Development of species-specific IgM antibodies and elevation of mucosal immune response in Labeo rohita using recombinant bicistronic nano DNA vaccine priming.

Abstract

Immunoglobulin (IgM) is the primary immunoglobulin essential for defense mechanisms in fish. It is difficult to reliably quantify IgM because a lack of standardization in methodology and limited availability of commercially reagents. In the present study, a polyclonal antibody was developed for the specific detection and quantification of IgM in Labeo rohita. Recombinant bicistronic NanoDNA plasmid (RBND Vac) encoding the glyceraldehyde - 3 - phosphate dehydrogenase gene of Edwarsiella tarda conjugated with poly (lactic - co - glycolic acid) - Chitosan (PLGA - Chit) was developed and its potential as a DNA vaccine, to prevent the infection of E. tarda in L. rohita was investigated. Two treatment groups [T1 - (PLGA - Chit - NPs - pDNA), T2 - (PLGA - NPs - pDNA) and one control group (T0 - 1 PBS)] were utilized. Polyclonal antibody was developed to estimate IgM titers in the serum and mucosal associated tissues (MAT) using Enzyme-linked Immunosorbent Assay (ELISA) technique. Additionally, immune gene expression was studied using qRT - PCR. Vaccinated groups also exhibited a significant increase in the total serum protein, globulin concentration and relatively less mortality was observed in T1 group. IgM level in serum and mucosal tissues (skin, gill and gut) increased significantly days post vaccination compared to control group, also non-specific immune parameters (myeloperoxidase and lysozyme levels) showed significant improvement in vaccinated fish. Furthermore, histopathological examination confirmed minor damage in physiological structure of kidney and liver tissues in vaccinated fish. Knowledge of the immunoglobulin in L. rohita primed with RBND Vac complex provides the better protection against E. tarda. The normal physiology findings of this study will aid in monitoring changes in the health status of fish, when the animals undergo vaccination by immersion method.