Identification of an Apically-Located Antigen That Is Conserved in Sporozoan Parasites

ABSTRACT. Sporozoan parasites of the phylum Apicomplexa all possess common apical structures. The current study used a monoclonal antibody (mAb-E12) to identify a conserved antigen in the apical region of merozoites of seven species of Plasmodium (including rodent, primate and human pathogens), tachyzoites of Toxoplasma gondii , bradyzoites of Sarcocystis bovis , and sporozoites and merozoites of Eimeria tenella and E. acervulina. The antigen was also present in sporozoites of haemosporinid parasites. Immunofluorescence studies showed that the antigen was restricted to the apical 3rd of these invasive stages. Using immunoelectron microscopy, labeling was demonstrated in the region of the polar ring, below the paired inner membranes of the parasite pellicle, and near the subpellicular microtubules radiating from the polar ring of merozoites and sporozoites of E. tenella . The majority of the antigen could be extracted with 1% Triton-X 100, but a portion remained associated with the cytoskeletal elements. The molecule has a relative rate of migration (M_r) of 47,000 in Plasmodium spp. and 43–46,000 in coccidian species. Since the epitope recognized by mAb-El 2 is highly conserved, restricted to motile stages, and appears to be associated with microtubules, this antigen could be involved in cellular motility and cellular invasion.     

The ultrastructure of penetrating stages of Babesia major infecting the ovary of Haemaphysalis punctata

The ultrastructure of penetrating stages of Babesia major merozoites responsible for causing trans-ovarian infection of the tick Haemaphysalis punctata was studied. These merozoites were different from forms of other Babesia species previously described in the tick in that they were highly pleomorphic. Most of the organelles found in these forms were similar to those found in other stages: however, the apical complex was found to be very active. Micronemes were found to be predominant in the cytoplasm of these merozoites and a cytoplasmic projection anterior to the polar ring and bounded by concentric rings was visible. A polar ring with radiating ribs, and microtubules was present. It is thought that the apical complex of these merozoites plays an important role in assisting the parasite to infect the ovary of the tick.     

Electron microscopic observation of cytoskeletal frame structures and detection of tubulin on the apical region of Cryptosporidium parvum sporozoites

Cryptosporidium parvum is an intracellular protozoan parasite belonging to the phylum Apicomplexa, and a major cause of waterborne gastroenteritis throughout the world. Invasive zoites of apicomplexan parasites, including C. parvum, are thought to have characteristic organelles on the apical apex; however, compared with other parasites, the cytoskeletal ultrastructure of C. parvum zoites is poorly understood. Thus, in the present study, we ultrastructurally examined C. parvum sporozoites using electron microscopy to clarify the framework of invasive stages. Consequently, at the apical end of sporozoites, 3 apical rings and an electron-dense collar were seen. Two thick central microtubules were seen further inside sporozoites and extended to the posterior region. Using anti-alpha and -beta tubulin antibodies generated from sea urchin and rat brain, both antibodies cross-reacted at the apical region of sporozoites in immunofluorescent morphology. The molecular mass of C. parvum alpha tubulin antigen was 50 kDa by Western blotting and the observed apical cytoskeletal structures were shown to be composed of alpha tubulin by immunoelectron microscopy. These results suggested that C. parvum sporozoites were clearly different in their cytoskeletal structure from those of other apicomplexan parasites.     

Ultrastructure of cytoplasmic and nuclear changes in Eimeria tenella during first-generation schizogony in cell culture.

