细粒棘球绦虫原头蚴组织化学的观察

本文对细粒棘球绦虫原头蚴的组织化学成份进行了较为系统和全面的观察,结果表明,该虫体内存在糖原、RNA、DNA、蛋白质结合的α-氨基、酪氨酸、色氨酸及组氨酸、碱性蛋白质、酸性粘多糖、胶原及网状纤维成分、AKP、ATP酶及ACP等生化物质。并对上述成分的定位,含量及其生理学的意义进行了重点讨论。     

Light microscopy and scanning electron microscopy of the In vitro evagination process of Echinococcus multilocularis protoscolices

Marchiondo A. A. and Andersen F. L. 1984. Light microscopy and scanning electron microscopy of the in vitro evagination process of Echinococcus multilocularis protoscolices. International Journal for Parasitotogy14:151–157. During histogenesis of the protoscolices of Echinococcus multilocularis, the apical portion of the protoscolex consisting of the suckers, rostellum and hook region develops as an introversion and invagination within the tissue of the basal portion. In vitro incubation of protoscolices in evagination fluid stimulates the emergence of the apical portion. The initiation of evagination is first detected by a surface change in the basal portion. The smooth contour of this surface which lacks microtriches becomes transformed into tegumental indentations that form transverse and longitudinal furrows within the basal tegument as the protoscolices contract and expand, respectively. An orifice formed at the site or junction where the apical portion is invaginated begins to expand laterally in order to allow emergence of the suckers. The hooks are arranged within the invaginated protoscolex with blades directed towards the basal orifice, the handles directed towards the peduncle and the guards directed laterally. This arrangement persists throughout the evagination of the suckers and rostellum until the apical dome of the hook region emerges, thereby rotating the blades laterally in the direction of the peduncle and rotating the handles and guards medially to assume a coronal arrangement. Evagination is an asynchronous event and therefore allows observation of individual protoscolices in various stages of emergence.     

In vitro effects of some herbs used in Egyptian traditional medicine on viability of protoscolices of hydatid cysts

The present work evaluated the effects of alcoholic extracts of salvia (Salvia officinalis), thyme (Thymus vulgaris), and 2 pure compounds (thymol and menthol) on the viability of Echinococcus granulosus protoscolices in vitro. Four different concentrations of each extract (2,500, 1,500, 1,000, and 500 μg/ml) and 3 different concentrations each of thymol and menthol (50, 10, and 1 μg/ml) were used. Concentration of 2,500 μg/ml of both extracts showed a significant protoscolicidal activity on the 6th day. Complete loss of viability of protoscolices occurred with 500 μg/ml concentration of both extracts at day 6 and day 7 post-treatment (PT), respectively. Pure compounds, i.e., menthol and thymol, showed potent effects with 50 μg/ml concentration at day 2 and day 5 PT, respectively. These effects were compared with those of albendazole sulfoxide (800 μg/ml), a commonly used treatment drug for hydatidosis. Krebs-Ringer solution and the hydatid cystic fluid at a ratio of 4:1 was a good preservative solution which kept the protoscolices viable for 15 days.     

Cellular organization and appearance of differentiated structures in developing stages of the parasitic platyhelminth Echinococcus granulosus

Echinococcus granulosus is the causative agent of hydatidosis, a major zoonoses that affects humans and herbivorous domestic animals. The disease is caused by the pressure exerted on viscera by hydatid cysts that are formed upon ingestion of E. granulosus eggs excreted by canine. Protoscoleces, larval forms infective to canine, develop asynchronously and clonally from the germinal layer (GL) of hydatid cysts. In this report, we describe the cellular organization and the appearance of differentiated structures both in nascent buds and developed protoscoleces attached to the GL. Early protoscolex morphogenesis is a highly complex and dynamic process starting from the constitution of a foramen in the early bud, around which nuclei are distributed mainly at the lateral and apical regions. Similarly, distribution of nuclei in mature protoscoleces is not homogenous but underlies three cellular territories: the suckers, the rostellar pad, and the body, that surrounds the foramen. Several nuclei are associated to calcareous corpuscles (Cc), differentiated structures that are absent in the earlier bud stages. The number of nuclei is similar from the grown, elongated bud stage to the mature protoscolex attached to the GL, strongly suggesting that there is no significant cellular proliferation during final protoscolex development. The amount of DNA per nucleus is in the same range to the one described for most other platyhelminthes. Our results point to a sequential series of events involving cell proliferation, spatial cell organization, and differentiation, starting in early buds at the GL of fertile hydatid cysts leading to mature protoscoleces infective to canine.     

