High prevalence of Acanthamoeba spp. in agricultural soil of barberry farms: a public health concern in eastern Iran.

Acanthamoeba spp. are ubiquitous environmental amoebae and opportunistic pathogens responsible for severe human infections such as keratitis and encephalitis. Their resilient cysts accumulate in soil, creating a reservoir of public health concern, especially in agricultural regions with high human activity. This study presents the first global assessment of Acanthamoeba spp. prevalence, genotypes, and phylogenetics in barberry agricultural soils. We collected soil samples from barberry fields in Birjand, Iran. Acanthamoeba spp. were isolated by filtration and cultured on non-nutrient agar with E. coli, then identified microscopically. DNA was extracted from positive cultures, and a diagnostic fragment of the 18S rRNA gene was amplified using JDP primers for sequencing and genotyping. Phylogenetic and population genetics analyses (Tajima's D, haplotype network) were performed using MEGA11 and DnaSP software. Acanthamoeba spp. were identified in 65 of 164 soil samples (39.6%). PCR confirmed all isolates, with sequencing of 10 isolates revealing exclusive T4 genotype identification. Population genetics (π = 0.02079, Tajima's D = 2.46922) indicated balancing selection within this population. Haplotype network analysis demonstrated genetic proximity between these soil isolates and strains from clinical (keratitis) and aquatic sources. This confirms a shared, circulating T4 population in the region. These findings highlight the urgent need for targeted public health interventions in endemic agricultural regions.