Localisation of three host-protective oncospheral antigens of Taenia ovis

Immunohistochemistry, confocal immunofluorescence and immunogold labelling were used to determine the localisation of the host-protective antigens To16, To18 and To45W in Taenia ovis oncospheres. During maturation of the adult tapeworm the antigens were initially seen as diffuse staining in the developing oncospheres but in mature oncospheres four distinct cells stained positively for the antigens. Confocal fluorescence microscopy using different fluorophores revealed that each of the antigens co-localises within the same cells in the oncosphere. No surface localisation was seen in non-activated or recently activated parasites. Immunogold labelling of non-activated oncosphere sections viewed in transmission electron microscopy revealed labelling of bilateral cells, however the identities of these cells was unclear due to deficiencies in the current level of understanding of oncosphere ultrastructure. Localisation of all the antigens changed dramatically after oncospheres were activated in vitro with each of the antigens being dispersed more generally throughout the parasite parenchyma. During development of the parasites in in vitro culture, surface localisation of the proteins was seen in parasites after 3 or more days in culture. All three antigens were found to be completely absent in parasites by 15 days of culture. The location of the host-protective antigens suggests that initially the invading oncospheres are not susceptible to vaccine-induced antibody and complement mediated attack, but that as the parasites mature, the host-protective antigens come to be associated with the parasite’s surface, rendering them susceptible to immune attack.     

Spontaneous worm expulsion and intestinal IgA response in mice infected by Vampirolepis nana

The expulsion of the gastrointestinal parasite Vampirolepis nana was examined in different mouse strains and in immunosuppressed mice infected to different degrees with eggs and cysticercoids. To investigate the immunological mechanism that regulates expulsion, surface-bound mouse immunoglobulins were examined on adult worms. The time to spontaneous expulsion of worms was dependent on strain (C57BL, BDF(1), B6C3F(1)相似文献   

Cortisone-sensitive, innate resistance to Hymenolepis nana infection in congenitally athymic nude rats

The innate resistance of the unnatural rat host to the mouse tapeworm Hymenolepis nana is cortisone sensitive but thymus independent. When congenitally athymic nude rats were orally given eggs, cysticercoids, or adult worms of H. nana, no lumenal adults were established except when they were treated with cortisone acetate during the expected lumenal development. The effect of cortisone to promote adult maturation in the rats was compared in nude and normal rats given eggs of H. nana. The fecundity of the worms (assessed by the fresh worm biomass and the number of infective eggs produced) was much higher in cortisone-treated nude rats than in cortisone-treated normal rats. When the nude rats reconstituted with thymocytes were given eggs and treated with cortisone, the fecundity of H. nana dropped to the same level as in cortisone-treated normal rats. It is strongly suggested that the unnatural rat host has thymus-independent cortisone sensitive resistance to an initial infection (which is the main component of the innate resistance and blocks the lumenal establishment of this parasite) and thymus-dependent resistance (which suppresses the established worms' fecundity and may be ascribed to acquired resistance to the ongoing infection).     

Echinostoma caproni in mice: ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode

The binding of mouse antibodies to the surface antigens of juvenile and 7 and 28 day old Echinostoma caproni was examined by transmission electron microscopy of thin sections of parasites, which were treated with antibodies in a double sandwich technique with ferritin-conjugated antibody. The surface of freshly recovered mature adult parasites was covered with an irregular but often rather intensive mouse antibody containing matrix, which probably represents a layer of mouse antibody/parasite antigen complexes. The complexes were lost after in vitro culturing of the parasites for 24 h, but incubation of the in vitro-maintained antibody-negative adult parasites with immune mouse serum led to reformation of a similar but less intensive cover with immune complexes. Juvenile and young stages of E. caproni, which had never been exposed to host antibodies, obtained a layer of immune complexes on their surface after incubation with immune mouse serum in vitro. In both young and mature parasites, the antibody-antigen complexes were observed to be rather loosely attached to the outer surface of the parasites, where the antigens probably constitute a part of the irregular glycocalyx of the organisms. It may also be that the antigens are present as isolated excretion along the surface of the parasites. Several sections indicated that the parasite surface antigens may be present in the tegument in vesicles which fuse with the outer membrane of the parasite whereby their contents are released to the exterior.     

