The Immune Response of Ranched Southern Bluefin Tuna Infected With Aporocotylid Blood Flukes

Aporocotylids, also known as fish blood flukes, are a significant health concern for the Australian Southern bluefin tuna (SBT) aquaculture industry. Cardicola forsteri and C. orientalis are the two species that parasitise SBT during ranching. Mortalities of SBT have been associated with severe Cardicola spp. infections. Cardicola spp. infect the circulatory system of SBT with the adult fluke infecting SBT hearts (C. forsteri) or gills (C. orientalis) and the eggs of both species infecting the gills. Most health concerns associated with these infections are related to Cardicola spp. eggs. The SBT industry manages infections with the anthelminthic praziquantel (PZQ). However, despite the economic importance of SBT, little is known about how the SBT immune system responds to Cardicola spp. as infection develops. This thesis focused on improving the understanding of the SBT immune response to Cardicola spp. throughout the duration of SBT ranching by addressing 3 aims. The first aim was to identify the relationship between SBT immune gene expression and the infection severity of adult or egg Cardicola spp. during standard commercial ranching. A targeted approach was used to quantify the expression of 6 immune genes (il1b, il8, igm, mhc2, tcrb and tnf2) at three ranching time points using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The expression of the immune genes was measured from the SBT anterior kidney to represent a systemic immune response, and from SBT gills to represent a localised immune response. Cardicola spp. infection severity was measured using three techniques. Adult flukes were collected and counted from SBT hearts, Cardicola spp. eggs were counted under a microscope from gill whole mounts and C. forsteri and C. orientalis specific rDNA (ITS-2) was quantified from SBT gills using qPCR. Gene expression was correlated to the diagnostic results and showed differences in correlations between the Cardicola spp. life stages and species diagnostics. Expression of igm, mhc2 and tcrb were correlated to the number of adult flukes; il8 and igm correlated to the number of eggs; and tcrb was negatively correlated to the quantity of C. orientalis rDNA (ITS-2) in SBT gills. Inflammatory cytokines (il1b and il8) expression decreased in the gills with ranching time and genes that represent an adaptive immune response (igm, mhc2 and tcrb) expression increased over time in the kidney. The second aim was to compare the gill and anterior kidney transcriptomes of C. forsteri infected SBT to uninfected SBT. The second aim expands on the first by using a total transcriptomic approach to understanding how all SBT genes are expressed during C. forsteri infection. SBT were collected during the commercial harvest from a single company. Cardicola spp. infection was diagnosed by the presence of adult flukes in SBT hearts and C. forsteri and C. orientalis rDNA (ITS-2) in SBT gills. The individuals that were positive for adult flukes and only C. forsteri rDNA (ITS-2) were considered as C. forsteri positive. RNA was extracted from the gill and anterior kidney of SBT, the mRNA was sequenced and aligned to the SBT genome. A count of the expressed genes was produced and normalised to 4 reference genes. Differentially expressed genes (DEGs) between C. forsteri positive and negative SBT for each organ were calculated with EdgeR and were considered significant when the p value adjusted for the false discovery rate was < 0.1. Immune system related transcripts in the gills of C. frosteri positive SBT showed an upregulation of the Notch signalling pathway. The immune system DEGs from the kidney showed C. forsteri positive SBT had a downregulation of the ubiquitin mediated proteolysis and 4 innate immune signalling pathways: NOD-like, C-type lectin, toll-like, and the RIG-I-like signalling receptor pathways. This was the first known study to document the transcriptome changes of SBT responding to a pathogen. The final aim was to investigate the relationship between infection severity and C. forsteri specific antibodies over an extended ranching period. The third aim expands on aim 1 and 2 by understanding the long-term immune response. To address this aim SBT were collected from a single commercial company that ranched SBT for 45 weeks, which is longer than industry standard of 12-26 weeks. SBT IgM was purified from serum and an anti-SBT IgM antibody was produced so C. forsteri specific antibodies could be measured through an enzyme linked immunosorbent assay (ELISA). Cardicola spp. infection severity was measured by the number of adult flukes in the heart and C. forsteri and C. orientalis rDNA (ITS-2) in SBT gills. At all time points studied, SBT had greater levels of C. forsteri specific antibodies than negative controls while Cardicola spp. infection intensity remained low after an infection peak. C. forsteri antibody levels were positively correlated to C. forsteri rDNA (ITS-2) in SBT gills at two time points and were negatively correlated to the number of adult flukes at the peak infection intensity. There is a time delay between infection and specific antibody production, identified as a 4-5 week delayed positive correlation between the initial C. forsteri infection and the C. forsteri specific antibodies in this study. This thesis contributes to understanding how the SBT immune system responds as Cardicola spp. infection develops within the ranching environment. The research has identified parallels to immune response dynamics seen in humans during schistosomiasis (disease caused by the human infecting blood fluke Schistosoma spp.). Immune response studies are important to understand how Cardicola spp. affects SBT which can help with improving health management for aquaculture industries troubled with aporocotylid infection.