CN104152368B - Rofe source of fish streptococcus agalactiae low virulent strain and application - Google Patents

Abstract

The invention relates to the disease prevention and control technology of aquaculture animals, in particular to the attenuated strain of Streptococcus agalactiae derived from tilapia and its application. The attenuated strain of Streptococcus agalactiae derived from tilapia is YM001, which is preserved in CCTCC, and the preservation number is: CCTCC M 2014045. After the attenuated strain was activated and fermented, the bacterial cells of the attenuated strain YM001 of 10 ⁶ ‑10 ⁹ CFU/mL were mixed with sterile phosphate buffer solution as an attenuated live vaccine for preventing and treating tilapia streptococcosis, and injected The administration concentration is 10 ⁵ ‑10 ⁸ CFU/tail, the oral administration concentration is 10 ⁸ ‑10 ⁹ CFU/tail, and the soaking administration concentration is 10 ⁷ ‑10 ⁸ CFU/mL. The invention relates to strain identification, virulence measurement, safety and stability of reversion, immunogenicity and immune protection effect of the attenuated strain. The tilapia streptococcal disease vaccine made with the strain has good immune protection effect and safety and strong stability.

Description

Attenuated Strain of Streptococcus agalactiae from Tilapia and Its Application

technical field

The invention relates to the disease prevention and control technology of aquaculture animals, in particular to an attenuated strain for the immune prevention and treatment of Streptococcus agalactiae infected Streptococcus agalactiae and its application.

Background technique

my country is the largest country in tilapia farming, and its annual output accounts for more than 50% of the world's total output. From 2001 to 2008, streptococcal infection occurred successively in tilapia farming in China, and caused a high mortality rate of 30% to 50% in individual farms. The main epidemic strain was Streptococcus iniae; 2009 to 2013 for 5 consecutive years , streptococcal disease has broken out in a large area of tilapia aquaculture in China, and the incidence area is expanding year by year, and the morbidity and mortality are increasing year by year. et al., 2011; Maixin Lu et al., 2010; Zhu Jinglin et al., 2010; Zhou Suming, 2008). Calculated according to the average annual tilapia production in my country, it is estimated that the direct economic loss caused by streptococcal disease to my country's tilapia industry is as high as 400 million U.S. dollars every year. At present, there is no effective method to prevent and treat the disease. Drugs can only play an auxiliary role in controlling the disease in the early stage. The disease outbreak and the subsequent product safety problems have seriously restricted the sustainable development of my country's tilapia industry.

The research and production application results at home and abroad show that the vaccine immunization prevention and control of tilapia streptococcosis is effective, safe and environmentally friendly. In 1995, Israeli scholar Eldar began to prevent tilapia streptococcal disease ( Streptococcus difficile ) through immunization, the United States (Klesius et al., 1999; Pridgeon et al., 2011), China (Sun et al., 2010; Chen Ming et al., 2010), Japan ( Many laboratories in countries such as Dumrongphol et al., 2009) and Korea (Shin et al., 2007) have successively carried out research on the inactivated vaccine against tilapia streptococcosis, and achieved good immune effects in the laboratory and production application demonstration. However, due to the inconvenient immunization route, short immune protection period and high cost of the developed inactivated vaccine, the vaccine has not been widely used in tilapia aquaculture production. The attenuated vaccine has the characteristics of low immune dose, good immune effect and long protection period. The development of attenuated vaccine plays an important role in the prevention and control of tilapia streptococcosis. Pridgeon and others in the United States obtained attenuated strains of Streptococcus iniae and Streptococcus agalactiae respectively through antibiotic screening methods, and proved the safety and feasibility of attenuated vaccines (Pridgeon et al., 2011). After many years of research, the present invention adopts the method of rapid culture and weakening of virulence by temperature difference subculture to obtain a tilapia-derived Streptococcus agalactiae attenuated strain YM001, which can be prepared into tilapia streptococcal disease vaccine, which can be injected, taken orally or soaked Immunizing tilapia with different routes of administration to prevent streptococcal disease has great development value for the prevention and treatment of tilapia streptococcal disease.