Hymenolepis microstoma: direct life cycle in immunodeficient mice

The mouse bile duct tapeworm Hymenolepis microstoma requires beetles as the obligatory intermediate host. However, when congenitally athymic NMRI-nu mice were infected with the mature tapeworm and allowed to eat their own faeces with tapeworm eggs, the oncospheres penetrated the intestinal tissue and developed to cysticercoids. After excysting, growth to adult worms occurs in the lumen of the small intestine and bile duct. Furthermore, the same happened when NMRI-nu mice, non-obese diabetic severe combined immunodeficiency (NOD/Shi-scid) mice and NOD/Shi-scid, IL-2 Rgamma(null) (NOG) mice were orally inoculated with shell-free eggs of this parasite. Differences between the cysticercoids of H. microstoma and H. nana developed in the mouse intestinal tissues were: (i) the time course for the development of fully matured cysticercoids of H. microstoma in mice was about 11 days but only 4 days for H. nana; and (ii) cysticercoids of H. microstoma developed in mice had a tail while those of H. nana had none.     

Antibody responses of experimentally infected lambs to antigens collected during in vitro maintenance of the adult, metacestode or oncosphere stages of Taenia hydatigena and Taenia ovis with further observations on anti-oncospheral antibodies

The antigenicity and specificity of crude antigens collected during the in vitro maintenance of Taenia hydatigena and T. ovis, excretory/secretory (ES) antigens, were assessed in a peroxidase microenzyme-linked immunosorbent assay (ELISA), using sera from lambs given experimental monospecific infections with T. hydatigena, T. ovis, Echinococcus granulosus or Fasciola hepatica. ES antigens of larval cysts of T. ovis and T. hydatigena were less reactive than those of adult or oncosphere stages. Strong interspecific cros-reactions occurred between all antigen preparations, and these antigens offered no better specificity than crude somatic extracts. IgG1 was the major immunoglobulin detected in sera from lambs experimentally infected with T. ovis or T. hydatigena using antigens prepared from sonicated oncospheres. Discrete peaks of anti-oncospheral antibodies were detected following initial and challenge infections with eggs (whether the homologous or heterologous species), when sera were assayed with a PBS sonicate or an ES antigen from oncospheres. However, when oncospheres solubilised with sodium deoxycholate were used, the antibody response was prolonged and resembled that reported previously when somatic extracts of adult and metacestode stages were used as antigen. The results showed that oncospheres share antigens in common with other life-cycle stages, but also support the notion that they may possess some unique stage-specific antigenic determinants.     

Trypanosoma cruzi: expression of antigens on the membrane surface of parasitized cells

A direct immunofluorescent antibody test with an anti-Trypanosoma cruzi F(ab')2 conjugate was used to demonstrate antigens of T. cruzi on the membrane surface of intact live or fixed macrophages and L929 mouse fibroblasts infected with the organism. Antigens were demonstrated in 5 to 50% of infected cells, and their presence was not directly related to the number of intracellular organisms. Cells with as few as four intracellular amastigotes had demonstrable surface antigens, whereas some cells with as many as twelve or more organisms did not. Capping of antigen-antibody complexes was noted to begin a few minutes after the addition of the anti-T cruzi F(ab')2 conjugate; by 30 min, most of the parasitized cells had eliminated the complexes, and no surface antigen of parasitic nature could be demonstrated. Although capping may have caused a negative result in a previously positive cell, other mechanisms may be involved, because antigens were not demonstrated in some heavily parasitized cells examined immediately after completion of the test. Treatment of the infected cells with trypsin or chymotrypsin resulted in the absence of demonstrable parasite antigens on the cell membrane surface. However, the antigens were again demonstrated 12 hr after the enzymes were removed. The reappearance of parasite antigens on the surface of infected cells was prevented by treatment of the monolayers with puromycin or tunicamycin. A T cell-enriched population of spleen lymphocytes from mice chronically infected with T. cruzi recognized the membrane-bound antigens and proceeded to destroy the host cell and the intracellular organisms. In this process, noninfected cells were also destroyed, possibly because they were coated with antigens released from intact infected cells or from infected cells that had been lysed by the action of the sensitized lymphocytes or their products.     

