Parasitophorous vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem)

We present a short insight into the problem of parasitophorous vacuole (PV) formation as a most peculiar kind of cell vacuolization occurring in the course of intracellular development of coccidian pathogens of the genera Eimeria, Isospora, Toxoplasma, Sarcocystis, Cryptosporidium, Epieimeria, and Karyolysus. The review focuses on the morpho-functional diversity of PVs in these parasites. By the present time, the PVs containing different parasite genera and species have been examined to different extent. The membrane of the PV (PVM) obviously derives from the host cell plasmalemma. But soon after parasite penetration, the morphofunctional organization and biochemical composition of the PVM drastically changes: its proteins are selectively excluded and those of the parasite are incorporated. As the result, the PV becomes not fusigenic for lysosomes or any other vacuoles or vesicles, because host cell surface markers necessary for membrane fusion are eliminated from the PVM during parasite invasion.The pattern of the PVs is parasite specific and demonstrates a broad diversity within the same genera and species and even at different stages of the endogenous development. The PV is far from being an indifferent membrane vesicle containing the parasite. Instead, it represents a dynamic system that reflects the innermost events of host-parasite relationships, thus promoting the accomplishing of the parasite life cycle, which, in its turn, is a necessary prerequisite of the parasite eventual survival as a species.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an intracellular parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other filamentous brown algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an Intracellular Parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other Filamentous Brown Algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

Formation and diversity of parasitophorous vacuoles in parasitic protozoa. The Coccidia (Sporozoa, Apicomplexa)

Data on parasitophorous vacuole (PV) formation in host cells (HC) harbouring different intracellular protozoan parasites have been reviewed and critically analysed, with special reference to the main representatives of the Coccidia. The vacuole membrane (PVM) is the interface between host and parasite, playing a role in nutrient acquisition by the parasite from the HC. The PV phenomenon is regarded as a generalized HC response to the introduction of alien bodies (microorganisms), which eventually reflects the evolutionary established host-parasite relationships at cellular, subcellular and molecular levels. Special attention has been paid to the existing morpho-functional diversity of the PVs within the same genera and species of parasites, and even at different stages of the parasite life cycle. The PVM is generally considered to derive from the HC plasmalemma, whose biochemical composition undergoes significant changes as the intravacuolar parasite grows. The original HC proteins are selectively excluded from the PVM, while those of the parasite are incorporated. As the result, the changed PVM becomes not fusigenic for HC lysosomes. For Toxoplasma gondii and other cyst-forming coccidia (Isospora, Sarcocystis), a definite correlation has been noticed between the extent of rhoptry and dense granule secrets released by a zoite during HC internalization, on the one hand, and the pattern of the PV that forms, on the other one. In T. gondii, tachyzoites, known to discharge abundant secrets, commonly force the development of PVs limited with a single unit membrane and equipped with a tubulovesicular network in the lumen. Unlike, bradyzoites known to be deficient in secretory materials trigger the formation of PVs with a three-membrane lining composed of the changed invaginated plasmalemma in addition to two membranes of endoplasmic reticulum. The two different types of PV harbour, respectively, exoenteric and enteric stages of T. gondii, the latter being confined to the cat intestine only. Unlike, all endogenous stages of the classic intestinal coccidia (Eimeria spp.) develop within PVs limited with a single membrane, with some invaginations extending into the PV lumen. Unusual PV patterns are characteristic of the extracytoplasmic eimerian coccidia (Cryptosporidium, Epieimeria) and adeleid haemogreagarines (Karyolysus). In cyst-forming coccidia, the PVM is actively involved in tissue cyst wall formation, thus protecting the encysted parasites from recognition by the host immune system. All this strongly suggests that the PV is far from being an indifferent membraneous vesicle containing a parasite, but represents a metabolically active compartment in infected cells. Since all the coccidia are obligate intracellular parasites, the mode of their intimate interaction with the HC, largely accomplished via the PV and its membrane, is vital for their survival as biological species.     

