:BackgroundDetection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci.ResultsIn this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe.ConclusionBy employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.

Dataset Information

Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum.

Publications

Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum.

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