Echinococcus granulosus: use of an intermediate host mouse model to evaluate sources of protective antigens and a role for antibody in the immune response.

A Balb/cJ mouse model was used to determine which stage of the E. granulosus life cycle possessed the most potent protective antigens. Mice were immunized with crude extracts of protoscoleces, brood capsules, cyst fluid, adult worm tissue, eggs or oncospheres and then challenged intraperitoneally with 600 activated oncospheres. Sonically disrupted oncospheres induced the highest levels of protection (greater than 90%) at doses greater than or equal to 10(3) oncosphere equivalents per mouse. High levels of protection were maintained when these preparations were solubilized in SDS. Immunization with Taenia ovis or T. hydatigena oncosphere preparations induced a maximum of 62 and 40% cross-protection, respectively. In passive transfer experiments, serum from triple-infected immune donors that were completely resistant to subsequent challenge induced 69% protection in naive recipients (P less than 0.01). Serum from mice that had been immunized with oncosphere sonicates that were shown to be highly immune, failed to induce statistically significant protection in recipients. A sheep trial confirmed the protective ability of prior infections. Immunization of sheep with a SDS solubilized oncosphere preparation produced 91% protection (P less than 0.01).     

Trypanosoma lewisi: stage-specific surface antigens and exoantigens recognized by monoclonal antibodies

The stage-specific expression of surface antigens by Trypanosoma lewisi was investigated using monoclonal antibodies directed against this parasite. Hybridomas secreting monoclonal antibodies were produced by the fusion of SP2/0-Ag 14 mouse plasmacytomas with spleen cells from rats infected previously with the Taliaferro strain of T. lewisi. Additivity enzyme-linked immunosorbent assays and indirect immunofluorescent antibody tests indicated the determinant recognized by monoclonal antibody TL40.3 (IgM) was different from those recognized by monoclonal antibodies TL40.1 (IgA), TL40.2 (IgM), and TL40.6 (IgG2 alpha). Monoclonal antibody TL40.3 agglutinated trypanosomes collected 3 days after parasite inoculation while monoclonal antibodies TL40.1, TL40.2, and TL40.6 agglutinated trypanosomes collected 6 days after inoculation. Since agglutinin titers against trypanosomes from irradiated (700 rad from a 60Co source) and nonirradiated rats were similar, expression of the antigens recognized by the monoclonal antibodies appeared to be independent of the immunological state of the host and the morphology of the parasite. The reproduction of T. lewisi in in vitro trypanostatic assays was inhibited only when the monoclonal antibodies were present in concentrations greater than or equal to those needed to agglutinate the trypanosomes. Monoclonal antibodies TL40.1 and TL40.3, but not TL40.2 and TL40.6, agglutinated erythrocytes collected later in the infection from irradiated, infected rats. None of the monoclonal antibodies agglutinated erythrocytes from nonirradiated, infected rats, from irradiated, noninfected rats or from nonirradiated, noninfected rats. This suggests that immunocompetent rats may make blocking antibodies against the exoantigens recognized by monoclonal antibodies TL40.1 and TL40.3.     

Trypanosoma cruzi specific antibody response in immunized C3H(He) mice during infection

C3H(He) mice previously immunized with live culture derived Corpus Christi strain T. cruzi are significantly protected (up to 100% survival) against challenge by Brazil strain blood trypanosomes. The antibody response, directed against the Brazil strain or the Corpus Christi strain, in these mice has been observed by comparing sera from mice immunized only, infected only, or immunized and infected. The anti- T. cruzi titers determined by both direct agglutination (DA) and indirect fluorescence (IFA) were routinely found to be highest for immunized and infected mice with immunized mice and infected mice following in decreasing order. The use of mercaptoethanol treatment of sera (DA) and isotope specific second antibody (IFA) showed that IgG is the major parasite specific immunoglobulin response through infection. Evidence of cross-reacting antigens on the two parasite strains was found. By both DA and IFA, 11 of 18 anti-Brazil strain monoclonal antibodies were found to react (IFA titers of 320 or greater) with both parasite strains. No evidence of localization of cross-reacting antigens (using mouse antisera) or antigenic determinants (using monoclonal antibodies) was found in that uniform fluorescence over the parasite was observed in all IFA tests.     

