Southern Bluefin Tuna and their blood flukes

Aporocotylid blood flukes Cardicola forsteri and C. orientalis are considered one of the most significant health concerns for Southern Bluefin Tuna (SBT) Thunnus maccoyii ranched in Port Lincoln, South Australia. Cardicola spp. infect the circulatory system of SBT, with adult C. forsteri typically found in the heart and adult C. orientalis typically found in the gills. Cardicola spp. eggs are released into the bloodstream and high severity of infection can cause blockages and lesions in the gills which can lead to mortalities. The anthelmintic praziquantel (PZQ) has been used by the SBT industry since 2012 and has reduced mortalities significantly. However, not all ranched SBT are PZQ treated, and reinfection after treatment can still occur. The effects of different treatment strategies implemented by commercial companies on blood fluke infections in ranched SBT is unknown. Blood flukes are currently diagnosed by microscopy and molecular detection using quantitative polymerase chain reaction (qPCR). qPCR is highly sensitive, highly specific, and allows for non-lethal diagnosis. However, its use requires specialised laboratory equipment and trained technicians, making it unsuitable for on-site diagnosis. There is an industry need for a rapid, highly sensitive and highly specific diagnostic tool for on-site detection of Cardicola spp. in SBT. The aims of this research are: 1. To determine the effects of different treatment strategies on blood fluke infection in ranched SBT, 2. To monitor the annual variability of blood fluke infection in ranched SBT, and 3. To develop a field-based molecular diagnostic tool for detection of Cardicola spp. in SBT samples. To address aims 1 and 2, prevalence and intensity of C. forsteri and C. orientalis infection in ranched SBT was reported during 2018, 2019 and 2021 commercial operations. Severity of infection was diagnosed by several criteria including adult fluke counts from hearts, egg counts from gill filaments and qPCR for detection of C. forsteri and C. orientalis ITS-2 DNA in SBT hearts and gills. In 2018, SBT from treated and untreated pontoons operated by a single company were sampled over several time points (transfer, pre and post treatment, and harvest). Cardicola forsteri was the dominant species detected, C. orientalis rarely detected and C. opisthorchis not detected. Intensity of C. forsteri infection in SBT was significantly higher in the untreated pontoon than the two PZQ treated pontoons from week 8 of ranching, however infection levels seen in untreated pontoons did not appear to decrease the condition of SBT or increase mortalities. No differences were seen between the pontoon treated week 2 and the pontoon treated week 6, indicating that treatment later into ranching is an effective method for controlling C. forsteri. In 2019, the study was expanded by comparing two companies and multiple ranching years to explore the effect of treatment, company and year. Results indicate that PZQ treatment remains highly effective against Cardicola spp. infection. Prevalence and intensity of Cardicola spp. infection in SBT was significantly higher in untreated pontoons than treated from week 12 of ranching (7 weeks post treatment) for Company B in 2019. When comparing treated pontoons between years, prevalence and intensity of Cardicola spp. infection was lower in 2019 than 2018 for Company A. When comparing untreated pontoons between companies, prevalence and intensity of Cardicola spp. infection was lower for Company A than Company B in 2019. A higher intensity of C. orientalis DNA was seen in SBT from a pontoon with higher mortalities than other pontoons in this study, suggesting differences in pathogenesis between species. qPCR demonstrated higher sensitivity when comparing diagnostic methods for C. forsteri in heart (McNemar's test, p < .0001), and higher specificity when comparing diagnostic methods for Cardicola spp. in gills. Finally, SBT were sampled from treated pontoons operated by seven commercial companies at harvest in 2018, 2019 and 2021. Sampling over three seasons allowed for exploration of longer-term trends with regards to blood fluke infections in ranched SBT. Variables included year, company, ranching duration, SBT condition index, and PZQ dose. Results aligned with studies from year one and two indicating that PZQ remains effective at controlling Cardicola spp. infection in ranched SBT. Cardicola forsteri continued to be the dominant species detected. Cardicola orientalis was rarely detected in 2018 and 2019, and not detected in 2021. Company A had the highest prevalence and intensity of Cardicola spp. infection in 2018, and Company G had the highest in 2019. No consistent pattern was seen when comparing companies in 2021. Overall, intensity of infection did not increase post treatment as ranching duration increased at harvest. Intensity of C. forsteri DNA in SBT heart decreased as PZQ treatment dose increased (Spearman's r = -.3553, d.f. = 167, p < .0001), and SBT Condition Index decreased as intensity of Cardicola spp. DNA in SBT gills increased (Spearman's r = -.2426, d.f. = 138, p = .0041). To address aim 3, a field-based molecular diagnostic tool for detection of Cardicola spp. in Southern Bluefin Tuna samples was developed. Recombinase polymerase amplification (RPA) is an isothermal technique which operates at constant low temperature (25-42ºC), and when coupled with a lateral flow (LF) strip, makes an ideal diagnostic tool for rapid, specific and sensitive identification of pathogens in field applications. RPA-LF assays were designed and validated for detection of C. forsteri and C. orientalis. For each assay, no cross-species amplification was seen and detection as low as 30-50 genome copy equivalents was achieved. Initial work demonstrates similar specificity and sensitivity (Kappa test, k = 0.881) on SBT samples when compared to qPCR, and demonstrated use of equipment-free incubation using body heat (37 ºC ) in Port Lincoln. Data collected in this research were shared in real time with the SBT industry and informed treatment strategies, with reduced treatment and costs for some companies. The RPA-LF diagnostic tool developed in this work will allow industry to assess blood fluke infection quicker, and deliver more targeted treatments to infected fish without the need for lab-based diagnostics. This research contributes to our understanding of blood flukes and the effect of PZQ on them, helping to improve the health of ranched SBT.