弓形虫感染家兔精液中虫体DNA检测初报

目的了解弓形虫感染家兔精液中是否有虫体存在，探明虫体在精液中动态变化情况。方法用RH株弓形虫速殖子不同剂量经腹腔感染家兔6只。在家兔感染前后分别采集精液，-40℃冻存备用，分别用普通PCR检测弓形虫感染家兔精液中弓形虫DNA。结果5只家兔在感染后的8～14d死亡，仅1只家兔在感染后的8d和72d，用PCR检测手段在精液中均可检测到弓形虫DNA。结论弓形虫感染雄兔精液中有虫体存在。     

弓形虫感染家兔精液中虫体含量的动态变化

目的检测弓形虫感染家兔精液中虫体DNA,观察虫体在精液中动态变化情况。方法用RH株弓形虫速殖子105个/只的剂量腹腔感染雄性家兔16只。在家兔感染前后分别采集精液,-40℃冻存备用,采用两种方法提取精液中总DNA,用实时定量PCR(quantitativereal-timePCR,QRT-PCR)检测弓形虫感染家兔精液中弓形虫DNA,依据对照计算虫体拷贝量,绘制感染后时间与精液中虫体含量的动态曲线图。结果16只雄兔感染前其精液中均不能检测到弓形虫DNA,精液中虫体含量在感染后7wk左右达到高峰期,然后虫体含量逐渐下降。在感染9wk~15wk维持较低水平。结论弓形虫感染雄兔精液中虫体含量随感染后时间不同而变化。     

应用双单克隆抗体ELISA夹心法检测循环抗原诊断弓形虫病实验研究

应用ELISA双单抗夹心法检测弓形虫不同感染度的家兔各临床期循环抗原(CA_g),并与双多抗法等检测CA_g进行比较,探讨该法检测CA_g诊断弓形虫病的敏感性和特异性。 人工感染弓形虫家兔分重度、中度及轻度感染组,分别感染13、6、15只。健康对照兔5只。各组同时分期采血。另设血吸虫感染兔42只。球虫感染兔8只,分别     

感染弓形虫家兔血清循环抗原、免疫复合物及抗体的消长规律

应用ELISA观察人工感染弓形虫家兔90d内血清循环抗原(C_Ag)、循环免疫复合物(CIC)及抗体(Ab)的消长规律,探讨检测C_Ag、CIC及Ab诊断弓形虫病的价值及诊断指标。 弓形虫感染组兔6只,对照组兔5只,两组同时分期采血分离血清。FLISA方法采用双单抗夹心法检测C_Ag;单抗二抗法检测CIC;一般间接法检测Ab. 一、家兔感染弓形虫的临床发病观察结果:接种后第1～3d无临床症状,可定为潜伏期。第4d有6兔     

弓形虫感染家兔血液中虫体的动态观察

目的探明虫体在家兔血液中动态变化情况。方法用RH株弓形虫速殖子105个/只的剂量腹腔感染雄性家兔16只。在家兔感染前后分别采集家兔血液,抗凝处理后,-40℃冻存备用,用实时定量PCR检测弓形虫感染家兔血液中弓形虫DNA,依据对照计算虫体拷贝量,绘制感染后时间与血液中虫体含量的动态曲线图。结果雄兔弓形虫感染后4d血液中即可检测到虫体DNA,血液中虫体含量为217.887×103个/ml,血液中虫体含量在感染后8 d达到高峰期,血液中虫体含量为248.019×103个/ml,然后虫体含量逐渐下降,在感染后121 d雄兔血液中虫体密度为1.45×103/ml。结论弓形虫感染雄兔血液中虫体含量随感染后时间不同而变化。     

弓形虫感染兔T淋巴细胞免疫反应的初步观察

近年来,众多的研究者认为,不能确定抗体滴度与抵抗弓形虫能力之间有任何关系,而证明在对弓形虫的免疫作用中,细胞免疫占有重要地位。本文应用淋巴细胞转化试验形态法,测定人工感染弓形虫兔外周血的淋转率,并与检测血清CAg进行同步试验,观察家兔感染弓形虫后不同临床期的细胞免疫反应变化。现将结果报告如下。 材料和方法 一、动物 新西兰纯种雌性兔19只,实验前经IHA及ELISA检测弓形虫抗体均为阴性。取14只为感染组,每兔腹腔接种弓形虫强毒株(CN)速殖子2万个,接种后分期采血。另5只兔为对照组,与感染兔同时采血。     