Eimeria tenella sporozoites were inoculated into primary cultures of chick kidney cells. Cells fixed from 1 1/2 to 54 hr later were examined with the electron microscope. At 1 1/2 and 24 hr, most intracellular sporozoites were fusiform and retained organelles typical of extracellular sporozoites. However, at 35 hr, rounded trophozoites were present without these structures; only a refractile body, nucleus, mitochondria, and endoplasmic reticulum remained. Binucleate parasites were also present at that time, but at 48 hr many multinucleate schizonts were present. Nuclei, with adjacent conoids, were at the periphery of these schizonts. Partly developed merozoites, each containing a conoid and a nucleus, protruded into the parasitophorous vacuole. At 54 hr, fully developed merozoites were separated from the residual body. Merozoites resembled sporozoites but lacked the large refractile bodies seen in sporozoites. Linear inclusions were present near the merozoite nucleus and in the residual body. Round vacuoles and ribosomes were also found in the residuum. Nucleoli were first seen in sporozoite nuclei at 1 1/2 hr. They were also present in merozoites but were more prominent in trophozoites and schizonts. Peripheral and scattered nuclear heterochromatins were prominent in intracellular sporozoites and diminished in trophozoites, but increased after several nuclear divisions and were again prominent in the merozoite. Small, distinct interchromatin granules were found in all stages. Intranuclear spindles, centrocones, and centrioles were found in connection with nuclear divisions. Ultrastructure of first-generation schizogony in cell culture was similar to that described for second-generation E. tenella in the chicken and to schizogony of other species of Eimeria.     

Characterization of the antigen SO7 during development of Eimeria tenella

The developmental expression of the antigen SO7, which has been previously shown to protect chickens against infection by several Eimeria species, was investigated. Using RT-PCR, mRNA for SO7 was found to be restricted primarily to unsporulated oocysts (0 hr). Western blot (WB) analysis with an antibody to recombinant SO7 (rbSO7) revealed expression of the protein from 6 to 72 hr (fully sporulated) of sporulation and in sporozoites (SZ). SO7 was absent in host-derived second-stage merozoites (MZ) and was present in culture-derived first-stage MZ but at a level of only 25% of that exhibited by SZ. During invasion of Madin-Darby bovine kidney (MDBK) cells by SZ in vitro, the level of SO7 within cells, as determined by WB analysis, remained relatively constant until 48 hr of development and then decreased by about 40% at the next time point (72 hr). The SO7 secreted into the culture media during in vitro development increased to a relative maximum at 48 hr and then decreased to about 20% of maximum at 72 hr. Immunostaining with anti-rbSO7 indicates that SO7 is highly concentrated in both refractile bodies (RB) of SZ, with some limited distribution in the apical complex. Anti-rbSO7 intensively stained the intracellular parasites and the first-stage schizonts during in vitro development of E. tenella in MDBK cells. Upon release from the schizonts, the first-stage merozoites stained with 1 or 2 bright spots typically at each end. The results suggest that SO7 is closely associated with the SZ RB and is developmentally regulated but may not play a direct role in cellular invasion.     

Effects of bile salt-stimulated lipase-treated triglycerides and free fatty acids on extracellular stages of Eimeria tenella

Normal human milk (NHM) has antiprotozoal activity unrelated to immunological components; this activity extends to sporozoites of Eimeria tenella. This activity may be due to free fatty acids (FFA) enzymatically hydrolyzed from triacyl glycerols by a bile salt-stimulated lipase (BSSL) found in NHM. Sporozoites were therefore incubated in the presence of several saturated and unsaturated FFA. Anticoccidial activity was observed for many unsaturated fatty acids and for some saturated fatty acids. In addition, sporozoites were added to solutions of triglycerides (trilinolein, triolein and trilinolenin) preincubated with BSSL and sodium cholate, which resulted in killing of the parasites. Triglycerides alone showed no anticoccidial activity. These results were duplicated with first generation merozoites. Intracellular stages of E. tenella were affected by FFA only at concentrations that inhibited host cells.     

Effects of Bile Salt-Stimulated Lipase-Treated Triglycerides and Free Fatty Acids on Extracellular Stages of Eimeria tenella

Normal human milk (NHM) has antiprotozoal activity unrelated to immunological components; this activity extends to sporozoites of Eimeria tenella . This activity may be due to free fatty acids (FFA) enzymatically hydrolyzed from tnacyl glycerols by a bile salt-stimulated lipase (BSSL) found in NHM. Sporozoites were therefore incubated in the presence of several saturated and unsaturated FFA. Anticoccidial activity was observed for many unsaturated fatty acids and for some saturated fatty acids. In addition, sporozoites were added to solutions of triglycerides (trilinolein, triolein and trilinolenin) preincubated with BSSL and sodium cholate. which resulted in killing of the parasites. Triglycerides alone showed no anticoccidial activity. These results were duplicated with first generation merozoites. Intracellular stages of E. tenella were affected by FFA only at concentrations that inhibited host cells.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Heat shock-like polypeptides of the sporozoites and merozoites of Eimeria bovis

