Human pathogenic microsporidia detection in agricultural samples : method development and assessment

A detection method was developed and assessed for the sensitive recovery of microsporidia from livestock fecal and manure-impacted environmental samples. Sensitive recovery of microsporidia was achieved when samples were subjected to 1) purification by sucrose floatation, 2) DNA extraction using the Qiagen QIAamp DNA Stool Mini Kit, 3) polymerase chain reaction (PCR) analysis using generic primers for microsporidia, and 4) DNA sequence analysis to identify which microsporidia were present in each sample. Livestock fecal and wastewater samples were inoculated with 1,000 and 100 Encephalitozoon intestinalis spores/g or ml of feces or wastewater. For cattle wastewater, ten of ten replicates were positive by PCR at concentrations of 1,000 spores/ml, and two of ten replicates were positive at concentrations of 100 spores/ml. For swine wastewater, ten of ten replicates were positive at concentrations of 1,000 spores/ml, and four of ten replicates were positive at concentrations of 100 spores/ml. For cattle feces, three of ten replicates were positive at the concentration of 1,000 spores/g. Several environmental samples were screened using this method, with two of 34 samples positive for human pathogenic microsporidia. To our knowledge, this is the first report of Encephalitozoon cuniculi detection in swine feces and wastewater.