应用生物素-亲和素系统检测弓形虫感染家兔特异性循环免疫复合物的实验研究

本文应用抗弓形虫单克隆抗体E_8株,亲和素和生物素标记的过氧化物酶复合物(ABC)为标记物的ABC-ELISA检测弓形虫感染家兔的特异性循环免疫复合物(CIC),并与常规ELISA、聚乙二醇沉淀试验(PEG)进行对比。 一、检测弓形虫感染兔(33只)及血吸虫感染兔(25只)血清CIC、ABC-ELISA检测CIC的阳性率为100％,PEG试验为96.9％:健康兔(29只)血清对照试验阴性符合率分别为100％及93.1％,但两法与血     

贵州省抑郁症患者刚地弓形虫感染状况调查及其基因型鉴定

目的了解贵州省抑郁症患者弓形虫感染状况及弓形虫基因型。方法 ELISA检测141例抑郁症患者和150例健康对照外周血弓形虫特异性抗体(IgG、IgM)和循环抗原(CAg),PCR扩增血样弓形虫高重复DNA片段(529 bp),多重-巢式PCR-限制性片段长度多态性(Mn-PCR-RFLP)方法鉴定弓形虫529 bp阳性样本的基因型。结果 ELISA检测结果显示,抑郁症患者组和健康对照组弓形虫抗体阳性率分别为21.3%(30/141)和7.3%(11/150),两者间差异有统计学意义(χ~2=11.674,P0.05);抑郁症患者组的IgG、IgM、CAg阳性率分别为18.4%(26/141)、1.4%(2/141)、1.4%(2/141);健康对照组IgG阳性率为7.3%(11/150),与抑郁症患者组间差异有统计学意义(P0.05),未检测到IgM和CAg。PCR结果显示,抑郁症患者组检测出1例弓形虫核酸阳性,经Mn-PCR-RFLP方法鉴定其基因型,结果为非典型Toxo DB#9(Chinese 1型)。结论贵州省抑郁症患者弓形虫抗体阳性率高于健康人群,1例抑郁症患者感染的弓形虫基因型为Chinese 1型。     

经胎盘感染日本血吸虫家兔血清抗体的动态变化

目的 研究日本血吸虫病经胎盘传播宿主的血清免疫反应。方法 5只怀孕晚期母兔，感染日本血吸虫尾蚴500条/只，仔兔出生后43d起，每隔2周采集血清1次，至仔兔发育到成兔（出生后13d)，然后以ELISA检测仔兔日本血吸虫特异性IgG,IgM抗体。结果 60%（12/20）仔兔经胎盘感染日本血吸虫，其中双性感染4只，肝脏均见虫卵结节；单性感染7只，仔兔血清IgM抗体均为阴性（A值为0.02-0.28，阳性对照为0.40-0.57)；仔兔血清IgG抗体，肝脏有虫卵结节的5只双性感染仔兔中，1只出生后57d起，2只出生后71d起，1只出生后85d起呈阳性，其余仔兔IgG抗体均为阴性。结论 经胎盘感染日本血吸虫家兔，其所产仔兔在短期内可产生免疫耐受性。     

ELISA双单克隆抗体夹心法检测弓形虫循环抗原的实验研究

用酶联免疫吸附试验(ELISA)双单克隆抗体夹心法,检测人工感染弓形虫家兔血清循环抗原(CAg)的累积阳性频率。在潜伏期内,重、中度感染兔CAg阳性率分别为30.8及11.1％;急性期、亚急性期和慢性早期的阳性率,中度感染兔分别为86.1及76.7％,轻度感染兔分别为43.3及32％。中、轻度感染兔的CAg阳性率在急性期逐步上升,而在亚急性、慢性早期逐步下降。健康兔、血吸虫病及球虫病兔的血清试验,均阴性。本法检测CAg的效果高于其他3种双抗体法,提示有较高的特异性、敏感性和重现性,可诊断弓形虫急性或活动性感染。     

弓形虫ROP2-P30重组真核表达质粒的构建及其免疫效应的初步分析

目的构建弓形虫棒状体蛋白2（ROP2）和膜表面蛋白1（P30）融合的重组真核表达质粒,观察融合抗原ROP2-P30以DNA免疫方式在体内的免疫学效应。方法以重组质粒pET28b/ROP2-P30为模板,利用分子克隆技术构建重组真核表达质粒pcDNA3.1/ROP2-P30,经PCR和酶切鉴定正确后,体外转染COS-7细胞,Westernblot检测ROP2-P30表达。重组质粒pcDNA3.1/ROP2-P30以每鼠100μg混合透明质酸酶10U的剂量肌肉注射免疫BALB/c雌性小鼠,以弓形虫虫体裂解抗原作包被抗原,ELISA法测定免疫小鼠血清IgG抗体,免疫结束2周后,以约100个弓形虫速殖子攻击感染小鼠,观察小鼠生存状况。结果PCR和酶切鉴定表明重组质粒pcDNA3.1/ROP2-P30构建正确;Westernblot显示该重组质粒在COS-7细胞中瞬时表达的产物可被重组ROP2-P30免疫兔血清识别;ELISA检测重组质粒免疫小鼠血清特异性IgG抗体水平升高;重组质粒免疫小鼠感染弓形虫后的存活时间较对照组有所延长。结论重组真核表达质粒pcDNA3.1/ROP2-P30构建成功,用该重组质粒DNA直接免疫小鼠,能够诱导产生特异的体液免疫反应,具有一定的免疫保护性;该重组质粒表达的融合抗原分子ROP2-P30具有免疫原性,可作为疫苗候选抗原深入研究。     

