Perkinsus olseni and P. chesapeaki detected in a survey of perkinsosis of various clam species in Galicia (NW Spain) using PCR–DGGE as a screening tool

DGGE profiles of Perkinsus species in a 8% w/v polyacrilamide gel. Lanes 1–4: LSU–PCR products obtained from in vitro cultured cells of P. marinus, P. chesapeaki, P. mediterraneus and P. olseni, respectively. Lanes 5–12 and 14–18: LSU–PCR products amplified from Manila clam Ruditapes philippinarum samples, indicating infection with P. olseni. Lanes 19 and 20: LSU–PCR products amplified from Manila clam R. philippinarum samples, indicating infection with P. olseni and P. chesapeaki. [Display omitted] •A perkinsosis survey involving 4 clam species was performed along the Galician coast.•A method for the identification of Perkinsus spp. using PCR–DGGE has been developed.•P. olseni PCR+cases were found in R. decussatus, R. philippinarum and V. corrugata.•Infection of Ruditapes philippinarum with Perkinsus chesapeaki was detected. A survey on perkinsosis was performed involving 15 locations scattered along the Galician coast (NW Spain) and four clam species with high market value (Ruditapes decussatus, Ruditapes philippinarum, Venerupis corrugata and Polititapes rhomboides). The prevalence of Perkinsus parasites was estimated by PCR using genus-specific primers. The highest percentage of PCR-positive cases for perkinsosis corresponded to clams R. decussatus and V. corrugata, while lower values were detected in R. philippinarum and no case was found in P. rhomboides. The discrimination of Perkinsus species was performed by PCR–RFLP and by a new PCR–DGGE method developed in this study. Perkinsus olseni was identified in every clam species, except in P. rhomboides, using both PCR–DGGE and PCR–RFLP. Additionally, Perkinsus chesapeaki was only detected by PCR–DGGE infecting two Manila clams R. philippinarum from the same location, reporting the first case in Galicia. P. chesapeaki identification was further confirmed by in situ hybridisation assay and phylogenetic analysis of ITS region and LSU rDNA.