艾美耳球虫子孢子从宿主细胞中逸出机制的初步研究

艾美耳球虫是一类重要的肠道病原,其裂殖生殖阶段的虫体逸出过程是造成畜禽肠道破坏的主要原因之一,但此逸出过程的机制仍鲜有报道。本研究以乙醇作为诱导剂研究柔嫩艾美耳球虫M2e株子孢子从宿主细胞中逸出的机制。结果显示,乙醇可诱导子孢子从MDBK细胞中逸出,此逸出过程依赖于虫体的运动能力;同时,乙醇可激发子孢子逸出相关的微线体蛋白2（Mic2）的分泌释放。进一步实验证实,螯合虫体内部钙离子明显阻断了子孢子逸出及Mic2蛋白的释放。本研究初步证实了与柔嫩艾美耳球虫逸出相关的蛋白和离子,为深入解析球虫致病的分子机制、研发新型抗球虫药物提供了新的研究方向。     

Involvement of CD 8+ and CD 3+ lymphocytes in the transport of Eimeria necatrix sporozoites within the intestinal mucosa of chickens

The phenotype of cells transporting sporozoites of Eimeria necatrix during a primary infection was determined using a panel of six monoclonal antibodies to various chicken lymphocyte surface markers. Sporozoites and cells harboring them were examined at 8, 12 and 18 h postinfection using two-color immunoflorescence and confocal microscopy. The majority of parasites observed within lymphocytes were found in CD 8 + (15%) or CD 3+ (13-22%) cells at all time periods examined. Smaller numbers were found within deltagamma TCR+ (5%) and alphabeta TCR+ (5%) lymphocytes. No sporozoites were found within CD 4+ or IgM+ lymphocytes at any of the time periods.     

Parasite exposure elicits a preferential T-cell response involved in protective immunity against Eimeria species in chickens primed by an internal-image anti-idiotypic antibody.

Polyclonal anti-idiotype 1073 (anti-Id 1073), raised against a monoclonal antibody specific for the protective epitope(s) of Eimeria tenella sporozoites, induced cell-mediated immune (CMI) responses in bursectomized chickens. Whereas alhydrogel-adsorbed anti-Id 1073 was sufficient to engender the CMI response at 4 h after injection, induction of the CMI response at 24 h required both alhydrogel and muramyl dipeptide sterol. Exposure of immunized chickens to live parasites prompted a dichotomous effect on the CMI response engendered by anti-Id in that the 4-h CMI response was preferentially stimulated and the 24-h CMI response was down regulated. Both types of CMI response were transferable to naive chickens by T cells from anti-Id 1073 immune donors or by parasite-specific T cells from clones 21 and 27. These T-cell clones were generated from chickens immunized by repeated infections with E. tenella and showed in vitro proliferative responses to anti-Id 1073. The abilities of T cells from clone 21 to selectively transfer the 4-h CMI response and to generate gamma interferon to activate macrophages for their cytotoxic effects on Eimeria sporozoites correlate with the preferential stimulation by parasites of the 4-h CMI response in chickens immunized with anti-Id 1073. These data show that anti-Id 1073 mimics the protective epitope(s) of the parasite and primes chickens for protective CMI responses. Cytotoxic T cells, equivalent to the mammalian T-cell subset of the Lyt2+ phenotype, appear to be the primary effector T cells in the CMI response engendered by anti-Id 1073 against Eimeria parasites.     

A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.     

Cross-reactivity among antisera raised against five avian Eimeria species in the natural host and in rabbits

Water- and SDS-soluble antigens were prepared from purified sporulated oocysts of Eimeria acervulina, E. maxima, E. necatrix, E. praecox and E. tenella. Reactivity of . chicken hyperimmune anti-Eimeria sera, rabbit anti-oocyst and rabbit anti-sporozoite sera with the homologous and heterologous oocyst antigens were determined and cross-reactivities were expressed as a percentage of those homologous sera. The results demonstrated that the antisera from chickens infected naturally with Eimeria species differed in their reactivities from those of the rabbit antisera. Occurrence of a high level cross-reactivity among the chicken antisera may suggest that the development of parasites inside the host cells, or the production of substances during the life-cycle, affect the extent of immune responses and that most non-protective antibodies are cross-reactive.     

The infectivity of Eimeria sporozoites recovered from monensin medicated chickens

Sporozoites of embryo-adapted lines of Eimeria tenella, E. brunetti and E. necatrix were recovered from monensin medicated chickens. The viability of these sporozoites was compared with that of sporozoites recovered from unmedicated birds by assessing their infectivity in chicken embryos. Sporozoites of the three species of Eimeria recovered from 100 parts/10(6) monensin-medicated birds produced infections in embryos which were 94 to 98% less severe than infections resulting from the inoculation of sporozoites recovered from unmedicated chickens. A concomitant scanning electron microscope study revealed no apparent ultrastructural damage to the exposed sporozoites. Timed medication experiments in chickens showed that medication with monensin for the period covering sporozoite penetration in vivo was almost completely effective, being comparable to continuous medication. Medication started before infection (to ensure tissue concentrations of monensin) but terminating 5 to 7 hours before the oocysts were inoculated was ineffective.     

