Analysis of Gene Expression of Immunological Markers in Nile Tilapia (Oreochromis niloticus) Subjected to DNA Vaccination against Francisella orientalis by intramuscular Route

• Grant number: 25/19190-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2025
• End date: September 30, 2026
• Field of knowledge: Agronomical Sciences - Fishery Resources and Fishery Engineering - Aquaculture
• Principal Investigator: Leonardo Tachibana
• Grantee: Eduarda Fernanda Nascimento da Silva
• Host Institution: Instituto de Pesca. Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 21/11955-8 - Solutions for emerging diseases in fish farming: diagnosis, vaccines and breeding, AP.CCD

Abstract

This work plan proposes the analysis of gene expression of immune markers in Nile tilapia (Oreochromis niloticus) subjected to DNA vaccination against Francisella orientalis, using the intramuscular route. The experiment will be conducted at the Interinstitutional Laboratory of Aquaculture Health at the Fisheries Institute, São Paulo.The main objective is to evaluate the expression of genes related to immune response and morphological changes in vaccinated fish using RT-qPCR. Specific objectives include quantifying the expression of genes associated with innate and adaptive immunity (IL-1¿, IL-6, TNF-¿, IFN-¿, TGF-¿, and MHC II) in target organs; correlating gene expression levels with histopathological changes; assessing the kinetics of immune response over time (0, 7, 15, and 30 days post-vaccination); and preliminarily determining the immunological efficacy of the vaccine based on transcriptional expression before and after experimental infection with the pathogen.For vaccination, recombinant plasmids containing bacterial antigen genes from F. orientalis will be propagated in E. coli and purified using commercial kits. Each fish will receive a dose of 50 µg of DNA vaccine administered intramuscularly. The experimental design will include six groups with five replicates: two negative controls (with and without infection), two positive controls (with inactivated vaccine), and two vaccinated groups (DNA vaccine and inactivated vaccine).Thirty days after vaccination, the fish will be challenged with F. orientalis via intraperitoneal injection. Infection will be monitored for 14 days, with mortality being verified by bacterial isolation and confirmation through PCR.Laboratory analyses will involve laparotomy and tissue collection (liver, spleen, and kidney). Samples will be divided for histopathological analysis (fixed in 4% formalin) and molecular analysis (stored in RNAlater). Total RNA will be extracted using Trizol®, and its quality verified by spectrophotometry and electrophoresis. cDNA synthesis will be carried out using 1 µg of RNA and reverse transcriptase. Gene expression of IL-1¿, IL-6, TNF-¿, IFN-¿, TGF-¿, and MHC II will be quantified by RT-qPCR, normalized using reference genes such as ¿-actin, 18S rRNA, or EF1¿.Gene expression data will be statistically analyzed using two-way ANOVA (time × treatment), with Tukey's post hoc test for multiple comparisons. If data are non-parametric, the Kruskal-Wallis test will be applied. The significance level will be set at p < 0.05.This study aims to contribute to the development of more effective vaccines against bacterial diseases in aquaculture. (AU)