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 <i>Leishmania major</i> (zoonotic cutaneous leishmaniasis, ZCL) and <i>L. tropica</i> (anthroponotic cutaneous leishmaniasis, ACL). This study investigated transmission drivers, including sand fly vectors, zoonotic and environmental factors, and the genetic diversity of <i>L. major</i>. A total of 1029 sand flies were collected indoors and outdoors in Shiraz from August to October using sticky traps. Samples from patient lesions (<i>n</i> = 30) and pooled female sand flies (<i>n</i> = 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 <i>Phlebotomus papatasi</i> (53.8%) and <i>Ph. sergenti</i> (38.7%). PCR detected <i>L. major</i> DNA in 80% of <i>Ph. papatasi</i> pools and both <i>Leishmania</i> species in <i>Ph. sergenti</i> pools; however, molecular detection alone does not confirm vector competence. All patient samples were confirmed positive for CL. ITS2 sequencing identified 22 haplotypes among <i>L. major</i> 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 (<i>Ph. papatasi</i> and <i>Ph. sergenti</i>) 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.