Diagnostic PCR assays to detect and differentiate Kudoa septempunctata, K. thyrsites and K. lateolabracis (Myxozoa, Multivalvulida) in muscle tissue of olive flounder (Paralichthys olivaceus)

Abstract

Kudoa infections in muscle tissue can be a serious problem for fisheries and aquaculture of olive flounder (Paralichthys olivaceus) due to post-mortem myoliquefaction that makes the fish unmarketable. Recently, cases of a new food poisoning of human associated with ingestion of raw olive flounder infected with Kudoa septempunctata have increased in Japan. In the present study, diagnostic PCR assays were developed to detect and distinguish Kudoa spp. from olive flounder muscle. Kudoa 18S and 28S rDNA were amplified from infected fish by universal primers and sequenced. Blast searches with the obtained sequences revealed the presence of not only K. septempunctata and K. thyrsites, but also K. lateolabracis which was detected for the first time in olive founder. Comparison of the 28S rDNA sequences obtained from K. thyrsites isolated in the present study with a previous isolate from GenBank indicated the existence of two genetic lineages of this species, both infecting olive flounder. Alignments of partial 28S rDNA sequences were used to design primers for each of the three Kudoa spp. Specificity of each primer pair was tested by PCR with DNA from different myxozoans and host DNA. For sensitivity testing, the target sequence was ligated into a plasmid vector and cloned. PCR was conducted with host DNA spiked with dilution series of the plasmid. Additionally, a practical test of the primers was conducted in comparison to classical diagnostic methods with 10 olive flounders from a fish farm suspected to be infected with Kudoa spp. All three primer pairs were specific for the respective parasite and amplified neither host DNA nor DNA of other tested myxozoans. Primers for K. septempunctata approximately amplified down to 0.06, those for K. thyrsites 0.001 and for K. lateolabracis 0.1 spores per reaction (240, 4 and 400 spores per 1g of muscle tissue). The diagnostic PCR assay developed in this study was shown to be more effective for detection of K. septempunctata from cultured olive flounder (10 out of 10 fish positive) than microscopic detection of spores in tissue homogenates (5 out of 10 fish positive). K. thyrsites and K. lateolabracis were not detected in the 10 flounders tested. This novel PCR assay will facilitate the screening and monitoring process for Kudoa infections and allow improved disease management in olive flounder aquaculture facilities. It will also improve food safety testing to avoid human consumption of Kudoa infected fillets.