Isolation of Theileria sergentipiroplasms from infected erythrocytes and development of an enzyme-linked immunosorbent assay for serodiagnosis of T sergentiinfections

A method of obtaining a pure suspension of Theileria sergentipiroplasms from infected bovine erythrocytes was developed and the resulting parasites used as antigen in an enzyme-linked immunosorbent assay (elisa). Piroplasms were freed from infected erythrocytes using the nitrogen cavitation technique and then separated from unbroken erythrocytes by centrifugation at 670 gmax. The parasites in the supernatant were then sedimented by centrifugation at 2700 gmaxand the purified fraction examined by electron microscopy. This examination showed that the isolated piroplasms were morphologically intact and that there were few contaminants. sds-pageand spectrophotometric analysis showed that this fraction contained little erythrocyte component. The piroplasms thus obtained were sonicated and treated with Triton X-100 then used for elisaantigen. The elisavalues had a high correlation with titres obtained using the indirect fluorescent antibody test on sera from cattle infected with T sergenti.