Infection with Eimeria tenella: Modulation of Lymphocyte Blastogenesis by Specific Antigen,and Evidence for Immunodepression1

ABSTRACT. The blastogenic effects of specific parasite antigen and of mitogens on the lymphocytes of chickens infected with Eimeria tenella were examined. Lymphocytes from infected chickens were stimulated to divide when cultured with parasite antigen, but their responses to the T-cell mitogen, phytohemagglutinin (PHA), were depressed throughout the period of infection. Responses to the B-cell mitogen, lipopolysaccharide (LPS), were depressed during the first week of infection but enhanced in the second week. The inclusion of plasma samples from infected chickens in the culture medium depressed the responses of normal spleen lymphocytes to PHA, suggesting that soluble suppressor factors are generated during infection.     

Production of monoclonal antibodies specific for Eimeria tenella microgametocytes

A panel of 4 monoclonal antibodies specific for Eimeria tenella, the causative agent of cecal coccidiosis of birds in the genus Gallus, was produced by standard techniques. The indirect fluorescent antibody (IFA) test demonstrated specificity of these 4 antibodies for the microgametocytes. Hybridoma TIA3B9 secreted a monoclonal antibody of subisotype IgG2b that was used throughout the course of this study. Immunologic potency of this antibody was demonstrated by in vitro experiments that revealed a greater than 50% reduction in oocyst production, indicating an apparent inhibition of fertilization.     

Eimeria tenella: specific reversal of t-butylaminoethanol toxicity for parasite and host by choline and dimethylaminoethanol.

t-Butylaminoethanol is an anticoccidial compound that is related structurally to the metabolically active substances, dimethylaminoethanol, and choline. Toxic effects of t-butylaminoethanol for chickens and Eimeria tenella are specifically overcome by feeding sufficient amounts of dimethylaminoethanol or choline. Dietary concentrations of the two above metabolites required to totally overcome toxic effiects of t-butylaminoethanol were determined and are expressed as the reversal ratio, inhibitor (t-butylamino-ethanol): metabolite. The inhibitor:choline ratio for total reversal of toxic effects of the inhibitor in chickens is approximately 1:10 over a concentration range of inhibitor from 0.019 to 0.05%. The inhibitor:choline ratio for reversal of antiparasitic effects is approximately 1:200 with a concentration of 0.01% inhibitor. The inhibitor:Dimethylaminoethanol ratio for reversal of toxic effects of the inhibitor in the chicken is approximately 1:7 with a concentration of 0.015% inhibitor. The inhibitor:dimethylaminoethanol ratio for reversal of antiparasitic effects is approxmately 1:20 wth a concentration of 0.01% inhibitor.     

Chicken T lymphocyte clones with specificity for Eimeria tenella. I. Generation and functional characterization

T lymphocyte clones reacting specifically with the antigenic components of Eimeria tenella were generated from splenic lymphocytes of immunized chickens and were maintained for 12 to 14 wk in vitro. These T cell growth factor-dependent T lymphocyte clones from bursectomized and normal chickens proliferated in vitro when stimulated with antigens from different developmental stages of homologous but not heterologous species of the parasite. Specific proliferative responses of the cloned T cells showed an absolute requirement for antigen presentation by histocompatible antigen-presenting cells. Some of the T cell clones exhibited functionally discrete interactions with syngeneic primed B cells; 25% of the T cell clones from immunized normal chickens and 7% of those obtained from immunized bursectomized chickens showed antigen-dependent helper activity and induced specific antibody production by syngeneic primed B cells. Of the T cell clones from immunized normal chickens, 19% showed suppression of in vitro antibody production in comparison to 7% of those isolated from immunized bursectomized chickens. The frequency of cloned T cells with ability to induce cytotoxic activity in macrophages against the sporozoites of E. tenella was much higher in those isolated from bursectomized chickens (80%) than in those isolated from normal chickens. Because both bursectomized and normal chickens can be immunized by repeated infections, differences in the distribution among cloned T cells suggest different effector mechanisms of immunity against coccidiosis in these chickens. Lack of B cells seem to affect the development of T cell immunity as reflected by slower development of immunity and enhanced activation of cytotoxic T cell function.     

Eimeria tenella: ginsenosides-enhanced immune response to the immunization with recombinant 5401 antigen in chickens

Three-day-old specific-pathogen-free chickens were subcutaneously immunized with Eimeria tenella recombinant 5401 antigen (100 microg per chicken) with (0.25, 0.5 or 1.0mg per dose) or without ginsenosides, and boosted with the same dosage 14 days later. The chickens were challenged with 6 x 10(4) homologous sporulated oocysts 14 day after the booster. The specific antibody response and lymphocyte proliferation in response to Con A were measured before and 7, 14, 21, 28, 35, 42 days after the immunization. Oocyst output, mortality, and lesion scores were measured to evaluate the protective effects of the immunization. The vaccine containing 0.5 or 1.0mg ginsenosides per dose induces higher antibody response and lymphocyte proliferation in response to Con A than the vaccine without ginsenosides or containing 0.25mg per dose. The oocyst output indicated that recombinant 5401 antigen with ginsenosides (0.5 and 1.0mg per dose) gave a protection rate of 59.38 and 62.5%, respectively. The lesion score in the group vaccinated with recombinant 5401 antigen with 0.5 or 1.0mg ginsenosides per dose were significantly lower than in group without ginsenosides or containing 0.25mg per dose. Therefore, we conclude that ginsenosides have strong adjuvant effects at a dose of 0.5 or 1.0mg when mixed with E. tenella recombinant 5401 antigen, and has a potential as an adjuvant in chicken vaccine.     