Cell: sporozoite interactions and invasion by apicomplexan parasites of the genus Eimeria

The site specificity that avian Eimeria sporozoites and, to a more limited degree, other apicomplexan parasites exhibit for invasion in vivo suggests that specific interactions between the sporozoites and the target host cells may mediate the invasion process. Although sporozoite motility and structural and secreted antigens appear to provide the mechanisms for propelling the sporozoite into the host cell,there is a growing body of evidence that the host cell provides characteristics by which the sporozoites recognise and interact with the host cell as a prelude to invasion. Molecules on the surface of cells in the intestinal epithelium, that act as receptor or recognition sites for sporozoite invasion, may be included among these characteristics. The existence of receptor molecules for invasion by apicomplexan parasites was suggested by in vitro studies in which parasite invasion was inhibited in cultured cells that were treated with a variety of substances designed to selectively alter the host cell membrane. These substance included cationic compounds or molecules, enzymes that cleave specific linkages, protease inhibitors, monoclonal antibodies, etc. More specific evidence for the presence of receptors was provided by the binding of parasite antigens to specific host cell surface molecules.Analyses of host cells have implicated 22, 31, and 37 kDa antigens, surface membrane glycoconjugates,conserved epitopes of host cells and sporozoites, etc., but no treatment that perturbs these putative receptors has completely inhibited invasion of the cells by parasites. Regardless of the mechanism,sporozoites of the avian Eimeria also invade the same specific sites in foreign host birds that they invade in the natural host. Thus, site specificity for invasion may be a response to characteristics of the intestine that are shared by a number of hosts rather than to a unique trait of the natural host. Protective immunity elicited against avian Eimeria species is not manifested in a total blockade of parasite invasion. In fact, the effect of immunity on invasion differs according to the eliciting species and depends upon the area of the intestine that is invaded. Immunity produced against caecal species of avian Eimeria, for example Eimeria tenella and Eimeria adenoeides, inhibits subsequent invasion by homologous or heterologous challenge species, regardless of the area of the intestine that the challenge species invade. Conversely, in birds immunised with upper intestinal species, Eimeria acervulina and Eimeria meleagrimitis, invasion by challenge species is not decreased and often is significantly increased.     

Ichthyophthirius multifiliis (Ciliophora) Invasion of Gill Epithelium1

The sequence of morphologic events associated with Ichthyophthirius rnultifliis invasion of gill epithelium began in the theront with differentiation of secretory mucocysts and the perforatorium. After escaping from the cyst the theront, which stained intensely with Mallory' stain, sought a host. As it approached the host epithelium, the contents of the mucocysts were discharged, enveloping the ciliate in sticky material, which made initial contact with the host epithelium. Rapid penetration by the theront caused disruption of one or more host cells and resulted in a focal necrosis associated with the anterior margin of the ciliate. Within five minutes postexposure, the parasite completed its invasion of the epithelial layer and stained less intensely. The remnants of host cells disrupted during its entry surrounded the trophont until they were ingested by the parasite. Within 40 min postexposure, synthetic activity of the parasite appeared to increase as evidenced by an abundance of organelles, particularly ribosomes and crystalline mucocysts. At this point, the overlying host epithelium appeared normal.     

Early events in the life cycle of the mouse coccidium Eimeria falciformis (Eimer, 1870) Schneider, 1875 in naive and immune hosts

The initial phases of invasion of mammalian coccidia of the genus Eimeria into host tissue are still poorly known. This process, including the passage of oocysts through the intestinal lumen, excystation of sporozoites, their penetration into epithelial cells and migration to the target site was studied in both naive and immune mice infected with Eimeria falciformis. After oral infection, the intact oocysts were transported with enteral contents to the large intestine, where the excystation of sporozoites and their penetration into superficial epithelium took place. The sporozoites subsequently migrated into the epithelium of crypts, which is the specific site of asexual multiplication. The immune status of the hosts did not affect the passage of oocysts, excystation and penetration of sporozoites. However, the migration of sporozoites towards their target site (crypts) was impeded in immune mice and sporozoites tended to remain in superficial mucosa rather than migrate to the crypts.     

