TH1903002198C3 - Method for the production of a two-serotype vaccine of Streptococcus agalactia (Streptococcus agalactiae) to prevent streptococcosis. streptococcosis in tilapia farming - Google Patents

Abstract

Method for the production of a two-serotype vaccine of Streptococcus agalactia (Streptococcus agaiactiae) to prevent streptococcosis. Streptococcosis in tilapia culture is a vaccine development that begins with the isolation of the pathogenic bacteria Streptococcus agalactiae (S. agaiactiae) that causes strep disease. coccosis Epidemic strepcoccosis in tilapia and ruby fish farming in Thailand and grouped by serotyping profile from virulence genes in the polysaccharide capsule cluster. (Capsular Polysaccharide Cluster [cps genes]) and found that in Thailand there is Streptococcus agalactia. Two serotypes of Streptococcus agaiactiae which has been prepared as a two-serotype combination vaccine for the prevention of streptococcosis (Streptococcosis) effective and can be used in tilapia farms in Thailand effectively. ------------ Page 1 of 1 Page Summary of the invention Method for the production of a two-serotype vaccine of Streptococcus agalactia (Streptococcus agalactioe) to prevent streptococcosis (streptococcosis) in tilapia culture is a vaccine development starting from Isolation of Streptococcus agalactiae (S. agalactiae) pathogenic bacteria Streptococcosis (Strepcoccosis) epidemic in tilapia and ruby fish farming in Thailand and made Serotyping profiles from virulence-regulating genes in the capsule group Polysaccharide Cluster (Capsular Polysaccharide Cluster [cps genes]) and was found in Thailand. Streptococcus agalactia Two serotypes of Streptococcus ogoloctioe prepared It is a two-serotype combination vaccine for the prevention of streptococcosis. Effective streptococcosis and can be used effectively in tilapia farms in Thailand.

Claims (1)

1. Production method of two serotype vaccine of Streptococcus agalactiae for the prevention of streptococcosis in raising tilapia A. Streptococcus agalactiae (S. agalactiae) bacteria were isolated from tilapia/ruby tilapia culture sites in Thailand. Sick and showing symptoms of streptococcoisis and dissection of fish to collect bacterial samples from the brain, liver, spleen and dorsal growth by raking the organisms from these organs onto B culture medium. Brain HeartInfusion (BHI) agar) with a total of 401 fish samples from the 4 regions of tilapia farming in Thailand. Let's confirm the type of bacteria Streptococcus agalactia (S. agalactiae) from microbiological testing. Preliminary biochemical properties (Biochemical test) with API 20 Strep test kit (API 20 STREP) and confirmation of the strain by Polymerase Chain Reaction gene technique; PCR)sixteen RNAs (16SrRNA)l for further sequencing studies. in which the bacteria Streptococcus agalactia (S. agalactiae) in 124 samples after identification of Streptococcus agalactiae (S. agalactiae) from diseased fish The serotypes were grouped according to the virulence regulating genes in the capsular polysaccharide cluster (cps genes). Standing specificity in the polysaccharide cluster capsule (cpsgenescluster) by multiplex PCR technique developed by this project. For the classification of serotypes of Streptococcus agalactiae (S.agalactiae) from the analysis of b. A total of 124 samples of Streptococcus agalactiae (S. agalactiae) can be isolated and can be classified according to virulence control genes in the predatory capsule group. Capsular Polysaccharide Cluster (cps genes) into 2 serotypes: serotype one (serotype la) and serotype III (serotype III). from both serotypes from To test the pathogenicity of the bacteria against disease in tilapia. It was found that Streptococcus agalactiae (S. agalactioe) in serotype III was more virulent than those in serotype oneage. Both serotypes were selected from the study in Article 1. d. One of each for the development of a Streptococcus agalactiae (S. agalactiae) vaccine from the selection of suitable bacteria (candidate antigen) for both serotypes. The preparation of the vaccine according to the conventional method is formalin-killed vaccine. (Formalin-killed vaccine) and tested the efficacy of the vaccine by dividing the vaccine into 3 experimental sets: Set 1) Streptococcus agalactiae (S. agalactiae) C vaccine serotype Ia (2nd series), vaccines from Streptococcus agalactiae (S. agalactiae), serotype III (3rd series) Streptococcus agalactiae (S. agalactiae) combined vaccine, both serotype Ia and serotype III (combini vaccine two) Serotype (Combine vaccine 2 serotypes) g. Select bacteria. Serotype 1 A (serotype la) from Khon Kaen (KKN 3/1) and serotype III (serotype III) from Ubon Ratchathani (UBN 6/2) as representatives for testing the