Insight from Molecular, Pathological, and Immunohistochemical Studies on Cellular and Humoral Mechanisms Responsible for Vaccine-Induced Protection of Rainbow Trout against Yersinia ruckeri

Abstract

bThe immunological mechanisms associated with protection of vaccinated rainbow trout, Oncorhynchus mykiss, against enteric redmouth disease (ERM), caused by Yersinia ruckeri, were previously elucidated by the use of gene expression methodology and immunochemical methods. That approach pointed indirectly to both humoral and cellular elements being involved in protection. The present study correlates the level of protection in rainbow trout to cellular reactions in spleen and head kidney and visualizes the processes by applying histopathological, immunohistochemical, and in situ hybridization techniques. It was shown that these cellular reactions, which were more prominent in spleen than in head kidney, were associated with the expression of immune-related genes, suggesting a Th2-like response. Y. ruckeri, as shown by in situ hybridization (ISH), was eliminated within a few days in vaccinated fish, whereas nonprotected fish still harbored bacteria for a week after infection. Vaccinated fish reestablished normal organ structure within a few days, whereas nonprotected fish showed abnormalities up to 1 month postinfection. Protection in the early phase of infection was mainly associated with the expression of genes encoding innate factors (complement factors, lysozyme, and acute phase proteins), but in the later phase of infection, increased expression of adaptive immune genes dominated. The histological approach used has shown that the cellular changes correlated with protection of vaccinated fish. They comprised transformation of resident cells into macrophage-like cells and increased occurrence of CD8␣ and IgM cells, suggesting these cells as main players in protection. Future studies should investigate the causality between these factors and protection.

Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model

“…It is well recognized that the spleen is a secondary lymphatic organ in fish (Manning ) and attract macrophages and lymphocytes in response to infection (Press & Evensen ; Deshmukh et al . ) and the organ is also involved in scavenging, antigen degradation as well as antibody production and retention (Dalmo, Ingebrigtsen & Bøgwald ; Press & Evensen ; Deshmukh et al . ).…”

“…) and the organ is also involved in scavenging, antigen degradation as well as antibody production and retention (Dalmo, Ingebrigtsen & Bøgwald ; Press & Evensen ; Deshmukh et al . ). Accordingly, spleen enlargement in fish has previously been documented during various defence responses like parasite infections and organ damage (Arnott, Barber & Huntingford ; Lefebvre et al .…”

Secondary immune response of rainbow trout following repeated immersion vaccination

“…Sections (4 μm) were cut, using a Leica RM2135 microtome (Leica Microsystems, Germany), mounted on SuperFrost UltraPlus glass slides (Menzel‐Glaser, Germany) and dried for 24 hr at 40°C. Slides were then deparaffinized, rehydrated in graded ethanol (99%, 96%, and 70%) and Tris‐buffered saline (TBS; Dako, Denmark) and used for immunohistochemical staining (Deshmukh et al., ). Before incubation with a specific trout monoclonal antibody, slides were pretreated with 1.5% H 2 O 2 for 10 min to quench endogenous peroxidase activity, and then, they were incubated at 80°C for 3 hr for antigen retrieval and finally incubated with 2% BSA in TBS for 10 min at room temperature for blocking non‐specific binding of antibodies.…”

Exclusion of IgD‐, IgT‐ and IgM‐positive immune cells in Ichthyophonus‐induced granulomas in rainbow trout Oncorhynchus mykiss (Walbaum)

“…Tissue sections (4 μm) were deparaffinized with xylene and subjected to immunohistochemical staining (Deshmukh et al . ). Specific monoclonal antibodies reacting against trout either IgM, IgT or IgD (Jørgensen et al .…”