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1.
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   

2.
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   

3.
A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.  相似文献   

4.
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   

5.
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   

6.
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   

7.
Seven monoclonal antibodies (MAB) generated against sporozoites of Eimeria bovis were tested for reactivity against immature and mature first-generation meronts, sexual stages, and oocysts in tissues from experimentally infected calves by use of an avidin-biotin peroxidase complex (ABPC) immunohistologic test. Three of the 7 MAB reacted in the ABPC test. One of these, MAB-4FB4, reacted only with mature E bovis meronts. The other 2 MAB, MAB-2AE7 and MAB-4AD7, reacted with all the developmental stages of E bovis tested. Asexual stages and sexual stages of E tenella from chickens and E papillata from mice also were examined in the ABPC test. Monoclonal antibodies MAB-2AE7 and MAB-4AD7 reacted with all stages of these eimerian protozoa. None of the other 5 MAB reacted with these parasites. Results of this study suggested that antigens are shared among the asexual and sexual stages of several diverse Eimeria species.  相似文献   

8.
For Apicomplexa (members) the host cell invasion is realized with the help of the organelles located at the apical tip of parasites. In this research paper the characterization of five chicken monoclonal antibodies (mabs) produced against Eimeria acervulina sporozoites is described. All mabs reacted with molecules belonging to the apical complex of chicken Eimeria sporozoites. On immunofluorescence assay (IFA) one mab, 8E-1, recognized an apical tip molecule present on all chicken Eimeria sporozoites, two mabs (8D-2 and HE-4) recognized an antigen present on the apical tip of the same two Eimeria species (E. acervulina and E. brunetti), another mab (5D-11) recognized an antigen present on the apical tip of other two species (E. acervulina and E. maxima) while one mab (8C-3) identified antigens present on the sporozoites and sporocysts wall of only E. acervulina. Besides the apical tip antigens, two mabs (HE-4 and 8D-2) recognized some proteins located in the anterior half of the sporozoites. Collectively, these mabs proved that the apical complex of chicken Eimeria sporozoites share one or more antigens that are expected to play a role in host cell recognition and invasion.  相似文献   

9.
A panel of monoclonal antibodies was produced and characterized by an indirect fluorescent antibody test (IFAT), an enzyme-linked immunosorbent assay (ELISA) and Western blotting with the aim of identifying antigens of Babesia bovis. After fusion, the resultant hybrids were selected by the IFAT, cloned, maintained in culture in vitro, and cryopreserved in liquid nitrogen. Ten clones producing monoclonal antibodies were found to react against the entire merozoites, three reacted on the surface of the merozoites, and one clone reacted against the polar region of the merozoites. All monoclonal antibodies reacted in ELISA, with the optical density varying from 0.368 to 0.502 (cut off = 0.022). The bands recognized by the monoclonal antibodies in Western blotting had molecular weights ranging from 162 to 19 kDa. Four clones recognized a single band of 73 kDa, and another four did not react in Western blotting.  相似文献   

10.
Norwegian sheep were investigated for globidial schizont infection in the abomasum. The frequency of infection was found to be 78.2 %. Light microscope studies of the various mature schizonts revealed the existence of four morphologically different merozoites, small A, small B, intermediate and long forms. Each globidial schizont was found to contain only one form of these merozoites. However, these four schizont types occurred in the same abomasum.The intermediate form of globidial schizont merozoites was investigated by the aid of an electron microscope, with the aim of comparing its internal morphology with that previously published for Eimeria species. A striking resemblance was observed between the fine structures of the intermediate merozoite and that of Eimeria species, particularly the first generation merozoites described in giant schizonts of Eimeria bovis.The present status of globidial schizonts infecting the abomasum of sheep was discussed. It was concluded that these four forms of merozoites could represent different generations of one Eimeria species or different E. species producing giant schizonts in the abomasum.Due to the practical difficulties in studying the life histories of the different Eimeria species infecting sheep, it was proposed that the in vitro propagation of the individual species in cultured cells may shed some light on the corresponding asexual, as well as the sexual, stages. This would offer a new approach to the study of the ultra-structure of the developing parasite.  相似文献   