Trichinella spiralis: recognition of muscle larva antigens during experimental infection of swine and its potential use in diagnosis

Longitudinal studies with Trichinella spiralis experimentally infected pigs were carried out to identify muscle larva antigens recognized during infection. This was approached using Western blot analysis and ELISA assays. Immunoblots of sera from experimentally infected pigs using total parasite extracts revealed five principal parasite antigens throughout infection. A similar pattern of antigen recognition was given by sera from backyard pigs in areas of Mexico, some of them endemic for Trichinella. Four of the five antigens recognized (MW 47, 52, 67, and 72 kDa) corresponded to surface/stichosomal antigens purified by monoclonal antibody NIM-M1. In addition, Western blots of excretions-secretions of muscle larva contained three (MW 52, 67, and 72 kDa) of the four surface/stichosomal components recognized by NIM-M1. Affinity-purified surface/stichosomal components, total soluble extracts, and excretory-secretory antigens of muscle larva were then evaluated in ELISA for detection of T. spiralis infections in experimentally infected, noninfected control, and 295 backyard pigs. These assays showed that purified surface/stichosomal components and excretory-secretory antigens increased the specificity of ELISA. These results suggest that muscle larva components purified by monoclonal antibody NIM-M1 are the major antigens recognized during infection of pigs with T. spiralis and therefore potentially useful for diagnosis of swine trichinellosis.     

Diagnostic Mr 31,000 Schistosoma mansoni proteins: requirement of infection, but not immunization, and use of the "miniblot" technique for the production of monoclonal antibodies

Antibodies directed against diagnostic Mr 31,000 polypeptide(s) of adult Schistosoma mansoni were already formed in mice during prepatency. In contrast, repeated immunization of mice with homogenates of adult schistosomes failed to elicit antibodies detectable in immunoblots in the Mr 31,000 region. Therefore, spleen cells of infected mice were used to produce hybridoma lines. The "miniblot technique" was developed in order to detect in hybridoma supernatants antibodies against schistosome Mr 31,000 components. Electrophoretically separated total S. mansoni proteins were transferred onto nitrocellulose, and the position of the Mr 31,000 components was determined with polyclonal antisera and immunoblotting. Pieces of about 3 square mm of nitrocellulose bearing the diagnostic proteins were incubated with about 100 microliter of hybridoma supernatant in microtitre plates and subsequently probed with peroxidase-conjugated antibody to mouse IgG. This screening technique identified hybridomas secreting antibody to the relevant S. mansoni antigens. It is applicable to other defined parasite antigens, which are, however, not available in biochemically purified form. The monoclonal antibodies produced against the proteins with diagnostic potential reacted with antigens localized in the schistosome gut.     

Identification and partial characterization of a parasite antigen in sera from humans infected with Wuchereria bancrofti

The purpose of this study was to identify and characterize soluble parasite antigens present in sera from humans infected with the filarial nematode Wuchereria bancrofti. Affinity chromatography and immunoblot methods were used to demonstrate a 200,000 m.w. circulating parasite antigen in sera from infected humans which corresponded to an antigen released by adult W. bancrofti during in vitro culture. Two monoclonal antibodies were produced to this antigen by immunizing mice with antigens from Dirofilaria immitis, a filarial parasite that is closely related to W. bancrofti, and screening cell fusion supernatants by enzyme immunoassay and counterimmunoelectrophoresis inhibition. The antibodies bound to a single repeated epitope (not phosphorylcholine) that was resistant to heat, acid, and protease treatments but sensitive to periodate oxidation. Immunoperoxidase studies showed that the epitope was concentrated in the cuticle and reproductive organs in D. immitis, and it was released in relatively large amounts by adult female D. immitis in vitro. The epitope is also present in antigens of other species of filarial and nonfilarial nematodes, but on the basis of preliminary studies, its presence in human serum appears to be specific for W. bancrofti infection.     

