[Effect of environmental temperature, cryopreservation and aging on Plasmodium vivax sporozoites developing into exoerythrocytic stages]

Abstract

After Anopheles stephensi mosquitoes with salivary infection of Plasmodium vivax were put in environments with temperatures of 30 +/- 1 degrees C, 26 +/- 1 degrees C or 13 +/- 1 degrees C for 5 d, their glands were aseptically dissected and sporozoites were collected and inoculated into HepG2-A16 cell monolayers. Seven days post-inoculation the cultured materials were harvested and the exoerythrocytic schizonts and hypnozoites were observed under the microscope by using immunoperoxidase staining technique. The results showed that the sporozoite developing rate of 30 +/- 1 degrees C group and 13 +/- 1 degrees C group was significantly lower than that of 26 +/- 1 degrees C group (0.33%, 0.35% and 0.75% respectively). The proportion of hypnozoites in the total number of EE forms was the highest in the low temperature group (62.5%) compared with 26 +/- 1 degrees C and 30 +/- 1 degrees C group (40.1% and 42.7% respectively). Suggesting that the low environmental temperature first affected the viability of tachysporozoites or the phenotype of sporozoites and thus resulted in heightened hypnozoite rate. This is parallel to the epidemiological data that in the regions of high latitute vivax malaria with long incubation period was more frequently observed. When the sporozoites within the body of mosquito were cryopreserved at -70 degrees C or in liquid nitrogen for 24 h or 5 d respectively, the proportion of hypnozoite increased 87.4% and 82.4%, respectively. However, cryopreservation did not inactivate all of the tachysporozoites, indicating that the resistance to ultralow temperature in bradysporozoite was much greater than that in tachysporozoites. Aging of sporozoites decreased their developing rate and the exoerythrocytic (EE) schizonts were found to grow sluggishly and asynchronously, indicating that the size of EE schizont and the age of sporozoites are in negative correlation. Meantime, proportion of the hyponozoite decreased significantly.