间接酶联免疫吸附试验检测北京市昌平地区体检人群中汉赛巴尔通体抗体

目的调查北京市昌平地区健康体检人群血清汉赛巴尔通体抗体阳性情况。方法用间接酶联免疫吸附试验（ELISA）和免疫荧光抗体（IFA）试剂盒检测体检人群血清汉赛巴尔通体抗体情况。结果以IFA为参考标准，间接ELISA方法的灵敏度为70．6％，特异度为91．6％；阳性预测值为82．2％（60／73），阴性预测值为84．9％。ELISA共检测了357份健康体检人群血清，汉赛巴尔通体抗体阳性率为34．5％，IFA共检测了239份体检血清，其汉赛巴尔通体抗体阳性率为35．6％。结论间接ELISA方法对于检测汉赛巴尔通体感染是一种快速、敏感、特异的方法，昌平地区健康体检人群中存在汉赛巴尔通体抗体阳性的情况。     

汉赛巴尔通体的研究进展

随着经济全球化和新发传染病在世界范围内的不断涌现，一种新发的虫媒传染病——巴尔通体感染，越来越引起人们的广泛关注。汉赛巴尔通体（Bartonella henselae）是巴尔通体属中重要的致病菌之一，直到20世纪90年代才被发现，由于其对动物和人的广泛致病性和复杂的临床特征，已引起国内外学者的兴趣。本研究针对汉赛巴尔通体的流行病学、临床诊断及治疗、实验室诊断、动物易感性及传播媒介等方面做一综述。     

山东省家猫检出汉赛巴尔通体

汉赛巴尔通体是一种可引起人类猫抓病的革兰染色阴性、营养条件要求苛刻兼性细胞内寄生的需氧杆菌,猫是汉赛巴尔通体的自然宿主,人通过猫抓伤或咬伤而感染患病.为了解家猫巴尔通体感染状况,我们于2005年4月在山东省采集家猫血液,进行巴尔通体分离培养及检测鉴定.     

首次在长角血蜱中检测到汉赛巴尔通体

目的检测长角血蜱中汉赛巴尔通体的携带情况。方法将采获于石家庄市灵寿县的长角血蜱分组,经无菌处理后研磨匀浆,一部分直接提取DNA进行PCR检测,另一部分接种于含5%去纤维羊血的胰酶大豆培养基上,在37℃,5%CO2的培养箱中进行培养后,挑取疑似菌落进行PCR检测。对所得阳性条带的PCR产物测序,并将所测核酸序列在Gen Bank中进行序列比对。结果菌落提取的DNA样品中有2个样品出现阳性条带,但测序未得结果;直接提取的DNA样品中有1个样品出现阳性条带,经过同源性比对后为汉赛巴尔通体。结论石家庄市灵寿县采集到的长角血蜱中存在汉赛巴尔通体感染,这是首次在长角血蜱中检测到汉赛巴尔通体。     

北京市宠物猫和流浪猫巴尔通体感染状况调查

目的调查北京市宠物猫和流浪猫巴尔通体感染状况。方法采集猫的抗凝血和血清并收集相关流行病学信息。将抗凝血用灭菌胰酶大豆肉汤按1∶4稀释后,取100μl接种于含5%去纤维羊血的脑心浸液培养基上,置于37℃、含5%CO2培养箱中分离培养至45 d。选择glt A、fts Z、rib C引物对分离到的疑似菌落进行PCR并测序,所测核酸序列进行同源性比较及系统发育分析,确定巴尔通体种。利用间接免疫荧光法检测血清样本汉赛巴尔通体抗体水平。利用SPSS 13.0软件分析实验室数据与现场采集的流行病学数据。结果北京市猫的巴尔通体血培养分离率为13.8%,获得的22株分离株全部为汉赛巴尔通体。血清抗体阳性率为39.4%。流浪猫(30.4%)、染蚤猫(36.6%)、幼猫(27.9%)的血培养阳性率较高,差异有统计学意义。染蚤猫的血清抗体阳性率(61.0%)也显著高于未染蚤猫(31.9%)。结论北京市宠物猫和流浪猫中巴尔通体感染率较高,且均为对人致病的汉赛巴尔通体,需做好宠物猫的防蚤除蚤、流浪猫的管理来预防人类巴尔通体感染。     

