贝氏隐孢子虫在鸡体内发育的形态学特征及其对寄生器官的影响

取 2日龄海兰雏鸡 5 0只 ,分为 5组 ,分别接种 0、 0 8× 10 6、 1 6× 10 6、 3 2× 10 6、 6 4× 10 6个鸭源贝氏隐孢子虫 (Cryptosporidiumbaileyi)卵囊。接种后在不同的时间间隔内剖杀雏鸡 ,取法氏囊、气管和喉头。扫描电镜观察发现贝氏隐孢子虫主要寄生在鸡的喉头、法氏囊、气管。裂殖体有 2种类型 :Ⅰ型裂殖体含 8个裂殖子 ,Ⅱ型裂殖体含 4个裂殖子。子孢子或裂殖子在钻入过程中 ,虫体逐渐由香蕉形过渡到鼓槌形 ,最后形成球形的滋养体。带虫空泡分为有球形残体的带虫空泡和无球形残体的带虫空泡。观察到小配子的释放和虫体寄生于杯状细胞的现象。贝氏隐孢子虫寄生于气管引起纤毛倒伏、排列紊乱、纤毛融合、脱落 ;致使法氏囊上皮肿胀 ,法氏囊粘膜表面形成皱褶 ,微绒毛脱落、融合、排列紊乱 ,粘液性分泌物增多 ,炎性细胞渗出     

贝氏隐孢子虫在珍珠鸡体内发育的扫描电镜观察

采用扫描电镜观察了贝氏隐孢子虫在珍珠鸡体内的发育。大量球形虫体镶嵌于气管和法氏囊微绒毛丛中。气管纤毛消失,微绒毛生发生融合。法氏囊粘膜表面可观察到宿主细胞突起,在突起的表面有数个虫体寄生。滋养体呈球状,平均大小为１．７μｍ。裂殖体拥有４个或８个香蕉状裂殖子。成熟大配子体大小为 ４.２ × ３．３μｍ,在其侧面可观察到锯齿状突起。偶尔能观察到卵囊,其表 面有一明显裂缝。虫体逸出后所留下的带虫空泡似弹坑状,根据其结构可将其分为两类,其中一类为裂殖子或小配子的形成场所,另一类为卵囊的形成场所。     

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。     

广东肝血簇虫(Apicomplexa:Haemogre-garinidae)感染试验和生活史的研究

作者利用致乏库蚊(Culex fatigans)作为实验媒介,把寄生在灰鼠蛇(Ptyas korros)的广东肝血簇虫(Hepatozoon guangdongensis)感染到无虫的灰鼠蛇(渔游蛇(Natrix piscator)、三索锦蛇(Elaphe radita)和中国水蛇(Enhydris chinesis);观察其生活史。首次描述了由x裂殖体产生的小裂殖子,进入红细胞,经滋养体和幼小配子母体阶段发育成配子母体的过程,以及由y裂殖体产生的大裂殖子继续在内脏进行无性生殖的细节;在对广东肝血簇虫生活史进行研究的基础上,提出一种新的肝血簇虫在脊椎动物体内发育的模式和血液时期各期虫体划分的形态学标准。     

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。     

微小泰泽球虫内生阶段虫体内多糖的细胞化学的研究

采用纯种微小泰泽球虫(Tyzzeria parvula)卵囊,人工感染四日龄雏鹅,定时剖杀,取肠道组织进行石蜡切片,细胞化学染色,观察微小泰泽球虫内生阶段虫体内多糖的分布。结果表明:滋养体、多核体、小配子体以及早期的大配子体内均不含多糖。裂殖子PAS反应阳性,但也有少数第二代裂殖子阴性。随着大配子体的发育,多糖逐渐在其体内合成,合子时期达到高峰。提示:微小泰泽球虫体内的多糖是一个合成—积累—消耗的过程。     