efficacy of formalin- Kill Vaccine (Formalin-killed vaccine) C. Bring Streptococcus agalactia (S. agalactiae), isolated colonies culture in BHI broth medium at 37 °C for 24 h. The bacterial cells were centrifuged, washed with sterile 0.85% sodium chloride solution and added with 1% formalin solution to kill bacterial cells, stored overnight at 4 °C. The apoptosis of bacterial cells was examined by incubating 100 μl of the agar solution as a total spread on Plate CountAgar (PCA) medium at 37 °C. Celsius for 18 - 24 h. If growth of Streptococcus agalactiae (S. agalactiae) is not observed on PCA medium, then the vaccine is suitable. For use as a veterinary vaccine, before using the vaccine, the bacterial sediment must be washed with sodium chloride solution. 0.8percent that have been sterilized The bacterial concentration was adjusted by measuring with a spectrophotometer at a wavelength of 600 nm with an absorbance of 0.667 to obtain approximately a bacterial cell of 10 to the power of 9 CFU/ml. CFU/ml)l.Then, the efficacy of the vaccine was tested in tilapia weighing approximately 102.3 A positive or a negative number of 3.9 grams by injecting into the abdominal area, volume 0.2 ml/body (Only 1 vaccination) to study the effect of vaccine ------------ 1. Production process of two serotype vaccine of Streptococcus agalae. Streptococcusagalactiae for the prevention of streptococcosis in tilapia farming. It consists of the following steps: a. Isolation of Streptococcus agalactiae (S. agaiactiae) bacteria from tilapia/ruby tilapia farms in Thailand was selected. of streptococcosis Fish were dissected to collect bacterial samples from the brain, liver, spleen and dorsal kidney. HeartInfusion (BHI) agar) with a total of 401 fish samples from tilapia farms in four regions of Thailand. b. Let's confirm the type of bacteria Streptococcus agalactia ('S. agalactiae) from microbiological testing. Initial biochemical properties (Biochemical test) with API 20 Strep test kit (API 20 STREP) and confirmed the species by polymerase chain reaction technique (Polymerase Chain Reaction ; PCR)sixteen RNAs (16SrRNA) for further sequencing studies. in which the bacteria Streptococcus agalactia (S. agalactiae) in 124 samples after identification of Streptococcus agalactiae (S. agolactiae) obtained from diseased fish The serotypes were grouped according to the serotypes from the virulence-controlled stands in the capsular polysaccharide cluster (cps genes) and designed primers containing Standing specificity in cpsgenescluster capsules by multiplex PCR technique developed by this project. For the classification of serotypes of Streptococcus agalactiae (S.agalactiae) c from analysis b. A total of 124 samples of Streptococcus agalactia (S. agoloctiae) were isolated and virulence-regulating genes could be identified in the predator capsule group. Capsular Polysaccharide Cluster (cps genes) into 2 serotypes: serotype one (serotype la) and serotype III (serotype III). from both serotypes from To test the pathogenicity of the bacteria against disease in tilapia. It was found that Streptococcus agalactiae (S. agaioctiae) in serotype III was more virulent than those in serotype 1a. Both serotypes were selected from the study in Article 1. d. One of each for the development of a Streptococcus agalactiae (S. agalactiae) vaccine from the selection of suitable bacteria (candidate antigen) for both serotypes. The preparation of the vaccine according to the conventional method is formalin-killed vaccine. (Formalin-killed vaccine) and tested the efficacy of the vaccine by dividing the vaccine into 3 experimental sets: Set 1) Streptococcus agalactiae (S. agalactiae) C vaccine Rotype one A (serotype la, series 2), vaccines from Streptococcus agalactiae (S. agalactiae), serotype III (serotype III) and series 3) Streptococcus agalactiae (S. agalactiae) combined vaccine, both serotype one A (serotype la) and serotype III (serotype III) (combined vaccine try Serotype (Combine vaccine 2 serotypes) g. Select bacteria Serotype 1 A (serotype la) from Khon Kaen (KKN 3/1) and serotype III (serotype III) from Ubon Ratchathani (UBN 6/2) as representatives for testing the efficacy of formalin- Kill Vaccine (Formalin-killed vaccine) C. Bring Streptococcus agalactia (S. agalactiae), isolated colonies culture in BHI broth medium at 37 °C for 24 h. The bacterial cells were centrifuged, washed with sterile 0.85% sodium chloride solution and added with 1% formalin solution to kill bacterial cells, stored overnight at 4 °C. The apoptosis of bacterial cells was examined by incubating 100 μl of the aqueous solution by total spread on Plate CountAgar (PCA) agar at 37 °C. Celsius for 18 - 24 h. If growth of Streptococcus agalactiae (S. agaiactiae) is not observed on PCA medium, the vaccine is suitable. for use as an animal vaccine k. Before use of the vaccine, the bacterial sediment must be washed with sodium chloride solution. 