Enhanced egress of intracellular Eimeria tenella sporozoites by splenic lymphocytes from coccidian-infected chickens.

Egress, which describes the mechanism that some intracellular parasites use to exit from parasitophorous vacuoles and host cells, plays a very important role in the parasite life cycle and is central to Eimeria propagation and pathogenesis. Despite the importance of egress in the intracellular parasite's life cycle, very little information is known on this process compared to other steps, e.g., invasion. The present study was conducted to investigate the interplay between the host adaptive immune system and Eimeria egression. Splenic lymphocytes or soluble immune factors were incubated with parasite-infected host cells for 3 or 5 h, and the percentage of egress was calculated according to an established formula. Viability of egressed parasites and host cells was tested using trypan blue exclusion and annexin V and propidium iodide staining, respectively. We found that premature egression of sporozoites from Eimeria tenella-infected primary chicken kidney cells or from chicken peripheral blood mononuclear cells occurred when the cells were cocultured in vitro with spleen lymphocytes from E. tenella-infected chickens but not when they were cocultured with splenocytes from uninfected chickens. Eimeria-specific antibodies and cytokines (gamma interferon [IFN-γ], interleukin-2 [IL-2], and IL-15), derived from E. tenella-primed B and T lymphocytes, respectively, were capable of promoting premature egress of sporozoites from infected host cells. Both egressed parasites and host cells were viable, although the latter showed reduced reinvasion ability. These results suggest a novel, immune-mediated mechanism that the host exploits to interrupt the normal Eimeria life cycle in vivo and thereby block the release of mature parasites into the environment.

Validation of the methylene blue test for assessment of viability of protoscolices from hydatid cysts.

Background: Methylene blue was used as a vital stain for the assessment of viability of protoscolices from hydatid cysts taking advantage of the chemical nature of the dye as a redox indicator and the kinetically distinct molecular transfer systems of Echinococcus protoscolex for uptake of materials across the tegument. Aim: The present study attempts to validate the application of methylene blue staining for assessment of viability of protoscolices. Methods: To validate the criteria by which viability is assessed, control tests were performed using normal protoscolices and protoscolices previously treated with distilled water at 60°C for 5 minutes. Performance of methylene blue was further studied at intervals over a period of 50 minutes after protoscolex exposure using 1% dye concentration. Results: Normal protoscolices were able to adsorb and reduce the dye and have, therefore, lost the blue color. Protoscolices previously treated with warm water on the other hand, being functionally dead, failed to reduce the adsorbed dye and permanently retained the blue color. Results also indicated that a clear distinction between dead and alive protoscolices can be made within 1 minute. Reading of the test after 10 minutes would be misleading giving a false result. Conclusion: These findings suggest that viability of protoscolices can be assessed on the basis of acquisition and loss/retaining of the dye blue color. Increasing the concentration of methylene blue to 1% was noticed to be associated with remarkable enhancement of contractility, sucker movement, and evagination. Such an excitatory action of the dye may be exploited in viability tests which adopt these criteria.