Ferguson D J P
Nuffield Department of Pathology, Oxford University, John Radcliffe Hospital, Oxford OX3 9DU, UK.
Int J Parasitol. 2004 Mar 9;34(3):347-60. doi: 10.1016/j.ijpara.2003.11.024.
Toxoplasma gondii has a complex life cycle involving definite (cat) and intermediate (all warm blooded animals) hosts. This gives rise to four infectious forms each of which has a distinctive biological role. Two (tachyzoite and merozoite) are involved in propagation within a host and two (bradyzoite and sporozoite) are involved in transmission to new hosts. The various forms can be identified by their structure, host parasite relationship and distinctive developmental processes. In the present in vivo study, the various stages have been evaluated by electron microscopy and immunocytochemistry using a panel of molecular markers relating to surface and cytoplasmic molecules, metabolic iso-enzymes and secreted proteins that can differentiate between tachyzoite, bradyzoite and coccidian development. Tachyzoites were characterised as being positive for surface antigen 1, enolase isoenzyme 2, lactic dehydrogenase isoenzyme 1 and negative for bradyzoite antigen 1. In contrast, bradyzoites were negative for SAG1 but positive for BAG1, ENO1 and LDH2. When stage conversion was followed in brain lesion at 10 and 15 days post-infection, tachyzoites were predominant but a number of single intermediate organisms displaying tachyzoite and certain bradyzoite markers were observed. At later time points, small groups of organisms displaying only bradyzoite markers were also present. A number (9) of dense granule proteins (GRA1-8, NTPase) have also been identified in both tachyzoites and bradyzoites but there were differences in their location during parasite development. All the dense granule proteins extensively label the parasitophorous vacuole during tachyzoite development. In contrast the tissue cyst wall displays variable staining for the dense granule proteins, which also expresses an additional unique cyst wall protein. The molecular differences could be identified at the single cell stage consistent with conversion occurring at the time of entry into a new cell. These molecular differences were reflected in the structural differences in the parasitophorous vacuoles observed by electron microscopy. Stage conversion to enteric (coccidian) development was limited to the enterocytes of the cat small intestine. Although no specific markers were available, this form of development can be identified by the absence of specific tachyzoite (SAG1) and bradyzoite (BAG1) markers although the isoenzymes ENO2 and LHD1 were expressed. There was also a significant difference in the expression of the dense granule proteins. The coccidian stages and merozoites only expressed two (GRA7 and NTPase) of the nine dense granule proteins and this was reflected in significant differences in the structure of the parasitophorous vacuole. The coccidian stages also undergo conversion from asexual to sexual development. The mechanism controlling this process is unknown but does not involve any change in the host cell type or parasitophorous vacuole and may be pre-programmed, since the number of asexual cycles was self-limiting. In conclusion, it was possible using a combination of molecular markers to identify tachyzoite, bradyzoite and coccidian development in tissue sections.
刚地弓形虫具有复杂的生命周期，涉及终末宿主（猫）和中间宿主（所有温血动物）。这产生了四种感染形式，每种形式都有独特的生物学作用。其中两种（速殖子和裂殖子）参与在宿主体内的繁殖，另外两种（缓殖子和子孢子）参与传播到新宿主。可以通过它们的结构、宿主-寄生虫关系和独特的发育过程来识别各种形式。在目前的体内研究中，使用一组与表面和细胞质分子、代谢同工酶以及分泌蛋白相关的分子标记，通过电子显微镜和免疫细胞化学对各个阶段进行了评估，这些分子标记可以区分速殖子、缓殖子和球虫发育。速殖子的特征是表面抗原1、烯醇化酶同工酶2、乳酸脱氢酶同工酶1呈阳性，而缓殖子抗原1呈阴性。相比之下，缓殖子SAG1呈阴性，但BAG1、ENO1和LDH2呈阳性。在感染后10天和15天跟踪脑损伤中的阶段转换时，速殖子占主导，但观察到一些显示速殖子和某些缓殖子标记的单个中间生物体。在更晚的时间点，也存在仅显示缓殖子标记的小群生物体。在速殖子和缓殖子中还鉴定出了一些（9种）致密颗粒蛋白（GRA1 - 8、NTPase），但它们在寄生虫发育过程中的位置存在差异。在速殖子发育过程中，所有致密颗粒蛋白都广泛标记寄生泡。相比之下，组织囊肿壁对致密颗粒蛋白的染色各不相同，并且还表达一种额外的独特囊肿壁蛋白。分子差异可以在单细胞阶段识别，这与进入新细胞时发生的转换一致。这些分子差异反映在电子显微镜观察到的寄生泡的结构差异中。向肠道（球虫）发育的阶段转换仅限于猫小肠的肠上皮细胞。虽然没有可用的特异性标记，但这种发育形式可以通过缺乏特异性速殖子（SAG1）和缓殖子（BAG1）标记来识别，尽管同工酶ENO2和LHD1会表达。致密颗粒蛋白的表达也存在显著差异。球虫阶段和裂殖子仅表达9种致密颗粒蛋白中的两种（GRA7和NTPase），这反映在寄生泡结构的显著差异中。球虫阶段也会从无性发育转变为有性发育。控制这一过程的机制尚不清楚，但不涉及宿主细胞类型或寄生泡的任何变化，并且可能是预先编程的，因为无性周期的数量是自我限制的。总之，使用分子标记的组合能够在组织切片中识别速殖子、缓殖子和球虫发育。
