First Report of Isolation and Characterization of Acanthamoeba spp. from the Milk Used for Calf Feeding.

Abstract

Acanthamoeba spp. can be found in natural and artificial environments, which reflects their high adaptability to different conditions. Based on the available data, there is scarce information about the isolation of amoeba from milk. This study aimed to investigate the probable presence of Acanthamoeba in milk used for calf feeding. 200 milk samples from 50 industrial and traditional farms were collected. The samples were filtered and cultured on the 1.5% Non-nutrient agar medium. The amoebic growth was examined with an inverted microscope daily. DNA was extracted from the positive plates, and a PCR reaction was undertaken using the primers amplifying the Acanthamoeba 18S rRNA gene. Five samples were purified and sequenced using specific primers. Maximum likelihood reconstructions were performed using the phylogenetic program MEGA software. The osmo and thermotolerance of isolated trophozoites were examined as well. Out of 200 milk samples, Acanthamoeba was isolated from 27 (13.5%). The phylogenetic tree represents that all the isolates belonged to the genotype T4. Results of thermo and osmotolerance tests showed that isolates could develop at 37 and 43 ◦C. Besides, trophozoites survived at 0.5M mannitol and 1M. For the first time, Acanthamoeba spp. were isolated from milk used to feed dairy calves. Due to Acanthamoeba's neglected role in pathogen persistence and survival, hygiene instructions should be reconsidered.