汉赛巴尔通体媒介和宿主的实验室研究进展

汉赛巴尔通体（Bartonella henselae）是一类革兰阴性的兼性厌氧菌，属于α-变形杆菌纲（Alphaproteobacteria）、根瘤菌目（Rhizobium）、巴尔通体科（Bartonellaceae）、巴尔通体属（Bartonella），该种又可分为2个亚型即：Houston型和Marseille型。汉赛巴尔通体为致病巴尔通体，可以引起猫抓病（CatScratch Disease，CSD）、慢性淋巴结病、心内膜炎、杆菌样血管肉瘤杆菌样紫瘢（BAP）、视神经病变等，其中以CSD最为普遍。     

不明原因发热病人钩端螺旋体和巴尔通体感染调查

目的了解不明原因发热病人钩端螺旋体和巴尔通体的感染状况。方法用显微镜凝集实验方法检测不明原因发热病人血清的钩端螺旋体抗体,用间接免疫荧光法检测血清的汉塞巴尔通体和五日热巴尔通体IgG抗体水平。结果共检测232份不明原因发热病人血清。感染率:汉塞巴尔通体(30.17%)五日热巴尔通体(18.10%)钩端螺旋体(4.74%),混合感染率:汉塞巴尔通体和五日热巴尔通体(14.22%)钩端螺旋体和汉塞巴尔通体(2.16%)钩端螺旋体和五日热巴尔通体(0.86%),1例病例三项抗体阳性。结论不明原因发热病人中有一定比率的病例是因为感染钩端螺旋体,和/或汉塞巴尔通体、五日热巴尔通体而引起的发热,感染率各不相同,混合感染较为普遍。在不明原因发热的诊断中,有必要进行钩端螺旋体病和巴尔通体病的诊断筛查。     

云南省不同环境鼠形动物巴尔通体感染情况的研究

目的 了解云南省不同地区、环境和气候条件下鼠形动物巴尔通体感染状况。方法 对2003年6～7月在云南省3种不同气候类型共5个县、市不同生境中捕获的鼠形动物采集股动脉血,用含5％去纤维兔血脑心浸液琼脂培养基置于35℃含5％CO_2培养箱中分离培养巴尔通体,疑似菌落用巴尔通体属特异性引物进行聚合酶链反应(PCR)扩增特异基因片段,电泳图中出现目标带即判断为阳性菌株。结果 捕获鼠形动物278只,接种鼠血标本176份,共检获巴尔通体疑似菌株79份,经PCR证实69份为巴尔通体。6种被检动物中有4种动物检出巴尔通体,感染鼠分属于家鼠属、绒鼠属、小鼠属。总检动物巴尔通体分离率为39.2％;其中以黄胸鼠分离率最高为42.0％。除大理市外,剑川、祥云、云龙和云县等县捕获的鼠类标本均分离到巴尔通体,鼠类生境涉及室内、庭院、溪场灌木丛、山地灌木丛,跨越暖温带季风气候、北亚热带季风气候、南亚热带河谷少雨气候三种气候类型。结论 研究再次证实云南鼠类宿主,特别是与人类关系密切的家栖鼠类巴尔通体感染率较高;巴尔通体在多种鼠类、不同地区、不同气候环境中均有分布,呈现出对不同地理和气候环境具有广泛适应性及宿主多样性特征。     

分子生物学技术检测鼠形动物巴尔通体

目的查明鼠形动物巴尔通体感染情况。方法在天台县采用夹夜法捕捉鼠形动物，采集该动物的肝脏，用PCR方法检测DNA，并对阳性产物克隆测序，计算感染情况。结果采集的55份鼠类肝脏阳性25份，其中黑线姬鼠阳性率为48．84％，黄毛鼠为33．33％，所检测到的巴尔通体与B．doshiae最接近。结论天台县鼠形动物有很高的巴尔通体DNA阳性率，存在传播给人的风险，必须采取措施加以控制。     

