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

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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.