Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water

Abstract

Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of Encephalitozoon intestinalis was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of Enterocytozoon bieneusi was confirmed in surface water; and the presence of Vittaforma corneae was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.

FIG. 1

Typical PCR screening of water concentrates showing several positive results after community DNA extraction. After such screening, PCR products are purified and sequenced and the species of the microsporidia are identified by computer database homology comparison. Lane 1, 100-bp DNA ladder; lanes 2 to 14, replicate PCRs of community DNA extracted from raw sewage samples; lane 15, negative control PCR derived from sterile double-distilled water and used to ensure that no cross-reactivity occurred during community DNA extractions and PCR setup. Several of the lanes show PCR products of the size predicted for microsporidia. Lanes 3, 4, 6, 9, and 11 are all presumptively positive, showing products between 250 and 270 bp. Lane 9 has a very faint band, of the size predicted for Enterocytozoon bieneusi, while the other lane have bands of the size predicted for Encephalitozoon intestinalis.