Molecular and phylogenetic evidences of dispersion of human-infecting microsporidia to vegetable farms via irrigation with treated wastewater: One-year follow up.

Background: Human-infecting microsporidia are a group of spore-forming eukaryotic microorganisms that can infect both animals and humans. Recent evidences indicate waterborne transmission of microsporidia spores to human via either drinking water or irrigation of vegetable farms with contaminated water resources. The current study aimed to evaluate the presence of human-infecting microsporidia in treated wastewater (TW) and vegetable farms irrigated with treated wastewater during a year.
Methods: Totally, twelve samples of each treated wastewater and vegetables were collected. In order to recover microsporidia spores, filtration using cellulose nitrate membrane (pore size 0.4 μm) and sedimentation were employed. DNA extraction was performed for all samples and genus/species were characterized using specific primers. In order to characterize genotypes, ITS fragment of E. bieneusi was amplified, sequenced and compared in GenBank database. Phylogenetic tree was employed to analysis the probable correlation between obtained genotypes with those E. bieneusi genotypes, which were previously obtained from human and animals from same region.
Results: After nested PCR, expected fragments of E. bieneusi and Encephalitozoon spp were observed among 5/12 (41.7%) and 1/12 (8.33%) of vegetable samples, respectively. From total of 12 TW samples, expected fragments of E. bieneusi and Encephalitozoon spp were amplified among 7/12 (53.8%) and 1/12 (8.33%) of TW samples, respectively. Genotypes D and E were characterized from both TW and vegetables samples. Phylogenetic analysis showed close-relationship between E. bieneusi from TW and vegetable samples with E. bieneusi from animals and humans obtained from the same region.
Conclusion: Our findings suggested the key role of animals in epidemiology of zoonotic transmission of E. bieneusi. Moreover, our findings revealed the occurrence of human-infecting microsporidia in treated wastewater because of either insufficiency of treatment process or distribution of microsporidia spores in wastewater treatment plant via animals.
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