Epitheliocystis: Development of PCR assay for the monitoring among the commercially important aquaculture species of Ukraine

Abstract

Epitheliocystis is an emerging disease of wild and cultured fish caused by a number of bacterial species, characterized by the presence of cytoplasmic bacterial inclusions in the epithelial cells of the gills, which contribute to the merging of the gill plates, and in some cases also spread to the skin of fish. This disease may manifest as hypertrophy and inflammation of the gills, white nodular lesions of epithelial tissue in the gills or skin, gasping on the surface of the water, lethargy, poor swimming and stunted growth. Among the commercially important aquaculture species of Ukraine, such as Atlantic salmon (Salmo salar), brown trout (S. trutta), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio) and gibel carp (Carassius auratus), Candidatus Clavochlamydia salmonicola and Candidatus Piscichlamydia salmonis are associated with epitheliocystis. There are currently no tools at the disposal of ichthyologists and veterinary laboratories in Ukraine to identify Ca. C. salmonicola and Ca. Piscichlamydia salmonis. Our basic concern was to develop a PCR assay of epitheliocystis diagnosis. We suggest the use of general primers for simultaneous detection of Ca. C. salmonicola and Ca. Piscichlamydia salmonis. The developed PCR assay for identification of Ca. C. salmonicola and Ca. Piscichlamydia salmonis has shown its suitability for amplifying control DNA. Confirmation of the amplification products identity was performed using selective recognition of the sequence by the TasI restriction endonuclease (Thermo Fisher Scientific, US). Analytical specificity verification of the PCR assay performed by amplifying the control DNA of 10 species of the Chlamydiales order showed the absence of PCR products, but observed in one. The designed PCR assay, after approbation on clinical material, can be used by researchers for extensive monitoring of epitheliocystis, doctors of veterinary medicine for diagnosis clarification, in addition to introduction into the practice of veterinary medicine laboratories and implementation in fish farm improvement programmes. The amplicon size of 197 base pairs theoretically permits application of this oligonucleotide primers pair for real-time PCR.