Characterization of rhoptry proteins of Eimeria tenella sporozoites: antigenic diversity of rhoptry epitopes within species of the genus Eimeria and among three asexual generations of a single species, E. tenella.

Rhoptry organelles from sporozoites of the apicomplexan parasite Eimeria tenella contain at least 60 independent polypeptides that can be resolved by two-dimensional gel electrophoresis. Rhoptries from three species of Eimeria that infect chickens share very few antibody cross-reactive epitopes, and there is poor conservation of epitopes among three distinct asexual generations of zoites within the developmental life cycle of a single parasite, E. tenella.     

X-irradiation of Eimeria tenella oocysts provides direct evidence that sporozoite invasion and early schizont development induce a protective immune response(s).

Sporulated oocysts of the protozoan parasite Eimeria tenella were attenuated by exposure to various doses of X-radiation to inhibit intracellular replication and thus determine whether sporozoites alone can induce a protective immune response. Exposure to doses greater than 15-kilorads had a significant effect on development, as indicated by the absence of oocyst production in chickens infected with parasites treated with 20 or 30 kilorads of radiation. Infection with nonirradiated or 15-kilorad-exposed parasites led to either normal or reduced oocyst shedding. Equivalent protection was afforded chickens inoculated with a minimum immunizing dose of either nonirradiated or 20-kilorad-irradiated E. tenella oocysts. Immunofluorescence staining of cecal tissue from chickens inoculated with 10(7) nonirradiated or 20- or 30-kilorad-irradiated oocysts with stage-specific monoclonal antibodies showed no significant difference in sporozoite invasion between treatment groups. Normal merogonic development was observed at appropriate times (48, 60, 72, and 96 h) postinfection in chickens inoculated with nonirradiated oocysts. In contrast, irradiated parasites exhibited minimal merogonic development at 48 h postinfection. Furthermore, no merogonic stages were observed at times of otherwise peak merozoite development (60, 72, and 96 h) in cecal tissue from chickens inoculated with irradiated parasites. Infection of chicken cells with irradiated or nonirradiated parasites in vitro corroborated these findings and indicate that events early after sporozoite invasion induce a protective immune response against this parasite.     

Ultrastructure of cryptosporidial life cycle in chicken host cells

Ultrastructural studies were conducted on cryptosporidia in various stages of the life cycle, attached to the epithelial cell surface of the bursa of Fabricius, caecum and trachea in six 5 and 6-week-old chickens fed faeces containing cryptosporidial oocysts. The stages observed were sporozoites or merozoites penetrating the host cells, trophozoites, schizonts, macrogametocytes, microgametocytes and oocysts. The cryptosporidia in the chickens were morphologically and developmen-tally similar to those reported previously in other animals and humans. All the life cycle stages were recognised in each organ examined and sporulating oocysts were found adherent to the host cells. Trophozoites, schizonts and macrogametocytes were common in each organ examined, while oocysts were found only occasionally and microgametocytes and sporozoites or merozoites were uncommon.     

Cellular pathology in mouse embryonic brain cells following in vitro penetration by sporozoites ofEimeria papillata

The pathology that occurs in mouse embryonic brain (MEB) cells that have been penetrated by sporozoites ofEimeria papillata was studied by light and electron microscopy. At the light microscopy level the greatest number of intracellular parasites was seen at 15 and 45 min postinoculation (PI). The monolayer of MEB cells had begun to round up by 45 min PI, and by 60 min PI most of the cells were stripped from the coverslip. Little ultrastructural damage was seen in MEB cells just penetrated by the parasites at 15 min PI, and no host cell membrane was seen around the sporozoites that had just entered the cells. Flexing and bending of the sporozoites within the MEB cell caused vacuolization of cell cytoplasm and in some cases rupture of host cell membrane. Sporozoites leaving the host cells at 15 min PI caused a rupture of the host cell membrane at the apical end of the parasite, and both host cell membrane and cytoplasm were attached to the surface of the parasite. MEB cells still attached to coverslips at 45 min PI demonstrated complete degeneration.     

A mechanism for secretory IgA-mediated inhibition of the cell penetration and intracellular development of Eimeria tenella.

The ability of Eimeria tenella sporozoites to develop normally in cultured chick kidney cells was used as an indicator of the anticoccidial effects of sera and extracts of caecal contents or tissue. Pre-treatment of sporozoites with normal serum globulin enhanced the frequency of intracellular development but pre-treatment in balanced salt solution, without protein, damaged sporozoites so that most had lost the ability to differentiate, even when they were able to invade host cells. The same inhibitory effect was seen when sporozoites were incubated in extracts of caecal contents from non-immunized chickens, although parasitic development was unaffected when sporozoites were pretreated in similar extracts of mucosae. Extracts of immune caecal contents impaired both cell penetration and subsequent development. These results show that sporozoites can lose the ability to differentiate before the ability to penetrate cells and provide evidence of a possible synergism between non-specific factors and secretory antibodies in anticoccidial immunity.     

