1. Introduction
canthamoeba spp, can cause severe and fatal diseases including encephalitis, skin granulomatous and keratitis [1]. Water can be a source of Acanthamoeba spp.; thus, contaminated waters can be a threat to high risk people such as contact lens wearers, patients with eye surgery or eye trauma, and immunocompromised patients [2]. There are numerous examples of microorganisms such as Vibrio cholera and Legionella which survive, grow, or proliferate inside amoebae [3]. Northern Iran including Guilan Province, attract many tourists due to its mediterranean climate and existence of many recreational beaches; however, there is no report regarding the presence of Acanthamoeba genotypes in coastal waters of Caspian Sea. 
The present study aims to investigate the presence of potentially pathogenic Acanthamoeba genus in coastal waters of Caspian Sea based on morphological criteria and thermotolerance assay. 
2. Materials and Methods
In this descriptive study, 60 water samples were collected from sea shore of Caspian Sea in Guilan, Iran in 2018. Samples were collected at a depth of 10-30 cm from the shores of Rudsar, Chamkhaleh, Kiyashahr, Zibakenar and Bandar-e Anzali. From each site, 12 samples of 500 mL were collected, filtered by cellulose nitrate membranes with 0.45 µm pore size, and cultured on Non-Nutrient Agar (NNA) according our previous studies [4, 5]. To determine potential pathogenicity of Acanthamoeba spp. grown in the agar by thermotolerane assay, culture positive sample were transferred to a new NNA plate and kept at 42°C. The presence of Free-Living Amoeba (FLA) examined daily for up to 14 days using a light microscope (100X). FLAs were identified by morphologic characteristics of both trophozoite and cyst.
3. Results
Out of 60 samples, FLA were grown in 30 samples (50%) at a room temperature, and in 8 samples (26.6 %) at a temperature of 42°C. Acanthamoeba were identified in all sampling sites, where 8 samples were potentially pathogenic using thermotolerance assay. 
4. Discussion and Conclusion
The study of FLA, especially in environmental waters, is dramatically increasing due to their increased prevalence and their capability to cause human diseases. The presence of FLA in water sources has been reported in various studies; however, there are few studies on the presence of these amoebae in seawater [6, 7, 8, 9, 10]. In Guilan Province, some studies have reported the presence of FLA in surface water [4, 5, 11]; however, no study has been conducted on the presence of FLA in the Caspian Sea water. The present study is the first study on the presence of FLA in the shores of Caspian Sea. FLA and the potentially pathogenic Acanthamba spp., were identified in 50% and 26.6% of water samples collected from recreational beaches of the Caspian Sea, respectively. In a study in Jamaica, West Indies, Acanthamoeba were identified in 49.6% of the sea-water samples, of which 40.4% were reported potentially pathogenic based on the morphologies of the cysts and trophozoites, and using thermotolerance and osmotolerance assays [7]. In Booton et al.’s study, Acanthamoeba strains were isolated from beach sand (n = 20) and nearly all beach isolates were genotype T4 [12]. 
Interestingly, in the present study, Acanthamoeba were isolated from high-salinity coastal water. It seems that some Acanthamoeba species can grow in waters with high osmolarity (high salinity) can be pathogenic [13]. Since Acanthamoeba spp. (pathogenic and non-pathogenic) can act as natural vectors for pathogenic microorganisms, it can has an impact on the public health sector [14]. The presence of pathogenic and non-pathogenic Acanthamoeba spp. in the Caspian Sea indicates a risk for human health. In this regard and considering that Guilan province attracts many tourists annually and all water samples were collected from recreational beaches where swimming activity usually takes place, it is necessary that high-risk people including contact lens wearers and immunocompromised patients in this area be warned by health organizations. 

Ethical Considerations
Compliance with ethical guidelines
The present study has been approved by the Ethics Committee of Guilan University of Medical Sciences (Code: IR.GUMS.REC.1397.079). 

Funding
This research didn’t receive any grant from funding agencies in the public, commercial, or not-for-profit sector.

Authors' contributions
Conceptualization: Mohammad Reza Mahmoudi, Keyhan Ashrafi; Sample collection and Methodology: Mohammad Reza Mahmoudi, Yasaman Vahedi; Supervision and funding: Mohammad Reza Mahmoudi; Writing: Mohammad Reza Mahmoudi, Keyhan Ashrafi.

Conflicts of interest
The authors declare no conflict of interest.

Acknowledgements
The authors would like to thank Ms. Behnam Rahmati.