COMPARATIVE DEVELOPMENT OF THE RED ALGAL PARASITES BOSTRYCHIOCOLAX AUSTRALIS GEN. ET SP. NOV. AND DAWSONIOCOLAX BOSTRYCHIAE (CHOREOCOLACACEAE,RHODOPHYTA)1

The development of two red algal parasites was examined in laboratory culture. The red algal parasite Bostrychiocolax australis gen. et sp. nov., from Australia, originally misidentified as Dawsoniocolax bostrychiae (Joly et Yamaguishi-Tomita) Joly et Yamaguishi-Tomita, completes its life history in 6 weeks on its host Bostrychia radicans (Montagne) Montagne. Initially the spores divide to form a small lenticular cell, and then a germ tube grows from the opposite pole. Upon contact with the host cuticle, the germ tube penetrates the host cell wall. The tip of the germ tube expands, and the spore cytoplasm moves into this expanded tip. The expanded germ tube tip becomes the first endophytic cell from which a parasite cell is cut off that fuses with a host tier cell. The nuclei of this infected host cell enlarge. As parasite development continues, other host-parasite cell fusions are formed, transferring more parasite nuclei into host cells. The erumpent colorless multicellular parasite develops externally on the host, and reproductive structures are visible within 2 weeks. Tetrasporangia are superficial and cruciately or tetra-hedrally divided. Spermatia are formed in clusters. The carpogonial branches are four-celled, and the carpogonium fuses directly with the auxiliary (support) cell. The mature carposporophyte has a large central fusion cell and sympodially branched gonimoblast filaments. Early stages of development differ markedly in Dawsoniocolax bostrychiae from Brazil. Upon contact with the host, the spore undergoes a nearly equal division, and a germ tube elongates from the more basal of the two spore cells, penetrates the host cell wall, and fuses with a host tier cell. Subsequent development involves enlargement of the original spore body and division to form a multicellular cushion, from which descending rhizoidal filaments form that fuse with underlying host cells. This radically different development is in marked contrast to the final reproductive morphology, which is similar to B. australis and has lead to taxonomic confusion between these two entities. The different spore germination patterns and early germ-ling development of B. australis and D. bostrychiae warrant the formation of a new genus for the Australian parasite.     

Molecular characterization of Encephalitozoon intestinalis (Microspora) replication kinetics in a murine intestinal cell line

Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. Most human infections are caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and result in chronic diarrhea. In order to determine the signals involved in microsporidial spore activation and invasion, kinetics of in vitro E. intestinalis replication were defined using real-time quantitative PCR. Segments of small subunit ribosomal RNA and polar tube protein 2 genes of E. intestinalis were used to quantify parasite gene copy number following infection in murine colon carcinoma cells. Parasite DNA was detectable in small but significant amounts within host cells as early as 4 h postinoculation, genome replication was completed by 36 h, and parasite progeny were released into the supernatant beginning 72 h postinoculation. Heat-treating spores did not prevent transfer of parasite DNA into cells, but did inhibit parasite replication. Treating cell cultures with albendazole suppressed but did not completely inhibit parasite replication. These results confirm observations that E. intestinalis completes its life cycle within the turnover time of its target host cells; invasion into susceptible host cells occurs independently of spore viability; and real-time quantitative PCR is a sensitive and reproducible method with which to monitor microsporidial infection under varying treatments or conditions.     

The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella

Eimeria spp. are a highly successful group of intracellular protozoan parasites that develop within intestinal epithelial cells of poultry, causing coccidiosis. As a result of resistance against anticoccidial drugs and the expense of manufacturing live vaccines, it is necessary to understand the relationship between Eimeria and its host more deeply, with a view to developing recombinant vaccines. Eimeria possesses a family of microneme lectins (MICs) that contain microneme adhesive repeat regions (MARR). We show that the major MARR protein from Eimeria tenella, EtMIC3, is deployed at the parasite-host interface during the early stages of invasion. EtMIC3 consists of seven tandem MAR1-type domains, which possess a high specificity for sialylated glycans as shown by cell-based assays and carbohydrate microarray analyses. The restricted tissue staining pattern observed for EtMIC3 in the chicken caecal epithelium indicates that EtMIC3 contributes to guiding the parasite to the site of invasion in the chicken gut. The microarray analyses also reveal a lack of recognition of glycan sequences terminating in the N-glycolyl form of sialic acid by EtMIC3. Thus the parasite is well adapted to the avian host which lacks N-glycolyl neuraminic acid. We provide new structural insight into the MAR1 family of domains and reveal the atomic resolution basis for the sialic acid-based carbohydrate recognition. Finally, a preliminary chicken immunization trial provides evidence that recombinant EtMIC3 protein and EtMIC3 DNA are effective vaccine candidates.     

