In this study,we established the growth curve of the paratyphoid live vaccine strain and obtained the parameter for bacteria cultivation,resulting in its application on the production of paratyphoid live vaccine using the synthetic medium.Salmonella enterica serovar Choleraesuis (S.Choleraesuis) strain CVCC79500 was cultured for 30 h in the test tube or flask,via either static culture at 37 ℃ or shaking culture at 37 ℃/200 r/min. Culturing samples were taken out for live bacteria counting at 4,8,12,18,24 and 30 h,to establish the growth curve.It was shown that the growth of the strain CVCC79500 would reach its stationary phase by static culture at 37 ℃ for 18 ~24 h or shaking culture at 37 ℃/200 r/min for 8 ~24 h.While inoculated with different amount, the growth of the strain CVCC79500 could reach its stationary phase by static culture at 37 ℃ for 24 h or shaking culture at 37 ℃/200 r/min for 12 h.The effect for proliferation of strain CVCC79500 in synthetic medium and conventional common broth were compared using the obtained parameter.While static culture at 37 ℃ for 24 h in synthetic medium,the obtained bacteria count was 27 ~31 ×108 CFU /mL and 33 ~41 ×108 CFU /mL in test tube and flask,respectively.While shaking culture at 37 ℃/200 r/min for 12 h in synthetic medium,the obtained bacteria count was 58 ~66 ×108 CFU /mL and 78 ~88 ×108 CFU /mL in test tube and flask,respectively. However,the obtained bacteria count was lower when cultured in common broth at same condition.Using different batches of medium,the difference among obtained bacteria count was larger while cultured in common broth than in synthetic medium.The S.Choleraesuis strain CVCC79500 was cultured in synthetic medium at 37 ℃/200 r/min for 12 h.The mice was inoculated with the harvested bacteria and showed a survival ratio (number of alive species /number of vaccinated species)of 100% (10 /10).The inoculated mice were challenged with virulent strain,and had a protective ratio (number of protected species /number of challenged species)of 80% ~100%(4 /5 ~5 /5).The safety and the protective efficacy of the strain cultured in synthetic medium were coincident with the strain cultured in common broth.The obtained bacteria count via shaking culturing was similar while the synthetic medium was kept in dark at 25 ℃ for 28 days,compared to fresh prepared synthetic medium. Therefore,the synthetic medium was evaluated under condition of obtained optimum parameter for production of paratyphoid live vaccine.The synthetic medium was better than the common broth,as higher bacteria count was obtained in the former than in the latter.Moreover,the synthetic medium had no influence on safety and immunogenicity of harvested bacteria.The preservation period of the synthetic medium could reach 28 days at room temperature and away from light.

10 lesioned lung tissues from typical swine mycoplasmosis were grinded,cleaned,and subjected to isolation,and a mycoplasma strain was obtained.The mycoplasma was proved to be Mycoplasma hyopneumoniae (Mhp)by culture character,serological identification,biochemical identification,PCR test and sequencing.The purefied isolated strain was named Mhp strain S.Afterwards,Homology comparison was performed on genetic sequence and amino acid sequence of P46 and P97 R1 region with the available sequence of other strains,the results show that the amino acid sequence of P46 gene is more than 99% homology with other strains,the number of P97 R1 region amino acid repeat sequence of the Mhp strain S is 11,different from other strains.The immunogenicity test results showed that the isolation strain had good immunogenicity.

For higher efficiency and lower cost of the protein production, response surface analysis(RSA) was used for culture optimization of the recombinant E. coli which could express Mycoplasma hyopneumoniae P46 protein, and the best ratio of each medium component was determined. Under the same culture conditions, the cell concentration of the optimized culture is twice higher than LB medium. The medium used in a bioreactor for the recombinant E. coli DE-pET-P46 culture fermentation, the wet bacteria weight of the culture is up to 39.5 g/L. SDS-PAGE electrophoresis showed that there is no obvious difference on the rP46 expression of the recombinant E. coli DE-pET-P46 between cultivated with bioreactor and with shaking incubator.

