Fine structure of immature cysts of Sarcocystis cruzi.

Sarcocystis cruzi forms cysts in striated muscle of the bovine host following schizogony. The fine structure of the immature cyst within muscle fibers of the ventricular myocardium was studied in relation to its development and to the multiplication of parasites within it. The young cyst is enclosed by a cyst wall containing numerous small protuberances. Metrocytes within the cyst are irregular in shape and are separated from each other and the cyst wall by a thin layer of ground substance. The parasite multiplies by endodyogeny within the metrocyte. As the cyst enlarges, the host muscle fiber is disrupted and large protrusions are present in the cyst wall.     

Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host

Cystic hydatid disease (CHD) is caused by infection with the Echinococcus granulosus metacestode and affects both humans and livestock. In this work, we performed a proteomic analysis of the E. granulosus metacestode during infection of its intermediate bovine host. Parasite proteins were identified in different metacestode components (94 from protoscolex, 25 from germinal layer and 20 from hydatid cyst fluid), along with host proteins (58) that permeate into the hydatid cyst, providing new insights into host‐parasite interplay. E. granulosus and platyhelminth EST data allowed successful identification of proteins potentially involved in downregulation of host defenses, highlighting possible evasion mechanisms adopted by the parasite to establish infection. Several intracellular proteins were found in hydatid cyst fluid, revealing a set of newly identified proteins that were previously thought to be inaccessible for inducing or modulating the host immune response. Host proteins identified in association with the hydatid cyst suggest that the parasite may bind/adsorb host molecules with nutritional and/or immune evasion purposes, masking surface antigens or inhibiting important effector molecules of host immunity, such as complement components and calgranulin. Overall, our results provide valuable information on parasite survival strategies in the adverse host environment and on the molecular mechanisms underpinning CHD immunopathology.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

The pathogenicity, localization, and cyst structure of echinostomatid metacercariae (Trematoda) infecting the kidneys of the frogs Rana clamitans and Rana pipiens

Light and scanning electron microscopy were used to examine the localization and pathogenicity of echinostomatid metacercariae infecting the kidneys of leopard frogs, Rana pipiens, and green frogs, Rana clamitans. Cysts occurred predominantly in the ventrolateral renal cortex, and at least some were confined to the lumen of the Bowman's capsules. Each vermiform metacercarial body was enclosed by a spherical cyst wall that had a uniform thickness. The wall was composed of a homogeneous material containing basic and keratinlike proteins, with sulfated acid mucopolysaccharides on the outer surface. Most cysts were enclosed by a fibrous capsule of host origin, or were surrounded by an inflammatory focus. Fibrosis was always focal, but its degree varied between individual hosts and between different cysts within the same host. Some heavily encapsulated cysts were darkened and contained disintegrating worms. In heavily infected kidneys, confluence of fibrotic or inflammatory foci resulted in the displacement of functional renal tissue. These data suggest that infection by echinostomatids may impair renal function and that the host's response affects parasite viability.     

The Giardia intestinalis filamentous cyst wall contains a novel beta(1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study

Assembly of a protective cyst wall by Giardia is essential for the survival of the parasite outside the host intestine and for transmission among susceptible hosts. The structure of the G. intestinalis filamentous cyst wall was studied by chemical methods, mass spectrometry, and (1)H nuclear magnetic resonance spectroscopy. Isolated cyst wall material contains carbohydrate and protein in a ratio of 3:2 (w/w), and the carbohydrate moiety is composed of a beta(1-3)-N-acetyl-D-galactopyranosamine homopolymer. Conformational analysis by molecular dynamics and persistence length calculations of GalNAc oligomers in solution demonstrated a flexible structure consisting of left- and right-handed helical elements. It is most likely that in the solid state, the polysaccharide forms ordered helices or possibly multiple helical structures having strong interchain interactions. The highly insoluble nature of the Giardia cyst wall must be due to these strong interchain interactions and, probably, a strong association between the carbohydrate and the protein moiety.     

