DNA probes for detection of the fish microsporidians Microgemma caulleryi and Tetramicra brevifilum

DNA probes were developed for the detection and identification of 2 microsp oridian parasites of marine fishes, Microgemma caulleryi (infecting the liv er of the greater sand-eel, Hyperoplus lanceolatus) and Tetramicra brevifil um (infecting muscle, intestine and liver of the turbot, Scophthalmus maxim us, 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 microsporidi an spores was isolated and digested with the restriction enzyme Hind III. T he fragments obtained were ligated into the vector pBluescript SK(+) and cl oned in Escherichia coli. Appropriate inserts were identified and then ampl ified by PCR, using primers specific for regions adjacent to the Hind III r estriction site in the vector sequence (and thus avoiding the need to devel op primers specific for the inserts themselves). The copies were labelled w ith digoxigenin, for subsequent use as probes, during PCR itself. The speci ficity of candidate probes was tested in dot-blot hybridization assays, wit h the target DNA being (a) genomic DNA of the microsporidian from which the probe had been obtained, or of another species, (b) the corresponding geno mic DNA in the phagemid, or (c) DNA from the corresponding host tissue. The se 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 de signed 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 D IG by enzyme immunoassay (i.e. using a phosphatase-coupled anti-DIG antibod y), and could no doubt be improved if a radioactive labelling and detection system were used. The probes developed in this study will greatly facilita te detection and identification of M. caulleryi and T. brevifilum in fish t issues, and may prove useful for identifying possible intermediate hosts us ed by these species.