广东艾美虫发育的研究

广东艾美虫寄生于乌鳢的消化道,其发育周期可分为裂体生殖、配子生殖和孢子生殖三个阶段。这三个阶段均在一个寄主体内完成。裂体生殖和配子生殖发生在幽门盲囊和前肠上皮细胞核之上方。成熟裂殖体为球形或椭圆形,含有8—14个香蕉形裂殖子。大配子的整个发育过程没有发现嗜伊红颗粒,嗜碱性颗粒在卵囊壁形成前后发生变化。成熟小配子母细胞内有许多新月形的小配子。孢子生殖在幽门盲囊和整条肠管内进行。       

毁灭泰泽球虫小配子形成及受精现象的扫描电镜观察

现今关于球虫超微结构方面的研究资料已相当丰富,其中涉猎最多的是文美耳属(Eimeria)的虫种,同样隶属于孢子虫纲真球虫目艾美耳科的泰泽属球虫(Tyzzeria)尚未见报道。作者以纯种毁灭泰泽球虫(T.Perniciosa)人工感染无球虫北京鸭,定时扑采取小肠组织,利用扫描电镜对虫体小配子的形成过程及其形态进行了描述,并观察了大小配子的受精现象。 小配子形成毁灭泰泽球虫的小配子发育在小肠上皮细胞的带虫泡(Parasito phorous vacuole)内进行,和已报道的肠道艾美耳属球虫相同。在感染严重的样品,一个上皮细胞内往往有几个大、小配子体     

柔嫩艾美耳球虫配子发生的超微结构

利用透射电镜对柔嫩艾美耳球虫配子生殖阶段的超微结构进行了,大配子体和小配子体于相邻的宿主肠上皮细胞内相产生,由末代裂殖子入后,长大变圆而形成,小配子的形成为直接分化型,首先细胞核分裂成为多核体,随后细胞核向周边移动,然后紧靠细胞处的限制向外突出,临近突出部位的限制膜下陷,在核上方形成中心粒,中心粒发育为基粒,鞭毛中的微管和附着微管,早期形成的小配子仍与小配子体的殖体相连,成熟的小配子与配子体分离,外型香蕉状,外被单位膜,内有一电子结构十分致密的细胞核,核的头端侧面有一个巨大的线粒体,小配子有鞭毛2根,每根鞭毛内有微管,组成为9＋2结构,此外,小配子至少有6根附着微管,大配子体和大配子外被单位膜,内部形成大量的成囊体1和成囊体2,并有大量的支链淀粉和脂肪体,中央有一个细胞核,卵囊臂有5层,细胞核位于细胞中央,细胞内有大量的支链淀粉和脂肪体。     

毁灭泰泽球虫Tyzzeria Perniciosa(Sporozoa:Eimeriidae)生活史各阶段的细胞化学研究:Ⅰ.多糖

本实验分别用过碘酸——雪夫氏剂染色方法(PAS)和乌洛托品——硝酸银染色方法在光镜和电镜下检验毁灭泰泽球虫生活史各时期体内的多糖及其分布。实验结果表明,子孢子内、各代裂殖体和裂殖子内都含有多糖。大配子和合子内除含有多糖外还含有成囊颗粒。成囊颗粒的成分是糖蛋白。无性世代的滋养体和多核体内未检出多糖。早期配子细胞,小配子体和小配子内也未检出多糖。本实验证明,毁灭泰泽球虫体内的多糖系由其自身合成,并在其发育过程中消耗。     

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.     