Heat shock proteins (hsps) are a group of highly conserved polypeptides found in a wide variety of organisms. Polypeptides of sporozoites and merozoites of Eimeria bovis, blotted onto nitrocellulose, were probed with antibodies to artificially constructed peptides representing portions of the cDNA-generated fragment of pf75, the 75K hsp of merozoites of Plasmodium falciparum. Polypeptide antigens of sporozoites and merozoites of E. bovis with molecular weights of 46K, 71-72K, and 75K reacted with antibodies against pf75, indicating that they are hsp70 (the 70K family of hsps) or hsp70 cognates (noninducible proteins homologous to hsps). Radiolabeling with 125I and treatment with antibodies against pf75 detected a 71K antigen on the merozoite surface. Hsps in sporozoites of E. bovis are either constitutive or evoked by treatment at 37 C for in vitro excystation. If hsp70 is mandatory for parasite survival, it may prove to be an appropriate antigen for a vaccine against bovine coccidiosis.     

Development of Babesiosoma stableri (Dactylosomatidae; Adeleida; Apicomplexa) in its Leech Vector (Batracobdella picta) and the Relationship of the Dactylosomatids to the Piroplasms of Higher Vertebrates

The sporogonic and merogonic development of Babesiosoma stableri Schmittner & McGhee, 1961 within its definitive host and vector, a leech Batracobdella picta (Verrill, 1872), was studied by light and electron microscopy. Gamonts released from frog erythrocytes in the blood meal of the leech associated in syzygy and fused; the gamonts were isogamous and only 1 microgamete was formed. The ultrastructural appearance of the resulting zygote was similar to that of the gamonts, but it was larger. The zygote had an apical complex (including a polar ring, conoid and 2 pre-conoidal rings and micronemes, but no recognizable rhoptries), triple-membraned pellicle, about 40 subpellicular microtubules and prominent stores of amylopectin. Zygotes penetrated the cells of the intestine and underwent sporogony directly within the cytosplasm of the ieech epithelial cell without the formation of a parasitophorous vacuole. Eight sporozoites budded simultaneously around the periphery of an irregularly shaped oocyst. No oocyst wall was formed. Each sporozoite had a complete apical complex (including rhoptries), abundant amylopectin inclusions and a triple-membraned pellicle with about 32 subpellicular microtubules. The sporozoites initiated merogonic replication primarily within the salivary cells of the leech although other tissues, such as muscle, were infected. Each meront produced 4 merozoites by simultaneous budding, forming a cruciform meront typical of the intraerythrocytic development of this parasite. The meront was located directly within the cytoplasm of the host cell. Merozoites, with abundant amylopectin, had a complete apical complex and triple-membraned pellicle with about 40 subpellicular microtubules. The merozoites either initiated a further cycle of replication, or they moved into the ductules of the leech salivary cells which extend to the tip of the proboscis. Observations on gametogenesis. syngamy and sporogony of B. stableri in its leech host indicate that the family Dactylosomatidae should be placed in the suborder Adeleina (Eucoccidiida: Apicomplexa). Babesiosoma stableri was transmitted to uninfected frogs (Rana spp.) by the bite of infected leeches. Prepatent periods ranged from 26 to 38 days at 25° C. Despite a directed search in laboratory reared tadpoles which had each been injected intraperitoneally with 150,000 merozoites, no pre-erythrocytic developmental stages were observed. Similarities in their biology suggest close phylogenetic affinities of the dactylosomatids, and other adeleid blood parasites, with the piroplasms of higher vertebrates.     

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites

Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB 1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.     

Flotillin-1 localization on sporozoites oF Eimeria tenella

In an attempt to identify parasite surface components involved in the interaction with the host cell, the present research focuses on the rafts of Eimeria tenella that might be involved in the host cell invasion process. To that end, this study was undertaken to investigate the expression of flotillin-1, which is an important component and marker of lipid rafts at the plasma membrane of sporozoites of E. tenella. The expression of this plasma membrane protein was identified by an antibody that specifically reacts with flotillin- and was studied by electron microscopy. Flotillin-1 was found to occur in patches on the surface of E. tenella sporozoites. Immunoblot analysis of the total proteins of the sporozoites showed only 1 band of approximately 48 kDa. This indicates that the antibody exclusively recognized the molecules of flotillin-1 expressed on the surface of E. tenella sporozoites. The presence of flotillin-1 on the cellular membrane of sporozoites predominantly at the apical tip suggests that flotillin-1 belongs to the invasion machinery of E. tenella.     

