In vivo induced genes of Tetracapsuloides bryosalmonae

Content of research project: Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease (PKD) in various species of salmonids in Europe and North America. The economic impacts of the disease make it an important factor for aquaculture. PKD is suspected to contribute to the decline of wild brown trout and salmon populations in Europe. T. bryosalmonae completes the life cycle in two hosts, an invertebrate host: bryozoan and vertebrate host: brown trout fish. The proliferation of parasite induces a granulomatous cellular response in the interstitial tissues, which leads to swelling of the kidney and spleen. The virulence mechanisms of T. bryosalmonae are not fully understood and nothing is known about the virulence genes of T. bryosalmonae, which are induced, expressed and involved in the pathogenicity within the fish host during the course of infection. Hypothesis: Our working hypothesis is that virulence genes of T. bryosalmonae are induced and expressed in vivo during parasite development in fish. These genes may be contributed to virulence or pathogenicity in fish during infection process. We will identify virulence genes of T. bryosalmonae that are in vivo expressed and cause pathogenicity in the brown trout during the course of infection. Methods: In this project, brown trout will be infected with T. bryosalmonae spores. Serum and organs from fish will be collected at different time points. cDNA library of T. bryosalmonae will be constructed and immunoscreening of cDNA library will be performed using adsorbed fish sera. Positive clones will be identified and validated by quantitative real-time PCR in the infected organs. Additionally, RNA sequencing of T. bryosalmonae will be generated for sequence homology analysis. New and unique aspect of the project: The project will represent the first investigation of virulence genes and antigens of T. bryosalmonae expressed in vivo in the course of parasite development. The results will provide insights into the mechanism of proliferative kidney disease in salmonids. The novel genes and antigens will be appropriate for consideration for therapeutic and vaccine applications. Additionally, transcriptome profiles of T. bryosalmonae will provide fundamental biological information and help us understand the disease mechanisms of this important fish parasite.

Proliferative kidney disease (PKD) is a malacosporean parasitic disease that significantly affects both farmed and wild salmonids fish in Europe and North America, causing economic losses and endangering wild fish populations. PKD is caused by the endoparasite Tetracapsuloides bryosalmonae, the life cycle of which is alternated between invertebrate freshwater bryozoan and vertebrate salmonid fish hosts. The parasite develops mostly in the kidney but has also been found in other tissues. Proliferation of the parasite in the kidney causes swelling by hyperplasia of the interstitial tissue and a granulomatous cellular reaction. During infection, virulence genes that are important for disease and pathogenicity are induced and expressed within the host. The aim of the proposed project was to identify T. bryosalmonae genes that are induced and expressed in vivo and involved in the pathogenicity in brown trout Salmo trutta during course of infection. This was achieved by using in vivo induced antigen technology, an immunoscreening technique that identifies parasite antigens expressed in vivo during infection in fish. The sera from brown trout exposed to T. bryosalmonae were collected and anti-T. bryosalmonae antibody was confirmed in each serum sample using indirect ELISA. The pooled sera were adsorbed against three forms of parasite sacs and E. coli XL1-Blue antigens to removing antibodies in the sera that react to antigens constitutively expressed by T. bryosalmonae in the bryozoan body cavity. The resulting adsorbed sera was used to screen a T. bryosalmonae cDNA phage expression library. Positive clones were screened and sequenced and their sequences analysed for homology to known genes. Quantitative real-time PCR was performed to assess the transcriptional levels of these genes in infected kidney and infected bryozoan. Additionally, RNA sequencing of T. bryosalmonae-infected bryozoan Fredericella sultana, posterior kidney of brown trout and parasite sacs was performed to generate transcriptome data. Results of this project demonstrate that 136 in vivo induced genes of T. bryosalmonae such as calmodulin, ras-related protein Rab-35, and vesicle-associated membrane protein 3 are induced and expressed in brown trout during parasite development. These genes are involved in several functions critical for the parasite such as signal transduction, transport, metal ion binding, and metabolic process. The identified in vivo induced genes might be used for the development of vaccines or might be utilized as drug targets for treating PKD in salmonids. The findings of RNA-seq analysis of infected F. sultana and infected kidney of brown trout offer new insights into the immune response of both invertebrate and vertebrate hosts against T. bryosalmonae. De novo transcriptome assemblies of parasite sacs and zooids of F. sultana will be a valuable resource for gene discovery and functional transcriptomic applications.