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1.
Neospora caninum, Toxoplasma gondii and Eimeria bovis are coccidian parasites of veterinary importance. Tachyzoites of N. caninum and T. gondii and sporozoites of E. bovis are able to invade and replicate in endothelial cells in vivo and in vitro. As it holds true for all eukaryotic cells, the survival of parasitized host cells and the parasites themselves should be dependent on ion balances, especially on extra- and intracellular calcium concentrations. Addition of the calcium ionophore A23187 reliably did release merozoites from mature N. caninum and T. gondii meronts grown in cultured primary bovine umbilical vein endothelial cells (BUVEC). Extent and time course of merozoite release depended on both, maturity of the meronts and concentration of the calcium ionophore. Attempts, however, to achieve synchronous release of merozoites from E. bovis first generation meronts by ionophore treatment failed, suggesting a different biological behaviour of this parasite. According to microscopical observations, the quite variable time of E. bovis macromeront maturation and a hampered merozoite exit owing to dense parasite-induced cytoskeleton elements surrounding the meront may be a reason for the lack of inducible synchronous release. Electronic supplementary materials The online version of this article (doi: ) contains supplementary material, which is available to authorized users.  相似文献   

2.
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3.
The merozoite surface antigens MSA-2 of Babesia bovis constitute a family of polymorphic GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes. These are therefore putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-2 antigens of the biologically cloned Mo7 strain are encoded by four tandemly organized genes: msa-2a1, a2, b and c, and (ii) at least one allele of each of these genes is present in the Argentine R1A strain with a moderate degree of polymorphism. The present work was aimed at defining neutralization-sensitive B-cell epitopes in the MSA-2 family, that are conserved among different B. bovis geographical isolates. To this end, msa-2a, b and c alleles from different isolates from Argentina, USA and Mexico were amplified by PCR, cloned and sequenced. Bioinformatic analysis by ClustalW alignments and B-cell epitope prediction algorithms performed on these sequences allowed the identification of several regions containing putative conserved B-cell epitopes. Four peptides representing these regions: (KDYKTMVKFCN from msa-2a1; YYKKHIS, from msa-2b; and THDALKAVKQLIKT and ELLKLLIEA from msa-2c) were chemically synthesized, conjugated to keyhole limpet hemocyanin and used to inoculate mice to obtain immune sera. Anti-peptide antibodies recognized B. bovis merozoite extracts in all cases in ELISA tests. In addition, these sera reacted with the surface of merozoites of an Argentine and a Mexican B. bovis strains in immunofluorescence assays, and sera against two of the selected peptides inhibited invasion of erythrocytes by in vitro cultured merozoites. Taken together, the results show that the peptide sequences selected by bioinformatic analysis represent expressed and geographically conserved B. bovis B-cell epitopes that might be strong candidates for development of subunit vaccines.  相似文献   

4.
Abstract

Edwardsiella ictaluri is the cause of enteric septicemia of catfish. A monoclonal antibody (MAb AA224) was used to identify a specific and predominant outer-membrane antigen of E. ictaluri. The MAb AA224 was produced by conventional cell fusion technology with spleen cells from mice immunized with an affinity-purified antigen. The affinity-purified antigen was obtained by immunoaffinity chromatography of an E. ictaluri extract with immunoaffinity purified immunoglobin from sera of channel catfish Ictalurus punctatus immune to E. ictaluri as a result of natural infection. The immunoaffinity-purified antigen was used for immunization and identification of the hybridoma producing MAb AA224 by enzyme-linked immunosorbent assay. The predominant antigen was purified by immunoaffinity chromatography with MAb AA224 as the immunoadsorbent. Immunoblotting and high-pressure liquid chromatography were used to determine that the relative sizes of the predominant antigens are 60 and 36 kilodaltons. Immunoelectron microscopy with MAb AA224 conjugated with colloidal gold localized the predominant antigen on the surface of the outer membrane of E. ictaluri  相似文献   

5.
The serological response to 12 purified Mycobacterium bovis antigens were examined in an ELISA assay. These antigens included the majority of M. bovis protein antigens described to date and in most cases they were very similar to the M. tuberculosis antigens of the same molecular mass.The purified antigens were tested against sera from M. bovis infected cattle, M. bovis culture-negative cattle from infected herds and animals infected with related microorganisms, mainly other mycobacterial species. All the antigens gave strong reactions with at least some sera from the M. bovis infected group and showed cross-reactivity with some of the sera from the other two groups. The antigen with the highest specificity reacted strongly with only 60% of the M. bovis infected sera. Antigens that reacted with most or all of the M. bovis infected sera also gave the highest cross-reactivity with sera from the other two groups. These results indicate that a serological test based on any one or a combination of these antigens, without removal of the cross-reacting epitopes, would be unsatisfactory.  相似文献   