0.8percent that have been sterilized The bacterial concentration was adjusted by measuring with a spectrophotometer at a wavelength of 600 nm with an absorbance of 0.667 to obtain approximately a bacterial cell of 10 to the power of 9 CFU/ml. CFU/ml) l.Then, the efficacy of the vaccine was tested in tilapia weighing about 102.3 positive or negative 3.9 g by intra-peritoneal injection at a volume of 0.2 ml/fish. (only 1 vaccination) to study the effect of the vaccine ------------ Claims 1. Method for the production of a two-serotype vaccine of Streptococcus agalactia. agalactiae) to prevent streptococcosis (streptococcosis) in tilapia culture consists of the following steps: a. (S. agaloctiae) from tilapia/tap tilapia farms in Thailand. Sick tilapia showing symptoms of streptococcosis were selected and dissected for collection. Bacterial samples from the brain, liver, spleen and kidney sections. A total of 401 fish were sampled from the tilapia aquaculture region in the four regions. of Thailand b. Take the sample obtained from item a. to confirm the type of Streptococcus agalactia bacteria (S. agaiactiaefe) by microbiological testing. Preliminary biochemical properties (Biochemical test) with API 20 strep test kit (API 20 STREP) and confirm the type of bacteria. stands for Polymerase Chain Reaction Technique; 16S rRNA PCR for sequencing in further isolation. 124 samples of Streptococcus agalactiae (S. agalactiae) were obtained after identification of Streptococcus agalactiae (S. agalactiae) obtained from diseased fish Classification of germs found according to serotypes. Based on the virulent control stand in the Capsular Polysaccharide Cluster (cps genes) group and designed specific primers in the Capsular Polysaccharide Cluster (cps genes) group. The cpsgenescluster multiplex PCR technique was developed by this project for the identification of streptococcus agalac serotypes. Tia(S. agalactiae) c. From the analysis from item b, it was found that a total of 124 samples of Streptococcus agalactiae (S. agaioctiae) could be isolated. Violence in the Capsular Polysaccharide Cluster (cps genes) is divided into two serotypes: serotype la and serotype III. ) d. selection of germs from both serotypes from C. To test the ability to cause pathogens of pathogens Pathogenicity to disease in tilapia It was found that Streptococcus agalactia (S. agalactioe) in group serotype III (serotype III) was higher than those in serotype onea. One of each of the two serotypes from study D. was selected for development. Streptococcus agalactia (S. agalactioe) vaccine was selected by selection of suitable bacteria (candidate antigen) of both serotypes by means of a convination preparation method. Conventional is a formalin-killed vaccine. (Formalin-killed vaccine) and testing the efficacy of the vaccine divided The vaccines were divided into three experimental series: series 1) vaccine from Streptococcus agalactia (S. agaioctiae), serotype 1 (serotype la), series 2) vaccine. from Streptococcus agalactiae (S. agaiactiae), serotype III (serotype III) and series 3). Lactia (S. agalactiae) both serotype one (serotype la) and serotype three (serotype III) (combine vaccine two serotypes) g. selection bacteria Serotype one A (serotype la) from Khon Kaen (KKN 3/1) and serotype III (serotype III) from Ubon Ratchathani (UBN 6/2) were used as representatives for efficacy testing. of the formalin-killed vaccine Formalin-killed vaccine S. brought Streptococcus agalactia (S. agaiactiae) isolated single colony feed on food BFHI broth was cultured at 37 °C for 24 h. Then, the bacteria obtained from C. were centrifuged, the bacterial cells were centrifuged, and the bacteria were washed. The sterile 0.85% sodium chloride solution was added to the 1% formalin solution to kill bacteria and stored overnight at 4 °C. 100 μl volume, total spread on Plate Count Agar (PCA), incubated at 37 °C for 18 - 24 h if growth was not observed. grow of Streptococcus agalactia (S. agalactiae) on PCA medium, indicating that the vaccine suitable for use as a veterinary vaccine k. Before use of the vaccine, the bacterial sediment must be washed with a sterile 0.8% sodium chloride solution. The bacterial concentration was adjusted by spectrophotometer. The spectrophotometer at a wavelength of 600 nm gave an absorbance of 0.667, giving approximately a bacterial cell of 109 CFU per ml. (CFU/ml) l. The efficacy of the vaccine was then tested in tilapia weighing about 102.3+-3.9 g by intra-peritoneal injection of 0.2 ml/head. (Only 1 vaccination) to study the effect of the vaccine.