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

牛马巴贝虫体外连续培养方法的建立及其在诊断方法中的应用

• 批准号: 06660397
• 负责人: IGARASHI Ikuo
• 金额: $ 0.32万
• 依托单位: Obihiro University Agriculture and Veterinary Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660397/
• 关键词: Babesia ovata; Babesia equi; Babesia caballi; In vitro culture; IFAT; Antibody survy; Monoclonal antibody; 酵素抗体法(ELISA); ウマバベシア; in vitro連続培養; 低酸素; 凍結保存

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.

牛和马中的巴贝病是内在的，在世界上大多数热带和亚热带地区造成了巨大的经济损失。 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培养基分别在低氧气气氛中补充了2 mM L-谷氨酰胺，20 mM HEPES和40％的成年马血清（2％O2,5％CO2和93％N2）。通过将培养物从低氧气转移到空气中的5％二氧化碳的转移来增强所有寄生虫的生长。与equi和caballi的体外培养物的成功建立，体外繁殖的b.equi和b. caballi感染的红细胞在1：80 serum dipution中用作1：80 serum dipution的抗原反应。 （（]sy。+ - 。[））病例被观察为伪阳性（（]SY。+ - 。[） - +），在B.Equi和/或b.cavalli的寄生虫周围的弱而尖锐的荧光中。抗体调查表明，蒙古中部的马匹和b.cavalli均是内在的，其中81.8％的马与两种感染都呈阳性。单克隆抗体（mAb）是针对b.equi和b.cavalli产生的。 Mabry认可的一种19kDa抗原的抗原，并抑制了B.Equi在体外的繁殖。现在，该mAb正在净化相应的抗原，以测试ELISA的抗原。

项目成果

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Avarzed, A.、五十岚, I.、Kanemaru, T.、Hirumi, K.、Hirumi, H.Omata, Y.、Saito, A.、Oyamada, T.、Nagasawa, H.、Toyoda, Y. 和 Suzuki

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