6.
Hybridoma antibodies (Hab) produced against sporozoites or merozoites of four species of Eimeria were tested for the ability to inhibit the invasion of cultured primary avian kidney cells by sporozoites of Eimeria. Five of 16 Hab that were tested showed inhibitory activity. All five of these Hab were produced against sporozoites and reacted with sporozoite surface antigens or surface/internal antigens. Four Hab produced against merozoites of E. acervulina cross-reacted with sporozoite surface antigens but failed to inhibit invasion. Similarly, Hab reacting with sporozoite anterior tips or refractile bodies had little effect on invasion. Collectively, the data suggest that surface antigens or surface/internal antigens that are unique to the sporozoite stage may influence or be part of the invasion process. Indirect immunofluorescent-antibody tests and ferritin (Fe) labeling combined with electron microscopy indicated differences in binding of two of the Hab to the sporozoite surface membranes. For example, after exposure to Hab 43A6 and a fluorescein-antimouse IgG conjugate, extracellular sporozoites of E. meleagrimitis fluoresced brightly but intracellular sporozoites exhibited little fluorescent label. Sporozoites labeled with Hab 43A6 plus a ferritin-antimouse IgG conjugate that were observed in the process of cell invasion had ferritin on the extracellular portion of the parasite but not on the intracellular portion. Extracellular aggregates of ferritin were observed near the site of invasion. The data suggested that antigens of the sporozoite surface that are recognized by Hab 43A6 are "scraped off" during the invasion of cells. In contrast, after exposure to Hab E5, both extracellular and intracellular sporozoites of E. tenella fluoresced. However, ferritin label was not observed on viable sporozoites, even when they were fixed immediately after the labeling procedure. The antigens recognized by Hab E5 may be associated with parasite secretory products rather than with an integral part of the sporozoite surface membrane.  相似文献   

7.
The characterization of five chicken monoclonal antibodies (mAbs) that were developed against apical complex antigens of Eimeria acervulina sporozoites is realized and the mAbs reactivity to merozoites belonging to this species is tested. Using immuno-fluorescence assay (IFA), one mAb (HE-4) that recognized apical antigens common to sporozoites of E. acervulina and E. brunetti bound antigens localized on the apical tip of merozoites from all stages of development examined. The mAb 8E-1, reactive with antigens found on the apical tip of all chicken Eimeria sporozoites, also showed binding to antigens common to merozoites from all generations. Another mAb, 8C-3, which identified an antigen shared by sporozoites apical tip and sporocysts wall of E. acervulina reacted very weak and inconstantly with the merozoites from all generations whereas the mAbs 5D-11 and 8D-2 that recognized antigens shared by the sporozoites of E. acervulina and E. maxima (mAb 5D-11) and E. acervulina and E. brunetti (mAb 8D-2) did not react with the merozoites from any generation. Collectively, these results showed that the invasive stages of chicken Eimeria share cross reactive apical complex antigens which are inter-species and inter-generation-specific that might be components of a potential recombinant vaccine.  相似文献   

8.
Merozoites of Eimeria bovis were harvested from bovine monocyte cell cultures and used to immunize BALB/C mice. Spleens from immunized mice were removed and the cells fused with mouse myeloma cells. Supernates from resulting hybridoma cell lines were examined for antibodies to first-generation E. bovis merozoites using an indirect immunofluorescent antibody (IFA) assay. Three positive cell lines were identified and cloned by limiting dilution. All three cell lines produced immunoglobulins of the IgG1 isotype that recognized antigens in the anterior half to two-thirds of the merozoites. Specificity of the monoclonal antibodies was examined with the IFA assay against sporozoites of E. bovis, sporozoites and merozoites of Eimeria papillata from mice and Eimeria tenella from chickens, sporozoites of Isospora suis from pigs, and tachyzoites of Toxoplasma gondii and Neospora caninum from cell cultures. Monoclonal antibodies from the three clones reacted with the anterior end of E. bovis sporozoites, but did not react with the other parasites examined. None of the monoclonal antibodies reacted with merozoite antigens in immunoblots.  相似文献   

