IgG responses to salivary gland extract of Ixodes ricinus ticks vary inversely with resistance in naturally exposed sheep

Enzyme-linked immunosorbent assay (ELISA) was used to investigate the antibody responses of control sheep, and sheep naturally exposed to Ixodes ricinus Linné (Acari: Ixodidae) ticks, to salivary gland extract (SGE) proteins of partially fed, adult I. ricinus. Comparisons between responses of control sheep and naturally infested sheep by Western blot analysis suggested that variations in IgG responses of I. ricinus-exposed sheep were mostly associated with specific responses to I. ricinus SGE antigens. Sheep IgG responses were positively related to the numbers of adult ticks feeding per sheep at the time samples were collected, were greater during the spring than the autumn periods of I. ricinus activity and were inversely related to sheep resistance to ticks measured by the weights of nymphal I. ricinus that engorged on the sheep. These findings suggest that sheep lose their resistance to ticks due to polarization of a Th1 type response to some tick antigens towards a Th2 type response when sheep are exposed to high, natural tick infestations, or to seasonal conditions of relative nutritional stress. Potential consequences for the epidemiology of tick-borne diseases are discussed.     

Use of direct solid-phase immunoenzyme analysis for assessing the immunological activity of a vaccine against tick-borne encephalitis

A high correlation between the current index of the effectiveness of tick-borne encephalitis vaccine, its protective activity in mice, and the results of the direct solid-phase enzyme-immunoassay has been established, which permits the use of this assay as an auxiliary method for the immunological evaluation of newly prepared commercial purified tick-borne-encephalitis vaccine.     

Effectiveness of the seroprophylaxis of tick-borne encephalitis depending on the titer of homologous gamma-globulin antihemagglutinins

To give a rationale for using homologous gamma globulin with antihemagglutinin titers of 1 : 20 to 1 : 80 for the prophylaxis of tick-borne encephalitis, 5-year observations covering all persons attacked by ticks in one of the intensive natural foci of the disease in the Western Urals have been made. The threefold statistically significant difference in the morbidity rate of tick-borne encephalitis between groups of persons immunized and not immunized with gamma globulin has been shown.     

Results of using the ELISA method in studying tick-borne encephalitis and rabies

The results of using the indirect variant of ELISA for the study of serum samples from humans and white mice for the presence of antibodies to tick-borne encephalitis virus and rabies virus are presented. The high sensitivity and specificity of this method have been confirmed.     

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.     

Echinostoma caproni in mice: shedding of antigens from the surface of an intestinal trematode

The surface antigens, which induce a serum antibody response during infection of mice with the intestinal trematode Echinostoma caproni, were examined. It was demonstrated that antigens are shed from the surface of juvenile and 4-week old adult E. caproni during in vitro culture. SDS-PAGE and Western blot analysis of in vitro shed and detergent solubilized surface antigens indicated that the four major antigens released from the surface of adult parasites had molecular masses of approximately 26,000, 66,000, 75,000 and 88,000. A modified ELISA technique showed the in vitro turn-over rate of the surface antigens to be very high, with a half-life of 8-15 min in both juvenile and adult E. caproni trematodes. Transmission electron microscopy of the surface of adult parasites revealed a highly active secreting tegument which was densely packed with membrane-bound vesicles, reflecting the high rate of shedding of the surface antigens. An attempt to immunize mice with detergent solubilized adult surface antigens failed to induce resistance to infection with metacercariae of E. caproni.     

Regional epidemiologic characteristics of combined foci of tick-borne encephalitis, endemic rickettsioses and leptospirosis in Western Siberia

The authors carried out complex study of combined foci of infections with natural foci in Western Siberia and their reflection in human pathology. The results of serological examination of 5917 persons and of 1743 of farm animals in respect to tick-borne encephalitis, Asian tick-borne rickettsiosis, Q-rickettsiosis, and leptospiroses are analysed. Affection of the population with all the four infections in all the landscape zones under study was shown; the intensity of this affection with different infections differed. Combined natural foci of the mentioned infections were found to be widespread; epidemiological significance of such combination was unequal in different ladscapes, this depending on the ladscape characteristics of the natural foci of infections under study and of different ways of transmission of their causative agents.     

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.     