Contents of the invention

The purpose of the present invention is to solve the problems of high cost of immunization prevention and control of tilapia streptococcosis inactivated vaccine, short immunization protection period, inconvenient immunization route, etc., to provide attenuated strains of Streptococcus agalactiae derived from tilapia and its application, and to develop tilapia Live attenuated streptococcal vaccine.

For realizing above-mentioned purpose of the invention, the technical scheme that the present invention adopts is:

The attenuated strain YM001 of Streptococcus agalactiae derived from tilapia is preserved in the China Center for Type Culture Collection CCTCC, and the preservation number is: CCTCC M 2014045.

The tilapia-derived Streptococcus agalactiae attenuated strain YM001 was stored in the China Center for Type Culture Collection CCTCC on February 26, 2014, at Luojia Mountain, Wuchang City, Wuhan City, and the preservation number is: CCTCC M 2014045, classified The name is Streptococcus agalactiae .

Application of attenuated strain of Streptococcus agalactiae derived from tilapia in the prevention and treatment of streptococcosis in tilapia.

The strain can be cultured with tryptone liquid medium (TSB). After the attenuated strain of Streptococcus agalactiae derived from tilapia is cultured through activation and fermentation, 10 ⁶ -10 ⁹ CFU/mL of Streptococcus agalactiae Bacterial cells of the attenuated strain are mixed with sterile phosphate-buffered saline solution as an attenuated live vaccine for the prevention and treatment of tilapia streptococcosis.

The injection concentration of the above-mentioned attenuated live vaccine is 10 ⁵ -10 ⁸ CFU/tail, the oral administration concentration is 10 ⁸ -10 ⁹ CFU/tail, the soaking administration concentration is 10 ⁷ -10 ⁸ CFU/mL, and the soaking time is 10 -30 min.

The advantages that the present invention has:

The present invention obtains tilapia-derived Streptococcus agalactiae attenuated strain YM001 by rapidly culturing and weakening the virulence by temperature difference subculture. The strain has the characteristics of low toxicity, strong immunogenicity, and good stability. Streptococcus vaccine has good immune protection effect, strong safety and stability, and can effectively protect experimental fish against the infection of highly pathogenic Streptococcus agalactiae strains derived from tilapia. The attenuated live vaccine prepared by the invention can be administered and immunized by injection, oral administration or immersion as required, so that the tilapia can obtain high-efficiency immune protection.

detailed description

Embodiment 1 Bacterial strain and medium used in the present invention

The wild type strain HN016 of Streptococcus agalactiae derived from tilapia was isolated from the brain of a tilapia farm where streptococcal disease broke out in southern my country. ).

The tilapia-derived Streptococcus agalactiae attenuated strain YM001 was obtained by attenuating the wild-type strain HN016 by using the rapid culture and attenuating method of temperature difference subculture. It is preserved in the China Center for Type Culture Collection CCTCC, and the preservation number is: CCTCCM 2014045.

Tryptone broth (TSB): tryptone 17.0 g/L, soybean peptone 5.0 g/L, NaCl 5.0 g/L, glucose 2.5 g/L, distilled water 1000 mL, adjust pH to 7.2, autoclave at 121 °C for 20 min, cooled at room temperature for later use.

Example 2 PCR and biochemical identification of tilapia-derived Streptococcus agalactiae attenuated strain YM001

The attenuated strain YM001 was identified by PCR identification method of tilapia-derived Streptococcus (Streptococcus iniae and Streptococcus agalactiae), API 20 Strep biochemical strip and VITEK 2 automatic microbial analyzer.