9.
Spleens from mice immunized with Eimeria bovis sporozoites were removed and the cells fused with mouse myeloma cells to produce hybridoma cell lines (HCLs). The resulting HCLs were examined for antibody (HAB) production against E. bovis sporozoites using an indirect immunofluorescent antibody test on air-dried sporozoites. Four fusions resulted in the production of 19 HCLs that produced HABs to E. bovis sporozoites. These 19 HCLs were further tested for reactivity with cell culture-grown merozoites of E. bovis and Sarcocystis cruzi of cattle; sporozoites of Eimeria tenella from chickens, Eimeria meleagrimitis from turkeys, Eimeria papillata and Eimeria vermiformis from mice; and bradyzoites of S. cruzi from calves. Six HCLs produced HABs that reacted only with E. bovis sporozoites and were species specific/stage specific. Two HCLs produced HABs that reacted only with E. bovis sporozoites and merozoites, and were species specific/stage cross-reactive. Seven HCLs produced HABs that reacted with the sporozoites of the other Eimeria species examined and were species cross-reactive/stage specific. Four of the HCLs produced HABs that reacted with all organisms tested and were species cross-reactive/stage cross-reactive. The results of this study suggest the conservation of some antigens throughout developmental stages and genera of Eimeriorina.  相似文献   

10.
In order to examine the antigenic similarity and specificity of the trail antigen of Eimeria stiedai and Etp 100, a microneme protein of Eimeria tenella, monoclonal antibodies to the trail antigen of E. stiedai sporozoites were selected by an indirect immunofluorescent antibody method. The monoclonal antibody of one clone, 3D10, reacted with the anterior portion of non-fixed sporozoites. By immunoblotting, the monoclonal antibody was found to react with a 100 kDa antigen of E. stiedai sporozoites, and a 117 kDa antigen of E. tenella sporozoites and merozoites. It was also found to react with a recombinant protein with thrombospondin-/properdin-like motifs homologous to E. tenella microneme protein Etp 100. The monoclonal antibody significantly inhibited the penetration of E. stiedai sporozoites into cultured rabbit hepatobiliary epithelial cells. These results suggest that E. stiedai sporozoites have a trail antigen, located in the anterior region on the outer surface of the sporozoites, which has an epitope with thrombospondin-/properdin-like motifs similar to E. tenella microneme protein Etp 100. This protein may play an important functional role in the process of penetration of host cells.  相似文献   

11.
Autonomous glycosylphosphatidylinositol (GPI) molecules (also protein-free GPIs or free GPIs) have been reported to be particularly abundant in some parasitic protozoa and mediate strong immunomodulatory effects on the host immune system. In the work at hand we have investigated the existence of free GPIs in Babesia bovis. Comparative thin layer chromatographic analysis of the protein-free glycolipid fraction of in vitro cultured B. bovis merozoites and erythrocyte membranes demonstrated the presence of an abundant parasite-specific band. Its chemical analysis revealed a GPI species containing a chain of two mannose residues, N-glucosamine and non-acylated inositol. The lipid moiety linked to inositol was diacylglycerol. The total fatty acid composition showed predominantly long-carbon chain molecules (12% of C22:0 and 45% of C24:0). The potential of B. bovis to assemble the presented free GPI species was verified by the existence of seven genes in its genome that putatively encode the following GPI biosynthetic enzymes: PI N-acetyl-GlcN-transferase (PIG-A and GPI-1), N-acetyl-GlcN-PI-de-N-acetylase (PIG-L), acyltransferase (PIG-W), dolichyl-phosphate mannosyl transferase (DPM-1), GPI mannosyltransferase I (PIG-M), and GPI mannosyltransferase II (PIG-V). GPI biosynthesis is vital for the intraerythrocytic parasite stage as mannosamine, an inhibitor of GPI biosynthesis, impaired in vitro growth of B. bovis merozoites. Absence of the vast majority of N-glycan metabolism encoding genes in the B. bovis genome underscores that the growth inhibitory effect of mannosamine is attributable to its interference with GPI biosynthesis and not with assembly of N-linked oligosaccharides, as has been described for higher eukaryotes. Elucidation of the structure and biosynthesis of GPI may allow to facilitate the development of future immune interventions against bovine babesiosis.  相似文献   

12.
The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite life cycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.  相似文献   

13.
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14.
The growth of Sarcocystis neurona, isolate UCD1, in continuous culture was examined in 10 cell lines to identify growth conditions and methods for the preparation of parasites free of gross host cell contamination for molecular studies. The unpredictable, slow release of merozoites in most cell lines prompted development of a method to synchronously release the parasites from infected host cells. The calcium ionophore A23187 at a concentration of 1 microM was found to release intracellular merozoites with a 40 min treatment at 37 degrees C. The release of merozoites en masse from attached host cells allowed for the rapid collection of relatively pure parasites from the culture supernatant. This release of merozoites occurred in five different host cell lines. The ionophore-released parasites were highly infectious for host cells and appeared to be morphologically identical to naturally released merozoites, except that the treated merozoites had an increased number of micronemes when examined by electron microscopy. The ionophore did not enhance the release of sporozoites from sporocysts, but freezing in the presence of 5% DMSO released sporozoites that were infectious to bovine monocytes in in vitro culture.  相似文献   

