Establishment of continuous in vitro cultivation of bovine and equine Babesia parasites and it's application to diagnostic methods

牛和马的巴贝斯虫病是地方性的，在世界上大多数热带和亚热带地区造成巨大的经济损失。在低氧、5%O_2、5%CO_2和90%N_2条件下，用含40%成年牛血清的199培养基在成年牛红细胞中诱导卵巴氏疟原虫增殖，并分别用M199和RPMI 1640培养基对马巴氏疟原虫和B.caballi进行体外连续培养。补充有2 mM L-谷氨酰胺、20 mM HEPES和40%成年马血清的动物模型。将培养物从低氧环境转移到5%CO2空气中可促进所有寄生虫的生长。随着B. equi和B.caballi体外培养物的成功建立，体外繁殖的B.equi和B.caballi感染的红细胞被用作IFAT中的抗原。未显示1：80份血清稀释度在日本的血清样品中，然而，19份微量或假阳性中有3份（（SY. ±. [))假阳性例（SY. ±.）。[）-+）在马B.equi和/或B. cabali寄生虫周围的弱而尖锐的荧光中。抗体调查表明，马B.equi和B.caballi在蒙古中部的马中流行，81.8%的马对这两种感染呈血清阳性。制备了抗马双歧杆菌和卡氏双歧杆菌的单克隆抗体。其中一株单抗能识别马B菌19 kDa抗原，并能抑制马B菌的体外增殖。该单克隆抗体目前正被用于纯化相应的抗原，以作为ELISA检测抗原。

Babesiosis in cattle and equine is endemic and causes enormous economic losses throughout most of the tropical and subtropical regions of the world. In this study, establishment of continuous cultivation of bovine and equine Babesia parasites were examined.Babesia ovata was initiated their multiplication within adult bovine RBC in Medium 199 supplemented with 40% adult bovine serum under a low oxygen atmosphere, 5% O2,5% CO2 and 90% N2.Babesia equi and B.caballi were continuously cultured in vitro in M199 and RPMI 1640 medium respectively, supplemented with 2 mM L-glutamine, 20 mM HEPES and 40% adult horse serum in a low oxygen atmosphere (2%O2,5%CO2 and 93% N2). All parasite growth was enhanced by transfer of cultures from a low oxygen to 5% CO2 in air.With the successful establishment of in vitro cultures for B.equi and B.caballi, in vitro propagated B.equi and B.caballi infected erythrocytes were used as antigen in the IFAT.None showed a positive reaction of a 1 : 80 serum dilution In the serum samples in Japan However, 3 of the 19 trace or false positive ((]SY.+-。[)) cases were observed as pseudo-positive ((]SY.+-。[) -+) in a weak and sharp fluorescence around the parasite of B.equi and/or B.caballi. Antibody survey indicated that both B.equi and B.caballi are endemic in horses in Central Mongolia with 81.8% of horses being seropositive to both infections. Monoclonal antibodies (MAb) were produced against B.equi and B.caballi. One of MAbrecognized 19kDa antigen of B.equi and inhibited the multiplication of B.equi in vitro. This MAb is now being utiuzed to purify the corresponding antigen in order to test as antigen for ELISA.