OBJECTIVE A new method was introduced for precise determination of the live cell titer of mycoplasma culture, and would be a candidate to replace the commonly used CCU (color change unit) assay. METHODS The CCU50 (50% color change unit ) was modified according to the method of TCID50 (50% tissue culture infective dose) assay used for viral titer assessment, and adopted to estimate the live cell titer of mycoplasma. Sensitivity and reproducibility of the CCU50 assay were assessed, and adaptability was checked with M. hyopneumoniae and M. synoviae. RESULTS The CCU50 assay showed better reproducibility, sensibility and adaptability than traditional CCU assessment approaches. CONCLUSION The method could be applied to accurate titration determination for mycoplasma, and might be considered as a useful tool for the research of high density fermentation of mycoplasma and development of vaccine.

为明确生产用培养基对猪丹毒杆菌G4T10株安全性及免疫原性影响,采用2种不同批次的肉肝胃膜消化汤(20120123批和130226批)及马丁琼脂(20120904批和130129批)进行猪丹毒杆菌G4T10株培养和选菌,然后进行安全及免疫原性试验.结果共筛选出4种不同优势菌落,其中一株的菌落(20120123批肉肝胃膜消化汤+20120904批马丁琼脂)形态最好,具有良好的安全性及免疫原性(小鼠全试验9/10存活,其免疫后攻毒达9/9保护);而有一株菌落(20120123批肉肝胃膜消化汤+130129批马丁琼脂)形态最差,其安全性及免疫原性试验结果差(小鼠安全试验仅6/10存活,其免疫后攻毒仅达2/5保护).试验表明,不同批次生产用培养基对猪丹毒活疫苗(G4T10株)质量影响大.

In this review article,we introduce the overview of etiology,epidemiology,clinical symptom and pathological change,pathogenesis,diagnosis,treatment and prevention,progress in immunization of NE in details.We also focus on the prospect in the development of research in the future.This review article provides knowledge for the basic research,prevention and control of NE.

To define the parameters in the synthesized culture medium for Clostridum perfringens C type and the ultrafiltration of cultured toxin,the experiment compared the toxin activity of Clostridum perfringens CVCC60102 cultured in the synthesized medium prepared with different pH,sterilisation temperature,water type,also under condition of different culture time and temperature,and designed two different methods to concentrate the toxin according to the molecular weight and the MWCO(Molecular Weight Cut Off) of the ultrafiltration membrane,and compared the harvest ratio and activity of toxin obtained by the previous two methods.Results showed that the optimal synthesized culture medium should be prepared with deionic water at the most suitable pH of 8.0~8.4 and autoclaved for 30 min at 116 ℃.For the strain(CVCC60102) of Clostridum perfringens,the toxic activity reached 500~1000 MLD/mL after growth for 18 hours.The toxicity did not increase with even longer growth time.The highest level of toxicity would be reached when cultured at 36 or 37 ℃.When concentrated by the 10 ku MWCO membrane,the harvest ratio of toxin was 68%,and the discarded effluent resulted in 2/2 lethal ratio on rat injected by 0.2 mL in vein.While by 8 ku MWCO,the harvest ratio was 80% and the discarded effluent had no lethal activity.Therefore,the ultrafiltration with membrane of 8 ku MWCO was suitable for the concentration of the cultured toxin.The above results provided the data support for the application of the synthesized culture medium for Clostridum perfringens C type,and these would be consulted for the manufactural application of corporations.

The optimal conditions for the synthesized culture medium of Clostridium perfringens A type is confirmed that the most suitable pH is about 8.0～8.5;the method of sterilisation in autoclave is 116 ℃,30 min;the used water is deionic water.For the strain(CVCC37) of Clostridium perfringens,the toxin activity attains 50～100 MLD/mL after growing in optimized medium for 6 hours,the toxic activity does not increase with even longer growth time;when compared with different culture temperature,the highest level of toxin activity at 36 ℃ or 37 ℃ is researched.Ultrafiltration with different membrane by MWCO(Molecular Weight Cutoff) is used to concentrate the culture supernatant,the harvest ratio of the 10 ku membrane by MWCO is 40%,and the discarded effluent results in 2/2 lethal ratio on rats injected by 0.2 mL in vein;while the harvest ratio of the 8 ku membrane by MWCO is 75% and the discarded effluent has no lethal activity;Therefore,the ultrafiltration with 8 ku membrane by MWCO is suitable for the ultrafiltration of cultured toxin produced by Clostridium perfringens A type.