Prophylactic measures in rainbow trout aquaculture: Further development of a DNA vaccine for proliferative kidney disease.

我们提出的工作将研究一种重要的疾病，影响虹鳟鱼养殖业在英国和中欧，称为增殖性肾脏疾病（PKD）。这种疾病是由一种与水母有远亲关系的微小寄生虫引起的，这种寄生虫在鱼类中引起严重的免疫反应，其特征是慢性肾脏病理。最近的研究发现，被称为苔藓虫的殖民地淡水无脊椎动物可以窝藏对鱼类感染的寄生虫阶段，水温升高与苔藓虫中寄生虫增殖增加之间存在明显联系。这导致传染性寄生虫孢子的释放增加，表明随着水温趋势因气候变化而上升，这种疾病在养殖和野生鲑鱼种群中可能变得更加严重。这一点已经在瑞士的野生褐鳟种群和挪威的野生大西洋鲑鱼种群以及近年来在英格兰南部鳟鱼养殖场日益流行的PKD中清楚地看到。目前还没有控制这种疾病的治疗方法或手段。然而，重要的是，开发一种治疗方法的潜力很大，因为已知在寄生虫感染后存活的鱼在再次接触寄生虫时对该疾病具有抵抗力。因此，对鱼类进行疫苗接种有望成为控制该病的一种方法。疫苗接种将基于发现从与鱼类免疫系统直接接触的寄生虫分泌的分子或在寄生虫表面发现的分子。这些分子可以使免疫系统在接触寄生虫时迅速作出反应，预防或减少慢性感染和相关的鱼类死亡。在最近的研究中，我们利用分子生物学技术从苔藓虫中分离出的寄生虫材料建立了一个“基因库”。从这个文库中创建的基因库使我们能够检查数千种寄生虫分子的能力，以启动鱼类免疫系统，防止疾病病理或减缓晚期临床疾病的发作。根据疫苗技术的最新进展，通过DNA疫苗接种对鱼类进行了不同的基因库接种，这是一种比使用传统的基于蛋白质的方法更经济有效的方法来发现合适的候选疫苗。每年在遭受这种疾病的商业虹鳟鱼养殖场，鱼都要接种疫苗。结果根据疾病病理程度和肾组织中寄生虫的丰度进行评估。经过连续两轮的细分和对最具保护性基因库的重新测试，我们能够识别出一批182个寄生虫分子，这些分子可以减缓肾脏病理的发生。这种疫苗研究的商业利用高度依赖于单个寄生虫保护分子的鉴定和充分表征。因此，在本提案中，我们的目标是采取下一步措施，通过细分和重新测试该基因批次，以识别和表征传递疾病保护的寄生虫分子，从而开发PKD疫苗。大多数寄生虫分子都存在于被感染的宿主中，少数是宿主特异性的。能够保护鱼类免受疾病侵害的寄生虫分子必须在两个宿主体内都发现，或者是鱼类特有的。根据我们之前的研究，我们已经从受感染的鱼中创建了一个寄生虫基因库。因此，我们进一步建议使用高通量分子生物学技术来确定相对于苔藓虫宿主，哪些寄生虫分子主要存在或仅存在于鱼宿主中，并测试那些预计从受感染鱼肾脏内寄生虫表面分泌或发现的“鱼特异性”分子。总的来说，我们将采取系统的方法进一步开发PKD疫苗，利用我们过去在发现单一保护性寄生虫基因库方面的成功，我们在分子生物学，PKD生物学和鱼类疫苗接种方面的现有专业知识。

Our proposed work will study an important disease, affecting the rainbow trout aquaculture industry in the UK and central Europe, called proliferative kidney disease (PKD). The disease is caused by a microscopic parasite distantly related to jellyfish, which causes a severe immune response in fish characterized by chronic kidney pathology. Recent studies discovered colonial freshwater invertebrates, called bryozoans, can harbour the parasite stage infective to fish with a clear link between increasing water temperatures and increased parasite proliferation in bryozoans. This causes an increase in the release of infective parasite spores suggesting that the disease is likely to become more problematic in farmed and wild salmonid stocks as water temperature trends increase due to climate change. This has already been clearly seen in wild brown trout populations in Switzerland and wild Atlantic salmon stocks in Norway and in the increasing prevalence of PKD in recent years in trout farms in Southern England. There are no current treatments or means of controlling the disease. Importantly, however, there is a great deal of potential to develop a treatment since it is known that fish surviving parasite infection are resistant to the disease upon parasite re-exposure. Thus, the vaccination of fish is expected to be one way to control the disease. Vaccination will be based on finding molecules secreted from or found on the surface of the parasite in direct contact with the fish immune system. Such molecules can prime the immune system to react quickly when exposed to the parasite preventing or reducing chronic infection and associated fish mortalities. In recent studies we used parasite material isolated from bryozoans in molecular biology techniques to create a "gene library". Gene pools created from this library enabled us to examine the ability of thousands of parasite molecules to prime the fish immune system and protect against disease pathology or to slow the onset of advanced clinical disease. Different gene pools were administered to fish by DNA vaccination in line with recent advances in vaccine technology, representing a more cost effective approach to discover suitable vaccine candidates than using conventional protein-based methods. Fish were vaccinated at a commercial rainbow trout farm that suffers from this disease every year. Results were assessed in terms of the extent of disease pathology and parasite abundance in kidney tissue. After two sequential rounds of subdivision and retesting of the most protective gene pool, we were able to identify a single batch of 182 parasite molecules slowing the onset of kidney pathology. The commercial exploitability of such vaccine studies is highly dependent on the identification and full characterization of single parasite protective molecules. So in this proposal we aim to take the next steps towards PKD vaccine development by subdividing and retesting this gene batch in order to identify and characterize the parasite molecules conveying disease protection. The majority of parasite molecules will be found in both infected hosts, with a minority being host-specific. Parasite molecules capable of protecting fish against the disease must either be found in both hosts or are fish-specific. From our previous studies, we have created a parasite gene library from infected fish. We, therefore, further propose to use a high throughput molecular biology technique to determine which parasite molecules are predominantly or solely found in the fish host relative to the bryozoan host and to test those "fish-specific" molecules predicted to be secreted from or found on the surface of parasites within the infected fish kidney. Overall, we will take a systematic approach to further vaccine development for PKD, exploiting our past success in uncovering a single protective parasite gene pool, our existing expertise in molecular biology, PKD biology and fish vaccination.