Malaria parasite pre-erythrocytic infection: preparation meets opportunity

For those stricken with malaria, the classic clinical symptoms are caused by the parasite's cyclic infection of red blood cells. However, this erythrocytic phase of the parasite's life cycle initiates from an asymptomatic pre-erythrocytic phase: the injection of sporozoites via the bite of a parasite-carrying Anopheline mosquito, and the ensuing infection of the liver. With the increased capabilities of studying liver stages in mice, much progress has been made elucidating the cellular and molecular basis of the parasite's progression through this bottleneck of its life cycle. Here we review relevant findings on how sporozoites prepare for infection of the liver and factors crucial to liver stage development as well as key host/parasite interactions.     

A Cysteine Protease Inhibitor of Plasmodium berghei Is Essential for Exo-erythrocytic Development

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.     

Development of Caryospora bigenetica n. sp. (Apicomplexa,Eimeriidae) in Rattlesnakes and Laboratory Mice1

During a survey of the coccidian parasites of reptiles from Iowa, three specimens of Crotalus horridus L., the Timber Rattlesnake, and one of Sistrurus catenatus (Rafinesque), the Massasauga Rattlesnake, were found to be passing oocysts of a Caryospora, here described as C. bigenetica n. sp. Since these snakes (family Crotalidae) are known to subsist mainly on small mammals, oocysts from one of the Timber Rattlesnakes were fed to laboratory white mice (Mus musculus L.) to determine if mammals might be involved as alternate hosts in the life cycle. At necropsy, tissues of the tongue and dermis of the mice revealed a sequence of stages which included mature male and female gamonts, fully sporulated sporocysts, “excysted” sporozoites, and “resting” sporozoites that lay individually in solitary, cyst-like host cells termed “caryocysts.” A coccidia-free Massasauga that was fed an infected mouse, at a time when caryocysts in the mouse would have been present, later passed oocysts similar to those of the original inoculum. These results, along with the discovery of endogenous stages (asexual and sexual) in the intestine of the Timber Rattlesnake and the experimentally infected Massasauga, suggest that this parasite has a heteroxenous life cycle pattern, with sexual stages occurring both in the ophidian and the mammalian hosts.     

PfSPATR,a Plasmodium falciparum protein containing an altered thrombospondin type I repeat domain is expressed at several stages of the parasite life cycle and is the target of inhibitory antibodies

The annotated sequence of chromosome 2 of Plasmodium falciparum was examined for genes encoding proteins that may be of interest for vaccine development. We describe here the characterization of a protein with an altered thrombospondin Type I repeat domain (PfSPATR) that is expressed in the sporozoite, asexual, and sexual erythrocytic stages of the parasite life cycle. Immunoelectron microscopy indicated that this protein was expressed on the surface of the sporozoites and around the rhoptries in the asexual erythrocytic stage. An Escherichia coli-produced recombinant form of the protein bound to HepG2 cells in a dose-dependent manner and antibodies raised against this protein blocked the invasion of sporozoites into a transformed hepatoma cell line. Sera from Ghanaian adults and from a volunteer who had been immunized with radiation-attenuated P. falciparum sporozoites specifically recognized the expression of this protein on transfected COS-7 cells. These data support the evaluation of this protein as a vaccine candidate.     

Eimeria necatrix virus: intracellular localisation of viral particles and proteins

The presence of the Eimeria necatrix virus was investigated in the following life cycle stages: sporocysts, sporozoites, merozoites, and macrogametes. Electron microscopy revealed virus-like particles (VLPs) in sporozoites, which were purified from sporozoite extracts and used to raise polyclonal antibodies. Viral proteins were identified as RNA polymerase (95 kDa) and the major capsid protein (80 kDa). Polyclonal antibody was used to detect the intracellular localisation of VLPs and proteins. Immunoelectron microscopy and immunohistochemistry identified a viral protein of 95 kDa in all the E. necatrix stages studied, whereas the 80 kDa protein was found only in sporocysts and sporozoites. In addition, no VLPs were found in sporocysts. These results indicate that the synthesis of viral capsid proteins takes place during the early events of sporulation, and is then packaged into novel viruses during the late events. No VLPs were seen and no capsid proteins were found in the merozoites and macrogametes, whereas the 95 kDa RNA polymerase was present in both these stages. In addition, no VLPs or proteins were detected in chicken tissues.     

