Purification of Enterocytozoon bieneusi from Stools and Production of Specific Antibodies

Abstract

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 m) 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.Enterocytozoon bieneusi is a widespread enteric microsporidium which probably infects most mammals. The infection was first identified two decades ago in individuals with AIDS with severe symptoms of chronic diarrhea and wasting (10) and has remained a significant pathogen in this subpopulation ever since (5,6,18). E. bieneusi is recognized as the most common and clinically significant organism among the microsporidia which infect humans who have immunodeficiencies (18,21) or who are receiving immunosuppressive therapy (13,22). E. bieneusi is only rarely symptomatic in immunocompetent individuals, possibly contributing to traveler's diarrhea (11). It is an intracellular microorganism which appears to infect the epithelium of the upper small intestine and the hepatobiliary tract (26), causing chronic diarrhea and cholangitis. The number of E. bieneusi cases has diminished considerably in developed countries due to the use of antiretroviral therapy in people with human immunodeficiency virus infection and AIDS (12). The number of cases, however, in such individuals in developing countries, where antiretroviral therapy is either not available or not affordable, remains very high (4, 14, 23).Scientific progress on E. bieneusi has been slow, largely because of a lack of in vitro and in vivo models for parasite propagation and laboratory investigations. While E. bieneusi did appear to infect cells in culture, the spore yields were low and continuous culture could not be maintained (25). Natural infections of immunologically competent and immunodeficient macaques have also been reported, and the distribution of infection and lesions are similar to those seen in infected humans (8,9,16,17,20). In macaques infected with simian immunodeficiency virus, lesions associated with E. bie...

Investigations into microsporidian methionine aminopeptidase type 2: a therapeutic target for microsporidiosis

“…Purified microsporidian DNA was dissolved in TE buffer and stored at -20°C. Enterocytozoon bieneusi DNA (gift of Dr. Saul Tzipori, Tufts University, School of Veterinary Medicine) was prepared from purified spores (purity confirmed by transmission electron microscopy) isolated from human faeces (cases were confirmed by PCR with specific primers to the E. bieneusi small subunit rRNA gene) (Sheoran et al 2005).…”

Production and Characterization of Monoclonal Antibodies against Enterocytozoon bieneusi Purified from Rhesus Macaques

“…(i) IFA. MAbs 8E2, 7G2, 7H2, and 12G8 were selected for further testing against purified spores and fecal samples of monkey and human origins and were confirmed by PCR and a polyclonal rabbit E. bieneusi-specific serum (5,33,40,41) (Fig. 3).…”

“…The spores were collected and resuspended in PBS. The recovery of spores at each step was monitored by an immunofluorescence assay (IFA) with rabbit polyclonal antibodies against human E. bieneusi as described previously (33).…”

“…(i) Salt-Percoll-sucrose centrifugation. Fecal specimens were processed as described previously (33), with the following modifications. Briefly, feces were homogenized in 0.01 M PBS, pH 7.2 to 7.4 (1:5 to 1:10), and serially filtered through American standard sieves (pore sizes, 425, 180, 100, and 63 m; Newark Wire Cloth Company, Newark, NJ).…”

“…Purification has not been easy because of the size of the spores. Several methods to purify spores from feces have been described by other laboratories (1,8,20) as well as by our group (33). Two monoclonal antibodies (MAbs) against human E. bieneusi have been reported (2), but they are unavailable commercially.…”