15.
Hybridoma antibodies (Hab) were produced against Eimeria acervulina merozoites that had been separated from extraneous intestinal material by a fiber column technique before injection into mice. The Hab demonstrated three different immunofluorescent-antibody (IFA) patterns of tip, surface, or surface-internal fluorescence in or on the merozoites. Some Hab reacted with round immature schizonts, which were also present in the fiber-cleaned merozoite material. Variations in cross-reactivity were seen with a number of Hab tested by IFA with merozoites, sporozoites, and immature schizonts of different coccidial species. Certain Hab were species- and stage-specific, whereas others cross-reacted with some or all stages or species tested. One Hab apparently reacted with only a small percentage of the E. acervulina merozoites in the fiber-cleaned material. The ferritin (Fe)-labeling technique showed that with one Hab, which gave a surface-internal IFA pattern, there was an irregular clumping of the Fe label along the surface of the immature schizont. A heavier deposit of Fe label was seen on the area of the schizont where the merozoite was beginning to form. A heavy uniform labeling of Fe was seen on the surface of the pellicle of the mature merozoites. These results demonstrate that stage-specific and cross-reactive antigens are present in or on the merozoites of E. acervulina, and as shown with one Hab, surface antigens present on the immature schizont are incorporated onto the mature merozoite.  相似文献   

16.
Macrophage activation in Theileria sergenti-infected calves was studied by testing the production of oxygen metabolites in macrophages following specific and non-specific stimulation with T sergenti merozoites or zymosan, respectively. Six calves were inoculated with merozoites and three calves with sporozoites. All showed significant macrophage activation within one month after inoculation (P less than 0.05). Activation of macrophages appeared earlier than parasitaemia or the peak of antibody titre against T sergenti. The highest chemiluminescence response, indicative of macrophage activation, was observed when the merozoites were opsonised with immune sera.  相似文献   

17.
The pathomorphological findings and the expression and distribution of variable surface protein antigens (Vsp) of Mycoplasma (M.) bovis were characterised immunohistochemically in lungs of eight calves following inoculation with a Vsp A-expressing clonal variant of M. bovis type strain PG45. Within 48 h post inoculation (p.i.) an innate immune response dominated by macrophages and neutrophils develops. The monoclonal antibodies (mAbs) 1A1 and 1E5 detected M. bovis Vsp antigens in paraffin tissue sections of seven calves. Vsp antigens were widely distributed and were already present at day two p.i. within macrophages and other lung compartments. Taken together, the results demonstrate that the bovine is unable to eliminate M. bovis during the time period examined. Based on the different immunohistochemical labelling patterns obtained with the mAbs, the results also support the speculation that the in vivo variability of Vsps together with immunological factors may contribute to the chronicity of pulmonary disease.  相似文献   

18.
Development of cell-mediated immunity (CMI) and comparative effectiveness of different stage-specific coccidia antigens in T cell activation during avian coccidiosis were evaluated in two inbred strains of chickens using a specific in vitro T cell proliferation assay. Lymphocytes from chickens infected with different Eimeria spp. showed proliferative response to sporozoites, merozoites or Eimeria soluble antigen (Esa) excreted by cultured parasites. Detectable CMI response was observed at 21 day P.I. in chickens infected with E. tenella and E. maxima. Generally lower T cell response was observed in chickens infected with E. acervulina. Merozoites were highly immunogenic compared to sporozoites. Esa prepared from cultured parasites was as effective as whole parasites in evoking a T cell response. Although strain variation in T cell response to parasites or Esa was observed during a primary infection, substantially enhanced T cell response was observed 3 days after a secondary infection in both strains of chickens. The results of the present investigation suggest that Esa may be a major parasite antigen released to the immune system during early stages of infection and relevant to the development of protective immunity.  相似文献   

19.
Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca2+ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca2+ concentration in the cytosol of the parasite cells. The increased intracellular Ca2+ concentration following these treatments was confirmed using live cell Ca2+ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca2+ signalling pathway in the egress of B. bovis merozoites.  相似文献   

20.
Two recombinant Mycobacterium bovis BCG (rBCG) strains carrying the Eimeria tenella rhomboid gene (Rho) delivered by extrachromosomal vector pMV261 and integrative vector pMV361 were evaluated for their ability to protect chickens against E. tenella challenge. The chickens were immunized intranasal with BCG, rBCG pMV261-Rho, or rBCG pMV361-Rho twice at a 2-week interval. All the recombinant BCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4+ and CD8+ cells compared to the control (P < 0.05). Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge. But vaccination with rBCG pMV261-Rho induced higher specific antibody titers and produced greater protection rate (56.04%) than rBCG pMV361-Rho group (P < 0.05). These results indicated that M. bovis BCG is a novel vaccine vector to express and present antigens of E. tenella, and rBCG has a potential as vaccine in chickens.  相似文献   

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