Genotyping of Toxoplasma gondii using GRA6 gene-based RFLP-PCR in raw milk from four livestock species in Sistan and Baluchestan Province, Iran

Background Toxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan Toxoplasma gondii , which is one of the primary causes of abortion, congenital infections, and stillbirth in animals and humans. In this study, we investigate the genetic diversity of T. gondii using the GRA6 gene in raw milk samples collected from various livestock in Sistan and Baluchestan Province, Iran. Methods A total of 224 raw milk samples (64 from cows, sheep, and goats; 32 from camels) were aseptically collected from animals in different herds across the province in 2024. Genomic DNA was extracted, and the presence of T. gondii was identified using nested-PCR targeting the GRA6 gene. Positive samples were genotyped using PCR-Restriction Fragment Length Polymorphism (RFLP) with the MseI enzyme. Representative amplicons were sequenced for phylogenetic confirmation. Results The molecular prevalence of T. gondii DNA was 11 samples (4.9%). RFLP analysis and subsequent sequencing revealed that all positive isolates belonged to genotype I of T. gondii . Conclusion This study confirms the presence of T. gondii DNA, particularly the potentially pathogenic genotype I, in raw milk from ruminants, especially small ruminants, in the region. Public health measures should urgently emphasize the risks of raw milk consumption.