环形泰勒焦虫裂殖体细胞培养及其免疫原性的研究

1.本实验中所试用的BLS和“乳叶”培养液,都能良好地支持环形泰勒焦虫裂殖体感染的成淋巴细胞贴壁和悬浮生长,在4天内细胞量增殖5倍以上,传代以后的成淋巴细胞95%以上都寄生有裂殖体。成淋巴细胞“乳叶”培养液中,已连续传代培养15个月以上,仍生长良好,初步证明某些培养液中所加入的多种维生素等成份,对促进寄生有裂殖体的成淋巴细胞的生长繁殖,不是必要的。 2.传代细胞内的裂殖体,仅见无性裂殖体一种。裂殖体的分裂方式与宿主细胞的有丝分裂是密切相关的,在成淋巴细胞分裂的后期,裂殖体核沿纺锤丝排列成一行或数行,然后几乎被均等地分配到两个子细胞中去。裂殖体是细胞分裂的一种强烈刺激物。 3.成淋巴细胞在明胶制剂中,于4—6℃下保存到40—50天仍不失其再生长繁殖的能力。 4.裂殖体具有坚强的免疫原性,接种牛体后可产生抗环形泰勒焦虫病的免疫力。传代培养45天,2个半月和5个半月的细胞接种牛体后,前二种细胞形成带虫免疫,后者为非带虫免疫,安全性皆表现良好。经蜱叮咬攻毒,17头牛100%得到保护。516×10~5—5×10~6细胞剂量均安全有效。用明胶制剂作为保护剂,所制的胶冻细胞苗在4—6℃下保存20天和30天,对牛体仍然可产生坚强的保护能力,即疫苗的有效保存期可达30天。     

感染环形泰勒焦虫(Theileria annulata)的牛外周血白细胞的形态观察

用光镜及扫描电镜观察了体外高代培养的含牛焦虫颗粒的牛外周血白细胞的形态及在细胞周期中细胞表面的特征性变化。这种经多年传代的含虫的牛外周血白细胞恢复了分裂和繁殖的能力,目前已成为较稳定的细胞系。细胞表面具多种伪足突起,如叶状、丝状及绒毛状。细胞周期中备期细胞表面的主要特征是:S期:细胞平扁,边缘具薄的时状伪足及丝状伪足;G_2期:细胞中部隆起,表面具少量绒毛状伪足;G_1期:绒毛状结构少或无,而出现丝状及小的叶状伪足,细胞仍保持球形;M期:细胞球形,表面密被以绒毛。作者根据扫描电镜的观察认为光镜下所观察的两类细胞,实际上是反映了一种细胞处于不同发育阶段时的特征。     

中华血簇虫的生活史研究(复顶门:孢子纲) Ⅱ.中华血簇虫在中华鳖中的发育

中华血簇虫在其无脊椎动物寄主中的发育已另有文描述。这里报道的是中华血簇虫在中华鳖中的发育。这一时期包括三个阶段:组织细胞内裂体增殖、深部血红细胞内的裂体增殖和外周血红细胞内的裂体增殖。组织细胞内裂殖体产生14—32个裂殖子。深部血红细胞内的裂殖体分为两类:一类是X裂殖体,它产生14—18个小裂殖子;另一类是Y裂殖体,它产生4—6个大裂殖子。外周血红细胞内的初期裂殖体可产生多至14个裂殖子,而随后的裂体增殖却产生越来越少的裂殖子,且裂殖体和裂殖子的大小也渐趋变小。外周血晚期的裂殖体只形成2个裂殖子。配子母细胞来源于Y裂殖子。营养体是由上一代裂殖子向下一代裂殖体发育的中间时期。     

广东肝血簇虫在宿主内脏裂体生殖时期超微结构的研究

本文详细描述了广东肝血簇虫(Hepatozoon guangdongensis)在实验宿主、中国水蛇肺部裂体生殖整个发育过程各期虫体的超微结构。成熟裂殖体内的裂殖子与肺部毛细血管内皮细胞内的裂殖子的超微结构是相似的。裂殖子(3.4×1.3μm)外被由外膜和内膜构成的表膜,它与球虫一样具有包括类锥体在内的完全顶复结构。内皮细胞内的裂殖子和滋养体都没有围虫泡和围虫泡膜包绕。具有内膜的长形滋养体变圆,并外被由宿主细胞产生的围虫泡和围虫泡膜包绕,转变成为圆形的幼期裂殖体,然后发育成为成熟的裂殖体。成熟裂殖体(23×10μm)内含30—50个裂殖子。裂殖体内没有观察到残余体存在。     

