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ARS Home » Plains Area » Lincoln, Nebraska » Agroecosystem Management Research » Research » Publications at this Location » Publication #191172
Title: HUMAN PATHOGENIC MICROSPORIDIA DETECTION: METHOD DEVELOPMENT AND ASSESSMENT OF AGRICULTURAL SAMPLES

Author
item KAHLER, AMY - FORMER GRAD STUDENT
item Thurston Enriquez, Jeanette

Submitted to: Journal of Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/7/2006
Publication Date: 12/15/2006
Citation: Kahler, A., Thurston Enriquez, J.A. 2006. Human pathogenic microsporidia detection: method development and assessment of agricultural samples. Journal of Parasitology 100:529-538.

Interpretive Summary: Microsporidia are intracellular parasites of humans and animals. Recently, those species that infect humans have been found in livestock feces and environmental samples. However, more sensitive detection methods are needed in order to better assess the occurrence of human pathogenic microsporidia in environmental samples. Additionally, there are currently no methods for the detection of microsporidia in livestock wastewater. A modified method was developed for the detection of human pathogenic microsporidia in environmental samples. Samples were concentrated and purified and the genetic material, deoxyribonucleic acid (DNA) was isolated from the sample using a commercial kit. Molecular methods were used to detect DNA from human pathogenic microsporidia and identify the microsporidian species. An initial comparison was made between two commercial kits used to isolate DNA, the Qiagen QIAamp DNA Stool Mini Kit and the Qiagen QIAamp DNA Mini Kit. The stool kit consistently recovered more DNA than the tissue kit. In order to evaluate this method, sensitivity studies were performed by inoculating livestock fecal and wastewater samples with known concentrations of microsporidia. For cattle and swine wastewater, the sensitivity was 100% at concentrations of 1,000 microsporidia/ml. At concentrations of 100 microsporidia/ml, the sensitivities for cattle and swine wastewater were 20% and 40%, respectively. For cattle feces, the sensitivity was 30% at a concentration of 1,000 microsporidia/g. Additionally, several environmental samples were screened using this modified method, with 6 of 34 samples positive for human pathogenic microsporidia.

Technical Abstract: Recently, microsporidia species that infect humans have been found in livestock feces and environmental water samples. However, more sensitive detection methods are needed in order to better assess the occurrence of human pathogenic microsporidia in livestock feces and wastewater. A modified method was developed for the detection of human pathogenic microsporidia in livestock feces and manure-impacted environmental samples. Sample concentrates were purified by sucrose floatation and DNA was extracted using the Qiagen QIAamp DNA Stool Mini Kit. Polymerase chain reaction (PCR) analysis was conducted using generic primers for microsporidia. DNA sequence analysis was performed for identification and confirmation of human pathogenic microsporidia species. Livestock fecal and wastewater samples were inoculated with 1,000 and 100 Encephalitozoon intestinalis spores/g or ml of feces or wastewater in order to asses the sensitivity of the method. For cattle wastewater, 10 of 10 replicates were positive by PCR at concentrations of 1,000 spores/ml, and 2 of 10 replicates were positive at concentrations of 100 spores/ml. For swine wastewater, 10 of 10 replicates were positive at concentrations of 1,000 spores/ml, and 4 of 10 replicates were positive at concentrations of 100 spores/ml. For cattle feces, 3 of 10 replicates were positive at the concentration of 1,000 spores/g. Several environmental samples were screened using this modified method, with 2 of 34 samples positive for human pathogenic microsporidia by PCR and DNA sequence analysis. To our knowledge, this is the first report of Encephalitozoon cuniculi detection in swine feces and wastewater.