Molecular Techniques for Detection, Species Differentiation, and Phylogenetic Analysis of Microsporidia

Abstract

SUMMARY Microsporidia are obligate intracellular protozoan parasites that infect a broad range of vertebrates and invertebrates. These parasites are now recognized as one of the most common pathogens in human immunodeficiency virus-infected patients. For most patients with infectious diseases, microbiological isolation and identification techniques offer the most rapid and specific determination of the etiologic agent. This is not a suitable procedure for microsporidia, which are obligate intracellular parasites requiring cell culture systems for growth. Therefore, the diagnosis of microsporidiosis currently depends on morphological demonstration of the organisms themselves. Although the diagnosis of microsporidiosis and identification of microsporidia by light microscopy have greatly improved during the last few years, species differentiation by these techniques is usually impossible and transmission electron microscopy may be necessary. Immunfluorescent-staining techniques have been developed for species differentiation of microsporidia, but the antibodies used in these procedures are available only at research laboratories at present. During the last 10 years, the detection of infectious disease agents has begun to include the use of nucleic acid-based technologies. Diagnosis of infection caused by parasitic organisms is the last field of clinical microbiology to incorporate these techniques and molecular techniques (e.g., PCR and hybridization assays) have recently been developed for the detection, species differentiation, and phylogenetic analysis of microsporidia. In this paper we review human microsporidial infections and describe and discuss these newly developed molecular techniques.

Detection of Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi in the brains of common voles (Microtus arvalis) and water voles (Arvicola terrestris) by gene amplification techniques in western Austria (Vorarlberg)

“…Primers NP6 and NP21 (Müller et al 1996) were used for the detection of N. caninum DNA. For evaluation of E. cuniculi, the primers MSP-3 and MSP-4B were used, which enable a species determination within the microsporidal LSU rRNA gene (Katzwinkel-Wladarsch et al 1996Franzen and Müller 1999). All gene amplifications were performed with a MolTaq Mastermix (Molzym GmbH & Co. KG, Germany) using previously reported protocols for T. gondii and E. cuniculi (e.g., Murphy et al 2007).…”

Molecular characterization of Enterocytozoon bieneusi in cattle indicates that only some isolates have zoonotic potential

“…In addition, E. bieneusi has been documented as a common microsporidian parasite in several mammals, including farm and companion animals, rhesus monkeys, and more recently in some wild mammals and a chicken Mathis et al 1999;Rinder et al 1999Rinder et al , 2000Chalifoux et al 2000;Dengjel et al 2001;Buckholt et al 2002;Reetz et al 2002;Fayer et al 2003;Sulaiman et al 2003). However, the transmission route of this parasite is still not clear (Franzen and Muller 1999;Rinder et al 2000;Sulaiman et al 2003). Spores of E. bieneusi-like organisms were detected in ditch, river, and lake water (Sparfel et al 1997;Dowd et al 1998;Fournier at al.…”

Prevalence of Enterocytozoon bieneusi in post-weaned dairy calves in the eastern United States

“…Microsporidia, reported worldwide, are one of the most frequently identified pathogens in HIV-infected patients (Franzen and Mu¨ller 1999). Enterocytozoon bieneusi is the microsporidian species most frequently found in human infections, which are characterized by chronic diarrhea (van Gool et al 1995;Kotler and Orenstein 1998).…”