玫瑰单胞菌新疆分离株致病性研究

目的利用BALB/c小鼠作为动物模型,对玫瑰单胞菌分离株的致病性进行实验研究,了解该菌的致病能力及其在BALB/c小鼠体内的主要侵袭部位以及病变特征。方法选取6周龄的BALB/c小鼠40只,腹腔注射感染剂量106/ml玫瑰单胞菌XTD509的细菌悬液0.5 ml,观察染菌动物的临床表现以及体温、体重变化情况,分别收集感染后不同时间的肝、脾、肺、肾、膀胱、脑、淋巴结以及血液等标本,进行细菌分离,观察感染后不同时间细菌在各组织脏器中的分布;进行病理切片,观察感染后不同时间细菌对动物各脏器的损伤情况;测定感染后不同时间动物血液中白细胞水平,以确定动物在感染后的免疫反应。结果 BALB/c小鼠在感染玫瑰单胞菌XTD509后,均表现出明显的精神不振、厌食、饮水减少、发热等症状,40%(12/30)BALB/c小鼠在48 h内死亡。病理检查发现感染小鼠的肝、脾、肾、膀胱、淋巴结及脑组织都有病变,早期的病变器官主要为泌尿系统的肾脏和膀胱,且病变呈进行性发展。在感染早期从染菌小鼠的病变器官及血液中都分离到了与感染菌株完全同源的菌株。结论根据动物实验结果及科赫法则,可确认玫瑰单胞菌分离株XTD509对BALB/c小鼠具有较强的...     

Immune responses and protection in different strains of aged mice immunized intranasally with an adjuvant-combined influenza vaccine.

Immune responses and protection against influenza virus infection were compared between young (2 months) and aged (18 months) BALB/c, C3H and C57BL/6 (B6) mice after intranasal vaccination. The mice were immunized with 2.5 microg protein of A/PR/8/34 (PR8) (H1N1) virus vaccine containing a cholera toxin adjuvant. In both the young and aged BALB/c mice, high levels of PR8-specific antibody-forming cell (AFC) responses were induced in the nasal-associated lymphoid tissue (NALT) 7 days after immunization. Nasal wash IgA and serum IgG antibody (Ab) responses to the PR8 haemagglutinin (HA) 4 weeks after immunization were slightly higher in the young mice than in the aged mice. The young mice showed complete protection against challenge infection, while the aged mice showed only a partial protection. In the C3H mice, NALT-AFC, and IgA and IgG Ab responses were higher in the young mice than those in the aged mice in parallel with the more efficient protection in the young mice than in the aged mice. Both the young and aged B6 mice showed no NALT-AFC responses, scarce IgA and IgG Ab responses and no protection. In the BALB/c mice, IgG1 and IgG2a levels were significantly lower in the aged mice. On the other hand, in the C3H mice, only IgG2a level was significantly lower in the aged mice. Similar results were obtained in terms of immune responses and protection between the young and aged mice of three different strains of mice after intra-nasal immunization with 0.1 microg of PR8 vaccine containing the adjuvant, two-times at 4-week intervals. In the B6 mice, the immune response was improved by immunization with a higher dose of the adjuvant-combined vaccine. These results suggest that local Ab responses, as well as systemic Ab responses, are downregulated in aged mice, although the degree of the downregulation of immune responses differs from strain to strain.     

Immunization with the Chlamydia trachomatis major outer membrane protein,using the outer surface protein A of Borrelia burgdorferi as an adjuvant,can induce protection against a chlamydial genital challenge