Ultrastructural observations on fertilization and sporulation in Goussia iroquoina (Molnar and Fernando, 1974) in experimentally infected fathead minnows (Pimephales promelas, Cyprinidae)

The ultrastructural features of fertilization and sporogony of Eimeria iroquoina are described from the intestinal epithelium of experimentally infected fathead minnows (Pimephales promelas). Intact microgametes were observed in the cytoplasm of macrogametes. Within immature macrogametes the microgamete was segregated from the cytoplasm of the former by the plasma membrane of each cell plus additional membranes. Within mature macrogametes, only the plasma membranes separated the gametes. Fertilization by fusion of the limiting membrane of both gametes occurred after the entire microgamete lay within the cytoplasm of the macrogamete. The cytoplasm of the zygote cleaved into sporoblasts within cisternae of endoplasmic reticulum. The sporocystic wall was composed of an outer electron-lucent layer and an inner, thicker layer with periodic striations at right angles to the surface of the sporocyst. The sporocysts were bivalved and joined by a continuous suture. The sporozoites were morphologically similar to sporozoites and merozoites of other Coccidia. Due to the structure of the sporocyst, Eimeria iroquoina Molnar and Fernando, 1974 is amended to Goussia iroquoina (Molnar and Fernando, 1974).     

Species of Eimeria in Nonepithelial Sites1

In a few homoiothermic host species, Eimeria spp. in a schizogonic stage, a gamogonic stage, or as resting sporozoites infect nonepithelial tissues. However, epithelium serves as a critical site in the cycle of these and all other species known from warm-blooded hosts. Eimeria funduli, infecting at least four different killifishes, undergoes both schizogony and gamogony in non-epithelial hepatic and pancreatic cells and requires an invertebrate host to complete its cycle. Oocysts are not released from the living fish into the environment. The cycle for this species, or aspects of it, may exemplify those found in several piscine species infecting nonepithelial (and possibly epithelial) cells. Eimeriu funduli differs from most species of Eimeria infecting homoiotherms in other respects, some of which may also characterize traits for piscine and other poikilotherm eimerians in general. Endogenous development is affected by temperature and host-age, sporogony occurs in the host, and infections occur in several related fishes. The presence of an intermediate host in at least E. funduli and the presence of sporozoites in macrophages of several eimerians of homoiotherms, as well as other features, suggest a closer relationship between eimeriids and Lankesterella, Schellackia, and even the Haemosporina than previously assumed.     

Modelling host cell availability and the crowding effect in Eimeria infections.

Within-host mathematical models of Eimeria maxima and Eimeria praecox infections of the chicken are presented and used to investigate the role of host cell availability as a possible determinant of the so-called 'crowding effect'; whereby the fecundity of the parasites decreases as infectious dose increases. Assumptions about the number of available host cells, the average lifespan of these cells and the age structure within the host-cell population were made and mathematical models were constructed and combined with experimental data to test whether these conditions could reproduce the crowding effect in the two species. Experimental data demonstrated that crowding during in vivo infections was apparent following very low infectious doses, but none of the models could adequately reproduce crowding at the same doses while maintaining realistic estimates of the dynamics of the enterocyte pool. However, both the size and lifespan of the enterocyte pool were demonstrated to have substantial effects on the fecundity of the infections, particularly at higher doses. These data indicate that host cell availability cannot be solely responsible for the crowding effect. Alternative factors such as the influence of the primary immune response to the parasite may also be explored using within-host models and other applications of these models are discussed.     

