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

机译:使用对重组新百日咳巴氏杆菌附着蛋白的黏膜反应设计抗阿米巴g病(AGD)的实验疫苗

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摘要

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.udIn 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:ud Investigate the mucosal and systemic immune response of Atlantic salmon against N. perurans, the causative agent of AGD.ud 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.ud Investigate vaccine formulations for AGD, using the recombinant protein described above.ud Investigate other mucosal components potentially involved in the host response against N. perurans.udThis 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.udFollowing 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.udWe 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.udFollowing 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.udThis 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.
机译:阿米巴bic病(AGD)是影响塔斯马尼亚鲑鱼产业的主要疾病,其他主要鲑鱼和鳟鱼生产国也描述了这种状况。 AGD是由Pereoraneoparamoeba引起的,该病的暴发在海洋成长期期间出现,特别是在水温升高时。通过基因表达研究以及通过其他针对抗紫苏猪链球菌,特别是IgM的抗体应答的研究,已经实现了针对该寄生虫的宿主免疫应答的某些表征。已经测试了多种处理方法,但是目前在塔斯马尼亚州广泛使用的唯一处理方法是淡水沐浴,这对行业构成了巨大的经济负担。因此,疫苗的开发仍然是鲑鱼生产者的高度优先事项,并且先前已经针对AGD对不同类型的疫苗进行了测试,但没有成功。 ud为了开发潜在成功的疫苗策略,请更好地理解与疫苗相关的抗体免疫反应疾病是必要的。为了实现这一总体目标,本论文研究了以下目的:ud研究大西洋鲑鱼对AGD的致病因子Perurans的粘膜和全身免疫反应。ud研究粘膜和全身性抗N抗体。 perurans抗体对变形虫假定的变形虫附着蛋白的反应,首先通过从该寄生虫生成cDNA文库来鉴定。ud使用上述重组蛋白研究AGD的疫苗制剂。ud潜在地调查其他粘膜成分 ud本论文介绍了旨在解决上述目标的几种不同实验的结果。首先,在反复感染猪痢疾奈瑟氏球菌后,在转录和抗体产生水平评估大西洋鲑的免疫反应。其次,进行了一次实验,评估了一次感染后的免疫反应,并给鱼类喂食了含有免疫刺激剂的商业开发日粮。我们表明抗体水平并不总是与在AGD ill损伤中鉴定的mRNA转录水平相关,这可能是由于细胞和组织中蛋白质和mRNA丰度之间存在弱相关性所致。此外,我们证明了使用含饮食的免疫刺激剂不会影响血清或皮肤粘液中IgM的水平,并且不能在AGD感染部位诱导IgM和IgT转录。大西洋鲑的全身和粘膜免疫反应使用两种蛋白-半抗原进行了研究。这项研究旨在评估在随后的实验中测试候选疫苗的最佳抗原递送方法。结果显示注射在FCA中乳化的免疫原是诱导全身和粘膜抗体应答的最佳递送方法。 ud我们描述了名为r22C03的重组蛋白的生产,该重组蛋白被鉴定为类似于甘露糖结合蛋白(MBP)的附着因子。其他变形虫和推定的Perurans附着因子。该蛋白能够诱导针对变形虫的全身和粘膜抗体应答,并且所产生的全身和粘膜抗体都能够结合福尔马林固定的Per。N. perurans表面。然后,按照以下原理测试重组蛋白作为抗AGD的候选疫苗:采用在粘膜表面存在的功能性抗体,可以阻断假定的猪坏死性奈瑟氏菌附着因子,并有可能降低AGD的严重性。使用两种不同的疫苗接种策略,用r22CO3对鱼进行免疫,然后用寄生虫攻击。在接种鲑鱼的血清和粘膜表面观察到了针对重组蛋白的强抗体反应,但在survival中未观察到存活曲线或病变大小的差异。然而,在实验过程中存在并发ruckeria ruckeri的感染,即使同时呈现两种病原体可能代表着与在商业农场上观察到的感染模式更紧密相关的情况,但仍需对寄生虫攻击后获得的存活结果进行检查。在针对百日咳猪笼草的疫苗效力方面要谨慎。 ud由于挑战未果,对受AGD感染的鱼类的皮肤和g黏液进行了nanoLC-MS / MS和蛋白质组学分析,作为鉴定反复感染变形虫后黏液蛋白质组变化的工具。已鉴定出先前与受AGD感染的g中的基因表达相关的蛋白质,以及先前未受AGD感染的鱼类中描述的蛋白质,并且建议未来的研究应集中于更好地理解这些成分在鱼中的作用。抵抗百日咳猪笼草的感染的反应。 ud本论文进一步了解了粘膜对AGD的反应。然而,直到完成对受AGD感染的鱼类的IgT应答的研究之前,尚无法完全理解粘膜抗体在针对AGD的应答中所起的作用,因为缺乏可用试剂阻碍了它的研究。最后,在AGD疫苗制剂中需要对经过专门设计以引起粘膜反应的佐剂进行全面测试。

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    Valdenegro Vega VAC;

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  • 年度 2014
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