Sexual and Asexual Development of Cryptosporidium parvum in Five Oocyst– or Sporozoite-Infected Human Enterocytic Cell Lines

ABSTRACT. The human enterocytic cell lines Caco-2, HT29, HCT8 and the Caco-2 clones TC7 and PF11 were studied for their ability to support Cryptosporidium parvum development. Following the addition in cultures of either oocysts or excysted sporozoites, immunofluorescent and transmission electron microscopy revealed the presence of all stages of the parasite life cycle by both procedures, and no difference in the ration of infected cells was found among cell lines. More oocysts were seen in cell monolayers infected with oocysts than with sporozoites (p < 0.0001). The number of meronts observed was the same after either oocysts or sporozoites inoculation. Data suggest that the two methods yield a same cell infection rate.     

Cryptosporidium parvum life cycle in suckling mice: a Nomarski interference-contrast study of a human-derived strain.

Cryptosporidiosis has emerged as one of the life-threatening opportunistic enteric infections in HIV-infected persons. To date, Cryptosporidium parvum is known to infect man via person-to-person or zoonotic transmission. We studied the sequential stages of the life cycle of C. parvum by Normarski interference-contrast microscopy in fresh gut specimens of newborn mice, infected with a strain derived from an AIDS patient with cryptosporidial diarrheal enteritis. Many 4- to 5-day-old suckling BALB/C mice were orally inoculated with 1 x 10(6) oocysts, obtained by acid flocculation of the patient's stools. The animals were sacrificed from 4 to 96 h post-infection and the ileum was examined microscopically. All stages of the asexual life cycle of C. parvum, from excysted sporozoites in the intestinal lumen through the development of type II mature meronts, 12- to 72-h post-infection, were documented by extemporaneous microscopic evaluation of fresh gut samples. The sexual cycle, characterized by the appearance of micro- and macrogametocytes, followed by a zygote developing into a sporulated oocyst, was documented as early 48-h post-infection. Our Nomarski interference-contrast observations on the life cycle of C. parvum yielded data comparable with those originally published by Current and Reese, and confirm the results of previous electron microscopic studies performed by several other authors.     

Cryptosporidium parvum merozoites share neutralization-sensitive epitopes with sporozoites

Sporozoites and merozoites are stages in the life cycle of Cryptosporidium parvum that can cyclically infect intestinal cells, causing persistent infection and severe diarrhea in immunodeficient patients. Infection by sporozoites can be neutralized by surface-reactive mAb. We show that merozoite infectivity can also be neutralized by surface-reactive mAb. To do this, viable C. parvum merozoites were isolated by differential and isopycnic. centrifugation, and distinguished from sporozoites by transmission electron microscopy. Differential reactivity with a panel of seven mAb was used to determine the amount of sporozoite contamination in isolated merozoite preparations. The isolated merozoites were distinguished from sporozoites (p less than 0.0001) by four sporozoite-specific mAb (16.332, 16.502, 17.25, and 18.357) in an indirect immunofluorescence assay. Three mAb (16.29, 17.41, and 18.44) consistently reacted with both merozoites and sporozoites. Isolated merozoites were infectious for neonatal mice when administered by intraintestinal injection. Infectivity for mice was significantly neutralized (p less than 0.05) when 1 to 2 x 10(5) merozoites were incubated with sporozoite-neutralizing mAb 17.41 or 18.44, before inoculation. Merozoites incubated with an isotype control mAb remained infectious for neonatal mice. We conclude that C. parvum merozoites share neutralization-sensitive epitopes with sporozoites.     

