Whole-Polinski-thesis.pdf (7.91 MB)
Application of molecular methods for determining immune responses and disease status in bluefin tuna
thesisposted on 2023-05-27, 07:01 authored by Polinski, M
Bluefin tuna (Thunnus spp.) are a globally threatened fish that currently constitutes one of the most economically important food fisheries in the world. Culture efforts have intensified in recent years to maximise fishery profitability and mitigate market dependency on wild stock; yet little is currently known concerning health and immune functions and best culture practices continue to be investigated. In an attempt to aid future aquaculture interests and to provide general tools for identifying immune responses and disease status for this group of fishes, this thesis investigated a number of immune and disease-identifying aspects of bluefin tuna through the use of molecular laboratory techniques centering on quantitative PCR.In the course of this research, six full length and thirteen partial mRNA transcripts were newly identified for two bluefin species [Southern bluefin tuna Thunnus maccoyii (SBT) and Pacific bluefin tuna Thunnus orientalis (PBT)] which represent a suite of genetic markers involved in immune, stress, growth, and inflammatory functions. As temperature is known to influence inflammatory signalling in mammals, in vitro stimulations and subsequent gene expression analyses were conducted on cells of SBT to identify the effects temperature might play during an immune response in these uniquely heterothermic fish. Results from these experiments suggested that temperature exerts influence in the timing but not the degree of an innate inflammatory response in bluefin tuna and also that different cell populations have differential responsiveness to heat shock in this heterothermic species. Additionally, heat shock co-activation of the IL-8 chemokine previously identified in mammals is also likely present in bluefin and suggests that increased immune cell trafficking has long been incorporated into the stress responses of vertebrates. Infections by three species of blood fluke from the genus Cardicola ‚Äö- C. forsteri, C. orientalis, and C. opisthorchis ‚Äö- represent the greatest disease concern for the sea-cage culture of bluefin tuna. Using real-time qPCR and novel applications of SYBR green nucleic acid dye in combination with a TaqMan probe common reporter system, sensitive quantitative detection of parasite species-specific DNA was achieved and was effective for identifying parasite in samples of host blood, gill, and heart of both SBT and PBT species. This identified infections earlier and more sensitively than either histology or microscopy methods previously employed. These detection techniques were used in conjunction with gene expression analyses to demonstrate that organ-specific transcriptive immune responses occurred in PBT during natural infection which mirrored the relative quantity of pathogenic load. Although ineffective at combating infection during primary exposure, this research demonstrates a cellular immune response is mounted in PBT as a potential rejoinder to future Cardicola exposure and/or mitigation against tissue destruction caused by the parasite. Further, IgM transcription was highly correlated to the relative abundance of C. orientalis but not C. opisthorchis DNA in gill samples, suggesting that host IgM transcription may be targeted to species or life stage specific antigen.Lastly, Praziquantel (PZQ), a drug long used in veterinary and human medicine for the treatment of helminth parasites and currently applied to treat Cardicola infections in tuna, has been shown to enhance humoral and cellular immune response in mammals. Here, PZQ was shown to induce gene transcriptional changes in immune-competent primary tissue cultures of both SBT and Atlantic salmon Salmo salar. Although expression profiles varied between species and tissue type, PZQ was observed to significantly induce both T cell receptor and IL-8 transcriptional expression in all cultures, as well as creating a general pattern of heightened antiviral signalling that included elevated transcription of both Type I (IFN˜í¬±) and Type II (IFN˜í‚â•) interferon along with elevated expression of MHC class I. These findings identify a direct immunomodulatory ability of PZQ in immune cells of fish and provide support for further investigation into the immunostimulant/adjuvant capabilities of this drug in fish culture. This work also aids in understanding the mode of action for this drug in the treatment of Cardicola infection in bluefin tuna.
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