Roles for the galactose-/N-acetylgalactosamine-binding lectin of Entamoeba in parasite virulence and differentiation

Entamoeba histolytica, an intestinal protozoan parasite, is a major cause of morbidity and mortality in developing countries. The pathology of the disease is caused by the colonization of the large intestine by the amoebic trophozoites and the invasion of the intestinal epithelium. Some of the trophozoites will eventually differentiate into the infectious cyst form, allowing them to be transmitted out of the bowel and into water supplies to be passed from person to person. Both the virulence of the organism and the differentiation process relies on a galactose-/N-acetylgalactosamine (GalNAc)-binding lectin that is expressed on the surface of trophozoites. The functional activity of this lectin has been shown to be involved in host cell binding, cytotoxicity, complement resistance, induction of encystation, and generation of the cyst wall. The role of the lectin in both differentiation and virulence suggests that it may be a pivotal molecule that determines the severity of the infection from a commensal state resulting from increased encystation to an invasive state. The lectin-glycan interactions that initiate these diverse processes are discussed with emphasis on comparing the binding of host ligands and the interactions involved in encystation.     

Expression of the proteinase specialized in bone resorption, cathepsin K, in granulomatous inflammation

BACKGROUND: The cysteine proteinase cathepsin K has aroused intense interest as the main effector in the digestion of extracellular matrix during bone resorption by osteoclasts. The enzyme is not a housekeeping lysosomal hydrolase, but is instead expressed with striking specificity in osteoclasts. In this work, we present evidence for the association of cathepsin K with the granulomatous reaction. Granulomas are inflammatory tissue reactions against persistent pathogens or foreign bodies. We came across cathepsin K while working on Echinococcus granulosus, a persistent tissue-dwelling, cyst-forming parasite that elicits a granulomatous response. MATERIALS AND METHODS: The walls of hydatid cysts from infected cattle were solubilized. Strong proteolytic activity was detected in the extracts. The proteinase responsible was purified by anion exchange and gel filtration. The purified protein was subjected to N-terminal sequencing, and its identity further confirmed by Western blotting, with a cathepsin K-specific antibody. The same antibody was used to localize the proteinase in paraffin-embedded sections of the parasite and the local host response. RESULTS: A proteinase was purified to near homogeneity from hydatid cyst extracts. The enzyme was unequivocally identified as host cathepsin K. Both the proenzyme and the mature enzyme forms were found. Cathepsin K was then immunolocalized both to the parasite cyst wall and to the epithelioid and giant multinucleated cells of the host granulomatous response. CONCLUSIONS: In the granulomatous response to the hydatid cyst, cathepsin K is expressed by epithelioid and giant multinucleated cells. We propose that, by analogy with bone resorption, cathepsin K is secreted by the host in an attempt to digest the persistent foreign body. Both processes, bone resorption and granulomatous reactions, therefore tackle persistent extracellular material (the bone matrix or the foreign body), and utilize specialized cells of the monocytic lineage (osteoclasts or epithelioid/giant cells) secreting cathepsin K as an effector.     

UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity

The protozoan parasite Giardia intestinalis has a simple life cycle consisting of an intestinal trophozoite stage and an environmentally resistant cyst stage. The cyst is formed when a trophozoite encases itself within an external filamentous covering, the cyst wall, which is crucial to the cyst's survival outside of the host. The filaments in the cyst wall consist mainly of a beta (1-3) polymer of N-acetylgalactosamine. Its precursor, UDP-N-acetylgalactosamine, is synthesized from fructose 6-phosphate by a pathway of five inducible enzymes. The fifth, UDP-N-acetylglucosamine 4'-epimerase, epimerizes UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine reversibly. The epimerase of G. intestinalis lacks UDP-glucose/UDP-galactose 4'-epimerase activity and shows characteristic amino acyl residues to allow binding of only the larger UDP-N-acetylhexosamines. While the Giardia epimerase catalyzes the reversible epimerization of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine, the reverse reaction apparently is favored. The enzyme has a higher Vmax and a smaller Km in this direction. Therefore, an excess of UDP-N-acetylglucosamine is required to drive the reaction towards the synthesis of UDP-N-acetylgalactosamine, when it is needed for cyst wall formation. This forms the ultimate regulatory step in cyst wall biosynthesis.     