Interaction of Eimeria tenella with intestinal mucin in vitro

The mucus gel layer overlying the gastrointestinal epithelium plays an important role in host-pathogen interactions. The initial interaction between the coccidian parasite Eimeria tenella and host cells of the intestinal epithelium must occur across this mucus interface. In this study, we examined the relationship between E. tenella and avian mucin, in particular the effect of purified intestinal regional mucin on parasite adherence and invasion in vitro. Secreted mucin from the chicken duodenum and cecum was purified by density gradient centrifugation and gel chromatography. Parasite invasion studies were performed in the Madin-Darby bovine kidney cell model. Eimeria tenella adherence to chicken duodenal mucin was detected, whereas adherence to cecal or bovine mucin was not shown. Parasite invasion into epithelial cells was not influenced by bovine mucin, whereas chicken mucin purified from the duodenum and cecum significantly inhibited invasion. Inhibition of E. tenella invasion into cells by mucin from the duodenum was marginally greater than that of the cecum, but this was not significant. This study demonstrated E. tenella interaction with native chicken intestinal mucin, which in turn inhibited parasite invasion into epithelial cells in vitro.     

Ultrastructural morphogenesis of dimorphic arcuate infection (gun) cells of Haptoglossa erumpens an obligate parasite of Bunonema nematodes

Haptoglossa is a genus of biflagellate organisms that has been placed in the oomycetes and is characterised by producing unique infective gun cells, which usually infect by physically rupturing the nematode cuticle. Haptoglossa erumpens is a parasite of Bunonema nematodes that produces arcuate infection cells and aplanospores that are discharged following the swelling and rupture of the thallus wall and distended host cuticle. Recent isolations of H. erumpens have revealed that the germinating aplanospores develop into two similar-sized but morphologically distinct infection cells. The uni-nucleate, convexly arcuate, gun cells were observed to fire in response to host nematodes, producing a cylindrical sporidium inside the host body. These gun cells had an apical missile chamber containing a needle with a unique arrangement of investing cones. Unlike previously described gun cells, the tube tail did not wind around the nucleus but continued into the basal vacuole where it terminated. The second type of infection cell was a concavely arcuate, bi-nucleate, cell that had an unusually large and elongate annulus component in the missile chamber. These modified bi-nucleate gun cells were never observed to fire in response to contact with Bunomena nematodes. The patterns of morphological and structural variations in these infection structures in this genus are reviewed in the light of these findings.     

A Model for Taxonomic Work on Homoxenous Coccidia: Redescription, Host Specificity, and Molecular Phylogeny of Eimeria ranae Dobell, 1909, with a Review of Anuran-Host Eimeria (Apicomplexa: Eimeriorina)

ABSTRACT. We attempt to extend knowledge of anuran Eimeria , and to provide a model for a complex approach to studies on coccidia. New host and geographic records of coccidia in European Anura are provided. In the second part, Eimeria ranae Dobell, 1909 is redescribed from European terrestrial frogs of the genus Rana based on light microscopic and ultrastructural data on both exogenous and endogenous developmental stages, host specificity, and molecular phylogenetic data. Results of experimental transmissions show for the first time that the host specificity of E. ranae is restricted to the genus Rana and that isolates from tadpoles and adults are conspecific. Disappearance of infection during metamorphosis was confirmed experimentally, suggesting that infections in adults result from reinfections. Poikilotherm-host Eimeria species possessing a Stieda body (SB) are for the first time included in a molecular phylogenetic analysis. Eimeria ranae and Eimeria arnyi from a colubrid snake form together a well-supported clade, basal to other SB-bearing coccidia. The other analysed reptile–host eimerians, Eimeria tropidura and Choleoeimeria sp., which possess bivalved sporocysts and lack a SB, represent a distinct basal lineage of the eimeriid clade. The third part of the article reviews anuran-host Eimeria . Three distinct oocyst morphotypes, apparently correlating with the character of endogenous development, are recognized and characterized among anuran eimeriids.     

Cryptosporidium molnari n. sp. (Apicomplexa: Cryptosporidiidae) infecting two marine fish species,Sparus aurata L. and Dicentrarchus labrax L

Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.