An Ultrastructural Study of Eimeria iroquoina Molnar & Fernando, 1974 in Experimentally Infected Fathead Minnows (Pimephales promelas,Cyprinidae) 3. Merogony1

Fathead minnows, Pimephales promelas, raised from eggs in the laboratory, were experimentally infected with oocysts of Eimeria iroquoina from either P. promelas or the common shiner, Notropis cornutus. Within intestinal epithelial cells, trophozoites thought to be derived from the sporozoites contained a prominent electron-dense refractile body. Merozoites dedifferentiated into trophic forms by losing components of their apical complex and pellicle. The inner membrane components of the pellicle appeared discontinuous, and the micronemes became enclosed within vacuoles. Prior to merozoite formation, multinucleate meronts were limited by a single membrane. Golgi complexes were associated with the nuclei of this stage. Merozoites were formed by ectomerogony in one generation and by endomerogony in the final generation. In both forms of merogony the final nuclear division was coupled with the onset of differentiation of the merozoites and featured eccentric mitotic spindles associated with centrocones located within the nuclear envelope and with the precursors of the apical complex. A Golgi complex was closely associated with the nucleus and apical tip of the forming merozoite. Unlike other Eimeria species, the complete pellicle of the merozoites of the final asexual generation of E. iroquoina was formed within the cytoplasm of the meront, without association with the limiting membrane, thus, all pellicular components are synthesized de novo. The inner membranes of the pellicle initially appeared as longitudinal strips, each of which was associated with a pair of the 22–24 subpellicular microtubules. Mature meronts of the final asexual generation averaged 9 μm in diameter and produced 13–16 merozoites. With the exception of the internal completion of the pellicle of the final generation merozoites, the basic processes of merogony in fish Eimeria species are similar to those recorded in terrestrial hosts.     

Ultrastructure of cytoplasmic and nuclear changes in Eimeria tenella during first-generation schizogony in cell culture.

Eimeria tenella sporozoites were inoculated into primary cultures of chick kidney cells. Cells fixed from 1 1/2 to 54 hr later were examined with the electron microscope. At 1 1/2 and 24 hr, most intracellular sporozoites were fusiform and retained organelles typical of extracellular sporozoites. However, at 35 hr, rounded trophozoites were present without these structures; only a refractile body, nucleus, mitochondria, and endoplasmic reticulum remained. Binucleate parasites were also present at that time, but at 48 hr many multinucleate schizonts were present. Nuclei, with adjacent conoids, were at the periphery of these schizonts. Partly developed merozoites, each containing a conoid and a nucleus, protruded into the parasitophorous vacuole. At 54 hr, fully developed merozoites were separated from the residual body. Merozoites resembled sporozoites but lacked the large refractile bodies seen in sporozoites. Linear inclusions were present near the merozoite nucleus and in the residual body. Round vacuoles and ribosomes were also found in the residuum. Nucleoli were first seen in sporozoite nuclei at 1 1/2 hr. They were also present in merozoites but were more prominent in trophozoites and schizonts. Peripheral and scattered nuclear heterochromatins were prominent in intracellular sporozoites and diminished in trophozoites, but increased after several nuclear divisions and were again prominent in the merozoite. Small, distinct interchromatin granules were found in all stages. Intranuclear spindles, centrocones, and centrioles were found in connection with nuclear divisions. Ultrastructure of first-generation schizogony in cell culture was similar to that described for second-generation E. tenella in the chicken and to schizogony of other species of Eimeria.     

In vitro development from sporozoites to first-generation merozoites in Eimeria contorta Haberkorn, 1971. A fine structural study.

The asexual development of Eimeria contorta from sporozoites to first-generation merozoites in tissue culture was investigated with the electron microscope. Sporozoites with a three-layered pellicle, 26 subpellicular microtubules, a conoid, 4-7 rhoptries, and an abundance of micronemes actively entered host cells and showed direct contact to the host cell's cytoplasm. Shortly after penetration, small vacuoles surrounding the parasite merged into a parasitophorous vacuole. Inside this vacuole, sporozoites assumed a definite U-shape before transformation into schizonts took place. This process was characterised by the occurrence of subpellicular microtubules exclusively in the anterior half of the sporozoite, by a degeneration of the 2 inner pellicular membranes, by an outpocketing of the parasite's surface, and by the arrangement of microtubules in clusters. About 25 merozoites were formed at the surface of mature schizonts, to which they remained attached at their posterior pole. A polar ring was present at that area. Anterior and posterior refractile bodies were conspicuous in merozoites and showed close association with mitochondria. The significance of a fibrillar substructure in rhoptries and micronemes is discussed, and special attention is drawn to the pathway of nutrient transport from host cell mitochondria and dictyosomes through intravacuolar folds, parasitophorous vacuole and crescent body into the parasite's food vacuoles.     

