Zoonotic transmission and genetic diversity of Leishmania major in Shiraz, Iran: An integrated entomological, clinical, and molecular study

Cutaneous leishmaniasis (CL), endemic in Shiraz, Iran, is caused by Leishmania major (zoonotic cutaneous leishmaniasis, ZCL) and L. tropica (anthroponotic cutaneous leishmaniasis, ACL). This study investigated transmission drivers, including sand fly vectors, zoonotic and environmental factors, and the genetic diversity of L. major . A total of 1029 sand flies were collected indoors and outdoors in Shiraz from August to October using sticky traps. Samples from patient lesions ( n = 30) and pooled female sand flies ( n = 40 pools) were examined by microscopy and screened by PCR targeting kDNA and ITS2 genes. Phylogenetic and haplotype analyses were conducted based on ITS2 sequences. The sand fly fauna was dominated by Phlebotomus papatasi (53.8%) and Ph. sergenti (38.7%). PCR detected L. major DNA in 80% of Ph. papatasi pools and both Leishmania species in Ph. sergenti pools; however, molecular detection alone does not confirm vector competence. All patient samples were confirmed positive for CL. ITS2 sequencing identified 22 haplotypes among L. major strains, 20 of which were detected in Iran, indicating relatively high haplotype diversity (Hd = 0.74). A dominant ancestral haplotype was shared across Asia, Africa, and Europe. These findings suggest that ZCL transmission in Shiraz is likely associated with peridomestic sand flies ( pH. papatasi and Ph. sergenti ) and environmental conditions that may facilitate transmission. Iran accounted for 91% of the identified haplotypes in this dataset, highlighting its substantial contribution to regional ITS2 diversity. Continued vector surveillance and improved environmental management may support more effective control strategies.