Identification of parasitic diseases affecting ranched Southern Bluefin Tuna (SBT), using conventional and molecular methods

In Australia, the Southern Bluefin Tuna (SBT) industry is regarded as one of the most profitable sectors of aquaculture. Even though SBT are relatively unaffected by infectious diseases, few still have an impact in SBT production, leading to economic losses. Two of the main diseases affecting ranched SBT are infections by blood flukes (Cardicola forsteri and C. orientalis) and swimmer syndrome. Until now, lethal sampling has been used for the detection of C. forsteri and C. orientalis, and in combination with conventional diagnostic methods, they are used as the gold standard‚ÄövÑvp for the diagnosis of Cardicola and Scuticociliates. Therefore, in an attempt to improve sampling and diagnostic tools for health management of SBT, the focus of this thesis was to: ‚Äö Compare different lethal and non-lethal sampling methods used with conventional and molecular diagnoses for the detection of C. forsteri and C. orientalis ‚Äö Determine the presence and identify Scuticociliates in samples from SBT showing swimmer syndrome ‚Äö Determine the presence of C. forsteri and C. orientalis in biofouling samples near SBT pontoons ‚Äö Identify the potential relationship between the substrate, the age of the biofouling sample, type of biofouling organisms and depth, with the presence of C. forsteri, and C. orientalis DNA in the biofouling To provide a better understanding of the diseases affecting ranched Bluefin Tuna, a review is presented in Chapter 2. In Chapter 3, lethal sampling and non-lethal sampling techniques as well as, conventional and molecular methods used for the diagnosis of C. forsteri and C. orientalis are compared. Lethal and non-lethal samples from SBT were collected over the course of three years. Results showed differences between lethal sampling used along with conventional diagnosis and non-lethal sampling and molecular diagnosis. Only 37% of the heart flushes were positive and 66% of gill filaments had egg counts, identification of Cardicola species proved to be too challenging. For the same SBT, real-time qPCR along with species specific primers and probes were used for the detection of C. forsteri and C. orientalis. Not all PCR-based techniques allowed the detection of animals positive for adult blood flukes of eggs, such is the case for serum samples where only 3% were real-time qPCR-positive for C. forsteri and 1% to C. orientalis. Gill mucus samples showed similar results to heart flushes (38% of the samples positive for C. forsteri and 28% to C. orientalis), nevertheless, the use of gill mucus and realtime qPCR presents the advantage of allowing the identification of Cardicola species. Gill snips, gill biopsies and gill filaments samples, all presented high sensitivity when compared to conventional diagnostic techniques, where 100% of gill snips and gill biopsies were positive for C. forsteri, 86% of the gill filaments were also positive for C. forsteri and 4% of gill filaments were positive for C. orientalis. All molecular techniques allowed differentiation between species of Cardicola. In Chapter 4, 16 olfactory rosettes from wild SBT and 23 cerebrospinal fluid (CSF) samples from ranched SBT which exhibited swimmer syndrome, were collected. CSF samples positive for Scuticociliates were identified firstly using microscopy methods observing motile cells with a pyriform shape and granular appearance. All samples were tested using end point PCR combined with primers amplifying fragments of the small subunit rDNA (SSU rDNA) and the mitochondrial cytochrome c oxidase subunit 1 (cox1), all the amplifications obtained were sequenced and compared against published data. Olfactory rosettes were considered negative for Scuticociliates as neither of them showed amplification using the SSU rDNA and cox1 primers. Sequencing of the PCR products of the CSF samples, evidenced presence of M. avidus, being 100% identical to sequences of M. avidus previously reported. This means that those swimmer syndrome cases investigated here were associated with M. avidus. In an attempt to identify possible sources of infection of C. forsteri, C. orientalis and M. avidus, and as a result of the data obtained from Chapter 3 and 4, biofouling samples plates and mesh for biofouling were placed at different depths near the tuna pontoons. Samples were placed at one and four metres of depth and collected after one and three months. Organisms were separated according to their taxonomic group and identified by molecular techniques species-specific primers and probes for real-time qPCR. The presence of C. forsteri and C. orientalis was detected in 4 samples, each one of them. C. forsteri was only detected in samples collected at a depth of four metres, while 75% of the positive samples collected at 1 metre depth were positive for C. orientalis. M. avidus prevalence showed an increase from 38% during the first month to 89% of the samples during the third month with no significant differences between depths. The results of this thesis identify different non-lethal sampling techniques that could be used along with molecular analysis as a monitoring and diagnostic tool for the detection of Cardicola in ranched tuna. Further studies need to be performed to evaluate the use and limitation of gill mucus as a non-lethal sample. The findings from this thesis also provide evidence of the advantages PCR and real-time qPCR have over conventional diagnostic methods, allowing the identification for the first time of M. avidus in ranched SBT, and associating M. avidus to swimmer syndrome. Finally, this work contributes to the understanding of the probable reservoirs of infection of Cardicola and U. nigricans in ranched SBT.