贝氏隐孢子虫在北京鸭体内发育的超微结构研究

贝氏隐孢子虫各期虫体均位于宿主粘膜上皮细胞的带虫空泡中。在虫体与上皮细胞接触处,虫体表膜反复折迭形成营养器。子孢子或裂殖子与粘膜上皮细胞接触后,逐步过渡为球形的滋养体;滋养体经2—3次核分裂、产生含4或8个裂殖子的两代裂殖体,裂殖体以外出芽方式产生裂殖子;裂殖子无微孔,顶端表皮形成3—4个环嵴,裂殖子进一步发育成为配子体;大配子体含有两种类型的成囊体。小配子呈楔形,无鞭毛和顶体,有一个致密的长椭圆形细胞核,小配子表膜内侧有9根膜下微管;孢子化卵囊内含四个裸露的子孢子和一个大残体。本文是有关鸭体内隐孢子虫超微结构的首次报导。     

Fine Structure of the Schizont and Merozoite of Isospora sp. (Sporozoa: Eimeriidae) Parasitic in Gehyra variegata (Dumeril and Bibron, 1836) (Reptilia: Gekkonidae)

SYNOPSIS. A study was made of the fine structure of some stages in the life cycle of an undesignated species of Isospora parasitic in a gecko. The merozoites which lay within a membrane-bound periparasitic vacuole in the host epithelial cell, had a striking similarity to Plasmodium, Lankesterella, Toxoplasma, Besnoitia, Sarcocystis, Eimeria and the M-organism. Each merozoite was invested with a triple-layered pellicle, the outer membrane of which was loosely applied. At the anterior end of the merozoite were conoid and apical rings; microtubules terminated in the posterior apical ring. Other organelles included nucleus, endoplasmic reticulum, mitochondria, micropyle, paired organelle, toxonemes and a variety of vacuoles. Although the sequence of development of the merozoite was not completely followed, some events in this process were recorded. The evidence suggests that anterior ends are formed early and that merozoites develop subsequently by a process of budding. The merozoite pellicle appears to be continuous with, altho structurally different from, the investing membrane of the parent cell.     

A circumsporozoite-like protein is present in micronemes of mature blood stages of malaria parasites

We demonstrate for the first time the presence of a circumsporozoite (CS)-like protein in invasive blood stages of malaria parasites. Immunogold electron microscopy using antisporozoite monoclonal antibodies localized these antigens in the micronemes of merozoites. Western immunoblot and two-dimensional gel electrophoresis of mature blood stage extracts of Plasmodium falciparum, P. berghei, P. cynomolgi, and P. brasilianum identified polypeptides having the same apparent molecular mass and isoelectric points as the corresponding sporozoite (CS) proteins. The CS-like protein of merozoites is present in relatively minor amounts, compared to the CS protein of sporozoites. Mice with long-term P. berghei blood-induced infections develop antibodies which react with sporozoites.     

Development of Eimeria tenella in MDBK cell culture with a note on enhancing effect of preincubation with chicken spleen cells

Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that E. tenella first penetrate into the mucosal intraepithelial lymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope-labelled uracil (3H-uracil). Third, the E. tenella sporozoites viability was assayed after preincubation of them with chicken spleen cells. E. tenella o?cysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (E) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schizogonic cycle of E. tenella in 3-4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merozoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48-60 hours, and decreased thereafter. The uptake amount of 3H-uracil depended not only upon the inoculum size of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of sporozoites with spleen cells for 4 or 12 hours, the 3H-uracil uptake was significantly increased compared with that of control group. From the results, it was inferred that, although the penetration of E. tenella sporozoites into the lymphoid cells such as IEL is not an essential step, it should be at least a beneficial one for the survival and development of sporozoites in the chicken intestine.