Nematospiroides dubius: passive transfer of protective immunity to mice with monoclonal antibodies

Nine hybridoma cell lines secreting monoclonal antibodies specific for Nematospiroides dubius were produced by fusion of the mouse myeloma cell line NS-1 to either spleen cells or mesenteric lymph node cells from mice repeatedly infected with N. dubius. Seven of the antibodies were identified as IgM and two as IgG1. Each monoclonal antibody bound to polypeptide epitopes on both infective larvae (L3) and adult worms. However, five antibodies bound preferentially to L3 and three to adult worms. All nine antibodies reacted with high molecular weight protein antigens. Passive protective immunity in Balb/c mice was demonstrated with monoclonal antibodies Nd2 and Nd3 in ascites fluid which stunted both male and female worms and reduced parasite fecundity.     

Identification of circulating antigens, including an immunoglobulin binding protein, from Toxoplasma gondii tissue cyst and tachyzoites in murine toxoplasmosis

Here we describe the identification of Toxoplasma gondii circulating antigens in sera of BALB/c mice experimentally infected with either the virulent RH strain, or the cystogenic WTD1 strain or with an isolate from a human patient. The circulating antigens were identified by immunoblot in tachyzoite (RH strain) and in tissue cyst (ME-49 strain) crude antigens, using antibodies produced by immunisation of BALB/c mice with homologous sera from infected animals. The most relevant tachyzoite antigen identified are in the following four clusters of 109-94, 67-57, 35-31 and 28-21 kDa. Tissue cyst-specific circulating antigens, like the 18 kDa one, were detected in sera from mice infected with the cystogenic strains. These immune sera, after depletion of tachyzoite specific antibodies, recognised three tissue cysts antigens with Mr of 120, 79 and 48 kDa, and a cluster of antigens in the range of 68-53 kDa. We produced monoclonal antibodies by fusion of myeloma cells with lymphocytes from the mouse immunised with circulating antigens from the RH strain. One of the clones (3A11/H12) obtained, secretes IgG(1) and recognises a peptide epitope from a tachyzoite 67 kDa protein. This parasite protein also binds irrelevant mouse IgG(1) as well as immunoglobulins from other species. The reactivity with non-specific antibodies was inhibited by preincubation with 2% normal mouse and goat serum, while the reaction with the monoclonal antibody 3A11/H12 was not. Furthermore, a biotinylated F(ab')(2) of an irrelevant mouse IgG(1) did not show any reactivity while the F(ab')(2) of the monoclonal antibody 3A11/H12 reacts specifically with the 67 kDa antigen suggesting that this circulating antigen is a putative Fc binding protein.     

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.     

Counter current immunoelectrophoresis, a new technique for the rapid serodiagnosis of porcine cysticercosis

Counter current immunoelectrophoresis was used to detect porcine cysticercosis and water soluble extracts of scolex and of scolex with cyst wall were used as antigens. Serum samples from 40 pigs infected with Cysticercus cellulosae, five infected with C. tenuicollis and five with hydatid cysts, and 15 healthy pigs were tested. A sharp and thick concave precipitin band was observed at the point of interaction of antigen and antibody within 90 min in 39 sera from infected pigs (97.5%). The precipitin reaction was better in barbitone buffer of pH 8.6 with the well distance at 6 mm. No false or cross reaction were found and the test was rapid and sensitive.     

Studies with Brugia pahangi. 16. Precipitation antibody in infected cats detected by counterimmunoelectrophoresis

In cats infected with normal, or irradiated, infective (L3) larvae of Brugia pahangi counterimmunoelectrophoresis revealed the presence of antibody to soluble antigens derived from microfilariae, adults and infective larvae of the same parasite. Infected cats with a persistently high to moderate microfilaraemia gave positive precipitin reactions to L3, microfilarial and adult worm antigens. Cats which had become amicrofilaraemic had antibody to L3 and microfilarial antigens but not to adult worm antigen. Serum from cats inoculated with irradiated L3 larvae produced a precipitin reaction only to the L3 antigen.