Development of a multiplex polymerase chain reaction technique for detection and discrimination of Eimeria spp. in cattle in Indonesia.

Background and Aim: Bovine eimeriosis is a disease caused by apicomplexan parasites of the genus Eimeria. It is one of the most important and widespread bovine illnesses in the world. Some of the identified species of bovine eimeriosis have morphologically similar oocysts that are difficult to differentiate. For the identification of particular Eimeria spp., diagnostic laboratories are increasingly turning to DNA-based technology. This study aims to develop a multiplex polymerase chain reaction (mPCR) technique based on the internal transcribed spacer-1 (ITS-1) gene for the simultaneous identification of pathogenic Eimeria spp. in cattle from Sulawesi Island, Indonesia. Materials and Methods: Genomic DNA was extracted by the DNAzol reagent from the purified Eimeria oocysts. Species-specific primers targeting the ITS-1 region were used to amplify the distinct Eimeria spp. Results: Using PCR ITS-1, this study showed that 36 of 120 fecal samples (30%) were infected by Eimeria spp. The multiplex PCR assay allowed for the simultaneous identification of six major Eimeria spp. in a single-tube reaction. The proportion of mixed Eimeria spp. infections was 100% (36/36). The maximum number of Eimeria spp. was five, and the minimum number was two. Conclusion: Identification of six pathogenic Eimeria spp. in cattle was successfully carried out by nested multiplex PCR using ITS-1 gene. In the future, a procedure to detect pathogenic Eimeria spp. in one tube reaction will offer economical and save diagnostic time.