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Vaccine potential of mutants of Francisella noatunensis subsp. orientalis for Nile tilapia


The goal of this project is to develop a live attenuated vaccine against Francisella noatunensis subspecies orientalis (FNO) that can be applied to Nile tilapia (Oreochromis niloticus). The project will have five specific objectives. i.) Produce at list one mutant strain of FNO deleting the wbtA, pilA, mglA, clpB, and tolC genes from a non-mutant Brazilian strain isolated in the Fish Institute laboratory (IP0012018). ii) Evaluate the efficacy of the mutant strains as vaccine by the experimental infection test with wild-type FNO as well as its influence on: iii) cell-mediated (CD4 + T lymphocytes and CD8 + T lymphocytes) and humoral (serum IgM) responses; iv) the expression of genes involved in immunomodulatory processes responsible for the synthesis of cytokines and chemokines; v) hematological characteristics (total and differential leukocyte counts) and plasma biochemistry (total protein, glucose, and lysozyme). The strain of FNO (IP0012018) will be subjected to the deletion of genes by "Lambda red" technique, and verified by PCR and DNA sequencing. Two in vivo tests will be conducted: one to assess the degree of immunization caused by the administration of the mutants produced; other to evaluate the cellular and humoral immune responses induced by the mutants. Both experiments will be in a completely randomized design with seven treatments and four replicates. The experimental groups are divided as follows: Nile tilapia inoculated with saline solution and challenged with wild type FNO (G1); Nile tilapia inoculated with mutant FNO strains, deleted on the operon gene (s) wbtA (G2), pilA (G3), mglA (G4), clpB (G5) and tolC (G6) and challenged with wild-type of FNO. Nile tilapia inoculated with saline and not challenged (G7). After 15 days of intraperitoneal inoculation, the G1-G6 groups will be challenged orally with pathogenic wild-type FNO. The first experiment will be taken up to 21 days post infection (DPI) to obtain the level of protection caused by the vaccine. In the second experiment serial collections will be performed the day before infection, and at 7, 14 and 21 DPI, when eight fish / group will be sacrificed for sampling. The population of CD4 + and CD8 + lymphocytes will be evaluated by immunohistochemistry and cytokines will be quantified by RT-qPCR in the cranial kidney and spleen; IgM levels will be measured by serum ELISA. Clinical or subclinical manifestation of the disease (symptomatology, macroscopic and microscopic lesions, reisolation) will be evaluated in both experiments. The data will be analyzed by ANOVA and compared by Tukey (P <0.05). After completion of the two stages of the project, it is expected to produce at least one live attenuated vaccine against FNO. (AU)

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