SYBR, TaqMan, or both: Highly sensitive, non-invasive detection of Cardicola blood fluke species in Southern Bluefin Tuna (Thunnus maccoyii)

tThree species of blood fluke from the genus Cardicola are known to parasitize and cause disease in BluefinTunas - C. forsteri, C. orientalis, and C. opisthorchis. Although initially believed to be separated by geogra-phy and host specificity, recent identification of at least two Cardicola spp. concurrently present withinall three Bluefin species has raised questions concerning pathogenicity, relative abundance, and distribu-tion of these parasites within Bluefin populations. Here, we present sensitive and differential real-timeqPCR nucleic acid detection of these Cardicola spp. by targeting the ITS2 region of the parasite rDNA forPCR amplification. A limit of sensitivity of 1-5 genome copy equivelents was achieved for each of thethree Cardicola species tested without cross-species or host genomic amplification. Similar sensitivitywas further achieved in the presence of up to 20 ng/uL non-target host gDNA using SYBR Green chem-istry alone, or in the presence of up to 160 ng/uL host gDNA through the utilization of a TaqMan probecommon-reporter detection system. These methods were subsequently used to positively identify both C.forsteri and C. orientalis DNA in preserved samples of serum, gill, and heart from ranched Southern BluefinTuna Thunnus maccoyii. Both methods were more sensitive for positively and differentially identifyingthe presence of Cardicola spp. than either histological or heart-flush microscopy techniques previouslyemployed, and also possess the ability to be applied in non-lethal blood sampling of these highly valuedfish. This is the first report for rapid and differential molecular quantitative detection of Cardicola, andopens the potential for effective monitoring of infection in cultured bluefin populations. Further, it isanticipated that the use of SYBR Green for melt-curve analyses in conjunction with a common-reporterTaqMan assay will present a flexible, accurate, and cost-effective approach for differential detection of avariety of other pathogens in future.