An indirect ELISA for detection of Theileria sergenti antibodies in water buffalo using a recombinant major piroplasm surface protein

In this study we investigated the prevalence and enzootic potential of Theileria spp. in water buffalo in the Hubei province in China. An indirect ELISA based on a recombinant major piroplasma surface protein was developed. The complete ORF of the 33-kDa major piroplasma surface protein (p33) was obtained from Theileria sergenti genomic DNA by PCR, cloned into the pET-28(a) vector and expressed in E. coli as a His-fusion protein. Then the recombinant p33 (rp33) was purified and used as the antigen to develop an iELISA. Specificity test showed that there was no cross-reaction with Babesia orientalis, Schistosoma japonicum, Anaplasma marginale and Toxoplasma gondii. 178 water buffaloes raised in different locations in Hubei province in China were detected by this iELISA, all samples were also examined by PCR and microscopy at the same time. The iELISA result showed a higher positive rate (27.5%) than PCR (22.5%) and microscopy (12.9%). This result indicated that the iELISA is a suitable method for the diagnosis of T. sergenti infection and could be used in serological surveys to map out the prevalence of the disease.