Production and partial characterization of monoclonal antibodies specific for the gamonts of Eimeria tenella.

Thirteen hybridomas secreting monoclonal antibodies (Mabs) specific for the sexual stages (gamonts) of Eimeria tenella were produced by fusing spleen cells of gamont-immunized RBF/Dn mice with FOX-NY myeloma cells. A Mab subisotype profile revealed 1 IgG2a and 12 IgG1. All Mabs demonstrated a similar binding pattern when incubated with parasitic gamonts as determined by the indirect fluorescent antibody test. Ascitic fluid containing Mab (GD9 (IgG1) was produced and used to immunize chicks passively per os. There was a 34% decrease (P less than 0.05) in oocyst output from immunized chicks when compared to control chicks. Passively immunized chicks also had reduced cecal lesion scores when compared to control chicks. These results suggest that Mab GD9 partially inhibited the fertilization process of E. tenella.     

Localization of a low molecular weight antigen of Eimeria tenella by use of hybridoma antibodies.

Three monoclonal antibodies (Mabs), found by western blot analysis to recognize 10-kDa bands of Eimeria tenella sporozoite preparations, were used with immunoelectron (IE) microscopy, immunogold-silver staining (IGSS), and indirect immunofluorescent antibody (IFA) light microscopy to determine the location and distribution of the antigens in or on extra- and intracellular parasites. All 3 of the Mabs (designated C3, E5, and 1231) were found by IE microscopy to label amylopectin granules of extracellular sporozoites. Additionally, these Mabs extensively gold-labeled the sporocyst wall. In cultured primary chicken kidney cells inoculated with sporozoites of E. tenella, IGSS showed surface labeling of the parasite and intense labeling of the infected host cells by 6 hr postinoculation (PI). At 24 hr PI, host cell vacuoles in infected and uninfected cells were labeled by the 3 Mabs by IFA. The E5 and C3 Mabs also were seen to label the host cell membrane of newly infected cells. The C3 and 1231 Mabs showed little label of the host cells by 48 hr PI, but the parasites still were labeled up to 96 hr PI. The E5 Mab had intense IFA labeling of infected host cells at 48 hr PI. The results of this study indicate that parasites apparently release antigenic material during the early stages of parasite development and that this material is found internally and/or on the surface of the infected host cells.     

Eimeria tenella: Local Antibodies and Interactions with the Sporozoite Surface

.Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10³ oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10³ oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and imtnunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results. The immunofluorescence studies reveal the extremely labile nature of the sporozoite surface antigens and support the hypothesis that naturally elaborated IgA antibodies are involved in the modulation of complexed sporozoite surface antigens.     

Eimeria tenella: accumulation and retention of anticoccidial ionophores by extracellular sporozoites

Extracellular sporozoites of Eimeria tenella were incubated at either 25 or 40 C in the presence of ¹⁴C-lasalocid or ¹⁴C-narasin, both anticoccidial ionophores. Liquid scintillation analysis shows that both ionophores are accumulated by the sporozoites outside their host cell. The relative degree of retention was significantly different for the two incubation temperatures and the concentration of lasalocid retained was consistently greater than that of narasin.     

Eimeria tenella: local antibodies and interactions with the sporozoite surface

Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10(3) oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10(5) oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and immunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results.(ABSTRACT TRUNCATED AT 250 WORDS)     

A genetic linkage map for the apicomplexan protozoan parasite Eimeria maxima and comparison with Eimeria tenella

Eimeria maxima is one of the seven Eimeria spp. that infect the chicken and cause the disease coccidiosis. The well characterised immunogenicity and genetic diversity associated with E. maxima promote its use in genetics-led studies on avian coccidiosis. The development of a genetic map for E. maxima, presented here based upon 647 amplified fragment length polymorphism markers typed from 22 clonal hybrid lines and assembled into 13 major linkage groups, is a major new resource for work with this parasite. Comparison with genetic maps produced for other coccidial parasites indicates relatively high levels of genetic recombination. Conversion of ∼14% of the markers representing the major linkage groups to sequence characterised amplified region markers can provide a scaffold for the assembly of future genomic sequences as well as providing a foundation for more detailed genetic maps. Comparison with the Eimeria tenella genetic map produced 10 years ago has revealed a less biased marker distribution, with no more than nine markers mapped within any unresolved heritable unit. Nonetheless, preliminary bioinformatic characterisation of the three largest publicly available genomic E. maxima sequences suggest that the feature-poor/feature-rich structure which has previously been found to define the first sequenced E. tenella chromosome also defines the E. maxima genome. The significance of such a segmented genome and the apparent potential for variation in genetic recombination will be relevant to haplotype stability and the longevity of future anticoccidial strategies based upon multiple loci targeted by novel chemotherapeutic drugs or recombinant subunit vaccines.     