References

1. Schuster FL, Visvesvara GS. Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. International Journal for Parasitology. 2004; 34(9):1001-27. [DOI:10.1016/j.ijpara.2004.06.004] [PMID]
2. La Scola B, Boyadjiev I, Greub G, Khamis A, Martin C, Raoult D. Amoeba-resisting bacteria and ventilator-associated pneumonia. Emerging Infectious Diseases. 2003; 9(7):815-21. [doi: 10.3201/eid0907.020760] [PMCID]
3. Feiz Haddad MH, Habibpour H, Mahmoudi MR. Isolation and molecular identification of free-living amoebae (Naegleria spp., Acanthamoeba spp. and Vermamoeba spp.) from mineral springs in Guilan Province, northern Iran. Journal of Water and Health. 2020; 18(1):60-6. [DOI:10.2166/wh.2020.191]
4. Gornik K, Kuzna-Grygiel W. Presence of virulent strains of amphizoic amoebae in swimming pools of the city of Szczecin. Annals of Agricultural and Environmental Medicine. 2004; 11(2):233-6. [PMID]
5. Patrick S. Free-living amoebae as human parasites and hosts for pathogenic microorganisms. Proceedings. 2018; 2(11):692. [DOI:10.3390/proceedings2110692]
6. Mahmoudi MR, Taghipour N, Eftekhar M, Haghighi A, Karanis P k. Isolation of Acanthamoeba species in surface waters of Gilan province-north of Iran. Parasitology Research. 2012; 110:473-7. [DOI:10.1007/s00436-011-2530-1] [PMID]
7. Mahmoudi MR, Kazemi B, Haghighi A, Karanis P. Detection of Acanthamoeba and Toxoplasma in river water samples by molecular methods in Iran. Iranian Journal of Parasitology. 2015; 10(2):250-7. [PMCID]
8. Mahmoudi MR, Rahmati B, Seyedpour SH, Karanis P. Occurrence and molecular characterization of free-living amoeba species (Acanthamoeba, Hartmannella, and Saccamoeba limax) in various surface water resources of Iran. Parasitology Research. 2015; 114:4669-74. [DOI:10.1007/s00436-015-4712-8] [PMID]
9. Munsona DA, Timothy AP. Distribution of Acanthamoeba in more and less polluted North Sea coastal sediments. Journal of Eukaryotic Microbiology. 2006; 53(S1):S12-4. [DOI:10.1111/j.1550-7408.2006.00157.x] [PMID]
10. Lorenzo-Morales J, Lindo JF, Martinez E, Calder D, Figueruelo E, Valladares B, et al. Pathogenic Acanthamoeba strains from water sources in Jamaica, West Indies. Annals of Tropical Medicine & Parasitology. 2005; 99(8):751-8. [DOI:10.1179/136485905X65215] [PMID]
11. Jonckheere JF. Molecular identification of free-living amoebae of the Vahlkampfiidae and Acanthamoebidae isolated in Arizona (USA). European Journal of Protistology. 2007; 43(1):9-15. [DOI:10.1016/j.ejop.2006.09.001] [PMID]
12. Sawyer TK, Visvesvara GS, Harke BA. Pathogenic amoebas from brackish and ocean sediments, with a description of Acanthamoeba hatchetti, n. sp. Science . 1977; 196(4296):1324-5. [DOI:10.1126/science.867031] [PMID]
13. Liu H, Ha YR, Lee ST, Hong YC, Kong HH, Chung DI. Genetic diversity of Acanthamoeba isolated from ocean sediments. The Korean Journal of Parasitology. 2006; 44(2):117-25. [DOI:10.3347/kjp.2006.44.2.117] [PMID] [PMCID]
14. Schroeder JM, Booton GC, Hay J, Niszl IA, Seal DV, Markus MB, et al. Use of subgenic 18S ribosomal DNAPCR and sequencing for genus and genotype identification of Acanthamoeba from humans with keratitis and from sewage sludge. Journal of Clinical Microbiology. 2001; 39(5):1903-11. [DOI:10.1128/JCM.39.5.1903-1911.2001] [PMID] [PMCID]
15. Khan NA. Acanthamoeba, biology and pathogenesis. 1 ^th ed. United Kingdom: Caister Academic Press; 2009. https://books.google.com/books/about/Acanthamoeba.html?id=7cx8zQEACAAJ&source=kp_book_description
16. Górnik K. Pathogenic properties of free-living amoebae isolated from natural and man-made bathing sites in the province of Western Pomerania. Annales Academiae Medicae Stetinensis. 2005; 51(1): 127-33. [PMID]
17. Łanocha N, Kosik-Bogacka D, Maciejewska A, Sawczuk M, Wilk A, Kuźna-Grygiel W. The Occurrence Acanthamoeba (Free Living Amoeba) in Environmental and Respiratory Samples in Poland. Acta Protozoologica. 2009; 48(3):271-9. http://agro.icm.edu.pl/agro/element/bwmeta1.element.agro-article-9b81eae1-714b-4516-b2f0-b571b6089514