Double PCR rapid detection of Streptococcus iniae and Streptococcus agalactiae: PCR primers refer to published papers by Li Jiong et al. (2010) and Zlotkin et al. (1998). The size of the fragment amplified by cocci-specific primers was 296 bp. The PCR reaction reagent was a PCR kit from TaKaRa Company, Dalian, China. The PCR system was as follows: 5.0 µL of 10× Ex Taq Buffer (Mg ²⁺ ), 2.0 µL of dNTP, 2.0 µL each of H1, H2, P1 and P2 primers, 2.0 µL of DNA template ( About 50 ng), 1.0 µL Takara Ex Taq (5 U/µL), 32.0 µL ddH ₂ O, total volume 50 µL; PCR reaction was performed on GeneAmp PCR system 9700 from ABI Company, USA; PCR reaction procedure: Step 1, 94°C , 5 min; second step, 94 ℃, 30 s, 58 ℃, 30 s, 72 ℃, 45 s, 35 cycles; third step, 72 ℃, 10 min; 1.2% agarose gel electrophoresis, after GeneGreen staining Use a gel imaging system to observe, and set up positive and blank controls. The results of rapid PCR detection showed that both the attenuated strain YM001 and the original wild strain HN016 could amplify a specific band (474bp). The results show that the attenuated strain YM001 of the present invention can be detected by the PCR method and identified as Streptococcus agalactiae, indicating that the basic attributes of the strain have not fundamentally changed compared with the original strong strain HN016.

Biochemical identification: Streak culture of the preserved wild virulent strain HN016 and attenuated strain YM001 on the blood plate, use an inoculation loop to pick a single colony growing on the solid medium, and inoculate it in 10 mL of TSB liquid medium at 28°C, 120 rpm Shake culture for 24 h. HN016 and YM001 were identified using API 20 Strep biochemical strip and VITEK 2 automatic microbial analyzer from BioMérieux France. The results of biochemical identification showed that except bacitracin resistance (BACI) HN016 was positive and YM001 was negative, other biochemical indicators were consistent. Bacitracin is a cyclic peptide produced by Bacillus subtilis and Bacillus licheniformis, which inhibits the synthesis of peptidoglycan on the cell wall of Gram-positive bacteria, has a bactericidal effect, and also inhibits the synthesis of glycoprotein core oligosaccharides. It is effective for Gram-positive bacteria and negative cocci, Pneumococcus, Staphylococcus, Neisseria gonorrhoeae, Neisseria meningitidis and spirochetes all have bactericidal effect. The anti-bacitracin of the attenuated strain was negative, indicating that the antibacterial effect of the strain was weakened after the virulence was weakened, which may be related to some inhibition of cell wall synthesis. The biochemical identification results show that the attenuated strain YM001 of the present invention can still be detected by the biochemical identification method, and it is identified as Streptococcus agalactiae. The biochemical indicators of this strain are consistent with those of the original virulent strain HN016 except for anti-bacitracin. , indicating that the biochemical properties of the attenuated strain YM001 have not changed greatly, which is conducive to maintaining the immunogenicity of the attenuated strain.

The PCR and biochemical identification results of the tilapia-derived Streptococcus agalactiae attenuated strain YM001 show that the gene and biochemical characteristics of the tilapia-derived Streptococcus agalactiae YM001 of the present invention have not changed greatly compared with the original strong strain HN016, and it is speculated that the immunity of the strain The originality is preserved, which is beneficial to the subsequent development of attenuated vaccine against streptococcal disease in tilapia.

Example 3: Determination of the median lethal dose LD ₅₀ of tilapia-derived Streptococcus agalactiae wild-type strain HN016 and attenuated strain YM001 to tilapia

Use TSB to culture tilapia-derived wild-type strain HN016 and attenuated strain YM001, respectively, at 25-30 °C, shake the shaker for 18-24 hours, and use the plate colony counting method to count the bacteria in the liquid. Eight serial dilutions of the culture solution were performed with a 10-fold gradient, and 20 tilapias were infected by intraperitoneal injection of each concentration of the bacterial solution, 0.1 ml/tail; the same volume of TSB medium was injected intraperitoneally into tilapias in the control group, 0.1 ml/tail . Both the infected group and the control group were kept in an inflatable aquarium with 40 L of water, 2/3 of the water was changed every 2 days, the water temperature was controlled at 30-32 ℃, and the fish were fed twice a day in the morning and afternoon based on 3% of the fish weight. . Feed and observe for 20 days after challenge, record food intake, morbidity and death every day, and use blood plate to isolate and identify bacteria from the brain and liver tissue of the dead test fish, calculate the cumulative number of deaths and mortality, and determine the median lethal dose of strains HN016 and YM001 ( LD50).

