Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: Molecular and immunohistochemical studies

Abstract

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.

Introduction

The parasitic ciliate Ichthyophthirius multifiliis (commonly referred to as Ich) infects a range of freshwater fish species causing significant economic losses to the aquaculture industry [1]. Fish surviving the infection develop a relatively long-lasting acquired protection against re-infection [2], [3], [4], [5]. The host immune factors contributing to the protection have been intensively studied [2], [6], [7], [8], [9], [10], [11] and include both specific and non-specific humoral and cellular factors. These comprise specific antibodies that agglutinate parasites in vitro [1], [2], [5]; non-specific humoral elements including complement factors [5], [8], [11], [12] and macrophage-like cells and neutrophils [7], [13], [14]. However, the relative contributions of these factors are still debated and the cells responsible for production of the humoral elements in the antiparasitic response have not been localized. The advent of new molecular and immunohistochemical techniques has enabled us to address these points in an experimental model involving rainbow trout. We studied the expression of immune genes in the gills of rainbow trout after immunization, immunosuppression and challenge infection. Immunohistochemical techniques based on monoclonal antibodies against the T-cell marker CD8, the macrophage marker MHC II, the teleost immunoglobulin IgT and the classical immunoglobulin IgM were used to localize immune cells in the gill tissue of rainbow trout exposed to this challenge scheme. Intra-peritoneal injection of live theronts was used for immunization of fish because this method has been shown as a reproducible way to confer protection [4], [11], [15].