水华蓝藻噬藻体对不同条件培养的宿主细胞感染性分析

在从武汉东湖水样中培养分离水华蓝藻噬藻体(Planktothrix agardhii Virus from Lake Donghu,PaV-LD)的基础上,对在不同条件培养的宿主蓝藻细胞中,PaV-LD增殖效率及裂解作用进行了测定分析。分别将PaV-LD接种到生长期、半连续培养更新率或光照不同的宿主蓝藻液中,并采用稀释培养计数(Mostprobable number,MPN)方法与电镜观察,测定子代PaV-LD释放量及宿主细胞的裂解作用。结果显示:对数生长期宿主蓝藻单个细胞中子代PaV-LD的平均释放量为350感染单位(Infectious Units,IU/cell),显著高于稳定生长期的平均释放量110 IU/cell。在用新鲜培养基更新率为0%、35%、50%和65%的半连续培养宿主蓝藻中,接种PaV-LD 5d之后,噬藻体的释放量分别约为50 IU/cell、70 IU/cell、220 IU/cell或310 IU/cell,表明子代PaV-LD释放率随培养基更新率的增加而显著提高。在光照条件下感染3—4d后,宿主蓝藻细胞充分裂解,并释放大量子代PaV-LD,滴度可由初始7.00×103IU/mL快速增加到8.56×107IU/mL;但在遮光条件下,同样感染的蓝藻细胞未见裂解,也检测不到释放的子代噬藻体。电镜观察显示,在光照条件下感染的蓝藻细胞类囊体膜结构消失,而大量子代PaV-LD颗粒主要分布在原有类囊体的部位。显然,宿主蓝藻细胞的培养条件和状态可能对获得噬藻体纯培养有决定性影响。     

日本血吸虫在离体培养中的产卵和虫卵发育过程的研究

1.本文证明日本血吸虫卵在适合的条件下,在离体培养中亦能发育至成熟,并孵出毛蚴,而并不一定需要经过一个宿主组织内发育阶段。日本血吸虫卵在离体培养中自产出到完全发育成熟所需的时间最少为12—13天。 2.日本血吸虫卵在离体培养中发育至成熟的必需条件为:培养液中必需有血清及红血细胞的存在,其中尤以红血细胞的存在更为重要。 3.本文应用相比差显微镜初步但较系统地观察和描写了血吸虫卵细胞的分裂发育过程。证明血吸虫卵细胞的分裂过程亦属不等裂的类型,与其它吸虫卵细胞分裂发育的过程类似。为了便于统计,本文把血吸虫卵的发育过程分为以下四个时期:1)单细胞期。2)细胞分裂期。3)胚胎发育期。4)毛蚴成熟期。本文并对发育各期的出现时间及增减趋势进行了初步观察。 4.本文证明凡是在宿主体内曾经合抱过的发育成熟的血吸虫雌虫,不论它在离体培养时是否处于合抱状态,雌虫均能产卵。其所产出的虫卵数量的多少与所用培养液的种类及雌虫在培养液中的存活时间有关。同时亦证明了血吸虫在离体培养的环境中不仅能将其子宫中已形成的虫卵排出,而且还有新的虫卵的形成。其产卵数量和培养时间有关,其产卵高峰期为培养后的第2—7天。     

贝氏隐孢子虫在北京鸭体内发育的超微结构研究

贝氏隐孢子虫各期虫体均位于宿主粘膜上皮细胞的带虫空泡中。在虫体与上皮细胞接触处,虫体表膜反复折迭形成营养器。子孢子或裂殖子与粘膜上皮细胞接触后,逐步过渡为球形的滋养体;滋养体经2—3次核分裂、产生含4或8个裂殖子的两代裂殖体,裂殖体以外出芽方式产生裂殖子;裂殖子无微孔,顶端表皮形成3—4个环嵴,裂殖子进一步发育成为配子体;大配子体含有两种类型的成囊体。小配子呈楔形,无鞭毛和顶体,有一个致密的长椭圆形细胞核,小配子表膜内侧有9根膜下微管;孢子化卵囊内含四个裸露的子孢子和一个大残体。本文是有关鸭体内隐孢子虫超微结构的首次报导。     

病原菌作用于细胞焦亡的策略:盾-矛之争

焦亡是一种细胞程序性死亡的形式,其特征表现为细胞的裂解并伴随细胞因子、损伤和病原体相关的分子模式的释放.细胞焦亡能够促进炎症免疫反应发生,消除细胞内的病原菌,在细胞抵御病原菌感染过程中发挥重要作用.但是,在长期的军备竞赛中,病原体已进化出抑制宿主细胞焦亡的机制,以增强它们生存和致病的能力.本文从细胞焦亡的分子机制及其在宿主防御中的作用,以及病原菌抵御宿主细胞焦亡的策略等方面进行了综述,将有助于人们进一步了解和探索细菌病原体和细胞焦亡相互作用的机制,并为将来开发基于细胞焦亡抵抗病原体感染的新药提供思路.     