Binding of fluorescein-labelled lectins on trophozoites and cysts of 3 strains of Toxoplasma gondii

The presence of carbohydrates on the surface of trophozoites and cysts of Toxoplasma gondii was investigated using 26 fluorescein labelled lectins. Three strains of parasites at different phases were tested; intra-cellular, intra-cystic and free trophozoites did not stain. However Soja hispida lectin intensly bind on cyst wall. The fluorescence could be eliminated by preincubation with certain carbohydrates, but not with trophozoite soluble antigen. These results suggest to the authors that cyst wall may be of host rather than of parasite origin.     

The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form

The encystation process is a key step in Toxoplasma gondii life cycle, allowing the parasite to escape from the host immune system and the transmission among the hosts. A detailed characterization of the formation and structure of the cyst stage is essential for a better knowledge of toxoplasmosis. Here we isolated cysts from mice brains and analysed the cyst wall structure and cyst matrix organization using different electron microscopy techniques. Images obtained showed that the cyst wall presented a filamentous aspect, with circular openings on its surface. The filaments were organized in two layers: a compact one, facing the exterior of the whole cyst and a more loosen one, facing the matrix. Within the cyst wall, we observed tubules and a large number of vesicles. The cyst matrix presented vesicles of different sizes and tubules, which were organized in a network connecting the bradyzoites to each other and to the cyst wall. Large vesicles, with a granular material in their lumen of glycidic nature were observed. Similar vesicles were also found associated with the posterior pole of the bradyzoites and in proximity to the cyst wall.     

A taxonomic re-appraisal of Sarcocystis nesbitti (Protozoa: Sarcocystidae) from the monkey Macaca fascicularis in Yunnan, PR China

The first detection of Sarcocystis nesbitti Mandour, 1969 in the Chinese mainland is reported and the morphology of the sarcocyst is described in detail. The parasite was detected in the monkey, Macaca fascicularis, maintained on a monkey farm in Yunnan Province; the infection may have occurred via faecal contamination from local rats, mice and/or birds. S. nesbitti was characterized as follows: a macroscopic sarcocyst, length of the cyst up to 2 mm; cyst wall smooth, thin and no perpendicular protrusion is seen under the light microscope; border of cyst wall wavy, primary cyst wall thin (38-65 nm) and invaginated; ground substance about 0.5-0.76 microm thick with electron-dense granules and concentric spherical bodies. The cyst wall is described as type 1 by electron microscopy. It is suspected that S. nesbitti may utilize Macaca mulatta, M. fascicularis, Cercocebus atys, and Papio papionis, as well as human as intermediate hosts. The taxonomy of S. nesbitti is re-appraised in the light of a consideration of possible experimental artefacts and examination of the past literature. Evidence is presented that S. nesbitti may be one of the species infecting humans in South Asia and that the monkey may be a potential reservoir host.     

Histopathology, ultrastructure and immunohistochemistry of Coregonus lavaretus hearts naturally infected with Ichthyocotylurus erraticus (Trematoda)

