Simultaneous differentiation between Theileria spp. and Babesia spp. on stained blood smear using PCR.

The tick-borne diseases of livestock constitute a complex of several diseases with different etiological agents. Theileriosis and babesiosis belong to this complex and are severe and often fatal protozoan tick-borne diseases of ruminants worldwide. This results in high economical losses yearly in Iran. The most common diagnostic method for the identification of piroplasms in Iran is Giemsa staining of blood smear, which is unspecific, accompanied by some technical problems and, in some cases, impossible, due to the carriers. In contrast, immunostaining is more specific and can only be performed with suitably prepared blood smears, but cannot be used also for the carriers. The most specific method for the differential diagnosis of piroplasms is the method of polymerase chain reaction. We extracted DNA from different sources of blood samples, including from already stained blood smears. The extracted DNA was subsequently amplified using specific primers derived from Theileria heat shock protein hsp70, Theileria lestoquardi ms1-2 gene, Babesia rhoptry protein gene and piroplasms hyper variable region V4 of 18S rRNA gene. The results show that it is possible to detect piroplasms in already stained blood smears as well enabling a simpler method to be developed for the collection of the samples. Furthermore, it is possible to analyse the already stained and registered blood smears from the patients with unclear differential diagnosis, e.g. in the carriers. In addition, the results revealed that using a primer designed from the hyper variable region V4 of 18S rRNA, it is possible to detect and differentiate simultaneously the genera Theileria and Babesia in DNA samples isolated from already stained blood smears.