不同环境温度、低温保存及衰老对间日疟原虫子孢子发育为红外期的影响

EFFECT OF ENVIRONMENTAL TEMPERATURE, CRYOPRESERVATION AND AGING ON PLASMODIUM VIVAX SPOROZOITES DEVELOPING INTO EXOERYTHROCYTIC STAGES

观察了蚊体内间日疟原虫子孢子进腺后，在不同温度的环境中继续生活5d，或将全蚊保存于－70℃或液氮中24h或5d，再取其子孢子接种HepG2－A16细胞，培养7d后，通过免疫酶染色观察红外期裂殖体与休眠体的发育情况。结果表明，30±1℃组及13±1℃组的子孢子发育率（0．33％。及0．35％。）均显著低于26±1℃组（0．75‰）；13±1℃组休眠体占红外期总数的比例显著高于另2组，分别为62．5％及40．1％或42．7％，提示较低的环境温度首先影响速发型子孢子的活力或影响其表现型，使休眠体的比例升高，与流行病学资料高纬度地区长潜伏期间日疟比例较大相一致。短时间的低温保存使子孢子发育为休眠体的比例上升：－70℃组为87．4％，液氮组为82．4％，但并不能使速发型子孢子全部灭活。子孢子日龄的增加使其在宿主细胞内的发育率显著下降并使红外期裂殖体发育迟缓，两者是负相关的线性关系，但休眠体的比例未见升高而明显下降。

After Anopheles stephensi mosquitoes with salivary infection of Plasmodium vivax were put in environments with temperatures of 30±1℃, 26±1 ℃ or 13±1 ℃ for 5 d, their glands were aseptically dissected and sporozoites were collected and inoculated into HepG2A16 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℃ group and 13 ± 1℃ group was significantly lower than that of 26 ± 1℃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 ℃ and 30±1℃ 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℃ 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 hypnozoite decreased significantly.