Ultrastructure of Cryptosporidium wrairi from the Guinea Pig

SYNOPSIS. The ultrastructure of the known tissue stages of Cryptosporidium wrairi Vetterling, Jervis, Merrill, and Sprinz, 1971 parasitizing the ileum of guinea pigs is described. Young trophozoites are surrounded by 4 unit membranes, the outer 2 of host origin, the inner 2 the pellicle of the parasite. Each trophozoite contains a vesicular nucleus with a large nucleolus. Its cytoplasm contains ribosomes, but eventually fills with cisternae of the rough endoplasmic reticulum. As the trophozoite matures the area of attachment of the parasite to the host cell becomes vacuolated, with vertical membranous folds. It is apparent that the parasite acquires nourishment from the host cell thru this area of attachment. As schizonts develop, (a) multiple nuclei appear, (b) the endoplasmic reticulum enlarges, (c) the attachment zone increases in area, (d) large vacuoles, which develop as endocytotic vesicles in the attachment area, are found in the cytoplasm and (e) the inner unit membrane of the parasite pellicle is resorbed around the sides of the developing schizont. Following nuclear division, merozoites develop from the schizont by budding. Merozoites have an ultrastructure similar to that described for other coccidia except that no mitochondria, micropores, or subpellicular tubules were observed. Merozoites penetrate the epithelial cell causing invagination of the microvillar membrane and lysing it. No unit membrane is formed between the parasite and the host cell. However, the cell produces one or 2 dense bands adjacent to the parasite attachment area. The macrogamete contains a nucleus, endoplasmic reticulum, attachment zone, and large vacuoles. It also contains a variety of granules, some of which are polysaccharide. The immature microgametocyte contains multiple compact nuclei. No mature microgametocytes or zygotes were found.     

Characterization of a Cryptosporidium parvum sporozoite glycoprotein.

Polyclonal and monoclonal antibodies directed against Cryptosporidium oocysts or sporozoites were developed to identify and characterize sporozoite pellicle and apical complex antigens. A very large glycoprotein of Cryptosporidium sporozoites was identified by three monoclonal antibodies that also reacted with intracellular merozoites. The glycoprotein was also identified by polyclonal antibodies that were affinity-purified on nitrocellulose-bound recombinant proteins expressed by four lambda gtll genomic clones.     

Ultrastructural development of first- to second-generation merozoites in Eimeria contorta Haberkorn, 1971.

The development of first-generation merozoites to second-generation schizonts and merozoites of Eimeria contorta in one of its natural hosts, the mouse, was investigated with the electron microscope. Merozoites inside a host cell show a marked U-shape and a degeneration of the inner-pellicular membrane complex prior to transformation into schizonts. These processes closely resemble those seen in transforming sporozoites. In young schizonts with about 3-5 nuclei, the Golgi-adjuncts (structures of unknown function) form a large interconnected network. Nuclear divisions in growing schizonts involve the formation of a centroc?ne, which develops in a pocket-like indentation of the nuclear envelope. At least one centriole is present immediately adjacent to this indentation. In a later stage, the centroc?ne forms a conical nuclear protrusion directed towards a merozoite-anlage. This developing merozoite contains anlagen of a conoid, of rhoptries, and of micronemes and a refractile body in addition to the nucleus, centrioles, and a Golgi-adjunct. The merozoite-anlage is limited by a triple unit membrane complex. Schizonts give rise to 8-15 second-generation merozoites. Interesting features of these merozoites are the high number of micronemes, the finding of one single large mitochondrion per merozoite, and the occurrence of 26 subpellicular microtubules, i.e. the same number as in sporozoites of E. contorta. At the end of their development, merozoites come into direct contact with the host cell cytoplasm as the parasitophorous vacuole breaks down.     