弓形虫ITS-1序列PCR诊断方法的建立

目的本文旨在为弓形虫感染的临床病例检测建立特异性PCR诊断方法。方法本研究以弓形虫ITS-1序列为种特异性遗传标记,采用有限稀释法稀释速殖子DNA,经PCR扩增,检测该方法的敏感性;用同一引物扩增鸡柔嫩艾美耳球虫原虫和弓形虫,检测PCR方法的特异性。人工感染小鼠和家兔,用组织触片法和PCR方法检测感染动物的组织,并检测10头临床疑似病猪的肺脏、肺门淋巴结、脾脏、肾脏和肝脏等组织,比较两者的敏感性。结果该PCR方法最低可以检测1pg弓形虫速殖子DNA(相当于10个速殖子的DNA);鸡柔嫩艾美耳球虫原虫未扩增出特异条带,仅弓形虫出现特异条带(300bp)。组织触片和PCR方法对人工感染小鼠组织DNA的检出率均为87.5%(7/8),对人工感染家兔的检出率分别为50%(3/6)和66.7%(4/6),对临床疑似弓形虫病猪检测的阳性率均为20%,两种方法的检查结果基本一致。结论本试验结果表明,PCR技术可以作为弓形虫感染动物的诊断与检测方法。     

Detection of Toxoplasma gondii in human placenta by PCR and placental histologic findings

Since isolation of Toxoplasma gondii from human placenta strongly correlates with fetal infection, the aims of the study were: to detect fragments of T. gondii B1 gene in human placentae by PCR and to evaluate their pathology. 36 placentae included in three groups were obtained: group I (n = 7) from pregnancies with prenatal diagnosis of fetal toxoplasmosis; II (n = 17) from women with serologic features of primary infection during pregnancy; III (n and 13) from pregnancies with fetal T. gondii infection based on clinical signs. T. gondii DNA was found in 2/4 samples from the I group and in 1/14 from the II group. Villitis was identified in 3/15 other placentae from the II group. In the III group we did not recognize neither T. gondii DNA nor villitis. We consider PCR and pathologic evaluations of placentae as the two complementary methods. PCR can be especially helpful in pregnancies not screened against T. gondii as positive result in placenta can confirm mother's primary infection.     

Toxoplasma gondii infection among chronic hepatitis C patients:A casecontrol study

Objective:To determine the detection rate of anti-Toxoplasma gondii(T.gondii) IgG and IgM in chronic HCV patients attending the Department of Tropical Medicine Mansoura University hospital in Egypt.Methods:This study included 120 adult chronic HCV patients.81 decompensate cirrhosis(late-stage)and 39 chronic HCV non cirrhotic patients(early-stage) and40 healthy blood donors as controls.Serum samples uere examined for anti-Toxoplasma IgM and anti-Toxoplasma IgG antibodies by ELISA.Real-time RT-polymerase chain reaction assay was done for quantitation of hepatitis C virus.Results:Anti-T.gondii IgG antibodies were detected in 75(92.6%) of 81 late-stage cirrhotic patients.30(76.9%) of the 39 chronic HCV non cirrhotic patients(early-Stage) and in 6(15ft) of 40 controls with statistically significant difference(P0.001).Anti-T.gondii IgM antibodies were found in 11(13.6%) in late stage patients,5(12.8%)in early stage and in 3(7.5%) of controls with no statistical significant difference(P=0.610).There was no correlation between stage of fibrosis and IgM or IgG antibodies positivity in our studied groups(P=0.526).High IgG levels significantly correlated with high viral load(P=0.026).Conclusions:Our findings suggest that the serious opportunistic T.gondii infection represent a potential significant risk for chronic HCV patients.So.toxoplasmosis should be considered in their investigations and follow-up.     

