TaqMan, or both: highly sensitive, non-invasive detection of Cardicola blood fluke species in southern bluefin tuna (Thunnus maccoyii)

<p>Three species of blood fluke from the genus<i> Cardicola </i>are known to parasitize and cause disease in Bluefin Tunas – <i>C. forsteri</i>, <i>C. orientalis</i>, and <i>C. opisthorchis.</i> Although initially believed to be separated by geography and host specificity, recent identification of at least two <i>Cardicola</i> spp. concurrently present within all three Bluefin species has raised questions concerning pathogenicity, relative abundance, and distribution of these parasites within Bluefin populations.</p> <p>Here, we present sensitive and differential real-time qPCR nucleic acid detection of these <i>Cardicola</i> spp. by targeting the ITS2 region of the parasite rDNA for PCR amplification. A limit of sensitivity was achieved to be between 1-5 genome copy equivalents for each of the three <i>Cardicola</i> species tested without cross-species or host genomic amplification. Similar sensitivity was further achieved in the presence of up to 20 ng/μL non-target host gDNA using SYBR Green chemistry alone, or in the presence of up to 160 ng/μL host gDNA through the utilization of a TaqMan probe common-reporter detection system. These methods were subsequently used to positively identify both <i>C. forsteri</i> and <i>C. orientalis</i> DNA in preserved samples of serum, gill, and heart from ranched Southern Bluefin Tuna <i>Thunnus maccoyii</i>. Both methods were more sensitive for positively and differentially identifying the presence of <i>Cardicola</i> spp. than either histological or heart-flush microscopy techniques previously employed, and also possess the ability to be applied in non-lethal blood sampling of these highly-valued fish.</p> <p>This is the first report for rapid and differential molecular quantitative detection of <i>Cardicola</i>, and opens the potential for effective monitoring of infection in cultured bluefin populations. Further, it is anticipated that the use of SYBR Green for melt-curve analyses in conjunction with a common-reporter TaqMan assay will present a flexible, accurate, and cost-effective approach for differential detection of a variety of other pathogens in future.</p>