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。     

Plasmodium lophurae: the ultrastructure of the exoerythrocytic stages

The fine structure of the exoerythrocytic stages of Plasmodium lophurae was studied. in specimens grown in tissue cultures of avian cells. Specimens were prepared for sectioning by a method which minimizes disturbance and permits precise selection and orientation specimens.Plasmodium lophurae is similar in many aspects to P. fallax. Merozoites are highly specialized and differentiated. Analysis of their ultrastructure revealed the polar complex to be a specialization of the pellicular envelope and its associated underlying microtubules. The polar rings may simply be a modification of the inner membrane of the pellicle and not discrete structures as previously reported. The electron-dense polar organelles are separated on morphological grounds into three groups: the large paired organelles and the small dense bodies which are both linked to microducts, and the transitional bodies, a third organelle being reported for the first time. Transitional bodies are without microducts, occur in fully mature merozoites and persist only for a short period. All three of these organelles appear to be related to and possibly even derived from internal membrane systems and ribosomes. The apolar end of the merozoite contains the mitochondrion and its associated spherical body. Detailed study of the latter shows it to be cylindrical.Upon entering the host cell, the parasite adds a third membrane at the interface between it and the cell. The merozoite becomes spherical and undergoes transformation into a trophozoite. During this reorganization phase, dedifferentiation occurs and is followed by a rapid growth phase. The end of the growth phase is signaled by the appearance of germinal clefts and nuclear division. The entire process of schizogony culminates in a highly synchronized formation of merozoites.Processes of the limiting membrane forming the host parasite interface were observed extending deply into the cytoplasm of the host cell and often appeared to form bridges between two or more parasites. The significance of this new observation is not yet established.     

Plasmodium berghei: infective exoerythrocytic schizonts in primary monolayer cultures of rat liver cells.

Suspensions of rat liver cells which included hepatic exoerythrocytic schizonts (HEX) of Plasmodium berghei were used to initiate primary monolayer cultures of rat hepatic cells. These cell suspensions were prepared by using an enzymatic method for the dissociation of the livers of rats that had been infected with sporozoites of P. berghei 3 to 10, 18 to 28, and 29 to 36 hr prior to the liver dissociation procedure. These cell suspensions included HEX which were infective for recipient rodents when inoculated intraperitoneally into the recipients. HEX were considered to have been successfully maintained if they retained their infectivity for rodents following the cultivation period. The relative number of infective HEX present in the liver cell suspensions before and after cultivation was determined by use of an infectivity assay. Using this infectivity assay, it was observed that less infective HEX were present in the cell population following cultivation than were present before cultivation. Infective HEX were recovered from culture in experiments in which the time in vitro ranged from 3 to 44 hr. Twelve of fifteen (80%) attempts to maintain infective HEX in culture for 21 to 28 hr were successful, while one of eight (12.5%) attempts to maintain HEX in culture for 36 to 48 hr were successful. Thus, these experiments have provided an 80% success rate for maintaining HEX for a period equivalent to over 50% of the incubation period of HEX of this parasite. This technique should be sufficient for studying in vitro the factors which influence the development of HEX, as well as for testing methods of causal prophylaxis.     

Plasmodium berghei: preparation of rat hepatic cell suspensions that include infective exoerythrocytic schizonts.

Employing an enzymatic method to dissociate rat liver, we prepared suspensions of liver cells from rats infected with sporozoites of Plasmodium berghei 3 to 10, 18 to 28, or 29 to 36 hr prior to liver dissociation. These suspensions of liver cells included hepatocytes, Kupffer cells, fibroblasts, and unidentified cells, as well as hepatocytes infected with exoerythrocytic schizonts (HEX) of P. berghei. These HEX were infective for recipient rodents when inoculated intraperitoneally into the recipients. The number of infective HEX present in the liver cell suspensions was quantitated by varying the number of HEX inoculated into recipients. This infectivity assay made it possible to compare the numbers of HEX in suspensions of liver cells from different donor rats. Infective HEX were obtained from donor rats in 35 of 41 experiments. The greatest number of infective HEX was obtained from donors injected with sporozoites 18 to 28 hr prior to liver dissociation. For morphological observation of mature HEX in cell suspensions, hepatic cells were prepared from donors infected with sporozoites 48 hr prior to liver dissociation. For experimental purposes, the preparation of infective HEX in suspensions of liver cells is superior to the preparation of infective HEX in liver fragments, because it is possible to quantitate the number of HEX which are present either visually or by means of the infectivity assay.     

Development and ultrastructure of Besnoitia oryctofelisi tachyzoites,tissue cysts,bradyzoites, schizonts and merozoites

Development and structure of different life cycle stages of Besnoitia oryctofelisi which has a rabbit-cat life cycle was studied by light and transmission electron microscopy. For light microscopy, Besnoitia oryctofelisi-infected tissues were stained with haematoxylin-eosin, periodic acid Schiff (PAS) reagent, and immunohistochemically with rabbit anti-B. oryctofelisi polyclonal antibodies and anti-BAG-1 antibodies. In vitro and in vivo-derived tachyzoites were 5-6 microm long and they were found to divide by endodyogeny. In tachyzoites, the nucleus was often central, and micronemes were few and located anterior to the nucleus. Earliest tissue cysts were seen in gerbils starting 12 days p.i. Early tissue cysts had an outer PAS-positive cyst wall, a middle PAS-negative host cell layer, and an inner PAS-negative parasitophorous vacuolar membrane. Organisms in early tissue cysts were PAS-negative, did not stain with anti-BAG-1 antibodies, and amylopectin granules and enigmatic bodies were absent. Tissue cysts beginning 17 days p.i. contained organisms that became PAS-positive and reacted with anti-BAG-1 antibodies, indicating they were bradyzoites. Immunoreactivity with polyclonal anti-B. oryctofelisi antibodies suggested that Besnoitia species bradyzoites are encapsulated by the host cell. Bradyzoites (10 microm) were about twice the length of tachyzoites and contained enigmatic bodies characteristic of Besnoitia bradyzoites. Unlike tachyzoites and tissue cysts, schizonts were located intravascularly in the lamina propria of the small intestine of cats. Merozoites were 5-6 microm long, had few rhoptries and amylopectin granules, had numerous micronemes and had a terminal nucleus.     

Establishment of Besnoitia darlingi from opossums (Didelphis virginiana) in experimental intermediate and definitive hosts,propagation in cell culture,and description of ultrastructural and genetic characteristics

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues. These schizonts measured up to 45 x 25 microm and contained many merozoites. A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B. darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA.     

Alkyne modified purines for assessment of activation of Plasmodium vivax hypnozoites and growth of pre-erythrocytic and erythrocytic stages in Plasmodium spp.

Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite’s ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesised that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalysed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.