11.
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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.
Four species of avian Eimeria invaded the intestine of foreign host birds in the same areas in which they invaded the natural host. Repeated inoculation (immunization) of chickens with the turkey coccidian, Eimeria adenoeides, partially protected the chickens against a subsequent challenge with 5.8 x 10(4) E. tenella oocysts. At 6 days post-challenge, the weight gain and feed conversion efficiency of the immunized chickens was significantly better than those of the chickens that were not immunized with E. adenoeides. Lesion scores and cellular invasion by the sporozoites were significantly lower in the immunized birds than in the unimmunized group. Electrophoresis and Western blot analysis identified changes in the serum antibody profiles of the chickens that appeared to be associated with the immunization and challenge programs. An antibody or antibodies recognizing a 60,000-molecular-weight antigen of E. tenella sporozoites disappeared when chickens immunized with E. adenoeides were challenged with E. tenella; an antibody or antibodies recognizing a 23,000-molecular-weight sporozoite antigen appeared within 6 days of challenge. Reciprocal studies, in which turkeys were immunized with E. tenella and challenged with E. adenoeides, showed little evidence of protection.  相似文献   

14.
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15.
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   

16.
Faecal Eimeria oocyst excretion and levels of antibodies to first generation merozoite antigen of E. bovis in sera and colostra were followed in 86 and 70 cow-calf pairs in northern (group EF) and central Germany (group H), respectively, over periods of 3 weeks before to 3 weeks after calving in cows and from birth to an age of 63 days in calves. Oocysts were found in 30 and 7.7% of cows in groups EF and H, respectively. They belonged to 10 (group EF) and four Eimeria spp. (group H) with E. bovis, E. ellipsoidalis, E. auburnensis and E. zuerni as the most frequently occurring species. Prevalence and intensity of oocyst excretion varied with time resulting in peak values around the date of parturition, particularly in the case of E. bovis. Peak values at the time of parturition were also seen in case of strongyle egg excretion. Seven (group H) and nine Eimeria spp. (group EF) were found in the calves. The predominant species E. ellipsoidalis, E. zuerni, E. bovis and E. auburnensis were detected for the first time earlier after birth (3-5 weeks) than the others. The prevalence of Eimeria infections increased to 67.1% (group EF) and 50.1% (group H) 9 weeks after birth. Specific IgM and IgA antibody levels (the latter only determined in group EF) in cow sera remained almost constant throughout the observation period, whereas IgG(1) and IgG(2) levels were reduced at the time of parturition. Levels of specific antibodies in sera and colostra were significantly correlated. Except IgM antibodies, significant inverse correlations were found in cows between intensity of infection with E. bovis and specific serum IgG (group H) and IgG(2) (group EF) antibodies. Antibodies to E. bovis were detected in calves sera only after colostrum intake with significant correlations between levels in calves sera and colostra. Levels decreased, starting within the first week of life (most conspicuously in case of IgM and IgA) until the third week. Subsequently, but except IgG(1) antibody concentrations increased until the end of the observation period. Interrelations between antibody levels and the total amount of E. bovis oocysts excreted by the calves until the ninth week of life varied with the age of the animals. Inverse relationships in the first 3 weeks of life as suggested by negative correlation coefficients could not be proven statistically. Thus, there is no unambiguous proof for immunoprotection of calves against E. bovis via maternal immunity. Considering antibody levels in the 3-9 weeks old calves significant direct correlations with E. bovis oocyst excretion were found in case of IgM, IgG(2) and IgA, reflecting an active immune response of young calves to coccidial infection.  相似文献   