Induction of secretion and surface capping of microneme proteins in Eimeria tenella

Micronemes are secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. We have examined the induction of microneme secretion in the coccidian Eimeria tenella. When sporozoites were added to MDBK cells in culture, microneme proteins were secreted, capped backwards over the parasite surface and deposited onto underlying host cells from the posterior end of gliding parasites. Induction of secretion was also achieved by the addition of foetal calf serum, or purified albumin, to extracellular sporozoites. Microneme secretion per se was not dependent on parasites being able to move or to invade host cells. However, in the presence of cytochalasin D, which disrupts actin polymerisation and prevents parasite movement, microneme proteins were secreted from the apical tip but were not capped backwards over the sporozoite surface. These observations support the hypothesis that microneme proteins function as ligands which, when secreted out onto the parasite surface, form a link, either directly or indirectly, between the sub-pellicular actin–myosin cytoskeletal motor of the parasite and the surface of target host cells.     

Passive protection of chickens against Eimeria tenella infection by monoclonal antibody.

Monoclonal antibodies reactive with the surface of Eimeria tenella sporozoites were produced in mice. This paper concerns one of these monoclonal antibodies, designated 1073.10, which agglutinated sporozoites in vitro and lysed the parasite in the presence of complement. This treatment neutralized sporozoite infections when the treated parasites were injected into the ceca of normal chickens. Passive transfer of ammonium sulfate-precipitated 1073.10 ascites fluid into 2- to 3-day-old or 3-week-old chickens conferred protection against challenge infection with E. tenella. These studies show that serum antibody may play a role in immunity to coccidiosis and that the sporozoite surface epitope recognized by 1073.10 is a possible vaccine candidate antigen.     

Intestinal immune responses to coccidiosis

Intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and production efficiency of livestock and poultry. Coccidiosis is an intestinal infection caused by intracellular protozoan parasites belonging to several different species of Eimeria. Infection with coccidia parasites seriously impairs the growth and feed utilization of chickens and costs the US poultry industry more than $1.5 billion in annual losses. Although acquired immunity to Eimeria develops following natural infection, due to the complex life cycle and intricate host immune response to Eimeria, vaccine development has been difficult and a better understanding of the basic immunobiology of pertinent host-parasite interactions is necessary for developing effective immunological control strategies against coccidiosis. Chickens infected with Eimeria produce parasite specific antibodies in both the circulation and mucosal secretions but humoral immunity plays only a minor role in protection against this disease. Rather, recent evidence implicates cell-mediated immunity as the major factor conferring resistance to coccidiosis. This review will summarize current understanding of the avian intestinal immune system and its response to Eimeria as well as provide a conceptual overview of the complex molecular and cellular events involved in intestinal immunity to coccidiosis. It is anticipated that increased knowledge of the interaction between parasites and host immunity will stimulate the birth of novel immunological and molecular biological concepts in the control of intestinal parasitism.     

Ultrastructural observations of host-cell invasion by sporozoites ofEimeria papillata in vivo

Scanning and transmission electron microscopy were used to study the invasion of mouse small-intestinal epithelium by sporozoites ofEimeria papillata. Some mice received oocysts by gavage and others received either sporocysts or sporozoites by direct injection into the small intestine. The highest concentration of invaded cells were found in ligated intestinal tissues studied at 5–45 min after the inoculation of sporozoites. Sporozoites actively invaded anterior end first, which resulted in extensive damage to the host cell. Such cells showed disrupted microvilli; protuberances of cytoplasm into the lumen, apparently the result of a disrupted plasma membrane; vacuolization of the cytoplasm; and damage to the mitochondria. These damaged cells were rapidly vacated as the sporozoite moved laterally into one or more adjacent intact host cells without entering the lumen. It is suggested that the host cell initially entered from the lumen becomes so severely traumatized that the parasite of necessity enters an adjacent cell as a prelude to further development. Various aspects of host-cell invasion by coccidia and malarial parasites are reviewed.     

Interactions in vitro between sporozoites of Eimeria tenella and host peritoneal exudate cells: Electron microscopal observations

Summary The interactions of sporozoites of Eimeria tenella with peritoneal cells from normal and from immunized chickens were examined in vitro. Although the uptake of sporozoites by cells from immunized birds was greater than by cells from susceptible birds, no differences were apparent in their appearance in electron micrographs. Entry into both macrophages and heterophils (comparable to mammalian neutrophils) was by phagocytosis. The findings are discussed.     

A toolbox facilitating stable transfection of Eimeria species

Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria tenella are described here, together with the identification of optimal parameters for the transfection process. A series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that confers resistance to pyrimethamine, were electroporated into sporozoites of the E. tenella Wisconsin strain and stabilised by selective passage through chickens. Very high transfection efficiencies of up to 25% sporozoites in transient transfection and up to 9% oocysts following a single round of in vivo selection were achieved. Crucial factors include the use of very freshly harvested parasites with the AMAXA nucleofection system (program U33 in a cytomix-buffered reaction) and linearised plasmid DNA. The use of a restriction enzyme mediated integration (REMI) protocol boosted overall efficiency and elevated insertion rate per genome. Successful development of methods to generate and isolate stable populations of transfected Eimeria parasites will now stimulate rapid expansion of reverse genetic studies in this important coccidian.