柔嫩艾美耳球虫早熟系的选育及其生物学特性研究

选育制备弱毒疫苗所需的柔嫩艾美耳球虫早熟系并对其生物学特性进行研究。运用Jeffers建立的早熟系选育方法对柔嫩艾美耳球虫山西株（E．tenella SX010323）进行早熟选育,并对其内生发育、致病性、繁殖力、免疫原性、稳定性进行观察和检测。结果表明：经过海兰白雏鸡15次传代之后,E．tenella SX010323潜隐期由141h缩短至120h,卵囊明显缩小。选育得到的柔嫩艾美耳球虫山西株早熟系（E．tenella SX010323P15）其内生发育表现第二代裂殖生殖不完全;亲本株与早熟系对11日龄雏鸡的半数致死量分别为7．52×10^4个／只、27．64×10^4个／只,早熟系在致病性与繁殖力上均较母株大幅度降低,但保留了母株的免疫原性,经早熟系免疫的雏鸡可抵抗亲本株半数致死量的攻击。对选育的早熟系进行放松选择传代10次,其潜隐期、OPG、致病性均保持了早熟系的特征。提示通过早熟选育得到了遗传相对稳定的柔嫩艾美耳球虫早熟系。     

Amylopectin Synthase of Eimeria tenella: Identification and Kinetic Characterization

ABSTRACT. A soluble enzyme amylopectin synthase (UDP-glucose-α 1,4-glucan α-4-glucosyltransferase) which transfers glucose from uridine 5'-diphosphate glucose (UDP-glucose) to a primer to form α-I,4-glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella . UDP-glucose and not ADP-glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis-Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km). with respect to UDP-glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing α-1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP-glucose-α-1,4-glucosetransferase EC 2.4.1.11).     

柔嫩艾美耳球虫HSP基因的克隆、表达及鉴定

为研究柔嫩艾美耳球虫热激蛋白(Heat shock proteins,HSPs)的生物学特性,应用RACE和RT-PCR技术,从柔嫩艾美耳球虫子孢子中首次克隆获得了EtHSP的全长cDNA(GenBank Accession No.FJ911605)。EtHSP包含一个1455 bp的开放阅读框,编码484个氨基酸,预测表达蛋白的分子量大小为53.5 kD。应用Real-time PCR对柔嫩艾美耳球虫不同发育阶段(未孢子化卵囊、孢子化卵囊、子孢子和裂殖子)表达量进行分析,发现该基因在子孢子阶段的表达明显高于其他阶段。同时,构建了原核表达重组质粒pET28a(+)-EtHSP,转化到大肠杆菌BL21(DE3)中,经IPTG诱导表达后,对表达产物进行SDS-PAGE及Western blotting分析。结果显示,重组质粒pET28a(+)-EtHSP在大肠杆菌中以包涵体形式表达,经1 mmol/L IPTG诱导6 h后的表达量最高,该蛋白可被抗柔嫩艾美耳球虫的多克隆抗血清识别,表明该蛋白具有较好的反应原性。本研究结果为进一步研究该基因的生物学功能奠定了基础。     

Ultrastructural localization of antigenic sites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and Eimeria tenella by monoclonal IgG and IgM antibodies

Immunoelectron microscopy was used to study the localization of monoclonal IgG (13.9 and 15.84) and IgM (10.84) antibodies generated against Eimeria tenella sporozoites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and E. tenella. A uniform layer of ferritin was present on sporozoites of E. tenella fixed chemically before the addition of 10.84, 13.90, or 15.84 (called prefixed), whereas postfixed (fixed chemically after exposure to monoclonal antibody) sporozoites lacked ferritin, indicating that the latter had capped immune complexes. Patches of ferritin were present on prefixed and postfixed sporozoites of E. acervulina exposed to 15.84, indicating that immune complexes containing 15.84 were not capped. Sporocysts of E. tenella exposed to 10.84 had a uniform layer of ferritin on their outer surface; ferritin was localized in patches on those exposed to 13.90 or 15.84. In E. acervulina sporocysts exposed to 15.84, ferritin was widely scattered on the outer surface but formed a uniform layer on the inner surface of the sporocyst wall. Patches of ferritin occurred on the inner layer of the oocyst walls of E. tenella and E. acervulina exposed to 10.84, 13.90, or 15.84. These findings indicate the shared antigen detected by 15.84 differed in relative amount, spatial distribution, and structural location in sporozoites and sporocysts of E. acervulina and E. tenella.