Table 1 Toxicity of wild-type strain HN016 and attenuated strain YM001 to tilapia

The above results showed that, compared with the original wild-type strain HN016 of Streptococcus agalactiae derived from tilapia, the attenuated strain YM001 had a significantly reduced virulence by more than 100,000 times.

Embodiment 4: the preparation of cultivation and attenuated vaccine of Streptococcus tilapia attenuated strain YM001

PBS buffer: NaCl 8 g/L, KCl 0.2 g/L, Na ₂ HPO ₄ ·12H ₂ O ₂ 3.58 g/L, NaH ₂ PO ₄ 0.24 g/L. The above ingredients were dissolved in 1 L of distilled water, autoclaved at 121 °C for 20 min, and cooled at room temperature for later use.

Preparation of live vaccine of attenuated strain YM001: Streak inoculation of attenuated strain YM001 stored at -80 ℃ on a blood plate, and incubate at 25-30 ℃ for 48 hours. Pick a single colony, inoculate in 10 mL of TSB medium, incubate on a shaker at 25-30 °C for 18-24 hours, inoculate in 500 mL of TSB medium according to the amount of 5-10 % (V/V), 25 Incubate in a shaker at -30°C for 18-24 hours. Bacteria were collected by centrifugation (7000 g, 10 min, 4°C), washed three times with PBS, resuspended in PBS buffer with a bacterial concentration of 10 ⁶ -10 ⁹ CFU/mL, and stored at -20°C for future use.

HN016 wild strain inactivated vaccine preparation: Streptococcus agalactiae wild strain HN016 from tilapia, which was stored at -80 ℃, was streaked and inoculated on blood plates, and cultured at 25-30 ℃ for 48 hours. Pick a single colony, inoculate in 10 mL of TSB medium, incubate on a shaker at 25-30 °C for 18-24 hours, and inoculate in 500 mL of TSB medium at 5-10 % (V/V), 25- Shake culture at 30°C for 18-24 hours, add 0.1-0.5% formalin solution, inactivate for 24-48 hours; collect bacteria by centrifugation (7000 g, 10 min, 4°C), wash with PBS three times, use The bacteria were resuspended in PBS buffer with a bacterial concentration of 10 ⁹ CFU/mL, and stored at -20°C for future use.

Example 5 Attenuated strain YM001 vaccine injection immune efficacy test

Use PBS to adjust the stock solution concentration of attenuated strain YM001 live vaccine and wild strain HN016 inactivated vaccine to 1.48×10 ⁹ CFU/mL, and adjust the stock solution, 1:1, 1:10, 1:100, 1:10 ³ , 1: 10 ⁴ , 1:10 ⁵ , and 1:10 ⁶ were diluted in 8 gradients, and each fish was injected with 0.1 mL; the size of the test fish was 25±5 g/fish, 20 fish/group, and the water temperature was 30-31.5°C. On the 15th day after immunization, the wild strain HN016 was used for intraperitoneal injection infection. The concentration of the original bacterial solution was 1.67×10 ⁹ CFU/mL. The original bacterial solution was diluted 10 times and injected with 0.1 mL per tail. The water temperature was 30-31.5°C. Feed and observe for 20 days after challenge, record food intake, morbidity and death every day, and use blood plate to isolate and identify bacteria from the brain and liver tissue of the dead test fish, and calculate the cumulative number of deaths, mortality and relative immune protection rate (see Table 2) .

