Global aquaculture production of turbot has rapidly increased worldwide in the last decade and it is expected to have even bigger growth in the next years due to new farms operating. The losses caused by pathogen infections have grown at the same time as the production of this species. Parasitological infections are among the main relevant pathologies associated with its culture and produce serious losses in aquaculture, reduce the growth rate in fish and may lead to unmarketable fish due to skeletal muscle abnormalities in cases with high intensity of infection. The microsporidian parasite Tetramicra brevifilum causes severe infections and generates major losses in farmed turbot. Infections are difficult to control due to spore longevity and its direct transmission. To facilitate the infection management, an effective tool for fast detection and identification of T. brevifilum is needed. This study provides a molecular methodology of fast Real-Time PCR for T. brevifilum detection to the aquaculture industry, useful for routine control of T. brevifilum at turbot farms. The method is characterized by its high specificity and sensitivity, and it can be applied to cultured turbot for parasite detection regardless of the life-cycle stage of the pathogen or the infection intensity.

This study investigated the spatial distribution of Tetramicra brevifilum spores in the musculature of infected turbot Scophthalmus maximus, with the aim of identifying the most appropriate body locations for diagnostic assays. A PCR protocol optimized for the detection of T. brevifilum spores in turbot muscle is also described. In fish showing low- and moderate-intensity infection, the spatial distribution of spores was best fitted by a negative binomial distribution, indicating a clumped spatial pattern; the negative binomial coefficient k was lower for fish with low-intensity infection, indicating a more markedly clumped pattern in these fish. In fish with high-intensity infection, the spatial distribution of spores was best fitted by the Poisson distribution, indicating a random pattern. In both low- and moderate-intensity infection, spores were present at highest density in the musculature adjoining the dorsal fins. Samples for PCR were therefore obtained from this location. PCR amplification was of the small subunit ribosomal DNA (SSUrDNA), using a pair of species-specific primers that amplify the 1250 bp product. The PCR protocol developed showed better sensitivity than microscopical techniques (detection rate by microscopy 25%, versus 42% by PCR), suggesting that it may be useful for routine screening for Tetramicra brevifilum infection in cultured turbot.

DNA probes were developed for the detection and identification of 2 microsporidian parasites of marine fishes, Microgemma caulleryi (infecting the liver of the greater sand-eel, Hyperoplus lanceolatus) and Tetramicra brevifilum (infecting muscle, intestine and liver of the turbot, Scophthalmus maximus, a commercially important species). The probe-development procedure used is fast and straightforward, and readily applicable to the development of probes for other microsporidian species. First, genomic DNA of microsporidian spores was isolated and digested with the restriction enzyme Hind III. The fragments obtained were ligated into the vector pBluescript SK(+) and cloned in Escherichia coli. Appropriate inserts were identified and then amplified by PCR, using primers specific for regions adjacent to the Hind III restriction site in the vector sequence (and thus avoiding the need to develop primers specific for the inserts themselves). The copies were labelled with digoxigenin, for subsequent use as probes, during PCR itself. The specificity of candidate probes was tested in dot–blot hybridization assays, with the target DNA being (a) genomic DNA of the microsporidian from which the probe had been obtained, or of another species, (b) the corresponding genomic DNA in the phagemid, or (c) DNA from the corresponding host tissue. These assays identified a ca 1180 bp probe for M. caulleryi, denominated C38, and a ca 1363 bp probe for T. brevifilum, denominated F9. Similar assays designed to assess sensitivity indicated that F9 showed detectable binding to as little as 500 ng of T. brevifilum genomic DNA, and C38 to as little as 125 ng of M. caulleryi DNA; these results were obtained with detection of DIG by enzyme immunoassay (i.e. using a phosphatase-coupled anti-DIG antibody), and could no doubt be improved if a radioactive labelling and detection system were used. The probes developed in this study will greatly facilitate detection and identification of M. caulleryi and T. brevifilum in fish tissues, and may prove useful for identifying possible intermediate hosts used by these species.