Two strains of mice C3H/HeN (H-2(k)) and BALB/c (H-2(d)) were immunized with the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) using the Borrelia burgdorferi outer surface protein A (OspA) as an adjuvant. As a control, groups of mice were inoculated with ovalbumin instead of MOMP. Female mice were immunized using three different routes: intramuscular (i.m.) plus subcutaneous (s.c.), intranasal (i.n.) and perivaginal and perisacral (p.vag.+p.sac.). Significant humoral and cell mediated immune responses developed particularly in mice inoculated by the i.m.+s.c. routes as determined by the levels of chlamydial specific antibody in the serum and genital secretions and a T-cell proliferative assay. Following immunization the animals were challenged in the genital tract with C. trachomatis MoPn and the course of the infection followed by vaginal cultures. Significant protection against infection was achieved in the C3H/HeN mice inoculated i.m.+s.c. with MOMP+OspA, as shown by the intensity and duration of vaginal cultures, and by the number of mice with positive cultures. On the other hand in BALB/c mice there was only a decrease in the number of animals with positive vaginal cultures. Six weeks after the challenge the mice were mated and the outcome of the pregnancy evaluated. In both the C3H/HeN and the BALB/c mice immunized i.m.+s.c. with MOMP+OspA there was significant protection against infertility as shown by the number of animals with bilateral fertility and number of embryos per uterine horn. In conclusion, immunization using C. trachomatis MOMP, and B. burgdorferi OspA as an adjuvant, can induce significant protection against a chlamydial genital challenge.     

Intranasal administration of a recombinant adenovirus expressing the norovirus capsid protein stimulates specific humoral, mucosal, and cellular immune responses in mice

Norovirus (NV) is a major cause of acute, epidemic nonbacterial gastroenteritis in individuals of all ages. The immunological mechanism of NV infection and the approaches used to prevent infection remain to be elucidated. In this study, the specific immune responses of BALB/c mice were assessed following intranasal immunization with a recombinant adenovirus vector expressing the genogroup II4 (GGII/4) norovirus capsid protein. Analysis of IgM, IgG, and IgA antibodies specific for the recombinant virus-like particles (VLPs) of NV demonstrated that a high level of humoral immunity developed following immunization. Mucosal immune responses were also detectable in stool, intestinal homogenates, lung homogenates, and lung lavage samples. Specific cellular immune responses were observed in NV VLPs-restimulated splenocytes by ELISPOT and Th1/Th2 cytokine cytometric array (CBA). Serum IgG subclass analysis showed that a balanced Th1- and Th2-like cellular immune response was induced in BALB/c mice following immunization with recombinant adenovirus. These findings demonstrate that the intranasal immunization of a recombinant adenovirus expressing the NV capsid protein is an efficient strategy to stimulate systemic, mucosal, and cellular Th1/Th2 immune responses in mice, and could serve as a novel approach for designing NV vaccines.     

人巨细胞病毒IE1核酸疫苗的免疫效果研究

[目的]用人巨细胞病毒(HCMV)IE1核酸疫苗pcDNA3.1(-)-IE1免疫BALB/c小鼠,初步研究其产生的体液免疫和细胞免疫应答水平。[方法]将pcDNA3.1(-)-IE1通过肌肉注射免疫BALB/c小鼠,通过PCR测定和免疫组化检测其在肌细胞中的表达,细胞因子测定、淋巴细胞转化实验检测免疫效果。[结果]PCR检测到与IE1大小一致的片段,免疫组化结果显示IE1基因在小鼠肌细胞中表达IE1目的蛋白。小鼠脾淋巴细胞经PHA刺激后,实验组IL-4、IL-2、IFN-γ含量以及淋巴细胞转化率显著增高,与对照组比较差异有统计学意义(P﹤0.05)。[结论]pcDNA3.1(-)-IE1核酸疫苗能在BALB/c小鼠肌细胞中稳定存在并能表达HCMV IE1蛋白,pcDNA3.1(-)-IE1核酸疫苗可诱导BALB/c小鼠脾细胞分泌IL-4、IL-2、IFN-r并刺激BALB/c小鼠脾细胞增殖。     

Comparison of the immune responses in BALB/c mice following immunization with DNA-based and live attenuated vaccines delivered via different routes

