Vaccination with recombinant protein (r22C03), a putative attachment factor of Neoparamoeba perurans, against AGD in Atlantic salmon (Salmo salar) and implications of a co-infection with Yersinia ruckeri.

Amoebic gill disease (AGD) affects salmonids during the marine grow-out phase in the Tasmanian industry and in other major salmonid producing countries. During the period post-transfer to seawater, the bacterial condition yersiniosis can also cause high levels of mortality in Atlantic salmon grown in Tasmania, in addition to the hatchery outbreaks. The recombinant protein r22C03, a mannose-binding protein-like (MBP-like) similar to attachment factors of other amoebae, was tested as a vaccine candidate against AGD in a large scale challenge trial. Fish were immunised with r22C03 combined with FCA via intraperitoneal (i.p.) injection, and given a booster five weeks later by either i.p. injection (RP group) or by a dip-immersion (mRP). Fish were then challenged twice with Neoparamoeba perurans: the initial challenge 16 weeks after primary immunisation was terminated due to presence of ulcerative lesions in the skin of salmon; the second challenge was carried out after five weeks of treatment with oxytetracycline. These skin lesions might have been associated with a concurrent infection with Yersinia ruckeri, which was detected by real-time qPCR in serum of a large proportion of moribund and survivor fish after the AGD challenge. Before and during the N. perurans infection, levels of antibodies against r22C03 were measured by ELISA in serum, skin mucus and supernatant from skin and gill explants. For the second challenge, the average size of AGD lesions was recorded from histology sections and survival curves were obtained. Before AGD challenge, r22C03 induced antibody responses in serum and explants with both vaccination strategies. At the end of the challenge, levels of antibodies were lower than before challenge irrespective of treatment. Both vaccinated groups presented increased serum antibody responses, while only mRP presented antibody responses in skin mucus, and no significant antibody responses were measured in the explants. Antibodies did not confer protection to N. perurans infection, as no difference was observed in the survival curves of the vaccinated and control groups, and there was no effect on the gill lesion size. The concurrent yersiniosis infection probably represented more closely infection patterns observed in commercial settings. However, it could have interfered with the survival results and with the ability of the fish to respond to the amoebae infection.

Effects of single and repeated infections with Neoparamoeba perurans on antibody levels and immune gene expression in Atlantic salmon (Salmo salar).

Amoebic gill disease (AGD) is the main health problem for the salmon industry in Tasmania, Australia and is now reported in most salmon producing countries. Antibody and gene expression responses to the pathogen, Neoparamoeba perurans, have been studied independently following primary exposure; however, the effects of sequential reinfection, which can often occur during net-pen culture of salmon, remain unclear. The association between the transcription of immunoglobulin (Ig) and their systemic and mucosal antibody levels in regards to AGD is unknown. Herein, we assessed the antibody responses as well as Ig transcription in the gills of Atlantic salmon infected only once and also sequentially with N. perurans. After four successive AGD challenges, no significant differences in plasma or skin mucus levels of IgM were observed between AGD-naïve and challenged fish. However, IgM gene expression in gill lesions of AGD-affected fish increased up to 31 d after infection, while no changes in IgT, TCR and CD8 transcription were observed. Changes at IgM transcription level did not match the lack of antibody response in mucus, which is possibly explained by weak correlations existing between protein and mRNA abundances in cells and tissues. In the second experiment, which investigated Ig responses to AGD at the transcriptional as well as antibody production level in salmon after a single infection, the levels of serum or skin mucus IgM antibody were not affected and no changes in the IgM or IgT transcription were induced.

Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease.

The external surfaces of fish, such as gill and skin, are covered by mucus, which forms a thin interface between the organism and water. Amoebic gill disease (AGD) is a parasitic condition caused by Neoparamoeba perurans that affects salmonids worldwide. This disease induces excessive mucus production in the gills. The host immune response to AGD is not fully understood, and research tools such as genomics and proteomics could be useful in providing further insight. Gill and skin mucus samples were obtained from Atlantic salmon (Salmo salar) which were infected with N. perurans on four successive occasions. NanoLC tandem mass spectrometry (MS/MS) was used to identify proteins in gill and skin mucus of Atlantic salmon affected by AGD. A total of 186 and 322 non-redundant proteins were identified in gill and skin mucus respectively, based on stringent filtration criteria, and statistics demonstrated that 52 gill and 42 skin mucus proteins were differentially expressed in mucus samples from AGD-affected fish. By generating protein-protein interaction networks, some of these proteins formed part of cell to cell signalling and inflammation pathways, such as C-reactive protein, apolipoprotein 1, granulin, cathepsin, angiogenin-1. In addition to proteins that were entirely novel in the context in the host response to N. perurans, our results have confirmed the presence of protein markers in mucus that have been previously predicted on the basis of modified mRNA expression, such as anterior gradient-2 protein, annexin A-1 and complement C3 factor. This first proteomic analysis of AGD-affected salmon provides new information on the effect of AGD on protein composition of gill and skin mucus. Future research should focus on better understanding of the role these components play in the response against infection with N. perurans.

Administration of recombinant attachment protein (r22C03) of Neoparamoeba perurans induces humoral immune response against the parasite in Atlantic salmon (Salmo salar).

This study investigated the use of a recombinant protein of Neoparamoeba perurans, the causative agent of Amoebic gill disease (AGD), as an immunogen to generate systemic and mucosal antibody responses against the parasite. Genes encoding N. perurans homologs of mannose-binding protein (MBP) from Acanthamoeba spp. have been identified. From these, a Neoparamoeba MBP - like EST has been identified and produced as a recombinant fusion protein. Attachment of N. perurans to the gill might be reduced by antibody-mediated interference of this protein, but this is dependent on the presence and level of functional antibodies in the mucus. Fish were immunized with the protein via i.p. injection with Freund's complete adjuvant (FCA); and serum and skin mucus samples were collected before and after immunization. Antibodies (IgM) present in samples were characterized via Western blot and their levels measured with an ELISA. The immunization was able to induce a systemic IgM response 8 weeks after primary exposure and a mucosal response 4 weeks post initial immunization, which were specific to the recombinant protein but not to antigens obtained from crude amoebic preparations. However, adherence of the antibodies to the parasite was observed using immunocytochemistry, and both, serum and skin mucus IgM, were able to bind the surface of formalin-fixed N. perurans. This finding may contribute to further research into the development of a vaccine for AGD.

Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar).

This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens - dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) - administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.