Efficient and reproducible experimental infections of rats with Blastocystis spp

Abstract

Although Blastocystis spp. infect probably more than 1 billion people worldwide, their clinical significance is still controversial and their pathophysiology remains poorly understood. In this study, we describe a protocol for an efficient and reproducible model of chronic infection in rats, laying the groundwork for future work to evaluate the pathogenic potential of this parasite. In our experimental conditions, we were unable to infect rats using vacuolar forms of an axenically cultivated ST4 isolate, but we successfully established chronic infections of 4 week-old rats after oral administration of both ST3 and ST4 purified cysts isolated from human stool samples. The infection protocol was also applied to 4 week-old C57BL/9, BALB/C and C3H mice, but any mouse was found to be infected by Blastocystis. Minimal cyst inoculum required for rat infection was higher with ST3 (105) than with ST4 (102). These results were confirmed by co-housing experiments highlighting a higher contagious potential of ST4 in rats compared to ST3. Finally, experiments mimicking fecal microbiota transfer from infected to healthy animals showed that Blastocystis spp. could easily infect a new host, even though its intestinal microbiota is not disturbed. In conclusion, our results provide a well-documented and robust rat model of Blastocystis chronic infection, reproducing "natural" infection. This model will be of great interest to study host parasite interactions and to better evaluate clinical significance of Blastocystis.

Fig 1. Method of cyst purification from human stools.

About 50 g of stools were suspended in 200 ml of distilled water and filtered (1mm) to remove larger particles. After washing by centrifugation in distilled water (1700 g for 10 min), cysts were concentrated by sucrose gradient (d = 1.20) followed by two Percoll gradients (d = 1.13). Then, cysts were incubated with an antibiotic cocktail composed of Vancomycin, Amoxicilin-clavunalic acid and Cefotaxim to eliminate bacteria. After washing to remove antibiotics, purified cysts were suspended in sterile PBS and quantified. This method was adapted from Yoshikawa’s protocol [18].

Fig 2. Blastocystis ST4 cysts purified from human stools.

(A) Purified cysts from human stools observed by light microscopy. Numerous cysts can be observed (asterisk). More than 4 million of cysts were counted after purification from 50 g of human stool. Cysts size ranged from 2 to 7 μm. (B) Purified cysts from human stools observed by immunofluorescence after labeling with polyclonal anti-Blastocystis ST4 antibodies (Green) and DAPI staining (Blue). Cysts contained two to four nuclei (white arrows, two on this picture).

Fig 3. Colonic sections from Blastocystis ST4-infected rats.

Colonic sections were stained with fluorescein phalloidin (Green), DAPI (Blue) and polyclonal anti-Blastocystis ST4 antibodies (Red). Vacuolar forms were detected in the intestinal lumen (A) and in close contact with the intestinal epithelium (B). Transmission electron micrographs of colonic sections from experimentally-infected rats Blastocystis vacuolar form surrounding by numerous bacteria localized in the intestinal lumen (C) and a granular form in close contact with an epithelial cell (D). M, mitochondrion-like organelle; N, nucleus, CV, central vacuole; EV, empty vacuole; V, vacuole; Bac, bacteria; EC, epithelial cell; MV, microvilli.