A retrospective evaluation of the role of T cells in the development of malaria vaccine

Due to the fact that the life cycle of malaria parasites is complex, undergoing both an extracellular and intracellular phases in its host, the human immune system has to mobilize both the humoral and cellular arms of immune responses to fight against this parasitic infection. Whereas humoral immunity is directed toward the extracellular stages which include sporozoites and merozoites, cell-mediated immunity (CMI), in which T cells play a major role, targets hepatic stages - liver stages - of the parasites. In this review, the role of T cells in protective immunity against liver stages of the malaria infection is being re-evaluated. Furthermore, this review intends to address how to translate the findings regarding the role of T cells obtained in experimental systems to actual development of malaria vaccine for humans.     

Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture.

This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle genotypes) in the HCT-8 cell line. Furthermore, for the first time the complete life cycle was perpetuated in vitro for up to 25 days by subculturing. The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-reinfection cycle of C. parvum. This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro. In addition, thin-walled oocysts of the cattle genotype were infective for ARC/Swiss mice but similar oocysts of the human genotype were not. This culture system will provide a model for propagation of the complete life cycle of C. parvum in vitro.     

Plasmodium pre-erythrocytic stages: what’s new?

The pre-erythrocytic (PE) phase of malaria infection, which extends from injection of sporozoites into the skin to the release of the first generation of merozoites, has traditionally been the 'black box' of the Plasmodium life cycle. However, since the advent of parasite transfection technology 13 years ago, our understanding of the PE phase in cellular and molecular terms has dramatically improved. Here, we review and comment on the major developments in the field in the past five years. Progress has been made in many diverse areas, including identifying and characterizing new proteins of interest, imaging parasites in vivo, understanding better the cell biology of hepatocyte infection and developing new vaccines against PE stages of the parasite.     

Automated classification of Plasmodium sporozoite movement patterns reveals a shift towards productive motility during salivary gland infection

The invasive stages of malaria and other apicomplexan parasites use a unique motility machinery based on actin, myosin and a number of parasite-specific proteins to invade host cells and tissues. The crucial importance of this motility machinery at several stages of the life cycle of these parasites makes the individual components potential drug targets. The different stages of the malaria parasite exhibit strikingly diverse movement patterns, likely reflecting the varied needs to achieve successful invasion. Here, we describe a Tool for Automated Sporozoite Tracking (ToAST) that allows the rapid simultaneous analysis of several hundred motile Plasmodium sporozoites, the stage of the malaria parasite transmitted by the mosquito. ToAST reliably categorizes different modes of sporozoite movement and can be used for both tracking changes in movement patterns and comparing overall movement parameters, such as average speed or the persistence of sporozoites undergoing a certain type of movement. This allows the comparison of potentially small differences between distinct parasite populations and will enable screening of drug libraries to find inhibitors of sporozoite motility. Using ToAST, we find that isolated sporozoites change their movement patterns towards productive motility during the first week after infection of mosquito salivary glands.     

Getting infectious: formation and maturation of Plasmodium sporozoites in the Anopheles vector

Research on Plasmodium sporozoite biology aims at understanding the developmental program steering the formation of mature infectious sporozoites - the transmission stage of the malaria parasite. The recent identification of genes that are vital for sporozoite egress from oocysts and subsequent targeting and transmigration of the mosquito salivary glands allows the identification of mosquito factors required for life cycle completion. Mature sporozoites appear to be equipped with the entire molecular repertoire for successful transmission and subsequent initiation of liver stage development. Innovative malaria intervention strategies that target the early, non-pathogenic phases of the life cycle will crucially depend on our insights into sporozoite biology and the underlying molecular mechanisms that lead the parasite from the mosquito midgut to the liver.     

Proteomics analysis and protein expression during sporozoite excystation of Cryptosporidium parvum (Coccidia, Apicomplexa)

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells.     

