Novel insights into Tetracapsuloides bryosalmonae virulence genes during proliferative kidney disease in trout through RNA-seq technology

Tetracapsuloides bryosalmonae is a malacosporean endoparasite that causes proliferative kidney disease (PKD) in salmonid fishes. This parasite poses a significant threat to both farmed and wild salmonid population in Europe and North America. The life-cycle of this parasite is intricately woven between invertebrate bryozoans and vertebrate fish hosts. Among the fish hosts, brown trout is known to acquire chronic infections while rainbow trout emerges as a dead-end host. A holistic understanding of both the host and the pathogen is the key to effective management strategies including vaccine development. Brown trout, serving as a host where T. bryosalmonae can successfully complete its life cycle, was our first point of investigation. We conducted a study to unravel the kinetics of the parasite-specific host's antibody response. In a controlled laboratory setting, brown trout were exposed to the spores of T. bryosalmonae. Blood, kidney and spleen were sampled from exposed and unexposed control groups at 2, 4, 6, 8-, 10-, 12- and 17-weeks post exposure (wpe). Samples were processed for indirect ELISA, histopathology, and qRT-PCR. Clinical signs of the disease such as renal hyperplasia, splenomegaly, and pale liver were observed in infected fish from 6 to 12 wpe. Our study showed the presence of T. bryosalmonae-specific antibodies from 4 wpe, which persisted until 17 wpe. Furthermore, we observed distinct regulation in the expressions of complement component C4A, and many B cell associated genes (CD34, CD79A, BLNK, CD74, BCL7, and CD22) within the critical immune organs, namely the kidney and spleen. To our knowledge, this marks the first investigation into the time-course of anti-T. bryosalmonae antibody response in brown trout. These findings offer valuable insights into the mechanisms governing alterations in B cell development, inflammation, and antibody production throughout the progression of PKD in brown trout. In the subsequent study, we investigated which virulence factors of T. bryosalmonae are induced during infection or interactions with host cells. By employing in vivo induced antigen technology (IVIAT), we identified 136 in vivo induced antigens of T. bryosalmonae. These antigenic genes, diverse in function, provided us with an invaluable map of the molecular landscape of this parasite during infection. Rainbow trout, in contrast, acts as a dead-end host, where the development of parasite is seemingly halted. This stark contrast prompted a research question: Does T. bryosalmonae adapt to different hosts? To this end, we implemented fluorescent activated cell sorting (FACS) for the isolation of parasites from brown trout and rainbow trout following experimental infection and then subjected the sorted parasite cells to RNA sequencing. This forms the first report of T. bryosalmonae isolation from infected kidney of fish host. This pioneering approach unveiled 1120 differentially expressed parasite transcripts between the two hosts, indicating the parasite's differential adaptations within different host species. Our study forms the first comparative transcriptome analysis of T. bryosalmonae between carrier and dead-end host. We observed an intriguing contrast: parasites from brown trout exhibited upregulated transcripts linked to cytoskeleton organization, cell polarity, peptidyl-serine phosphorylation while those from rainbow trout exhibited shifts in translation, protein refolding ribonucleoprotein complex biogenesis and subunit organisation, non-membrane bounded organelle assembly, regulation of protein catabolic process. In this study, we have introduced a novel method involving the use of FACS-based isolation to separate T. bryosalmonae cells from the infected kidneys of fish. This approach not only advances research in this field but also enables the identification of parasite transcripts that exhibit differential expression between carrier and dead-end fish hosts for the first time. Our study has generated significant knowledge on anti-T. bryosalmonae antibody response, in vivo induced genes and differentially expressed transcripts of T. bryosalmonae between hosts with contrasting fate for the parasite. The information generated from our study opens up avenues for a better understanding of host-T. bryosalmonae interactions, development of diagnostic tools, potential candidates for development of therapeutics and vaccines against T. bryosalmonae in salmonids.