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一种地方性疾病，在世界大部分热带和亚热带地区造成巨大的经济损失。本研究对牛巴贝斯虫和马巴贝斯虫的连续培养进行了研究。在低氧环境，5% O2,5% CO2和90% N2条件下，在添加40%成牛血清的培养基199中培养卵形巴贝斯虫，使其在成年牛红细胞内增殖。在低氧环境（2%O2、5%CO2和93% N2）中，分别在M199和RPMI 1640培养基中，添加2 mM l -谷氨酰胺、20 mM HEPES和40%成年马血清，对马巴贝斯虫和卡巴利贝斯虫进行体外连续培养。将培养物从低氧环境转移到空气中5%的二氧化碳环境中，所有寄生虫的生长都得到了增强。随着马b.b.和caballi体外培养的成功建立，体外繁殖的马b.b.和caballi感染的红细胞被用作IFAT的抗原。在日本的血清样本中，没有一例血清稀释率为1:80的阳性反应。然而，19例微量或假阳性中有3例(1)SY.+-。[))例观察为伪阳性([]SY.+-。[) -+]在马b.a i和/或caballi b.a i的寄生虫周围发出微弱而尖锐的荧光。抗体调查结果显示，马双歧杆菌和卡巴利双歧杆菌在蒙古中部马中均有流行，81.8%的马对这两种感染呈血清阳性。制备了抗马b.a equi和caballi b.a的单克隆抗体（MAb）。其中一种抗体在体外可识别马布氏杆菌19kDa抗原，抑制马布氏杆菌的增殖。该单抗目前正用于纯化相应抗原，以便作为ELISA抗原进行检测。

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.