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Using the mucosal response to recombinant Neoparamoeba perurans attachment proteins to design an experimental vaccine against amoebic gill disease (AGD)


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Valdenegro Vega, VAC 2014 , 'Using the mucosal response to recombinant Neoparamoeba perurans attachment proteins to design an experimental vaccine against amoebic gill disease (AGD)', PhD thesis, University of Tasmania.

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Amoebic gill disease (AGD) is the main disease affecting the Tasmanian salmonid industry and the condition has also been described in other major salmon and trout producing countries. AGD is caused by Neoparamoeba perurans, and outbreaks of the disease appear during the marine grow-out phase, in particular when water temperature rises. Some characterisation of the host immune response against the parasite has been achieved through gene expression studies and through others investigations which focused on antibody responses against N. perurans, particularly IgM. A variety of treatments have been tested, but currently the only treatment option widely used in Tasmania is freshwater bathing, which represent a high economic burden for the industry. Therefore, the development of a vaccine remains a high priority for salmon producers and different types of vaccines have been previously tested against AGD without success.
In order to develop a potentially successful vaccine strategy, a better understanding of the antibody immune response associated with the disease is necessary. To address this general objective, the followings aims were studied in this thesis:
 Investigate the mucosal and systemic immune response of Atlantic salmon against N. perurans, the causative agent of AGD.
 Investigate mucosal and systemic anti-N. perurans antibody responses to a recombinant putative attachment protein of the amoeba, first identified by the generation of a cDNA library from the parasite.
 Investigate vaccine formulations for AGD, using the recombinant protein described above.
 Investigate other mucosal components potentially involved in the host response against N. perurans.
This thesis presents the results obtained from several different experiments aimed at addressing the above stated aims. Firstly, an experiment where the immune responses of Atlantic salmon were assessed at transcription and antibody production levels, after repeated infections with N. perurans. Secondly, an experiment where immune responses were assessed after a single infection and fish were fed commercially developed diets containing immunostimulants. We showed that antibody levels do not always correlate with mRNA transcription levels identified in AGD gill lesions, which is possibly explained by weak correlations existing between protein and mRNA abundances in cells and tissues. Additionally, we demonstrated that the use of immunostimulants containing diets did not affect the levels of serum or skin mucus IgM and were unable to induce IgM and IgT transcription at the site of AGD infection.
Following from this experiment; the systemic and mucosal immune responses of Atlantic salmon were studied using two protein-hapten antigens. This study aimed at evaluating the best delivery method of antigens to be used in the testing of a vaccine candidate in subsequent experiments. The results showed that i.p. injection of immunogens emulsified in FCA was the best delivery method for inducing systemic and mucosal antibody responses.
We described the production of a recombinant protein named r22C03, identified as a mannose-binding protein-like (MBP-like) similar to attachment factors of other amoebae, and a putative attachment factor of N. perurans. This protein was capable of inducing systemic and mucosal antibody responses against the amoebae and both systemic and mucosal antibodies produced were able to bind the surface of formalin-fixed N. perurans. The recombinant protein was then tested as a vaccine candidate against AGD, following the rationale that by using functional antibodies present in mucosal surfaces, the putative attachment factor of N. perurans might be blocked and the severity of AGD could potentially be reduced. Fish were immunised with r22C03 using two different vaccination strategies and then challenged with the parasite. A strong antibody response against the recombinant protein was observed in serum and mucosal surfaces of vaccinated salmon, but no differences in survival curves or size of lesion in the gills were observed. However, a concurrent infection with Yersinia ruckeri was present during the experiment, and even though the simultaneous presentation of both pathogens could represent a situation more closely related to infection patterns observed on commercial farms, survival results obtained after the parasite challenge had to be examined with caution in the context of vaccine efficacy against N. perurans.
Following from the unsuccessful challenge, nanoLC-MS/MS and proteomics analyses were used on skin and gill mucus of AGD-affected fish, as a tool to identify the changes in the proteome of mucus after repeated infection with amoebae. Proteins that have been previously related to gene expression in AGD-affected gills as well as proteins that have not been previously described in AGD-affected fish were identified and it was proposed that future research should focus on better understanding the role these components play in the response against infection with N. perurans.
This thesis provided further understanding into the mucosal responses to AGD. However, the role mucosal antibodies play in responses against AGD cannot be completely comprehended until the study of IgT responses in AGD-affected fish can be completed, as it has been hampered by the lack of available reagents. Finally, adjuvants that have been designed specifically to elicit mucosal responses need to be fully tested in AGD vaccine formulations.

Item Type: Thesis - PhD
Authors/Creators:Valdenegro Vega, VAC
Keywords: amoebic gill disease, mucosal immunity, atlantic salmon
Copyright Holders: The Author
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Copyright 2014 the Author

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