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

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.

Proliferative kidney disease is one of the most devastating diseases affecting the UK salmonid aquaculture industry. Due to the impact of elevated water temperatures on parasite development in bryozoans, increasing water temperatures attributed to climate change processes are likely to exacerbate PKD outbreaks. There are no current ways to treat the disease, although vaccination remains a possibility since recovering fish are immune to re-infection. Given recent advances in fish DNA vaccinology, we previously used expression library (plasmid DNA) immunization, based on a bryozoan-derived parasite library, to aid the identification of protective antigens. After 3 rounds of ELI, we were able to identify a single protective gene pool (R14) comprising of 182 contiguous sequences. The need to identify parasite antigens conveying protection for commercial exploitation has provided the impetus for our current proposed studies. In the first instance, we will further fractionate R14 into groups of 5-6 antigens and test the protective efficacy of each group. We will attempt to further improve the efficacy of the most immune protective genes in this study by cloning each gene into a newly developed fish secretory vector and by assessing the protective efficacy of the corresponding recombinant proteins. Although our previous work was successful in identifying a protective gene pool, we are unable to account for disease protection that could be offered by fish-specific parasite antigens. Using a newly developed fish-derived parasite library, we are now able to identify fish-specific parasite antigens by comparing the transcriptomes of both parasite stages using a next generation sequencing strategy. Roche 454 GS FLX plus Titanium technology will be used to sequence and assemble host-specific parasite transcriptomes. Subsequent assembly of a consensus transcriptome will enable the identification of host-specific parasite antigens for downstream testing alongside R14 antigens.

We believe our research has the potential to provide tangible impact towards long-term economic and societal benefits by paving the way towards the future development of a vaccine to control a devastating disease affecting fish farming industries in the UK and throughout Europe. Hence, in the (highly probable) event individual protective parasite antigens are identified, the main thrust of research impact will be felt by the Aquaculture industry and, ultimately, the consumer. Aquaculture plays a key role in food security worldwide in meeting the ever-growing demand for fresh health-promoting food to sustain the growing human population. Fish disease represents the single largest threat to farmed fish production, highlighting the importance of developing an in-depth knowledge of fish immunology and the development of highly efficacious vaccines to protect fish from disease. Whilst many diseases have been brought under control by vaccination, no vaccines exist for PKD, considered the most serious disease issue facing the UK trout industry. PKD reduces the profitability of fish farming practices and their ability to compete in the market place. Inevitably, this leads to downstream impacts on job security with losses passed onto the consumer to allow farms to remain viable. Fish is a highly nutritional food that can improve health and quality of life. Our work has the potential to aid fish production by reducing fish losses and reduce the cost of fish to the consumer. In turn, increasing consumer awareness and understanding through the promotion of fish consumption will help to tackle health issues in UK society. It is, therefore, clearly in the best interests of both the Aquaculture industry and the consumer that effective means of controlling diseases like PKD are developed. Fish diseases which incur substantial fish losses are a major animal welfare issue. Parties involved in the rearing and consumption of fish (the industry, regulatory bodies/government agencies, and general public) have a responsibility to improve animal welfare wherever possible. The development of a commercially viable PKD vaccine would reduce fish losses and improve fish welfare.
Recently PKD has spread North and to higher altitudes in Europe, with substantial losses seen in brown trout populations in Switzerland, Atlantic salmon fisheries/smolts in Norway and outbreaks in Scottish farmed smolts. Given the temperature-driven nature of PKD, research has linked these outbreaks with increasing water temperature, which may be exacerbated further as a consequence of climate change processes. This has serious implications concerning the occurrence of PKD in wild salmonid stocks in the UK. The understanding of disease risk to wild fisheries requires a thorough understanding of the population dynamics and spread of the definitive host (bryozoans) and the PKD parasite. In the absence of such information, the vaccination of fish to be used for restocking could be a solution if similar increases in the prevalence of PKD occur in UK fisheries. Thus, PKD may become an important consideration for the Environment Agency, the Scottish Environment Protection Agency and the leisure (angling) industry. CEFAS, with remits over both farmed and wild salmonid stocks, have a long standing record in the study of PKD. The successful implementation of a PKD vaccine for farmed fish could potentially free-up monies set aside for monitoring PKD prevalence in farms, allowing more focus into the protection of wild stock.
Finally, effective vaccination against PKD could have a significant bearing on the protection of fish infected with closely related parasites. This is exemplified by Whirling disease in the USA and Canada, where research funded by foreign environment/ government agencies may benefit from the effective control of PKD.