畜牧兽医学报 ›› 2008, Vol. 39 ›› Issue (8): 1142-1146.doi:

• 研究简报 • 上一篇    

猪附红细胞体的体外连续培养

李晓云1,贾杏林3,石德时1,肖运才1,胡思顺1,刘梅1,袁宗辉1,毕丁仁1, 2   

  1. 1.华中农业大学动物医学院, 武汉 430070; 2.农业微生物学国家重点实验室, 武汉 430070;3.湖南农业大学动物医学院,长沙 410128
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2008-08-24 发布日期:2008-08-24
  • 通讯作者: 毕丁仁

Continuous in vitro Cultivation of Mycoplasma suis

LI Xiao-yun1,JIA Xing-lin3,SHI De-shi1,XIAO Yun-cai1,HU Si-shun1,
LIU Mei1,YUAN Zong-hui1, BI Ding-ren1, 2
  

  1. 1.College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China;2. State Key Laboratory of Agricultural Microbiology, Wuhan 430070, China;3.College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128,China
  • Received:1900-01-01 Revised:1900-01-01 Online:2008-08-24 Published:2008-08-24
  • Contact: BI Ding-ren

摘要: 临床疑似猪附红细胞体病猪红细胞,经Giemsa染色镜检及PCR扩增,证实感染猪附红细胞体(M.suis)。PCR扩增片段(781 bp)与M.suis已知序列(AJ504999)同源性达99.4%。经预试验对红细胞接种量、血清添加量、pH等条件优化后,选用添加新生牛血清、酵母浸出液、葡萄糖等成分的PPLO肉汤,将染虫红细胞接种到细胞培养板上,置于37 ℃、含5%CO2的培养箱培养,定期更换培养液,约72 h后红细胞溶血变为带虫血影(Ghost)。除首次接种外不添加任何红细胞,连续培养达280 d以上,染虫率维持近100%。对培养的带虫血影进行了光镜及扫描电镜观察、PCR鉴定、感染阴性红细胞试验、抗生素敏感性试验,证实与接种物为同一病原,并保持有感染阴性红细胞的能力。

Abstract: Organisms obtained from symptomatic pig with clinical signs typical of porcine eperythrozoonosis, were Mycoplasma suis (M. suis), as was confirmed by microscopic examination of Giemsastained peripheral blood smears as well as by PCR. A fragment of 781 bp was amplified and its similarity to a known sequence of M. suis(accession numbers AJ504999)was 99.4%. Continuous culture of the pathogen was established from the infected blood sample. The culture medium was PPLO broth medium supplemented with newborn calf serum, yeast extract, glucose ,etc.. The optimal dilution of red blood cell (RBC), ratio of serum and pH during incubation were evaluated. Cultivation was initiated in an atmosphere containing 5% CO2 at 37 ℃. The infected RBCs became ghosts with parasites after 72 h of cultivation. No host erythrocyte was supplied in the cultures except initiation. Cultures were maintained in medium ,with a percentage of parasitized erythrocytes(PPE) nearly 100% for more than 280 days. The cultured organisms adhering to ghost was identified to be M. suis on the basis of light and electron microscopic examinations, identical gene sequences, infect negative RBCs test, antibiotics sensitivity test.