Development Of Continuous Cultivation In Vitro And An ELISA For Diagnosis Of Babesia Bovis

Babesiosis is the general term of hematic protozoosis which is caused by parasites in Babesiagenus parasitizing in host red blood cell（RBC）. It is a tick-born diease with great significance to thedevelopment of animal husbandry. In China there are five Babesia spp. parasitizing in cattle， B. bovis，B. bigemina， B. ovata， B. major and Babesia sp. in which B. bovis is one of the most pathogenic. Inthis study the continuous cultivation of B. bovis was established. An ELISA was developed to detect theantibody level by using antigens from culture supernatants. The C-terminal truncated recombinantantigens of B. bovis was expressed to establish a new diagnostic method. The major results werepresented as following:1. The continuous cultivation of Babesia bovis Shanxian maintained in an incubator at37℃and50mL/L CO2for more than9month by changing the culture medium and supplying RBCs， thepercentage of parasitized erythrocytes(PPE) peaked at13.5%. The morphology of B. bovis is the samein vitro and in vivo. Growth characteristics in vitro were measured by a microtiter-basedspectrophotometric method and from the PPE. The generation time in bovine erythrocytes was between33.66h and38.51h. Furthermore， the host range of parasite was identified with in vitro culture.Erythrocytes of cattle，humans，donkey and sheep could be invaded into by B.bovis Shanxian in vitro，and the parasites could only propagate in cattle erythrocytes. Goat erythrocytes were refractory to theinvasion of B. bovis in vitro.2. An ELISA was developed with merozoite antigens of B. bovis Shanxian purified from in vitroculture. It demonstrated a specificity of98.1%and a sensitivity of96.2%. Except for Theileria sergenti，there was cross-reaction between merozoite antigens and positive sera from B. bigemina， B. ovata， B.major and Babesia sp. Xinjiang，therefore the method has the versatility to detect Babesia. A total of1692serum samples from12provinces in China were detected by this method， the results show that:1)the positive sera samples were found in every provinces， the average positive rate was6.7%，2)Henan and Guangdong exhibited35.9%（28/78）and21.3%（23/108） positive rate respectively，3)Qinghai and Tibet has the lowest positive rate with1.91%（4/209）and0.89%（2/225）.3. The C-terminal truncated recombinant antigens of B. bovis Shanxian rhoptry-associatedprotein-1(BoRCT) were expressed in prokaryotic system. Western blot result showed that therecombinant protein has a good antigenicity with B. bovis and no cross-reaction with B. bigemina. Insummary the BoRCT could be used in ELISA to identify B. bovis and B. bigemina.