Bovine (Bos taurus) humoral immune response against Echinococcus granulosus and hydatid cyst infertility

Echinococcus granulosus, the agent of hydatid disease, presents an indirect life cycle, with canines (mainly dogs) as definitive hosts, and herbivores and human as intermediary ones. In intermediary hosts fertile and infertile cysts develop, but only the first ones develop protoscoleces, the parasite form infective to definitive hosts. We report the presence of bovine IgGs in the germinal layer from infertile cysts (GLIC), in an order of magnitude greater than in the germinal layer from fertile cysts (GLFC). When extracted with salt solutions, bovine IgGs from GLIC are associated with low or with high affinity (most likely corresponding to non specific and antigen specific antibodies, respectively). Specific IgGs penetrate both the cells of the germinal layer and HeLa cultured cells and recognize parasitic proteins. These results, taken together with previous ones from our laboratory, showing induction of apoptosis in the germinal layer of infertile hydatid cysts, provide the first coherent explanation of the infertility process. They also offer the possibility of identifying the parasite antigens recognized, as possible targets for immune modulation.     

Construction of In Vivo Fluorescent Imaging of Echinococcus granulosus in a Mouse Model

Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation.     

Echinococcus multilocularis: Proteomic analysis of the protoscoleces by two-dimensional electrophoresis and mass spectrometry

Echinococcus multilocularis is an important parasite that causes human alveolar echinococcosis. Identification and characterization of the proteins encoded by E. multilocularis metacestode might help to understand the complexity of the parasites and their interactions with the host, and to identify new candidates for immunodiagnosis and vaccine development. Here we present a proteomic analysis of E. multilocularis protoscolex (PSC) proteins. The proteins were resolved by 2-DE (pH range 3.5-10), followed by MALDI-TOF MS analysis. Fourteen known Echinococcus proteins were identified, including cytoskeletal proteins, heat shock proteins, metabolic enzymes, 14-3-3 protein, antigen P-29 and calreticulin. To construct a systematic reference map of the immunogenic proteins from E. multilocularis PSC, immunoblot analysis of PSC 2-DE maps was performed. Over 50 proteins spots were detected on immunoblots as antigens and 15 of them were defined. The results showed that cytoskeletal proteins and heat shock proteins were immunodominant antigens in alveolar echinococcosis.     

A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis

Macpherson C. N. L. and Mcmanus D. P. 1982). A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis. International Journal for Parasitology12: 515–521. The soluble enzyme extracts from protoscoleces obtained from hydatid cysts of human, camel, cattle, sheep and goat origin were compared on the basis of their isoenzyme patterns for GPI and PGM using isoelectric focusing. Consistent GPI and PGM isoenzyme patterns were obtained for larvae of human, camel and sheep material. Cattle material varied occasionally in having an additional cathodic band in some of the GPI patterns. Two distinct isoenzyme patterns were evident in the goat material for both enzymes. The more common goat patterns were similar to those of human, cattle and sheep (Kenya, U.K. and Argentina) material, which were similar to each other. The rare goat patterns were similar to those obtained for camel material. Cyst location in the various intermediate hosts had no effect on the zymograms obtained. Additionally, no alteration in the major banding patterns was observed between the larvae and homologous adults produced by experimental infections. Of 26 naturally infected dogs, 19 produced adult GPI zymograms resembling human/ sheep/goat (common form) experimental infection patterns, three were similar to experimental cattle infections and four had camel/goat (rare form) patterns.     

Formation of Echinococcus granulosus laminated membrane in a defined medium

Echinoccocus granulosus protoscoleces were digested from brood capsule material using artificial gastric fluid, and were cultured for 60 days in medium NCTC 135. Vesiculation occurred within 7 days, and the first laminated membranes were observed after 17 days of culture. Some contaminating sheep antigens appeared to be lost after 21 days.