牛病毒性腹泻病毒的成熟和释放

试验中用电镜观察了牛病毒性腹泻病毒ＯｒｅｇｏｎＣ２４Ｖ株在感染新生牛睾丸细胞中的形态发生。成熟的病毒颗粒是直径约为５０ｎｍ的球形颗粒,内含直径约为３０ｎｍ的核心。病毒在宿主细胞的胞质内复制,通过糙面内质网膜出芽成熟。病毒可以通过外排或在细胞死亡后含有病毒颗粒的空泡崩溃而释放到胞外。     

病原体逃逸宿主固有免疫的新策略：抑制细胞焦亡

细胞焦亡是一种由Gasdermin家族蛋白介导的新型程序性细胞死亡。当宿主细胞感应病原体感染或其他危险信号时，Gasdermin家族蛋白被切割活化并诱导细胞焦亡。细胞焦亡过程往往伴随大量炎性细胞因子释放，这些炎性细胞因子在宿主清除病原体过程中发挥着至关重要作用，而病原体在与宿主长期“博弈”过程中也进化出抑制细胞焦亡的策略以实现免疫逃逸。本文介绍了细胞焦亡的发现历程及其在抗感染免疫中的重要功能，并总结了病原体抑制细胞焦亡的多种新策略及其相关研究进展。深入理解细胞焦亡的发生及调控机制，可揭示相关感染性疾病的发病机制并有助于开发有效的抗感染治疗策略。     

Development of Eimeria ninakohlyakimovae from Sheep In Cell Cultures*

SYNOPSIS. Monolayer cell line cultures of ovine trachea, thyroid, thymus, and kidney cells, as well as an established cell line (Madin-Darby) of bovine kidney cells, were inoculated with sporozoites of Eimeria ninakohlyakimovae and observed for a maximum of 24 days. Sporozoites were seen penetrating cells within 5 minutes after inoculation, as well as 2 and 3 days after inoculation, and leaving cells 3 days after inoculation. Transformation from sporozoites to trophozoites occurred by a widening or by a lateral outpocketing of the sporozoite body. Trophozoites and schizonts were first seen 3 days after inoculation in all ovine cell types. Large numbers of immature schizonts were observed, but only an estimated 0.4–4.3% of these became mature in the different kinds of cells. Usually, mature schizonts were first seen 10–11 days after inoculation in the ovine cells, but they sometimes occurred as early as 8 days. More mature schizonts were seen in the ovine kidney and trachea cells than in the others; the smallest number occurred in the bovine cells. The nucleoli of cells harboring large schizonts in each type of culture were enlarged and the chromatin clumps normally seen in the nuclei of non-infected cells were not visible. The cytoplasm of some infected cells was vacuolated. The formation of merozoites occurred by a budding process from blastophores, from the surface of schizonts, and/or from infoldings and invaginations of this surface. Merozoites were observed leaving host cells, but were not seen penetrating new cells. Intracellular first-generation merozoites were observed 13 and 15 days after inoculation in lamb trachea and kidney cells, respectively. No evidence of further development of such merozoites was found.     

Gametocyte formation by the progeny of single Plasmodium falciparum schizonts

Gametocytogenesis of the malaria parasite Plasmodium falciparum was studied in monolayers of erythrocytes attached to tissue culture dishes. Merozoites produced by single schizonts in erythrocytes overlaying the monolayer infected the attached erythrocytes and produced clusters of progeny. Parasites in these readily indentifiable clusters then underwent either asexual growth or sexual differentiation. The progeny of most schizonts yielded no gametocytes. However, the progeny of those schizonts that did yield gametocytes showed a marked tendency to produce multiple gametocytes. Gametocytogenesis, therefore, was not random. Instead, the progeny of certain schizonts were committed to produce gametes. However, even those clusters containing several gametocytes also contained asexual forms. Therefore, not all merozoites of a single schizont were committed to gametocytogenesis. In those cells infected with two or more merozoites the formation of a gametocyte was usually associated with a block in the further development of other parasites.     