Trichinella britovi human infection in Spain: antibody response to surface,excretory/secretory and somatic antigens

A third outbreak of Trichinella britovi with 140 people involved, occurred in Granada Spain (December 1998). The source of infection was sausage made from uninspected wild boar meat. Fifty-two patients agreed to participated in this study. An elevated eosinophil level (> 5%) was detected in 59.6% of patients, and persisted in most of these cases for two months. A moderate IgG response was observed. At the onset of symptoms, Western blot (WB) test detected more positive cases than Enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF). Six months from infection, ELISA revealed fewer positive cases than the other two tests. It would appear that the response to somatic antigens starts earlier than those to cuticular and excretory/secretory (ES) antigens and that the response to ES antigens is the first to decrease.     

抗猪鼻支原体单克隆抗体的研制及双抗体夹心ELISA检测法的建立

目的 制备多种抗猪鼻支原体的单克隆抗体,建立双抗体夹心ELISA方法用于该病原体的检测。方法用猪鼻支原体CVCC361免疫BALB／c小鼠,采用杂交瘤技术和酶联免疫吸附实验筛选出抗该病原体的单克隆抗体;运用免疫双向扩散试验、Western blotting确定I异G亚类及针对抗原的相对分子质量;筛选出配对抗体,建立双抗体夹心ELISA的检测方法,并评价其灵敏度和特异性。结果共筛选出17株单克隆抗体,抗体亚类分别为IgG1、IgG2a、IgG2b、IgG3,免疫印迹结果表明单抗ZB1、ZB2及ZB16与相对分子质量为35×103的抗原有特异性结合,而ZB3和ZBIO与相对分子质量为70×10^3的抗原有特异性结合。确定了2个配对抗体（ZB1-ZB1-HRP和ZB1-ZB2-HRP）,可检出最小抗原量为30ns／mL,检出猪鼻支原体活菌8．34×10^2CFU／mL,与人呼吸道常见的致病菌及支原体均无非特异性反应。结论筛选的单克隆抗体具有较高的特异性和敏感性,应用双抗体夹心ELISA方法可用于猪鼻支原体的检测。     

Serological survey of a captive macaque colony in China for antibodies to simian type D retroviruses.

Sera from 510 macaques consisting of Macaca mulatta, Macaca assamensis, Macaca fascicularis, Macaca nemestrina, and Macaca arctoides were investigated for antibodies to simian AIDS type D retrovirus (SRV) by ELISA and Western blot with viral antigens purified from supernatants of SRV-1 infected cell cultures. Of these monkeys, 104 were seropositive by ELISA; only 23 were confirmed by Western blot. The true positive reaction to SRV was found in 15 of 463 (3.2%) M. mulatta and eight of eleven (72.7%) M. assamensis.     

Identification of antigenic proteins encoded by human herpesvirus 8 and seroprevalence in the general population and among patients with and without Kaposi's sarcoma

To establish a sensitive and specific antibody assay, potent antigenic proteins encoded by human herpesvirus 8 (HHV8) were studied. Fifteen recombinant HHV8-encoded proteins were produced as glutathione S-transferase fusion proteins. The sera from AIDS-associated Kaposi's sarcoma (KS) patients reacted with four proteins encoded by open reading frames (ORFs) K8.1, 59, 65, and 73 in a Western blot assay. An enzyme-linked immunosorbent assay (ELISA) using these four proteins as antigens (mixed-antigen ELISA) revealed that all 26 sera derived from KS patients (24 with and 2 without human immunodeficiency virus infection) became positive for anti-HHV8 antibodies. The presence of HHV8 was demonstrated in 14 (1. 4%) of 1,004 sera from the Japanese general population and 10 (1.9%) of 527 sera from patients without HHV8-associated diseases. The presence of immunoglobulin G (IgG) and IgM antibodies against HHV8 examined further by the mixed-antigen ELISA and Western blotting revealed IgG antibody in all ELISA-positive sera, while IgM antibody against ORF K8.1 was absent. These data suggest that the ORF 73 and 65 proteins are potent antigens for a sensitive serological assay.     

