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.
Highlights
Bacteria belonging to the genus Flavobacterium are non-fermentative, catalase- and oxidase-positive, gram-negative bacteria that occur in abiotic and biotic compartments of many ecosystems
Stable and accurate results were obtained using the two F. psychrophilum probes; the addition of the Pan-Flavo probe was, crucial to obtain the optimal fluorescence at which these bacteria can be seen through the microscope
To test whether or not the improved staining results, with the combination of the three probes, is due to a potentially cumulative fluorescence caused by the simultaneous presence of two types of fluorochromes (CY3 and FAM), we prepared a helper oligonucleotide probe with the same sequence as the Pan-Flavo probe but without a fluorescent label: this led to the same results as with the two fluorochromes
Summary
Bacteria belonging to the genus Flavobacterium are non-fermentative, catalase- and oxidase-positive, gram-negative bacteria that occur in abiotic and biotic compartments of many ecosystems (e.g. soil, fresh and marine water, fish). The Rainbow Trout Fry Syndrome (RTFS) is a severe systemic infection that occurs in general when bacteria accumulate in the liver or spleen of salmonids. It causes high mortalities in cultured fish stocks, primarily when the infection occurs in small rainbow trout [7]. It is not yet clear, whether RTFS is the result of a systemic infection or an advanced form of a superficial infection
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