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

Abstract

Approximately 14 of the more than 1,000 species of microsporidia infect humans, only two of which, Enterocytozoon bieneusi and Encephalitozoon intestinalis, cause intestinal microsporidiosis. Clinical isolates of three microsporidia species, E. intestinalis, Encephalitozoon hellem, and the insect parasite, Anncaliia (Brachiola, Nosema) algerae were used in a spore germination assay, and enterocyte attachment and infection assays were performed to model the potential roles of gastric and duodenal environments and host temperature in determining why only one of these microsporidia species causes intestinal microsporidiosis. Enterocyte infection with A. algerae spores was 10% that of the Encephalitozoon species, a difference not attributable to differences in spore attachment to host cells. Prior spore treatment with pepsin in HCl, pancreatic enzymes, or ox bile did not inhibit germination or enterocyte infection by the three microsporidia species. While the Encephalitozoon species differentiated to mature spores within 3 days, the time taken for many enterocytes to turn over, A. algerae took 3-5 days to produce mature spores, near the upper limit for enterocyte turnover in vivo. Thus, host temperature may contribute to A. algerae not causing human intestinal microsporidiosis, but none of the factors tested account for the inability of E. hellem to cause such an infection.

Figure 1

A. Number of infected C2Bbe1 cells per field 3 days PI with spores of E. intestinalis, E. hellem or A. algerae. B. Attachment of E. intestinalis, E.hellem and B. algerae spores to differentiated C2Bbe1 cells 4 hours after inoculation. Means ± SEM. (N=4). * Significantly different from the other two parasite species, p < 0.05.

Figure 2

Number of infected differentiated C2Bbe1 cells per field 3 days after inoculation with E. intestinalis, E. hellem or A. algerae spores that had been exposed to PBS (control), 1 mg/ml pepsin in 100 mM HCl, 1 mg/ml pancreatin in PBS, or 10 mg/ml ox bile in PBS . Means ± SEM. (N=3)

Figure 3

A, B. Transmission electron microscopic appearance of C2Bbe1 cells cultured on transwells at 37°C, 3 days PI. A. Several parasitophorous vacuoles containing E. intestinalis developing stages and matures spores (S). Brush border (BB). B. Large parasitophorous vacuole containing E. hellem developing stages and mature spores. C. Empty A. algerae spores in cells 5 days PI, cultured at 37°C. Arrow indicates a site near polar tube extrusion. D. A. algerae developing stages and mature spores in 33°C culture, 3 days PI. Germinating intracellular spore (inset). Bars, 2μm.

Figure 4

Number of infected cells (open bars) and number of spores (closed bars) detected in fields of C2Bbe1 cell cultures 3 and 5 days after inoculation with A. algerae spores. Cultures were maintained at 33°C or 37°C. Means ± SEM. (N=4).

Figure 5

Effect of incubation temperature on the germination of A. algerae spores. Spores were stimulated to germinate using a H₂O₂ germinating solution or a pH 9.5 germinating solution. Means ± SEM. (N = 4).