Histopathological, ultrastructural and immunohistochemical investigations were conducted on 26 specimens of powan Coregonus lavaretus (L.) from Loch Lomond (Scotland). The hearts of all 26 powan (15 females and 11 males) investigated harboured metacercariae of the digenean trematode Ichthyocotylurus erraticus (Rudolphi, 1809). The vast majority of metacercariae were located either singly or as an aggregation of white cysts on the surface of the bulbus arteriosus. The intensity of infection ranged from 2 to 200 larvae heart(-1), although the number of metacercariae found on male powan did not exceed 13. Histochemically, the parasite cyst wall gave a strong positive reaction with periodic acid schiff (PAS) and a faint positive signal with Azan-Mallory stain. All the metacercariae cysts were embedded in a granulomatous proliferation of heart epicardium tissue, forming a reactive fibroconnective capsule around the parasite. The capsule enclosing the parasite (produced by the host's reaction to the parasite) measured 13.57 to 90.20 microm (37.43 +/- 3.56) in thickness. Within the capsule wall, eosinophilic granular cells (EGCs), granulocytes, melanocytes and, in some instances, partially degenerated or vacuolated epithelioid cells were observed in close proximity to the cyst wall. Pigment-bearing macrophages were scattered throughout the granulomatous host-tissue reaction and as macrophage aggregates (MAs) within the capsules surrounding parasites. Immunohistochemical tests were applied to infected heart sections using 12 different antisera. Nerve fibres immunoreactive to bombesin, substance P (SP), and atrial natriuretic peptide (ANP) antisera were observed in close proximity to the parasite larvae. The presence of a serotonin-like substance was also observed within host immune-cells surrounding trematode cysts. Large cells of the epicardium were found to be immunoreactive to met-enkephalin and vasoactive intestinal peptide (VIP) antisera but not immunoreactive to anti-protein gene-product 9.5 (PGP9.5) sera.     

Histochemistry and ultrastructure of the metacercarial cyst of Bolbogonotylus corkumi (Trematoda: Cryptogonimidae).

Histochemical and ultrastructural studies were conducted on the metacercarial cyst of the cryptogonimid trematode Bolbogonotylus corkumi from the muscle tissue of fantail darters Etheostoma flabellare. The metacercarial cyst consisted of an outer host capsule and an inner parasite cyst. The host capsule was composed of an outer region of fibroblasts, collagen, macrophages, and unidentified cells, and an inner region containing degenerating cells. The parasite cyst was thin, homogenous, and noncellular in nature. The host capsule stained strongly for connective tissue and proteins and moderately for lipids, nucleic acids, nonspecific esterase activity, and acid and alkaline phosphatase activities. The parasite cyst stained intensely for acid mucopolysaccharides and moderately for acid phosphatase activity. A thick glycocalyx occurred between the parasite cyst and metacercarial tegument.     

Toxoplasma gondii: role of the phosphatidylcholine-specific phospholipase C during cell invasion and intracellular development

The effect of D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C, was investigated on cyst development of the Prugniaud strain of Toxoplasma gondii in vitro. Following treatment with the inhibitor 24 h after cell infection, cyst development was affected as assessed by staining with the bradyzoite-specific mAb CC2: the CC2-reactive antigen was shown to be differently located (in the wall versus the matrix under control conditions). This correlated with a decrease in parasite multiplication induced by D609. Pretreatment of the parasites with D609 inhibited their entry into the host cells, whereas pretreatment of the host cells enhanced the intracellular multiplication of the para sites, without any effect on cell invasion or cyst formation. Our results suggest a crucial role for phosphatidylcholine-specific phospholipase C in the pathophysiology of toxoplasmosis.     

Pathology of experimental Sarcocystis falcatula infections of canaries (Serinus canarius) and pigeons (Columba livia)

Sarcocystis falcatula is an apicomplexan parasite with a broad range of avian intermediate hosts. The pathology and pathogenesis of infection with this parasite has been studied experimentally in the budgerigar (Melopsittacus undulatus). The present study quantitatively examines the pathology of this parasite in canaries (Serinus canarius) and pigeons (Columba livia) and compares it with that found in budgerigars. The general progression of merogony and cyst formation is similar qualitatively to that seen in budgerigars, but it differs quantitatively. The principal site of precystic merogony is in pulmonary endothelial cells. The magnitude of pulmonary meront burdens (at similar inoculated dosages) varies in different intermediate host species. Merogony is less persistent than in budgerigars. Among the various species of birds, the magnitude of precystic merogony correlates differently with the magnitude of skeletal muscle cyst burdens. The distribution of cyst burdens among various muscles also differs. The composition of inflammatory cells differs among various avian species' response to S. falcatula. Pathologic changes quantitatively parallel tissue meront burdens (except possibly in the liver of canaries), resulting in an interstitial pneumonitis, hepatitis, and mild inflammatory lesions of other organs.     