17.
A single-chain antibody library against Eimeria tenella sporozoites was constructed by phage display. Antibody-displaying phage was selected in five panning rounds against cryopreserved E. tenella sporozoites. A 1000-fold increase in phage output and a 3000-fold enrichment were obtained after three rounds of panning, as the binding clones became the dominant population in the library. Ten clones were randomly selected from the last selection round, and their nucleotide sequences were aligned and compared to chicken germ-line sequences. Analysis of the light chain variable regions revealed possible donor pseudogenes which act as donors in gene conversion events, and contribute to the diversification of the V(L) immune repertoire. Possible somatic hypermutation events, a consequence of affinity maturation, were also identified. Soluble antibody was produced in a non-suppressor E. coli strain, purified by nickel affinity chromatography, and characterized by immunoblotting. In an immunofluorescence assay, this recombinant antibody showed specific binding to E. tenella sporozoites.  相似文献   

18.
Host immune responses conducted against antigens of Eimeria bovis are key factors for the development of protective immunity against this protozoan disease. In this study we investigated the expression of E. bovis-derived antigens on the host cell surface membrane during E. bovis first merogony in vitro. Host cells carrying E. bovis-meront I stages expressed E. bovis host cell surface antigens (EbHCSAg) on their surface membrane which were recognised by hyperimmune sera of calves and by sera from rats immunized with E. bovis merozoites I, when tested by indirect immune fluorescent antibody test (IIFAT), laser scanning confocal microscopy (LSCM) and immune electron microscopy. Expression of EbHCSAg on permissive host cells was earliest detected 7 days p. i., thus coinciding with the onset of the parasite replication. Membrane-associated EbHCSAg were removed from infected host cells by proteinase K, partially by Triton X-100, Triton X-114 and Triton X-405, but not by 1 M NaCl, CHAPS or phospholipase C treatment. Antibodies, affinity-purified on paraformaldehyde/glutardialdehyde (PAGA)-fixed E. bovis meront I-infected bovine host cells bound to the surface meront I-carrying cells and to merozoites I (IIFAT, LSCM) but, in contrast to untreated sera, not to sporozoites. When tested on methanol-fixed merozoites I and sporozoites by IIFAT, affinity-purified antibodies bound to structures in the apical complex area of merozoites I, but not to sporozoites, whilst untreated sera caused diffuse labelling of internal structures of both parasite stages. Immune electron microscopy demonstrated binding of affinity-purified antibodies to micronemes and dense granules of merozoites I. Although the function of EbHCSAg is still unknown, results of this study might suggest an involvement in the development of protective immunity against E. bovis infections.  相似文献   

19.
利用原核表达的柔嫩艾美耳球虫棒状体蛋白（EtRP）的重组蛋白免疫BALB／c小鼠，经4次免疫后取脾脏制备免疫脾细胞，与小鼠骨髓瘤细胞SP2／0融合，经间接ELISA和Western blot筛选获得一株抗EtRP蛋白的特异性单克隆抗体细胞株，命名为2E3，亚类鉴定为IgG2a。细胞上清和小鼠腹水的效价分别为1：40和1：2．56×10^4。利用该单抗对EtRP在柔嫩艾关耳球虫子孢子中的分布进行分子定位，结果显示该蛋白主要分布于子孢子的顶端；而当子孢子在DMEM培养基中41℃孵育1h后，该蛋白则分布于整个虫体。研究结果表明，成功制备了EtRP单克隆抗体，为进一步研究球虫相关蛋白功能奠定了基础。  相似文献   

20.
以抗堆型艾美耳球虫子孢子的单抗EASP-3G3作为工具,对鸡各段消化道上皮细胞切片和子孢子进行免疫组化染色,并利用蛋白质印迹技术来检测单抗所识别子孢子可溶性抗原的分子量,来确定子孢子和十二指肠上皮细胞之间是否存在共同抗原。结果表明单抗只与十二指肠上皮细胞发生反应,而与其他肠段无染色反应。而且单抗所识别的可溶性抗原分子量为35~48 ku。抗子孢子的单抗同时与十二指肠上皮细胞和子孢子反应,而不与其他肠段反应,证明堆型艾美耳球虫寄生的位点特异性与十二指肠上皮细胞表面的某种抗原分子有内在的关系。  相似文献   

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