Role of the spleen in the immune response of turbot (Scophthalmus maximus) to vaccination with the ciliate parasite Philasterides dicentrarchi

Trabajo presentado en la 3rd Conference of the International Society of Fish & Shellfish Immunology (ISFSI), celebrada en Las Palmas de Gran Canaria (España) del 16 al 20 de junio de 2019.Fish spleen is rich in lymphocytes, particularly in B lymphocytes, and plays an important role in the adaptive immune response after vaccination. After vaccination of fish by intraperitoneal injection, there is a strong migration of antigen-containing cells to the spleen, where antigen presentation occurs. In the present study, we analysed the B cell populations (IgM, IgT or IgD positive cells) and gene expression (IgM, IgT, IgD, MHCII, and several immune-related genes) in the spleen of turbot immunized with a vaccine containing an oleous adjuvant and a particulate Philasterides dicentrarchi antigen. Fish were immunized on days 0 and 30, and samples were obtained on days 3, 7, 33, 37 and 60. The vaccine provoked a significant increase in specific and total serum IgM at 37 dpi and at 60 dpi, but the specific IgT levels did not vary significantly in vaccinated fish. No significant regulation of sIgT, mIgT, sIgM, mIgM and IgD occurred before 37 dpi, whereas at 37 and 60 dpi, overexpression of sIgT and mIgT was detected in fish injected with adjuvant alone or with the vaccine. Immunofluorescence analysis enabled classification of the B lymphocytes into four groups: IgM+IgD-IgT- (the majority of IgM+ cells), IgM+IgD+IgT-, IgM-IgD+IgT- (very few cells, probably corresponding to lymphocytes with low levels of IgM) and IgM-IgD-IgT+ cells. The IgM+ and IgT+ cells were scattered throughout the parenchyma, and grouped around large vessels and surrounding melanomacrophage centres (MMC). Cell proliferation was estimated using a combination of anti-IgT, anti-IgM and anti-PCNA antibodies. Proliferation of both IgM+ and IgT+ B cells was observed in several areas of the spleen. In addition, vaccinated fish showed a mass of vaccine and cells (CVM) associated to the spleen. The CVM contained scattered PCNA+/IgM + and PCNA+/ IgT+ B cells, possibly indicating its importance during vaccination, above and beyond its role as a site for phagocytosis and material exchange.This work was funded by EU H2020 program through ParaFishControl Project (634429), by the Ministerio de Economía y Competitividad (Spain) and FEDER (European Union) (AGL2017-83577-R) and by grant ED431C2017/31 from the Xunta de Galicia. I.E. was contracted under APOSTD/2016/037 grant by the “Generalitat Valenciana”, and F.F. was contracted by the Xunta de Galicia