The formula for calculating the relative immune protection rate is:

Relative immune protection rate (RPS) = (mortality rate in control group - mortality rate in immune group) / mortality rate in control group

Table 2 Comparison of injection immune efficacy of attenuated vaccine and inactivated vaccine

The results of vaccination immunity showed that as the vaccination dose increased, the vaccine immunity protection rate also increased. The injection dose of inactivated vaccine above 10 ⁷ CFU/tail can well prevent the infection of Streptococcus agalactiae from tilapia, but the injection dose lower than 10 ⁴ CFU/tail has almost no immune protection effect. The attenuated live vaccine injection dose of 10 ² ～l0 ⁸ CFU/tail showed a good effect on the prevention of Streptococcus agalactiae infection from tilapia, and the injection dose of 10 ⁵ ～l0 ⁸ CFU/tail showed an excellent effect on the prevention of tilapia source Streptococcus agalactiae infection. Effects of Streptococcus agalactiae infection. The results showed that the attenuated strain YM001 had strong immunogenicity, and the prepared live vaccine had a lower immunization dose than the inactivated vaccine, which could effectively save the immunization cost.

Embodiment 6 Attenuated Vaccine and Inactivated Vaccine Oral Immune Effectiveness Comparative Test

The live vaccine of the attenuated strain YM001 and the inactivated vaccine of the wild strain HN016 were diluted in 4 gradients of stock solution (1.48×10 ⁹ CFU/mL), 1:10, 1:100, and 1:1000, corresponding to 0.67 mL per fish The amount of bacterial liquid, each gradient bacterial liquid was sprayed on the feed, the feed of the control group was sprayed with the same volume of PBS, and the ratio of the feed amount was 4:2:1 and fed three times (each interval was 4 h), the size of the test fish was 25 ±5 g/tail, 20 tails/group, feeding water temperature 30-31.5°C. On the 15th day after immunization, the wild-type strain HN016 was used for intraperitoneal injection infection. The concentration of the original bacterial solution was 1.67×10 ⁹ CFU/mL. The original bacterial solution was diluted 10 times and injected with 0.1 mL per tail. The water temperature was 30-31.5°C. Feed and observe for 20 days after challenge, record food intake, morbidity and death every day, and use blood plate to isolate and identify bacteria from the brain and liver tissue of the dead test fish, calculate the cumulative number of deaths and mortality, and calculate the relative immune protection rate (see Table 3 ).

The formula for calculating the relative immune protection rate is:

Relative immune protection rate (RPS) = (mortality rate in control group - mortality rate in immune group) / mortality rate in control group

Table 3 Comparison of oral immunity efficacy between attenuated vaccine and inactivated vaccine

The results of oral immunity of the vaccine showed that the oral immunity of the inactivated tilapia streptococcosis vaccine prepared by the original wild strain HN016 was poor, and the immune protection rate of the attenuated live vaccine prepared by the YM001 strain increased with the increase of the inoculation dose, and in The oral immunization dose range of 10 ⁸ ～l0 ⁹ CFU/tail showed a good effect on the prevention of Streptococcus agalactiae infection from tilapia.

Example 7 Comparison test of attenuated vaccine and inactivated vaccine immersion immune efficacy

The attenuated strain YM001 vaccine stock solution (10 ⁹ CFU/mL) and the wild strain HN016 inactivated vaccine stock solution (10 ⁹ CFU/mL) were prepared with ordinary tap water exposed to the sun for 48 h to a concentration of 10 ⁸ CFU/mL, 10 ⁷ CFU/mL mL, 10 ⁶ CFU/mL vaccine solution, and the control group was a 10-fold dilution of PBS. The tilapia in each test group were soaked in different concentrations of vaccine solution or PBS dilution solution for 20 minutes, and then moved into the test water tank for feeding. The size of the test fish was 25±5 g/tail, 20 fish/group, and the water temperature was 30-31.5°C. On the 15th day after immunization, the wild strain HN016 was used for intraperitoneal injection infection. The concentration of the original bacterial solution was 1.42×10 ⁹ CFU/mL. After the original bacterial solution was diluted 10 times, 0.1 mL per tail was injected, and the water temperature was 30-31.5°C. Feed and observe for 20 days after the challenge, record food intake, morbidity and death every day, and use blood plate to isolate and identify bacteria from the brain and liver tissue of the dead test fish, calculate the cumulative number of deaths and mortality, and calculate the relative immune protection rate (see Table 4 ).