Oral immunization with a live recombinant attenuated Salmonella typhimurium protects mice against Toxoplasma gondii

The natural site of infection for T. gondii is the mucosal surface of the intestine, so the protective immunity obtained after natural infection with T. gondii points to the importance of developing a vaccine that stimulates mucosal defences. In this study, an aroA- and aroD- attenuated strain of Salmonella typhimurium (BRD509) has been used to deliver the recombinant eukaryotic plasmid pSAG(1-2)/CTA2/B expressing a multi-antigenic gene encoding SAG1 and SAG2 of T. gondii linked to A2/B subunits of cholera toxin as a candidate oral T. gondii vaccine. Immunoblot analysis showed compound gene expression in HeLa cells in vitro and intragastric immunization of mice with the recombinant salmonella resulted in the induction of humoral and Th1 type cellular immune responses and afforded protection against RH strain T. gondii challenge. Anti-T. gondii IgG values increased markedly in the BRD509/pSAG(1-2)-CTA2/B immunized group; these values were significantly higher than those in the negative controls (P = 0.008). With CTA2/B genetic adjuvant, the T. gondii-specific response was predominantly Th1, indicating that the CTA(2)/B genetic adjuvant was able to overcome the strong Th2-bias of the antigen (IgG2a > IgG1). Antigen-specific T cell proliferative responses and CTL activity were significantly enhanced when cholera toxin CTA2/B genetic adjuvant was used (P = 0.009; P = 0.006). Culture supernatants from antigen-stimulated splenocytes from mice in these groups were also examined by ELISA for Th1- and Th2-type cytokines; mean IFN-gamma levels produced after oral immunization with BRD509/pSAG(1-2)-CTA2/B were about nine-fold higher than after immunization with BRD509/pSAG(1-2) (P = 0.007). On the other hand, the levels of IL-4 were low for all groups and no increase was seen in the presence of CTA2/B genetic adjuvant. When the immunized mice were intraperitoneally challenged with 10(3) tachyzoites of the highly virulent RH strain, the survival time of the mice immunized with BRD509/pSAG(1-2)-CTA2/B was markedly longer than other groups (P = 0.003) and a 40% survival rate was achieved. This is the first report that demonstrates that an oral attenuated salmonella DNA vaccine can induce protective immunity against the acute phase of T. gondii infection.     

小鼠口服弓形虫DNA混合疫苗的免疫保护反应

目的 观察携带弓形虫表面抗原复合基因重组沙门氏菌经口免疫BALB/c小鼠所诱导的免疫反应。 方法 构建弓形虫主要表面抗原SAG1、SAG2复合基因真核表达质粒，将其转入减毒鼠伤寒沙门氏菌BRD509 (BRD509/pSAG1/SAG2)，经口服免疫BALB/c小鼠，以携带空载体质粒的减毒沙门氏菌BRD509及生理盐水（NS）组为对照，分别于每次免疫前和免疫后断尾取血，并于末次免疫后第4周取小鼠脾脏测定T淋巴细胞增殖活性、天然杀伤细胞（NK细胞）活性和T细胞亚群；ELISA法测定IgG抗体及血清中的细胞因子干扰素（IFN-γ），白介素-4（IL-4）。与末次免疫后4周，每只小鼠腹腔注射0.1 ml(10⁵)弓形虫RH株速殖子攻击，观察小鼠存活时间。 结果 免疫组小鼠的IgG抗体水平明显提高，滴度为1∶100；NK细胞杀伤活性和T细胞增殖活性也明显增强，其中NK细胞杀伤活性为85%±7%，T细胞增殖指数为2.83，与对照组相比差异有统计学意义(P < 0.05)。免疫鼠攻击感染后平均存活时间比对照组长5 d。 结论 弓形虫口服DNA混合疫苗可诱导小鼠产生保护性免疫。     

microRNA?155在弓形虫感染调节巨噬细胞极化中的作用

目的 探讨弓形虫感染对宿主细胞内microRNA?155（miR?155）表达的影响及其对巨噬细胞极化的作用。 方法 利用miRNAs基因芯片检测弓形虫感染宿主细胞内miRNAs表达水平,应用实时定量聚合酶链反应技术（qPCR）检测miR?155表达水平。采用脂质体转染法将pEGFP?miR?155导入人巨噬细胞,利用流式细胞术检测转染效率。采用流式细胞术、qPCR、酶联免疫法检测弓形虫感染组、pEGFP?miR?155过表达组以及miR?155抑制组诱导巨噬细胞表面分子CD86及诱导型一氧化氮合酶（iNOS）和白细胞介素（IL）?12表达水平。结果 基因芯片和qPCR检测结果显示,随着弓形虫感染时间的延长,miR?155表达量上升;pEGFP?miR?155转染宿主细胞的转染效率可达82.6%。弓形虫感染组和pEGFP?miR?155过表达组CD86表达水平显著高于对照组和miR?155抑制组,iNOS和IL?12基因表达量显著升高。结论 弓形虫感染能够通过上调miR?155表达参与驱动人源巨噬细胞向M1型偏移。