| Swine streptococcal disease is the general terms for pig infectious diseases caused by bacteria of the genus Streptococcus.Streptococcus suis(SS)is the most important pathogen causing Swine streptococcal disease in the world.According to different characteristics of the SS bacteria capsular antigen,it can be divided into 35 serotypes(1~34 type and 1/2 type).Type 2(SS2)spreads widelyand the pathogenicity of SS2 is also the strongest,causing severe economic losses on the pig industry as well as posing a threat to public health.Vaccine immunity is effective in preventing and controlling swine streptococcosis,but it often exists immune failure,and the exact reasons are complex.Besides,the different potential virulence in different regions of SS2 is remarkable.In this study,43 strains of SS2 from Anhui Province were screened for SS2 vaccine strains by pathogenicity,antigenicity and stability tests.By further comparing the types of medium,nutrients and culture methods,the optimal culture conditions for SS2 strain-producing strains were determined.Theaim is to provide technical reserves for the development of inactivated vaccineand its multivalent and multi-linked vaccines with better immune protection.According to the test results of virulence factor(CPS/EF/MRP/SLY)identification,hemolytic determination and the pathogenicity test of Kunming mice and zebrafish,6strains of SS2 virulent strains(numbered as HF1,XC1,HF2,HF3,BZ1,and BB1)were initially selected as test strains.(1)Use the plate counting method and the modified Karber's method to determine the growth curve of strains and median lethal dose(LD50).(2)By using formaldehyde to inactivate the whole bacteria,positive serum was prepared from immunized rabbits and Kunming mice and the antibody titer was detected by ELISA.The inactivated whole bacteria were used to immunize Kunming mice and zebrafish,and the challenge protection experiment was conducted to measure the immune protection rate,then the organs of Kunming mice were collected to make pathological tissue sections.(3)The tested strains were cultured for 30 generations,and the tenth,twentieth,thirtieth generation strains were taken to detect the LD50,serum antibody titer and immunological protection rate,then the pathological tissue sections of the organs of Kunming mice were made.Based on the above test results,the bacterin-producing strains of SS2 were screened out.(4)The optimum culture conditions were determined by measuring the growth curvesof the screened strains of SS2 strains in 3 culture medium(TSB-YE?BHI?THB),different glucose content(0.2%~1.2%)and serum content(2%~6%)and two culture modes.The results:(1)The LD50 of HF3,BZ1,HF2,HF1,XC1,BB1 for Kunming mice was3.24×108 CFU/mL?3.31×108 CFU/mL?5.01×108 CFU/mL?1.00×109 CFU/mL?1.58×109CFU/mL?3.72×109 CFU/mL.The LD50 for zebrafish are 6.64 ×105 CFU/mL?1.05×105CFU/mL?5.60×105 CFU/mL?3.09×106 CFU/mL?2.70×106×109 CFU/mL?1.17×106×109CFU/mL.(2)The serum IgG antibody titer after immunization of rabbits with inactivated whole organism HF3,BZ1,HF2,HF1,XC1,BB1 was 1:102400,1:102400,1:204800,1:102400,1:51200,1:204800(The ultrasonic SS2(AH10-8)of whole organism lysate as antigen)and 1:6400,1:6400,1:1600,1:1600,1:800,1:3200(The recombinant SLY protein as antigen to detect).The serum IgG antibody titer after immunization for Kunming mice was 1:12800?1:25600?1:25600?1:3200?1:1600?1:6400(The ultrasonic SS2(AH10-8)of whole organism lysate as antigen)and 1:400?1:3200?1:400?1:400?1:100?1:800(The recombinant SLY protein as antigen to detect).The immune protection rates of HF3,BZ1,HF2,HF1,XC1 and BB1 strains for the Kunming mice and zebrafish were 60%,100%,100%,60%,40%,60% and 86.7%,66.7%,80%,40%,60% and 60% respectively.The observation of pathological changes showed that the pathological changes in the HF1,XC1,and BB1 groups were more severe than those in the HF2,HF3,and BZ1 groups.(3)The LD50 of HF2 and HF3 in Kunming mice was up to 1.07 ×109 CFU/mL and 2.19 ×109CFU/mL in the 10 passages,the LD50 of zebrafish reached 7.76×106 CFU/mL and9.97×106 CFU/mL,and both tending to stabilize to 30 generations,and the BZ1 passed down to the 10 th,20th and 30 th generations.The antibody titers of inactivated whole bacteriophage HF2 and HF3 decreased from 1:51200 in the 10 th generation to 1:12800 in the 30 th passage.The serum antibody titer of the inactivated whole organism BZ1 decreased from the 10 th generation 1:12800 to the 30 th generation 1:6400.The immune protection rates of HF2,HF3,and BZ1 in the 10 th to the 30 th generation in Kunming mice were 100% to 80%,80% to 60%,and 80% to 40% respectively,and they were immune protective for zebrafish,the rates are 66.7%~60%,80%~60%,60%~53.3% respectively.The observation of pathological changes in the efficacy test of immunization challenge showed that the 10 th to 30 th generation of BZ1 strain was more severe in pathological changes than HF2 and HF3.(4)The strains HF2 and HF3 screening of SS2 strains were most suitable for growth in BHI medium,reaching 2.68×109 CFU/mL and 2.07×109CFU/mL.When the glucose content reaches 0.4%,the maximum number of living bacterium is 3.85×109 CFU/mL.When the serum content reached 4%,the maximumnumber of living bacterium was 4.35×109 CFU/mL.The maximum number of living bacteria of HF2 and HF3 in oscillating culture and stationary culture was 4.35×109CFU/mL?3.90×109 CFU/mL and 2.70×109 CFU/mL?2.94×109 CFU/mL,respectively.Conclusion: According to the test results of pathogenicity,antigenicity and stability tests,HF2 and HF3 were screened out as SS2 strain.According to the optimized test results of the culture conditions of SS2 vaccine strains,it was determined that the BHI medium(containing 0.4% glucose and 4% serum)combining with the mode of oscillating culture was the best condition for the cultivation of HF2 and HF3. |
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