DNA detection of Gyrodactylus spp. in skin mucus of Nile tilapia Oreochromis niloticus.

Monogeneans Gyrodactylus von Nordmann 1832, cause outbreaks of gyrodactylosis in aquaculture settings worldwide. Detection of Gyrodactylus spp. is based on the morphological identification of isolated parasites after fish necropsy. Contributing to the diagnosis of gyrodactylosis, in this study, a non-destructive PCR assay was standardized; the PCR was first performed using genomic DNA of Gyrodactylus spp. isolated from the surface of the Nile tilapia Oreochromis niloticus (Linnaeus 1758), and subsequently tested with mucus samples of infected and uninfected Nile tilapia fish. The primers (Ekgyro1) were designed from the ribosomal Internal Transcriber Spacer (ITS) RNA region (ITS1, 5.8S and ITS2 rRNA gene) of Gyrodactylus cichlidarum Paperna 1968. The positive control group included the DNA of 30 monogeneans Gyrodactylus spp. The heterologous control group included 75 monogeneans Cichlidogyrus Paperna 1960, 75 protozoans Ichthyophthirius multifiliis Fouquet 1876 and 75 Trichodina Ehrenberg 1830. PCR products of each parasite and from the external mucus samples (described as P and M respectively), were sequenced. The average DNA concentration of the ectoparasites was of 13.5 ng/μl. The PCR test had an analytical sensitivity of 0.0039 ng μl-1 of DNA of Gyrodactylus spp. No cross-reactions were observed with the heterologous group. The sensitivity and specificity of the PCR test were of 100% either with genomic DNA or with DNA from mucus samples. Six DNA consensus sequences with sizes ranging from 568 bp to 571 bp were obtained and the BLAST analysis matched with DNA sequences of G. cichlidarum.