The objective of this study was to compare immune responses induced in BALB/c mice following immunization with pcDNA-GPV-VP2 DNA by gene gun bombardment (6 μg) or by intramuscular (im) injection (100 μg) with the responses to live attenuated vaccine by im injection (100 μl). pcDNA3.1 (+) and physiological saline were used as controls. Peripheral blood samples were collected at 3, 7, 14, 21, 28, 35, 49, 63, 77 and 105 d after immunization. T lymphocyte proliferation was analyzed by MTT assay and enumeration of CD4⁺, and CD8⁺ T cell populations in peripheral blood was performed by flow cytometric analysis. Indirect ELISA was used to detect IgG levels. Cellular and humoral responses were induced by pcDNA-GPV-VP2 DNA and live virus vaccines. No differences were observed in T cell proliferation and CD8⁺ T cell responses induced by the genetic vaccine regardless of the route of delivery. However, CD4⁺ T cell responses and humoral immunity were enhanced in following gene gun immunization compared with im injection of the genetic vaccine. Cellular and humoral immunity was enhanced in following gene gun delivery of the genetic vaccine compared with the live attenuated vaccine. In conclusion, the pcDNA-GPV-VP2 DNA vaccine induced enhanced cellular and humoral immunity compared with that induced by the live attenuated vaccine.     

Cytokine mRNAs in the nasal-associated lymphoid tissue during influenza virus infection and nasal vaccination

Intranasal immunization with a current inactivated influenza vaccine together with an adjuvant (cholera toxin B subunit supplemented with a trace amount of whole toxin, CTB*) was confirmed in BALB/c mice to mimic influenza virus (A/PR/8/34, H1N1) infection with respect to mucosal IgA antibody responses, in which IgA antibody-forming cell responses in the nasal-associated lymphoid tissue (NALT) were involved with a peak around 7 days after infection or vaccination. Next, the expression of various cytokine mRNAs in the NALT was compared in mice either infected with viruses or immunized with CTB*-combined vaccine, to examine Th cell and cytokine regulation of mucosal IgA antibody responses. In infected mice, strong IL-2, weak IL-4, strong IL-6 and strong IFN-gamma mRNA expressions were induced during early days of infection; especially, IFN-gamma mRNA was expressed by both CD4(+) and CD8(+) T cells around 7 days after infection. In mice given CTB*-combined vaccine, weak IL-2, strong IL-4, strong IL-6 and weak IFN-gamma mRNA expressions were induced during early days of vaccination; especially, IL-4 mRNA was expressed by CD4(+) T cells. Thus, IL-6 mRNAs were expressed strongly in both infected and vaccinated mice. The IFN-gamma-rich cytokine mRNA profiles in the infected mice were reflected upon serum IgG2a-rich Ab responses, while the IL-4-rich profiles in the vaccinated mice were reflected upon the IgG1-rich Ab responses. Thus, influenza virus infection and CTB*-combined nasal vaccine induced Th1 dominant and Th2 dominant cytokine profiles, respectively, while the similarity of mucosal IgA antibody responses between infection and vaccination could be explained by the appearance of IL-6 mRNAs.     

Baculovirus as an avian influenza vaccine vector: differential immune responses elicited by different vector forms

Baculovirus is an enveloped virus that infects insects in nature and has emerged as a novel vaccine vector. We previously constructed a recombinant baculovirus displaying the hemagglutinin protein (HA) of avian influenza virus (AIV) on the viral envelope (Bac-HA64), and demonstrated the induction of humoral responses in immunized mice. To improve the vector design and explore how the vector forms influence the vaccine efficacy, we constructed two more baculoviruses Bac-CHA and Bac-CHA/HA64. Bac-CHA expressed HA after transducing the host cells while Bac-CHA/HA64 not only expressed HA but also displayed HA on the envelope. After administration into BALB/c mice, all three vectors elicited HA-specific humoral (IgG1, IgG2a and hemagglutination inhibition titers), mucosal (IgA titers) and cellular (interferon (IFN)-γ and IL-4 producing T cells and IFN-γ(+)/CD8(+) T cells) immune responses. Intriguingly, the magnitudes and types of responses hinged on the vaccine form and administration route. Via intranasal (i.n.) and subcutaneous (s.c.) inoculation, the HA-displaying vectors Bac-HA64 and Bac-CHA/HA64 triggered stronger humoral and mucosal responses than Bac-CHA, but upon intramuscular (i.m.) injection the HA-expressing vectors (Bac-CHA and Bac-CHA/2HA64) elicited more robust humoral and cellular responses than Bac-HA64. Via either administration route, the dual form vaccine Bac-CHA/HA64 gave rise to superior or at least comparable HA-specific immune responses than the other two vaccine forms, implicating the potential of Bac-CHA/HA64 as a vaccine candidate against AIV infection.     

