Molecular Characterization of Encephalitozoon intestinalis (Microspora) Replication Kinetics in a Murine Intestinal Cell Line

Abstract

Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. Most human infections are caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and result in chronic diarrhea. In order to determine the signals involved in microsporidial spore activation and invasion, kinetics of in vitro E. intestinalis replication were defined using real-time quantitative PCR. Segments of small subunit ribosomal RNA and polar tube protein 2 genes of E. intestinalis were used to quantify parasite gene copy number following infection in murine colon carcinoma cells. Parasite DNA was detectable in small but significant amounts within host cells as early as 4 h postinoculation, genome replication was completed by 36 h, and parasite progeny were released into the supernatant beginning 72 h postinoculation. Heat-treating spores did not prevent transfer of parasite DNA into cells, but did inhibit parasite replication. Treating cell cultures with albendazole suppressed but did not completely inhibit parasite replication. These results confirm observations that E. intestinalis completes its life cycle within the turnover time of its target host cells; invasion into susceptible host cells occurs independently of spore viability; and real-time quantitative PCR is a sensitive and reproducible method with which to monitor microsporidial infection under varying treatments or conditions.

Animal cell cultures in microsporidial research: their general roles and their specific use for fish microsporidia

“…The in vitro approach can be used to determine the timing of intracellular events and to study the effect of variables, such as temperature, on proliferation and sporogony. The replication kinetics of E. intestinalis in a mouse intestinal cell line (CMT-93) was studied by real time PCR (Wasson and Barry 2003). Elevated temperature was shown to impeded proliferation and the onset of sporogony for B. algerae in rabbit kidney cells (Lowman et al 2000).…”

Quantitative PCR to detect, discriminate and quantify intracellular parasites in their host: an example from three microsporidians in Daphnia

“…Despite its general use in molecular biology, including quantification of human pathogenic microsporidia (e.g. Hester et al 2002 ;Menotti et al 2003 a, b;Wasson and Barry, 2003), qPCR is not often applied in studies on the ecology and evolution of host-parasite interactions (Cheesman et al 2003 ;Mouton et al 2004 ;Mucklow et al 2004). Here, empirical research lags behind theory : one reason for this is that the techniques to analyse experiments on host-parasite interactions are limited.…”

Effects of host temperature and gastric and duodenal environments on microsporidia spore germination and infectivity of intestinal epithelial cells

“…In an in vitro murine model E . intestinalis has been shown to be able to complete its life cycle well within this time frame (Wasson and Barry, 2003). Using transmission electron microscopy of transwell-cultured C2Bbe1 cells maintained at 37°C we were frequently able to detect all parasite stages, including mature spores, in both E. intestinalis - and E. hellem -infected cells 3 days after spore inoculation (Figure 3).…”