Development of Polymerase Chain Reaction Assays for Detection of the Kinetoplastid Azumiobodo hoyamushi, the Causative Agent for Soft Tunic Syndrome in the Ascidian Halocynthia roretzi

摘要

PCR assays were developed for the rapid and sensitive detection of Azumiobodo hoyamushi, the protistan pathogen that causes soft tunic syndrome in the cultured ascidian Halocynthia roretzi. Two sets of A. hoyamushi-specihc primers based on the18S rRNAand beta-tubulin gene sequences, respectively, of the flagellate were designed and validated for the detection of A. hoyamushi. In both PCR assays, the flagellate genes were detected in diseased ascidians, but not in apparently healthy ones. When DNA were extracted from flagellate suspensions obtained from infected tunics, the detection limits of the PCR assays using primers for the18S rRNA and beta-tubulin were 3.4 x10 cells/mL and 3.4 x102 cells/mL, respectively. Even at the early stage of infectionwhen the slight clinical signs were observed only in the siphons, A. hoyamushi was detected from the affected tissue using18S rRNA PCR, but not beta-tubulin PCR. Thus, the18S rRNA PCR is a rapid, sensitive and specific assay for diagnosing soft tunic syndrome.