Newly recognized mosquito-associated viruses in mainland China, in the last two decades

There are four principal arboviruses in mainland China. Two kinds of them are mosquito-borne viruses, namely Japanese encephalitis virus and dengue virus, which lead to Japanese encephalitis, and dengue fever/dengue hemorrhagic fever respectively; the other two are tick-borne viruses, namely tick-borne encephalitis virus and Crimean-Congo hemorrhagic fever virus (also known as Xinjiang hemorrhagic fever virus), which contribute to tick-borne encephalitis and Xinjiang hemorrhagic fever respectively. With exception of these four main arboviruses, many other mosquito-associated viruses have been isolated and identified in recent years. These newly isolated and identified mosquito-associated viruses are probably responsible for human and animal infections and diseases. The purpose of this review is to describe the newly isolated mosquito-associated viruses in mainland China which belong to five viral families, including their virological properties, phylogenetic relationships, serological evidence, as well as to appeal the public health concentration worldwide.     

Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits

The interaction of viral proteins with host cell components plays an important role in antiviral immune response. One of the key steps of antiviral defense is the formation of immunoproteasomes. The effect of nonstructural protein 1 (NS1) of tick-borne encephalitis virus on the immunoproteasome formation was studied. It was shown that cell expression of NS1 does not reduce the efficacy of the immunoproteasome generation in response to interferon-γ stimulation and even increases the content of the immunoproteasome subunits without the interferon-γ treatment. Thus, NS1 of tick-borne encephalitis virus activates, rather than blocks the mechanisms of immune defense in the cell.     

Impact of immunization technology and assay application on antibody performance--a systematic comparative evaluation

Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost ("DNA immunization"; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.     

An assessment of the immunoepidemiological efficacy of a liquid cultured killed vaccine against tick-borne encephalitis virus strain 205 in the Maritime Territory]

Inactivated vaccine against tick-borne encephalitis (TBE), prepared on the basis of strain 205, is characterized by epidemiological (53%) and immunobiological activity. The appearance of a few TBE cases among the vaccinees is probably due to different maturation rate of immune response to various strains (different specificity of immune response). A suggestion has been made that no inactivated vaccine prepared from a single strain can produce a reliable protective effect because of pronounced heterogeneity of the population of TBE virus.     

蜱传脑炎病毒非结构蛋白NS2B-NS3与NS5的研究进展

蜱传脑炎病毒是引起严重的中枢神经系统疾病蜱传脑炎的病原体,每年在欧洲、俄罗斯远东地区、日本和中国北部报道的蜱传脑炎病例数约为10000-12000例,且在我国和多个欧洲国家的发病率逐渐增高,正成为人类健康的潜在危害。主动免疫是预防蜱传脑炎的有效措施,包括我国在内的多个国家已研制出安全性较高的疫苗,但在我国流行省份的疫苗接种较为有限,特异性抗病毒药物的研发或许是治疗蜱传脑炎病毒感染的研究方向之一。蜱传脑炎病毒非结构蛋白NS2B-NS3与NS5因为在病毒基因组复制、加帽和宿主免疫调节中的重要作用,成为关键的抗病毒药物研发靶点。本文综述了蜱传脑炎病毒非结构蛋白NS2B-NS3与NS5的三维结构和抑制剂研发工作,为深入探究该病毒感染的分子机制和抗病毒药物研发提供参考。     

Correlation of TBE Incidence with Red Deer and Roe Deer Abundance in Slovenia

Tick-borne encephalitis (TBE) is a virus infection which sometimes causes human disease. The TBE virus is found in ticks and certain vertebrate tick hosts in restricted endemic localities termed TBE foci. The formation of natural foci is a combination of several factors: the vectors, a suitable and numerous enough number of hosts and in a habitat with suitable vegetation and climate. The present study investigated the influence of deer on the incidence of tick-borne encephalitis. We were able to obtain data from deer culls. Using this data, the abundance of deer was estimated and temporal and spatial analysis was performed. The abundance of deer has increased in the past decades, as well as the incidence of tick-borne encephalitis. Temporal analysis confirmed a correlation between red deer abundance and tick-borne encephalitis occurrence. Additionally, spatial analysis established, that in areas with high incidence of tick-borne encephalitis red deer density is higher, compared to areas with no or few human cases of tick-borne encephalitis. However, such correlation could not be confirmed between roe deer density and the incidence of tick-borne encephalitis. This is presumably due to roe deer density being above a certain threshold so that availability of tick reproduction hosts has no apparent effect on ticks'' host finding and consequently may not be possible to correlate with incidence of human TBE.