Myxobolus cycloides on the swimbladder of chub Leuciscus cephalus: a controlled,host-specific localisation

Of 150 wild stock chub, Leuciscus cephalus L. captured in Lower Austrian watercourses, 112 revealed disc like plasmodia of Myxobolus cycloides Gurley, 1893 on the caudal chamber of the swim bladder. Other cyprinid species from the same waters lacked M. cycloides or other myxosporeans in this specific localisation. In chub, the intensity of infection (number of discs on the swim bladder) showed a logarithmic, age-dependent increase. The plasmodia of M. cycloides were situated in the connective tissue--mainly along blood vessels--and exhibited a delicate envelope of host tissue, thus forming a characteristic myxosporean cyst. Occasionally single trophozoites seemed to merge. A general process of fibroblast proliferation leading to encapsulation and degradation of the parasite was observed. This process was initiated by the formation of small multiple encapsulations within the spore containing trophozoid, before thickening of the outer cyst wall occurred. The general non-inflammatory course of the M. cycloides infection, and the obvious good health of the investigated chub suggest that this myxosporean in its host specific localisation cannot be regarded as a serious pathogen--on the contrary: parasite multiplication and degradation seemed to occur in a well-defined equilibrium controlled by the host fish.     

Oral immunization of BALB/c mice by intragastric delivery of Streptococcus gordonii-expressing Giardia cyst wall protein 2 decreases cyst shedding in challenged mice

Giardia lamblia (Giardia duodenalis or Giardia intestinalis) is a protozoan parasite of vertebrates with broad host specificity. Specific antibodies directed against cyst antigens can interfere with the cyst wall-building process. In this study, we engineered Streptococcus gordonii to express a 26 kDa fragment of cyst wall protein 2 (CWP2), containing a relevant B cell epitope, on the cell surface. This is the first report of S. gordonii expressing a protein of parasite origin. As S. gordonii was intended for intestinal delivery of CWP2, it was determined that this oral commensal bacterium is able to persist in the murine intestine for 30 days. Immunization with recombinant streptococci expressing the 26 kDa fragment resulted in higher antibody levels. Specific anti-CWP2 IgA antibodies were detected in fecal samples and anti-CWP2 IgG antibodies were detected in serum demonstrating the efficacy of S. gordonii for intragastric antigen delivery. In a pilot challenge experiment, immunized mice demonstrated a significant 70% reduction in cyst output.     

Identification and characterization of a novel secretory granule calcium-binding protein from the early branching eukaryote Giardia lamblia

Giardia lamblia is a flagellate protozoan that infects humans and other mammals and the most frequently isolated intestinal parasite worldwide. Giardia trophozoites undergo essential biological changes to survive outside the intestine of their host by differentiating into infective cysts. Cyst formation, or encystation, is considered one of the most primitive adaptive responses developed by eukaryotes early in evolution and crucial for the transmission of the parasite among susceptible hosts. During this process, proteins that will assemble into the extracellular cyst wall (CWP1 and CWP2) are transported to the cell surface within encystation-specific secretory vesicles (ESVs) by a developmentally regulated secretory pathway. Cyst wall proteins (CWPs) are maintained as a dense material inside the ESVs, but after exocytosis, they form the fibrillar matrix of the cyst wall. Little is known about the molecular mechanisms involved in granule biogenesis and discharge in Giardia, as well as the assembly of the extracellular wall. In this work, we provide evidences that a novel 54-kDa protein that exclusively localizes to the ESVs is induced during encystation similar to CWPs, proteolytically processed during granule maturation, and able to bind calcium in vitro. The gene encoding this molecule predicts a novel protein (called gGSP for G. lamblia Granule-specific Protein) without homology to any other protein reported in public databases. Nevertheless, it possesses characteristics of calcium-sequestering molecules of higher eukaryotes. Inhibition of gGSP expression abolishes cyst wall formation, suggesting that this secretory granule protein regulates Ca(2+)-dependent degranulation of ESVs during cyst wall formation.