PRODUCTION OF POLYCLONAL ANTIBODIES ANTI - IgM OF NILE TILAPIA, Oreochromis niloticus FOR STANDARDIZATION OF IMMUNOENZYMATIC ASSAY

Abstract

Dear Editor,According to Anuário 2021 Peixe BR da Piscicultura, in 2020 the Brazilian fish culture reached 802,930 tons, maintaining growth of around 5.93% compared to the previous year. Tilapia is the most popular aquaculture species item farmed in Brazil, representing 60,6% of the national production, with 486,155 tons in 2020, an increase of 12.5% over the previous year. As a result, Brazil consolidates itself in the 4 th position among the largest Tilapia producers in the world. Among the largest producing states in Brazil, Paraná stands out, which leads the national ranking, with 166,000 tons, followed by São Paulo, with 70,500 tons, and Minas Gerais with 42,100 tons.The intensification of fish production increases outbreaks of infectious diseases that affect the aquaculture sector. Streptococcosis is a fish disease that is horizontally transmitted by direct contact between infected and healthy animals or indirect contact with water and fomites, resulting in disseminated disease in fish production systems (MIAN et al., 2009). Streptococcus species are considered the pathogenic bacterium with the highest occurrence and economic importance for intensive fish culture (MISHRA et al, 2018).One of the most used ways to treat and prevent bacterial infections in a fish farm is the administration of antibiotics; however, the abuse of these drugs represents a risk to the environment, besides causing drug resistance to future infections (CARUSO, 2016), A good alternative strategy for the control of this disease is the administration of vaccines in animals, which has proven to be a successful strategy (SECOMBES & BELMONTE, 2016). Fish immunization can be performed by intraperitoneal injection, immersion, or oral administration (DADAR et al, 2017). Although these methods have the same purpose, there are advantages and disadvantages concerning the efficiency practicality, side effects, and cost/benefit of immunization. Therefore, it is interesting for fish producers to develop methods capable of identifying and thus confirming those animals that have been immunized.In this letter, we describe the development of an immunodiagnostic assay capable of detecting specific IgM in the blood circulation of experimental Nile tilapia that received the vaccine against S. agalactiae.Rabbit immunization protocol: The Immunization procedure was approved by the Animal Ethics Committee of the Butantan Institute (CEUA Protocol number 3124080618). A female rabbit, weighing 2.5 kg, was immunized intramuscularly with a solution containing 500 μg of purified tilapia IgM/500 μL of PBS, emulsified in 500 μL of Marcol-Montanide  . After 30 days, the animal was injected intramuscularly with 500 μg of IgM, following the same protocol as before. At the end of 30 days after the second immunization, two more doses of 150 μg of previously purified IgM/300μL of PBS were injected intradermally (i.d.) at intervals of 15 days. Blood was collected after the last immunization by central artery ear bleed. The serum was separated from the red cells by centrif...

Oral Administration of Probiotics (Bacillus subtilis and Lactobacillus plantarum) in Nile Tilapia (Oreochromis niloticus) Vaccinated and Challenged with Streptococcus agalactiae

“…The plates were rewashed, and 200 µL of Nile tilapia anti-IgM polyclonal antibody developed in a rabbit (1:1000-in-house produced) were transferred to each well, followed by incubation at 37 • C for 1 h. After the incubation period, 200 µL of anti-rabbit IgG (whole molecule), and peroxidase-conjugated, (1:1000; Sigma, St. Louis, MO, USA) were added and the plates were incubated at 37 • C for 1 h. After three washes, the colorimetric substrate tetramethylbenzidine (TMB) (BD™, Franklin Lakes, NJ, USA) was added to the wells, and the solution was blocked with sulfuric acid at 30%. The absorbance was read at 450 nm [28]. The head kidney samples (N = 6) of fish at 63 days were removed from the −80 • C freezer and immediately macerated in a mortar containing liquid nitrogen.…”