Isolation and identification of free-living amoeba from the hot springs and beaches of the Caspian Sea

Abstract

Free-living amoeba (FLA) such as Acanthamoeba, Naegleria, Balamuthia, and Vermamoeba have been identified from both natural and human-made environments such as Hot springs and spa. Naegleria fowleri causes Primary Amoebic Meningoencephalitis (PAM), while Acanthamoeba and Balamuthia cause chronic granulomatous encephalitis. Acanthamoeba also can cause cutaneous lesions and Amoebic Keratitis (AK) that is associated with contact lens use or corneal trauma. FLA are known to serve as host of and vehicles for diverse intracellular organisms. This study aimed was to identify the presence of FLA in the hot springs and beaches of the Caspian Sea in Ramsar tourist town located in the northern part of Iran. Water samples were collected in sterile bottles and were transferred to the laboratory. One litre of each sample passed through the nitrocellulose membrane filter. Each filter insert was then placed in non-nutrient agar plates already seeded with lawn culture of Escherichia coli. Positive samples were analyzed by morphological keys and Polymerase chain reaction (PCR) using 18S rDNA gene and ITS region to identify amoeba isolates. A total of 81 water sampled were tasted. After identified using the morphological key and PCR assay, 54 (66.6%) of the samples were positive for FLA. Ten of the samples were identified as Acanthamoeba (belong to T3, T4, and T5 genotypes), three as Vermamoeba vermiformis, four as Naegleria (3 N.australiensis and 1 N.grubery). Only one sample was positive Vahlkampfia. The presence of thermotolerant FLA in the Hot springs and beaches of the Caspian Sea as places for recreational purposes or wellness may be a potential health risk.

Keywords: Caspian Sea; Free-living amoeba; Hot spring; Ramsar.

Fig. 1

Location of hot springs and beaches close to the hot springs in the Ramsar tourist city, in northern Iran.

Fig. 2

Free-living amoeba in NNA (magnification 20x) A) The trophozoites of Acanthamoeba spp. b) The Cysts of Acanthamoeba spp. c) The trophozoites and Cysts of Vahlkampfiids. d) The cysts of Vermamoeba sp.