Optimization of a molecular technique for diagnosis of bovine Eimeria species based on Internal transcribed spacer region

The most economically significant disease of cattle throughout world is Bovine coccidiosis caused by Eimeria infection. There have been no molecular techniques for detection of this parasite in IRAN yet. Hence, the purpose of the current study was to set up a molecular method-based on ITS1 gene for detection of this pathogenic agent in calves samples.In first step, 164 available ITS1 nucleotide sequences from eleven Eimeria species were collected from GenBank. Complete ribosomal DNA sequences with 2984 bp includes 18srRNA, ITS1, 5.8srRNA, ITS2 and 28srRNA were compared between different Eimeria species. The conserve and hypervariable regions were identified.A set of novel degenerated primers was designed in conserved sequences of flanking hypervariable region of internal transcribed spacer 1 based on multiple alignments of all species of Eimera ITS1 nucleotide sequences. Ten stool samples were collected from infected bovine from Alborz province. Oocysts were separated from the fecal debris and concentrated by flotation technique using saturated sucrose solution.Genomic DNA was extracted by MBST kit. The PCR condition was optimized. The presence of Eimeria genus genomic DNA was tested by PCR amplification of the ITS1 sequence using the universal primer.The results of our study were showed that these Novel degenerated primers might be used for amplification of hypervariable region of ITS1 and diagnosis of Eimeria.This is the first intention for the identification of Eimeria parasites in the genomic level in Iran thus provide as useful methods for diagnosis and help implement strategies to control the parasites.