Eimeria tenella enolase and pyruvate kinase: a likely role in glycolysis and in others functions

Two cDNA codings for glycolytic enzymes were cloned from a cDNA library constructed from the schizont stage of the avian parasite Eimeria tenella. Enolase and pyruvate kinase cDNA were fully sequenced and compared with sequences of enzymes from other organisms. Although these enzymes were already detected in the sporozoite stage, their expression was enhanced during the first schizogony in accordance with the anaerobic conditions of this part of the life cycle of the parasite. Under activating conditions, microscopic observations suggest that these glycolytic enzymes were relocalised inside sporozoites and moreover were in part secreted. The enzymes were also localised at the apex of the first generation of merozoites. Enolase was partly observed inside the nucleus of sporozoites and schizonts. Taken together, these results suggest that glycolytic enzymes not only have a function in glycolysis during anaerobic intracellular stages but may also participate in the invasion process and, for enolase, in the control of gene regulation.     

Comparative Development of Eimeria tenella from Sporozoites to Oocysts in Primary Kidney Cell Cultures from Gallinaceous Birds

Eimeria tenella completed its endogenous life cycle in primary cultures of kidney cells from 2- to 3-week-old-chickens, guinea fowl, partridges, pheasants, quail, and turkeys. Similarity in percentage of infection at 4 hr suggested that sporozoites entered cells from all birds in equal numbers. Development was better, however, in chicken cells in that the percentage of survival and of developmental stages during the first 2 days were greater, developmental stages occurring after 2 days usually were found earlier, mature 2nd-generation schizonts and oocysts were larger, and oocyst production was far greater than in nonhost cells. Multinucleate macrogametes, which sometimes reached sizes 3–4 times greater than normal oocysts, are reported for the first time.     

中华血簇虫的生活史研究 Ⅰ.中华血簇虫在鳖穆蛭中的发育

中华血簇虫是湖北产的中华鳖体内发现的一种寄主虫。在其生活史发育中,存在着两种寄主的交替。本文只介绍它在无脊椎动物寄主——鳖穆蛭体中的发育情形。这一时期包括两个阶段:配子生殖和孢子生殖。配子生殖的特点是,两性配子母细胞先融合,然后才进行配子分化,产生4个雄配子核,其中1个核与雌配子核受精,形成合子核。孢子生殖以单核卵囊的核分裂开始,最后形成含8个裸子孢子的成熟卵囊,并解体释放出子孢子。整个过程都发生在蛭消化道内。脊椎动物寄主的感染,很可能是因为吃下含成熟子孢子的无脊椎动物寄主而引起的。     

Studies on the Life History of a Neogregarine Parasite Found in Leptothorax Ants from North America

Pupal stages of Leptothorax ants collected near West Yellowstone, MT, USA, displayed striking signs and symptoms of disease, i.e., grey to black coloration, irregular pigmentation of compound eyes and toothless mandibles. Light microscope studies revealed heavy infections by a neogregarine, the life history of which is described. The life cycle of the pathogen includes micronuclear and macronuclear schizogonies, gametogony and sporogony. Schizonts of both types vary in size depending on the number of nuclei which is usually defined by doubling, thus giving rise to 8, 16, 32, 64 or even 128 uninucleate merozoites. In smears and sections, micronuclear merozoites are typically arranged in rosettes. In the early transformation of zygotes, sickle-shaped developmental stages have been encountered, so far undescribed from neogregarines. Two spores (oocysts), each developing eight sporozoites, evolve from each gametocyst, as is typical of the genus Mattesia. Mature lemon-shaped spores measure 13.8 9.3 μm in fresh preparations. Infections can be readily transmitted to healthy colonies and to other Leptothorax species by feeding crushed infected pupae. Vegetative life cycle stages grow and multiply in the haemocoel, only to some extent they infect fat body cells. Macronuclear merozoites invade the hypodermis and the fat body but also settle extracellularly in the haemocoel. The disease process terminates with the death of the pupae that harbour abundant spores. Infections of adults have not been observed. Despite some minor differences that may result from development of the pathogen in this host, from the type, sequence and morphology of life cycle stages and from the signs and symptoms of disease, this Mattesia species is identified with M. geminata, first discovered in the tropical fire ant, Solenopsis geminata (Fabricius).