Development of a attenuated vaccine for control and prevention of infections caused by Francisella noatunensis subspecies orientalis in fish farms

• Grant number: 16/25347-1
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Start date: November 01, 2017
• End date: July 31, 2018
• Field of knowledge: Agronomical Sciences - Fishery Resources and Fishery Engineering - Aquaculture
• Principal Investigator: Miguel Frederico Fernandez Alarcon
• Grantee: Miguel Frederico Fernandez Alarcon
• Company: Prevet Laboratório de Diagnóstico, Desenvolvimento e Sanidade Aquícola Ltda. - ME
• City: Jaboticabal
• Pesquisadores principais: André Marcos Santana
• Associated researchers: Oliveiro Caetano de Freitas Neto
• Associated scholarship(s): 17/24653-4 - Production of three mutant strains of Francisella noatunensis orientalis by deletion of the virulence genes igl and pdp, BP.TT; 17/23269-6 - Development of an attenuated vaccine for control and prevention of infections caused by Francisella noatunensis subspecies Orientalis in Fish Farms, BP.PIPE

Abstract

The ultimate goal of this project (PIPE) is to produce a live attenuated vaccine against Francisella noatunensis subspecies orientalis to be used on fish farms for control and prevention of infections caused by this agent. For this, the project was divided into three phases by rules established by FAPESP, as follows: Phase 1: The objective of this phase is to produce three Francisella noatunensis orientalis mutant strains from a not mutant Brazilian strain isolated in PREVET laboratory (36163D). The proposal is to delete the virulence genes igl and pdp, that belong to Francisella pathogenicity island. According to studies, these genes play a central role in intracellular survival and proliferation of the bacteria. Therefore, it is expected that the mutant strains can be used for the preparation of a live attenuated vaccine, not yet on the market. For this, the first step will be to accomplish full sequencing and annotation of the genome of the Brazilian strain isolated in PREVET laboratory (36163D), in order to identify sets of genes from the "operons" igl and pdp, which will be deleted so to produce the mutant strains proposed in the study. Then, the deletion of the genes will be performed, using the Brazilian strain (36163D), by the "Lambda red" technique, to create the mutant strains. The mutation of the bacteria strains produced will be verified by PCR and full sequencing and analysis of the genomes of the mutants. Stage 2: After checking the quality of the three mutant strains produced, the second phase of the experiment will be proposed, where an experimental infection using Nile tilapia (Oreochromis niloticus) will be performed by comparing five experimental groups, so to test the reliability of use of mutant strain for vaccine production against Francisella noatunensis orientalis. The experimental groups will be divided as follows: Nile Tilapias experimentally infected with a unmutated strain of Francisella noatunensis orientalis (G1); Nile tilapia experimentally infected with a mutant strains of Francisella noatunensis orientalis gene with deletion of gene(s) from "operon" igl (G2), pdp (G3) and igl + pdp (G4). Not infected healthy Nile tilapias (G5). In this step, tests will be performed to evaluate the immune response of Nile tilapias upon challenge and the effect of each strain regarding clinical or subclinical manifestation of the disease (symptoms, gross and microscopic lesions, biochemical and serum profile for evaluating lesions in organs, etc.). Stage 3: Upon success in the first two steps, the final stage will be proposed, aiming the marketing of the product generated. This step will be made possible through partnerships with large companies already partners of PREVET in other studies, such as Virbac, BIOVET and MSD. In conclusion, after the three phases of the project are completed, it is expected to produce a live attenuated vaccine against Francisella noatunensis orientalis, not yet on the market, that can be used in fish farming for the control and prevention of infections caused by this agent. Moreover, the importance of the study of such vaccines in fish farming must be emphasizes, as the inappropriate use of antibiotics has serious disadvantages, such as the spread of plasmids that can confer resistance and possible damages to the environment. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

FERNANDEZ-ALARCON, M. F.; SANTANA, A. M.; VIADANNA, P. H. O.; MANZINI, B.; NATORI, M. M.; ISHIKAWA, C. M.; DIAS, D. C.; RANZANI-PAIVA, M. J. T.; SQUASSONI, G. H.; COUTO, F. D.; et al. Nile tilapia (Oreochromis niloticus) challenged infection by Francisella noatunensis subsp. orientalis via an intragastric route protocol. Aquaculture, v. 510, p. 380-385, AUG 15 2019. (16/25347-1, 17/23269-6, 17/24653-4)