T-cell and antibody response characterisation of a new recombinant pre-S1, pre-S2 and SHBs antigen-containing hepatitis B vaccine; demonstration of superior anti-SHBs antibody induction in responder mice.

The incidence of non-responders to hepatitis B (HB) virus SHBs antigen (Ag) vaccines has prompted the development of pre-S containing vaccines. The aim of this study was to characterise the murine immune response to a novel recombinant particle (Hepagene) (Medeva plc) containing pre-S1, pre-S2 and SHBsAg components. Hepagene induced potent in vitro spleen T-cell proliferative responses in both BALB/c (maximum stimulation index (SI) = 38) and SWR/J (maximum SI = 43) strains of mouse, following immunisation. High concentrations of interferon-gamma and low concentrations of interleukin-10 were detected in the media of spleen cells stimulated with Hepagene. The anti-Hepagene antibody response was higher in SWR/J mice and alhydrogel adjuvant significantly improved the titres. Anti-pre-S1 antibody was detected in both strains of mouse, whereas antipre-S2 antibody was only detected in SWR/J mice. IgG subclass analysis of the anti-Hepagene response revealed a Th2-type response in BALB/c mice and a mixed Th1/Th2 response in SWR/J mice. Hepagene induced higher anti-SHBs antibody responses than Engerix-B (11097 and 1276 IU/ml, respectively) in BALB/c mice. Hepagene therefore, stimulates strong cellular and humoral immune responses in murine models. The high anti-SHBs antibody response suggests that Hepagene is an improved hepatitis B virus vaccine.     

Potent antigen-specific immunity to Toxoplasma gondii in adjuvant-free vaccination system using Rop2-Leishmania infantum Hsp83 fusion protein

The results of this study describe the immunostimulatory properties of Leishmania infantum Hsp83 (83) to elicit humoral and cellular response against the Toxoplasma gondii Rop2 protein in an adjuvant-free vaccination system. The analysis was performed by immunizing three different mice strains (BALB/c, C57BL/6 and C3H). Mice immunized with fusion Rop2-83 elicited a stronger humoral and cellular response in comparison to mice immunized with Rop2 alone, or a mix of LiHsp83 and Rop2. The fusion protein induced a Th1 type response, with predominance of specific IgG2a/IgG2c isotype and IFN-gamma secretions, whereas Rop2 alone or mixed with LiHsp83 produced a Th1/Th2 mixed response. Vaccination with fusion protein conferred a remarkable resistance against oral infection with ME49 cysts in C57BL/6 and C3H mice, in comparison to mice immunized with Rop2 alone or the protein mixture. Following lethal challenge, a significant survival rate was observed in Rop2-83 immunized Balb/c and C57BL/6 mice in comparison to control groups.     

Molecular adjuvant C3d3 improved the anti-hCGbeta humoral immune response in vaginal inoculation with live recombinant Lactobacillus expressing hCGbeta-C3d3 fusion protein

To enhance the contraceptive efficiency of human chorionic gonadotrophin (hCG)-beta contraceptive vaccine, we coupled hCG-beta gene with molecular adjuvant C3d3, and cloned into live Lactobacilli (Lb.) to express fusion protein hCGbeta-C3d3. The recombinant Lb. could survive in BALB/c murine vagina for at least 3 weeks. After inoculating BALB/c and C57BL/6 mice via vagina, we found that the antibody titer peaks induced by the Lb.hCGbeta-C3d3 inoculation were higher significantly than the Lb.hCGbeta. T and B cells in spleen and vagina were significantly increased, and anti-hCGbeta IgG and IgA antibody-secreting cells in uterus and vagina were significantly increased compared to the control in different strain mice. Our study shows that the C3d3 can display apparent adjuvant efficiency to induce more powerful humoral response to the hCGbeta antigen in vaginal mucosal immunization.