Development of a rapid and cost-effective Allele-Specific PCR assay targeting the 18S rRNA gene for differential detection of Sarcocystis species in Cattle and Water Buffalo, and phylogenetic analysis of macrocyst-forming species in cattle in Iran.

Sarcocystis species are highly prevalent in bovine intermediate hosts, represent an important veterinary and economic concern. The most documented losses are related to condemnation of Sarcocystis infected carcasses because of macroscopic sarcocysts or infection associated BEM (Bovine eosinophilic myositis) lesions. Previous studies have indicated some similarities between Sarcocystis species in cattle and water buffaloes. Differentiation of morphologically or genetically similar species in related hosts, remains a key diagnostic challenge. Most of the current distinguishing methods are complicated or expensive. Moreover, sanger sequencing of PCR amplicons alone not suitable for species identification in mixed infections. Although different loci such as 28SrDNA, ITS or cox1 are widely used for phylogenetic analyses, the 18 S rRNA gene remains a reliable marker due to its conserved and variable regions. So, this study aimed to develop a rapid and cost-effective allele-specific PCR (AS-PCR) method based on polymorphic sites within the 18 S rRNA gene to differentiate between species in cattle and water buffalo. Specific primers were designed based on differences found at the proper position as SNPs (single nucleotide polymorphisms). SNPs were identified based on multiple sequence alignment of GenBank published 18 S rRNA gene sequences of 65 isolates and clones of 12 recognized Sarcocystis species in cattle and water buffalos. The method uses one forward and two reverse primers, allowing clear molecular separation between macrocyst-forming species in both cattle and water buffalo. Notably, this AS-PCR enables molecular differentiation of these macrocyst-forming species from at least eight microsarcocyst-forming species without the need for sequencing. The assay successfully distinguished S. hirsuta and S. fusiformis in their respective hosts. Interestingly, a sequence closely related to S. fusiformis was also detected in some cattle samples. Phylogenetic analysis revealed that this isolate clustered closely with S. fusiformis but in a distinct clade, leading to its identification as S. fusiformis-like. This is the first molecular report of such an isolate in cattle in Iran.