The life cycle of Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) infecting chickens

The life cycle and morphology of a previously undescribed species of Cryptosporidium isolated from commercial broiler chickens is described. The prepatent period for Cryptosporidium baileyi n. sp. was three days post oral inoculation (PI) of oocysts, and the patent period was days 4-24 PI for chickens inoculated at two days of age and days 4-14 for chickens inoculated at one and six months of age. During the first three days PI, most developmental stages of C. baileyi were found in the microvillous region of enterocytes of the ileum and large intestine. By day 4 PI, most parasites occurred in enterocytes of the cloaca and bursa of Fabricius (BF). Mature Type I meronts with eight merozoites first appeared 12 h PI and measured 5.0 x 4.9 micrometers. Mature Type II meronts with four merozoites and a large granular residuum first appeared 48 h PI and measured 5.1 x 5.1 micrometers. Type III meronts with eight short merozoites and a large homogeneous residuum first appeared 72 h PI and measured 5.2 x 5.1 micrometers. Microgamonts (4.0 x 4.0 micrometers) produced approximately 16 microgametes that penetrated into macrogametes (4.7 x 4.7 micrometers). Macrogametes gave rise to two types of oocysts that sporulated within the host cells. Most were thick-walled oocysts (6.3 x 5.2 micrometers), the resistant forms that passed unaltered in the feces. Some were thin-walled oocysts whose wall (membrane) readily ruptured upon release from the host cell. Sporozoites from thin-walled oocysts were observed penetrating enterocytes in mucosal smears. The presence of thin-walled, autoinfective oocysts and the recycling of Type I meronts may explain why chickens develop heavy intestinal infections lasting up to 21 days. Oocysts of C. baileyi were inoculated orally into several animals to determine its host specificity. Cryptosporidium baileyi did not produce infections in suckling mice and goats or in two-day-old or two-week-old quail. One of six 10-day-old turkeys had small numbers of asexual stages only in the BF. Four of six one-day-old turkeys developed mild infections only in the BF, and sexual stages of the parasite were observed in only one of the four. All seven one-day-old ducks and seven two-day-old geese developed heavy infections only in the BF with all known developmental stages present.     

Eimeria tenella: use of a monoclonal antibody in determining the intracellular fate of the refractile body organelles and the effect on in vitro development

A monoclonal antibody, which recognizes the refractile body of Eimeria sporozoites, was used to study the developmental fate of this organelle during asexual development of E. tenella and to determine the effect of this monoclonal antibody on in vitro development of the parasite. Through use of immunofluorescent antibody and gold-labeling techniques at the light and electron microscopy level, the refractile body at 48 to 96 hr postinoculation was found to separate into 6 to 10 small globules, then diffuse throughout the schizont cytoplasm, and eventually reconcentrate as a small dot of material in each of the mature first-generation merozoites. The schizont did not develop to maturity if diffusion of the refractile body did not occur. The refractile body material was quickly lost as the merozoite left the schizont and invaded new cells and was not detected in any later developmental stages. The in vitro development of first- and second-generation schizonts of E. tenella was greatly inhibited (up to 100%) with exposure to the monoclonal antibody. There was an increase in the number of schizonts with nondispersed refractile body in the monoclonal antibody-treated cells when compared to the untreated controls, and the few mature schizonts seen had up to a 50-fold decrease in the number of merozoites. Immunofluorescent antibody labeling of the refractile body of intracellular sporozoites and schizonts treated in vitro with the monoclonal antibody for 24-96 hr postinoculation indicated that the antibody had crossed the host cell and parasite plasma membrane during incubation.