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Journal of Clinical Microbiology, January 2005, p. 387-392, Vol. 43, No. 1
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.1.387-392.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Purification of Enterocytozoon bieneusi from Stools and Production of Specific Antibodies

Abhineet S. Sheoran,¹ Xiaochuan Feng,¹ Sabrina Kitaka,² Linda Green,³ Christine Pearson,^1, Elizabeth S. Didier,³ Susan Chapman,¹ James K. Tumwine,² and Saul Tzipori^1*

Division of Infectious Diseases, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts,¹ Department of Pediatrics and Child Health, Mulago Hospital, Makerere University Medical School, Kampala, Uganda,² Department of Microbiology, Tulane Regional Primate Research Center, Covington, Louisiana³

Received 21 May 2004/ Returned for modification 24 June 2004/ Accepted 23 August 2004

Enterocytozoon bieneusi is clinically the most significant of the microsporidia in humans, causing chronic diarrhea wasting and cholangitis in individuals with human immunodeficiency virus infection and AIDS. Little progress on this infection has been made because of the inability to propagate E. bieneusi in vitro and in vivo, which limits the source of parasite spores to the stools of infected human patients. Given the size and shape of the E. bieneusi spores (1.1 to 1.6 by 0.7 to 1.0 µm) and the lack of specific immune reagents, the identification and purification of large quantities of spores from feces are technically challenging. Consequently, diagnosis relies entirely on PCR, a labor-intensive approach that requires highly skilled personnel. We describe a method for the purification of E. bieneusi spores from human stools and the production of rabbit-specific antisera. Spores were purified by a combination of isopycnic Percoll gradient centrifugation and continuous sucrose gradient centrifugation. Specific polyclonal antibodies raised in mice and rabbits reacted by indirect immunofluorescence with E. bieneusi but not with Encephalitozoon spp., Candida albicans, Staphylococcus aureus, Escherichia coli, or other forms present in human stools.

Present address: Division of Comparative Pathology, New England Primate Research Center, Southborough, MA 01772.

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