In Vitro Cultivation of the Vascular Phase of Sarcocystis singaporensis

To establish an in vitro culture system for the precystic phase of Sarcocystis singaporensis, we initially tested various excysting fluids for sporocysts. An excysting fluid containing 2.5% bovine taurocholate and 10% bile of the specific intermediate host, Rattus norvegicus, in RPMI medium was the most suitable resulting in excystation of 80% of the sporozoites. Subsequently, we identified brain endothelial cells and pneumonocytes of the rat to promote growth of sporozoites to schizonts. Hepatoma, fibroblastic, or myoblastic cells were not suitable for the parasite's development. First-generation schizonts were seen at days 3-10 postinoculation (PI); a distinct second peak of schizogonic development only occurred in endothelial cells at days 14-18 PI. First-generation schizonts were 26.0 (± 3.8) μm in diameter and contained 32-50 merozoites, second-generation schizonts measured 34.4 (± 10.6) μm and contained 54-72 merozoites. Merozoite yield at large-scale culture conditions (75 cm² flasks) using pneumonocytes as host cells was relatively low. Ultrastructurally, sporozoites and merozoites were quite similar to corresponding stages of other Sarcocystis species. With regard to host cell specificity and developmental kinetics, in vitro cultivation showed close similarities to the situation in vivo.     

Schizogonic Development of Leucocytozoon smithi

ABSTRACT The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.     

Schizogonic development of Leucocytozoon smithi.

The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.     

Time-dependent loss of invasive ability of Plasmodium berghei merozoites in vitro.

The invasive ability of Plasmodium berghei merozoites in vivo was studied following their artificial removal from parasitized mouse red cells using complement-mediated immune lysis in vitro and in vivo. Time-course experiments revealed that lysed preparations contained two components contributing to the parasites' infectivity in mice. One component, presumed to be free merozoites released from mature schizont-infected cells, rapidly lost infectivity with time at 1 to 2 C. A second minor component appeared to have more stability at this temperature, and could be accounted for as intact parasitized cells containing mature schizonts not lysed by the complement in vitro, but lysed by the recipients' plasma complement in vivo. Further experiments revealed that suspension of parasitized cells in an isotonic diluent and centrifugation at moderate speeds substantially removes the number of invasive free merozoites insolable from a given sample of infected blood by immune hemolysis. Conclusions: merzoites, either contained within the confines of mature schizont-infected cells, or artificially removed from host cells, rapidly lose the ability to invade susceptible erythrocytes in vivo when suspended in an isotonic medium and held at 1 to 2 C in vitro.     

Development of Eimeria meleagrimitis Tyzzer from sporozoites and merozoites in turkey kidney cell cultures

Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P less than 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P less than 0.05) than their in vivo counter parts.     

The life cycle of Eimeria falciformis var. pragensis (Sporozoa: Coccidia) in the mouse, Mus musculus

The life cycle of Eimeria falciformis var. pragensis, established from a single oocyst, is described in experimentally infected mice (Mus musculus). The coccidium had a prepatent period of 7 days and a patent period of 10--16 days. Oocysts were spherical to ellipsoidal in shape and measured 21.2 x 18.3 micron. Sporulation time was 3 to 3.5 days. Sporocysts measured 12.2 x 7.2 micron and contained a circular to avoid granular sporocyst residuum measuring 5.5 X 5.0 micron. One, 2 or 3 circular to rectangular polar granules were observed within each sporulated oocyst. The endogenous stages developed primarily in the cecum and colon and only occasionally in the lower ileum. Four generations of schizonts were found. Mature 1st-generation schizonts, first observed 48 hr postinfection (PI), measured 17.8 x 12.3 micron and had 12 merozoites that measured 13.3 x 2.0 micron. Mature 2nd-generation schizonts appeared 78 hr PI. They measured 10.2 x 9.3 micron and had 8 merozoites measuring 5.0 x 1.6 micron. Mature 3rd-generation schizonts appeared first at 114 hr PI and measured 17.5 x 10.2 micron and had 10 merozoites that measured 12.4 x 1.8 micron. Mature 4th-generation schizonts appeared first at 144 hr PI. They measured 18.2 x 15.3 micron and had 18 merozoites. The merozoites of the 4th-generation schizont were 4.5 x 1.2 micron. Mature macrogamonts and microgamonts developed simultaneously appearing at 156 hr PI. Macrogamonts measured 16 x 14.5 micron and microgamonts were 18.2 x 15.3 micron. In experimentally infected rats (Rattus norvegicus), development of E. falciformis var. pragensis progressed only as far as mature 1st-generation schizonts.     

In vitro cultivation of Sarcocystis neurona from the spinal cord of a horse with equine protozoal myelitis

Asexual stages of Sarcocystis neurona were seen in cultured bovine monocytes (M617) inoculated with tissue homogenates from the spinal cord of a horse with naturally acquired protozoal myelitis. Organisms first were observed as intracytoplasmic schizonts and later as motile extracellular zoites capable of infecting surrounding M617 cells. Parasites most often occurred as clusters of merozoites dispersed throughout the host cell cytoplasm; however, schizonts also contained merozoites arranged in a radial fashion surrounding a prominent residual body. Schizonts divided by endopolygeny. The parasite has been maintained beyond 280 days in the laboratory by serial passage of infected M617 cells.