A new alternative in vitro method for quantification of Toxoplasma gondii infectivity.

An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of T. gondii RH tachyzoites was inoculated in 25-cm² flasks containing a 90% confluent monolayer of human foreskin fibroblasts. Lysis kinetics was monitored for infection ratios ranging from 1∶106 to 1∶10; we defined 106 tachyzoites/ml⁻¹ as the threshold value for parasite egress. Results allowed us to build a calibration curve relating the initial infection ratios to the time needed to reach 106 tachyzoites/ml⁻¹. Finally, we validated the method using a known mixture of dead and live parasites. This method was found to estimate with accuracy the initial ratio of infection of the unknown parasite suspension. This easy-to-use method is reproducible and can be applied to any T. gondii tachyzoite RH strain, genetically modified or not. This method is also suitable for testing promising candidates for an effective live vaccine.

Cryptosporidium parvum isolate-dependent postinfectious jejunal hypersensitivity and mast cell accumulation in an immunocompetent rat model.

Cryptosporidium spp. are a cause of self-limited diarrhea in immunocompetent hosts. In immunocompetent rats, Cryptosporidium parvum infection induced digestive hypersensitivity, a key pathophysiological factor in functional digestive disorders such as irritable bowel syndrome (IBS). In such a rat model, we sought to document whether jejunal hypersensitivity depends on C. parvum isolate and is associated with a mast cell accumulation. Five-day-old rats were orally administered 10(5) oocysts of either Nouzilly (NoI) or Iowa (IoI) C. parvum isolate. NoI-infected rats exhibited the lowest food intake on days 7 and 14 postinfection (p.i.). On day 7 p.i., small intestine villus atrophy, crypt hyperplasia, and inflammatory cell infiltration were prominent in NoI-infected rats, with higher numbers of Cryptosporidium forms than in IoI-infected rats. Compared to uninfected control rats, jejunal intraepithelial lymphocytes (IELs) were increased only in NoI-infected rats on day 14 p.i. On day 50 p.i., jejunal hypersensitivity to distension was found only in NoI-infected rats; this hypersensitivity is associated with activated mast cell accumulation. The number of mast cells in the jejunal lamina propria was increased from day 36 p.i. in NoI-infected rats and only at day 120 p.i. in IoI-infected rats. Our data suggest that both the severity of infection (weight loss, reduced food intake, villus atrophy, and IEL accumulation) and the onset of a jejunal hypersensitivity after infection in association with an activated mast cell accumulation are isolate dependent and related to NoI infection. This cryptosporidiosis rat model is a relevant model for the study of underlying mechanisms of postinfectious IBS-like symptoms.