Abstract
BackgroundBovine babesiosis and theileriosis is an important hemoprotozoal disease in cattles and yaks in tropical and subtropical regions leading to significant economic losses. In the field, the risk of co-infection between the bovine Babesia and Theileria species is very high. Thus, it is necessary to develop a simple, accurate, rapid and cost-effective method for large-scale epidemic investigation, in particular for the detection of co-infection in field.MethodsIn this study, DNA sequences of a ribosomal protein S8 (RPS8) gene from eight species of cattle piroplasms in China were used to develop a species-specific PCR-RFLP diagnostic tool. The eight Theileria and Babesia species could be differentiated by digesting the RPS8 PCR product with Mbo I.ResultsThe sensitivity of the PCR assays was 0.1 pg DNA for Babesia species but 1 pg DNA for Theileria species. The clearly different size of the PCR-RFLP products allowed for a direct discrimination between eight bovine Theileria and Babesia species (T. annulata, T. sinensis, T. sergenti, B. ovata, B. bovis, B. bigemina, B. major and Babesia species Kashi isolate).ConclusionOur results indicated that the established method based on the RPS8 gene was a reliable molecular diagnostic tool for the simultaneous detection and identification of bovine Babesia and Theileria species in China, which could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.
Highlights
Bovine babesiosis and theileriosis is an important hemoprotozoal disease in cattles and yaks in tropical and subtropical regions leading to significant economic losses
polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis PCR amplification of ribosomal protein S8 (RPS8) gene from the DNA yielded a product of 709 bp for T. annulata isolates, 713 bp for T. sergenti Lushi isolate, 707 bp for T. sinensis isolates
RFLP will clearly distinguish among Babesia- and Theileria- infected cattles
Summary
DNA extraction The calves, aged between 12 and 24 months old, were infected by inoculating 5 ml of cryopreserved infected blood stock of these Babesia and Theileria isolates into the jugular vein. Parasite DNA was extracted by conventional phenol/chloroform for deproteinization of the aqueous solution containing the desired nucleic acid. Each PCR mixture (total volume, 50 μl) contained 5 μl of 10 × PCR buffer, 6 mM MgCl2, deoxynucleoside triphosphate at a concentration of 200 μM each, primer at a concentration of 200 nM each, 2.5 U of Taq polymerase, and 20 ng of DNA template. 20 μl of the PCR products was digested in a 50-μl reaction mixture containing 20 U of MboI (Takara) and 5 μl of the appropriate restriction buffer at 37 °C for 1 h, under conditions recommended by the supplier. Image analysis of the electrophoretic gels was performed with 1day Manager Software (TDI, Madrid, Spain)
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