Brucellosis, a zoonotic disease caused by Brucella spp. globally, is of great significance not only to livestock but also to public health. The most significant of the twelve species is Brucella melitensis. This article is devoted to the endemic region of Iran and aims to uncover the molecular epidemiology of B. melitensis. Biotyping, AMOS-PCR, Bruce-ladder PCR, and the in silico method of MLVA were employed to test 40 B. melitensis isolates from humans, cows, sheep, goats, camels, and horses which are found in thirteen Iranian provinces throughout the years 2015 to 2022. The data from the MLVA-8 analysis showed that there were seven genotypes that could be identified, and the most commonly identified genotype was genotype 63. The data from the MLVA-10 analysis showed that there were seven genotypes, with genotype 213 being the most prevalent in Iran. The data from the MLVA-11 analysis showed that there were eight genotypes, with genotype 111 being the most prevalent in Iran. The MLVA and SNP analysis results showed that the bacteria were grouped into two main groups, known as the Eastern Mediterranean and American groups. Moreover, the outcomes from these analyses have added considerably to our understanding of the genetic/historical relationships among the isolates. Our study indicates a high prevalence of B. melitensis biovar 1 in Iran, accounting for 82.5% of the isolates. The study provides insight into such matters as the complex epidemiology of B. melitensis in Iran, suggesting different ways of transmission and sources of infection. This research points out the vital significance of the continuation of the surveillance and curation of B. melitensis in the diverse species of animals and humans. The simplicity and efficiency of MLVA-based molecular epidemiology offer information on the geographic distribution and genetic diversity of B. melitensis and, therefore, help in the devising of targeted strategies for the prevention of disease in animals.
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