Host response to amoebic gill disease in Atlantic salmon and blood fluke infection in Pacific bluefin tuna

Amoebic gill disease (AGD), caused by Neoparamoeba perurans, and Cardicola spp. infection represent a great concern for the sea-cage culture of Atlantic salmon in Australia, and Pacific bluefin tuna (PBT) in Japan. These fish parasitoses have been linked to mortality of the affected individuals if treatment is not provided. To date the only treatments used by industries to mitigate the outbreaks of these parasitic diseases and avoid mortality are the freshwater bath and the hydrogen peroxide bath (Northern hemisphere) of the AGD-affected Atlantic salmon stocks and the administration of the antihelminthic drug praziquantel (PZQ) to bluefin tuna infected by Cardicola spp. Several studies have been carried out on AGD and Cardicola spp. infection, nevertheless little is known about the host immune response to these infectious diseases. To deepen our knowledge of the fish immune response to AGD and Cardicola spp. infection is of utmost importance to contribute to the development of immune-based health strategies or other practical solutions to improve the fish health in mariculture. In this thesis, to provide a better understanding of the host inflammatory and immune response to N. perurans and Cardicola spp. , several genes were investigated at the transcriptional level by using quantitative real-time PCR. In particular, research focused on the fish gills as this organ has been linked to host mortality due to lack of oxygen in both parasitic diseases. Other techniques used to assess and describe the severity of pathology in the fish gills were routine histology for both AGD and Cardicola spp. infected individuals and gross gill score and image analysis for AGD-affected fish. Furthermore, a quantitative molecular technique was used to determine the species-specific relative prevalence of Cardicola spp. DNA in the fish organs and describe the natural infection timeline. In Chapter 2 the characterisation of pro-inflammatory (IL-1β and TNF-α) and immune (TCR-α chain, CD8, CD4, MHC-IIα, MHC-I, IgM and IgT) related genes at the mRNA level in the gills of AGD-affected Atlantic salmon was performed at 10 days post-exposure to N. perurans. A significant up-regulation of the mRNA expression was detected in the AGD-affected gills, suggesting that the parasite elicits a classical inflammatory response in the host and most importantly that the immune gene expression within AGD-lesions misrepresents the cellular immune response in the gills during AGD. Chapter 3 focused on investigating the Atlantic salmon immune response to N. perurans following reinfection. The targeted pro-inflammatory and immune-related genes were IL-1β, TCR-α chain, CD8, CD4, MHC-IIα, MHC-I, IgM and IgT. Gross gill score was used to assess the AGD-severity during the trial and determine the fish categories for gene expression. Histopathology and image analysis were used to further assess the AGD-severity. Overall the expression at the mRNA level of the immune-related genes analysed showed little change in AGD-affected gills of experimentally reinfected fish. In Chapter 4 the inflammatory and immune response in the gills of cultured juvenile PBT during early infection with Cardicola spp. at the transcriptional level was described by using a quantitative real-time PCR. Furthermore, C. orientalis and C. opisthorchis DNA within host gills and heart was quantitatively detected using qPCR. An increase of immune-related genes, namely IgM, MHC-I, TCR-β and IL-1β was observed in the PBT gills infected by Cardicola spp. Most notably, IgM and MHC-I transcription was strongly up-regulated in gill samples infected by C. orientalis relative to uninfected fish but not by C. opisthorchis. Cardicola-specific DNA was first detected in the host 14 days post transfer (DPT) to sea-cages and 55 days earlier than the first observation of parasite life stages with microscopy and histology. PZQ treatment did not have an immediate effect on parasite DNA presence in the host. A reduction in Cardicola spp. DNA was detected 24 days post PZQ in the host heart. Altogether these findings indicate the involvement of an immune reaction in the host gills during early Cardicola spp. infection although the response was not effective at clearing or mitigating the primary infection.