Molecular cloning, mRNA expression analysis of immune-related genes and innate immune response of freshwater African catfish (Clarias gariepinus) vaccinated with heat-killed Aeromonas hydrophila.

Innate immunity is vital to vertebrates as first line of defence during infections but more critical for fish species that rely on innate immune system when they are infected. Unfortunately, innate immune pathways, including PRRs and associated cytokines, have not been characterized in African catfish. In this study, PCR and qPCR techniques were employed to assess immune gene expression in both untreated and vaccinated African catfish. Transcripts of nod1, nod2, tlr3, mhcii, il1β, cox2 and mpo genes were identified, cloned, and characterized. mRNA levels of nod1, nod2, tlr3 and mhcii genes were further compared in two fish groups distinguished by body weight. Following vaccination, expression profile of nod1 and nod2 was assessed in 72 fish. In healthy fish, transcripts of the genes were widely expressed across tested tissues. mRNA expression levels varied significantly among tissues. Between the two fish groups, expression levels of nod1 and nod2 differed in spleen, whereas tlr3 expression varied significantly in gill and liver. After vaccination, expression of nod1 and nod2 in spleen was significantly altered across treatment groups at 24 h and 7 days post-treatment. Highest and lowest expression levels were observed in vaccine group at 24 h and bacterin group at 7 days, respectively. In contrast, all treatments triggered continuously increased expression of both receptors in gill, reaching a peak level at 34 days in bacterin group. Whereas expression was clearly downregulated at 7 days in vaccine group. Furthermore, at 34 days, nod1 expression in gills of vaccine group was significantly lower compared to other treatment groups, whereas nod2 expression in bacterin group was greater than that in vaccine group. Overall, the findings demonstrate that African catfish express immune genes much like other teleost species, and that both heat-killed whole-cell A. hydrophila vaccines and mineral oil-based adjuvants can stimulate African catfish innate immune system.