The formula for calculating the relative immune protection rate is:

Relative immune protection rate (RPS) = (mortality rate in control group - mortality rate in immune group) / mortality rate in control group

Table 4 Immune potency test of attenuated strain YM001 vaccine soaking

The results of vaccine immersion immunization efficacy showed that the immersion immunization effect of the inactivated tilapia streptococcosis vaccine prepared by the original wild strain HN016 was poor, while the immersion immunity protection rate of the attenuated live vaccine prepared by the YM001 strain increased with the increase of the inoculation dose. Immunization with immersion concentration of 10 ⁷ ～l0 ⁸ CFU/mL showed a good effect on preventing Streptococcus agalactiae infection from tilapia.

Example 8 Attenuated strain YM001 reversion safety and stability test

After using TSB to cultivate the attenuated strain YM001, mix it according to the bacteria solution: glycerol = 4:1 (V/V), then divide it into cryopreservation tubes, 1 ml/tube, store in -80 ℃ refrigerator, take 0 months, 1 The strains were preserved for 1 month, 3 months and 6 months, and it was tested whether the strain YM001 was stable and did not return to the virus. Take out the preserved strain from the -80 ℃ refrigerator, inoculate it on a blood plate, and incubate at 25-30 ℃ for 48 hours. Pick a single colony, inoculate in 10 mL of TSB medium, shake and culture at 25-30 ℃ for 18-24 hours, inoculate in 500 mL of TSB medium according to the amount of 5-10 % (V/V), 25 Incubate in a shaker at -30°C for 18-24 hours. Prepare 6 groups of tilapias, coded as A, B, C, D, E and F, 6 fish/group, and the water temperature for feeding is 32-33°C. Cultivate attenuated strain YM001 to more than 10 ⁸ CFU/mL, inject 0.5 mL per fish in group A, isolate bacteria from the livers of 6 test fishes in 48 h, homogenate the liver tissues in a mortar, add PBS 4 mL, average Divided into 6 parts to inject the test fish of group B to challenge the virus. According to the above method until the challenge to group F, the test fish in group F were fed and observed for 30 days. The virulence stability of the attenuated strain YM001 was determined according to the above method for 0 months, 1 month, 3 months and 6 months. Streptococcus could be isolated from the liver, but none of the test fish showed symptoms of streptococcus disease, and no test fish died. The test fish showed symptoms and died 24 hours after being challenged with the wild strong strain. The fishes in group F were fed and observed for 30 days after being challenged with the virus, and no tilapias showed symptoms of disease and died. The test results show that the attenuated strain YM001 is not easy to return to the virus when stored or stored in tilapia for a long time, and the vaccine prepared by using the attenuated strain YM001 has higher stability and safety.

Claims (2)

1. The attenuated strain YM001 of Streptococcus agalactiae derived from tilapia is preserved in the China Center for Type Culture Collection CCTCC, and the preservation number is: CCTCC M 2014045. 2. the application of the tilapia source streptococcus agalactiae attenuated strain YM001 according to claim 1, it is characterized in that: after the tilapia source streptococcus agalactiae strain YM001 is through activation culture and fermentation culture, 10 ⁶ -10 ⁹ Bacterial cells of CFU/mL Streptococcus agalactiae strain YM001 were mixed with sterile phosphate-buffered saline solution to prepare attenuated live vaccines for preventing and treating tilapia streptococcosis.