Development of a test to identify species of disease-causing bacteria in fish farms through environmental DNA (eDNA)

• Grant number: 22/04185-4
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Start date: September 01, 2022
• End date: May 31, 2023
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Preventive Veterinary Medicine
• Principal Investigator: Tiago Milanin
• Grantee: Tiago Milanin
• Company: Tiago Milanin
• CNAE: Pesca em água doce ; Aqüicultura em água doce
• City: Pirassununga
• Associated researchers: Antonio Augusto Mendes Maia ; Heidge Fukumasu ; Marcia Ramos Monteiro da Silva ; Mirele Daiana Poleti ; Ricardo Luiz Moro de Sousa
• Associated scholarship(s): 22/15656-8 - Development of a test to identify species of disease-causing bacteria in fish farms through environmental DNA (eDNA), BP.TT; 22/12712-4 - Development of a test to identify species of disease-causing bacteria in fish farms through environmental DNA (eDNA), BP.TT; 22/12140-0 - Development of a test to identify species of disease-causing bacteria in fish farms through environmental DNA (eDNA), BP.PIPE

Abstract

The cultivation of aquatic organisms is the agricultural sector that has grown the most in recent years, with Brazil being one of the world's leading producers of freshwater fish. Along with this increase in production, there is also an increase in the frequency and intensity of disease outbreaks, leading to serious economic losses. In an attempt to reduce such losses, management within fish farming is often done inappropriately. In this context, in many cases, the fish farmer is unaware of the etiological agent of the disease, which can culminate in the inappropriate use of massive amounts of chemicals in fish farms, resulting in the burden of processes and the emergence of resistant bacterial strains, a strong threat to fish farming and the public health. The traditional diagnosis of diseases caused by bacteria in fish is time consuming, since it depends on manpower to collect the sick specimens and send these fish to diagnostic laboratories. The tests still depend on the euthanasia of the fish, exposure of the organs and cultivation of the bacteria to identify which species is related to the pathology and death of the fish. These bacterial identification methods take time and the lack of an early diagnosis can compromise the entire breeding system, causing serious economic losses. Therefore, it is essential to develop a diagnostic test to identify the main species of bacteria and the presence of virulence genes related to diseases in fish. In this way, the fish farmer will be able to anticipate the problem by taking prophylactic measures, ensuring that their ponds are suitable for raising fish and demanding that the specimens acquired also have the certification of origin from an environment free from contamination by pathogenic bacteria. The objective of the project is to develop a diagnostic test to identify the main species of bacteria that infect fish from fish farms through environmental DNA (eDNA) and quantitative PCR (qPCR) through multiplex, and that is consistent with the needs of the Brazilian market. The first and very important step is to optimize a water sampling protocol that makes it possible to detect the main species of bacteria that infect farmed fish. For this, two filtering methodologies will be evaluated. After filtering, DNA will be extracted from the samples and, to verify the presence of bacteria of the species Aeromonas hydrophila, Francisella noatunensis subsp. Orientalis and Streptocoocus agalactiae and their virulence genes, a multiplex (mPCR) will be performed using specific probes and primers. (AU)