Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 16 2 2021 05 28 295 304 EN Asal Tanzifi Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran Yousof Dadimoghadam Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran Samira Dodangeh Department of Medical Parasitology and Mycology, Medical Microbiology Research Center, Qazvin University of Medical Scienc-es, Qazvin, Iran Ahmad Daryani Toxoplasmosis Research Center, Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran Shahabeddin Sarvi Toxoplasmosis Research Center, Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran Shirzad Gholami Toxoplasmosis Research Center, Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran Seyed Abdollah Hosseini Toxoplasmosis Research Center, Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran Tooran Nayeri Chegeni Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran Zahra Hosseininejad Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran 2020 04 03 2021 03 08 Background: Free-living amoeba (FLA) belonging to Acanthamoeba spp., Naegleria, and Balamuthia mandrillaris are the soil-born protozoa. This study aimed to survey the occurrence of FLA, including Acanthamoeba spp., B. mandrillaris, Vermamoeba spp., and Naegleria spp., in soil samples collected from various districts of Mazandaran Province (Northern Iran) from July to December 2018. Methods: Overall, 118 soil samples from the recreational and public places were surveyed for the existence of Acanthamoeba spp., Vermamoeba, Naegleria, and B. mandrillaris using both morphological key and molecular tools with genus-specific primers of JDP1, NA, ITS1, and Bal, respectively. To verify the taxonomic status of isolated amoeba, the phylogenetic tree was made based on sequences of 18S rRNA by MEGA (5.05) software with the maximum likelihood model. Results: Overall, 61/118 samples (51.6%) were contaminated with FLA, and based on the sequencing data, 29 isolates were successfully sequenced. Among the samples, all isolated Acanthamoeba (52.4%) belonged to the T4 genotype with amplification of the DF3 region (18S rRNA gene). Internal transcribed spacer (ITS) sequencing revealed the presence of one strain of Naegleria americana. Twenty-eight V. vermiformis were also confirmed based on Nuclear SSU rDNA. Morphological survey and PCR assay did not show any positive samples for B. mandrillaris. Conclusion: The present study indicates the occurrence of FLA in soil sources of the recreational and public places in Mazandaran province that it can be a severe risk to human health. Thus, more studies are expected to survey the infection source in patients with FLA-related diseases. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/2875 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/2875/1135