弓形虫感染实验动物模型的特点及建模方案的选择

弓形虫感染对人类生活和畜牧业发展构成严重威胁。弓形虫感染实验动物模型是进行弓形虫学相关研究的基础条件之一。在实际研究工作中,根据不同的实验目的、选取不同的实验动物和以不同的实验方法所建立的实验动物模型,呈现出复杂和多变的特点。这一方面可以满足不同实验的需要,但同时也在实验结果的评价上导致一定程度的不足。根据弓形虫感染实验动物模型的不同特点,针对特定的实验目的,选择适合方法建立适合的实验动物模型,是进行相关弓形虫学研究的有效基础和前提。     

大熊猫布鲁氏菌病、弓形虫病以及心丝虫病的血清学调查研究

为摸清大熊猫布鲁氏菌病、弓形虫病以及心丝虫病的血清学感染资料,采用虎红平板凝集试验、试管凝集试验以及外膜蛋白BCSP3基因PCR扩增检测布鲁氏菌病;同时采用弓形虫间接血凝试验和犬心丝虫抗原快速诊断对样品进行检测。结果表明,6只大熊猫RBPT检测为阳性,进一步采用SAT和血液细菌BCSP31基因PCR扩增结果均为阴性,排除了布鲁氏菌感染;大熊猫蜀兰弓形虫抗体检测呈阳性,复查也为阳性,表明存在弓形虫感染;所有大熊猫心丝虫抗原检测结果均为阴性,表明无心丝虫感染。本次检测的3种疾病中,仅发现1只熊猫(蜀兰)存在弓形虫感染,布鲁氏菌病和心丝虫病均为阴性,表明目前成都大熊猫繁育研究基地内大熊猫疾病的预防工作成果显著。     

香菇多糖对急性弓形虫感染过程中CD4+CD25+Foxp3+调节性T细胞的调节作用研究

目的 探讨香菇多糖(Lentinan,Lent)对急性弓形虫感染BALB/c小鼠CD4+ CD25+ Foxp3+调节性T细胞(Tregs)数量和功能的调节作用.方法 对RH强毒株感染的BALB/c小鼠进行不同时间点的Lent预处理,动态观察用药后各组感染小鼠的生存率;在感染后第0、3、5、8和10天提取小鼠的脾细胞,FACS检测Tregs细胞数量的动态变化,ELISA法检测脾细胞培养上清中IL-10的分泌水平.结果 感染前6 d 1 mg/kg Lent用药组与药物未处理组相比显著提高了弓形虫感染小鼠的生存率;具有免疫抑制功能的Tregs数量于感染后8d达峰值,同时免疫应答中关键细胞因子IL-10的分泌水平也于感染后8d和10d显著增加.结论 对急性弓形虫感染的BALB/c小鼠采用Lent预处理之后能有效的调节Tregs的数量和功能,从而调控Th1/Th2之间的动态平衡达到治疗弓形虫的作用,为弓形虫病的临床治疗提供的新的理论依据.     

宠物弓形虫的感染及其防控

弓形虫病是一种世界性分布的人兽共患寄生虫病,对人类,尤其是妇女、儿童危害很大,估计全世界有1/3人受到该病的威胁。孕妇感染弓形虫后导致早产、流产、胎儿发育畸形;弓形虫是免疫功能低下患者的主要死亡原因之一。犬、猫是弓形虫的中间宿主和终末宿主,是人类感染弓形虫的主要来源。随着我国经济的迅速发展和人民生活水平的不断提高,城市中饲养犬、猫作为宠物的人越来越多,人、宠物间的亲密接触增加了弓形虫病传播给人的机会。加强对宠物犬、猫弓形虫病的研究及防控势在必行。本文就弓形虫的危害、宠物犬猫弓形虫感染及其防控措施作以综述。     

潘氏细胞在昆明小鼠弓形虫病发病过程中的变化

每只昆明小鼠灌胃1×104个弓形虫卵囊,分别在灌胃后1,3,5,6 DAI(days after inoculation),采用HE方法和免疫组织化学方法对小鼠回肠潘氏细胞的变化以及弓形虫在回肠的分布和数量进行研究,探讨潘氏细胞(paneth cells,PCs)在小鼠弓形虫病感染期间的变化。结果发现,回肠隐窝总数、含PCs的隐窝数、PCs总数及颗粒总数呈先增加后迅速减少至消失的趋势,回肠后段表现明显,6 DAI各参数与对照组比较差异显著(P0.05)。弓形虫在回肠中的分布面积呈增大趋势,6 DAI与1 DAI、3 DAI相比差异显著(P0.05)。该研究结果表明,弓形虫卵囊感染昆明小鼠可减少回肠潘氏细胞及其分泌颗粒的数量,为以小肠潘氏细胞为基础的弓形虫病的防治提供了实验依据。     

迟钝爱德华菌感染大鲵的研究

目的 研究迟钝爱德华菌对大鲵的致病性,为防治提供实验依据.方法 用不同浓度(102～108 cfu/ml)迟钝爱德华菌,通过不同的感染途径(喂养的水环境、灌喂及注射)感染大鲵,观察病原菌的致病条件及大鲵各组织器官的细菌分布(皮肤、消化道、肝、脾、血液)和病理变化及敏感抗生素的治疗作用.结果 喂养的水环境不能感染,大量灌喂(106～108 cfu/ml)出现轻微感染症状,注射感染(104～107 cfu/ml)出现明显感染症状,且部分死亡.感染后细菌分布于皮下、血液、肝、脾、胃等器官.用敏感抗生素处理后病鲵好转.结论 迟钝爱德华菌对大鲵可能是一种机会致病菌,病菌感染大鲵需要合适的入侵途径和数量,感染后用敏感抗生素进行治疗有较好效果.     

弓形虫主要表面抗原基因克隆的初步研究

弓形虫是能够引起人、畜感染的专性细胞内寄生的原虫,世界各地人群感染率一般在25％～50％,正常人感染可不显示任何临床症状而成为带虫者,但是胎儿感染常可导致胎儿畸形、神经系统发育障碍、流产甚至死胎;免疫功能低下或免疫功能缺陷的病人(如恶性肿瘤、接受器官移植、AIDS病人等)并发弓形虫感染,尤其神经系统感染常是引起死亡的主要原因之一。因此,弓形虫感染的早期诊断研究及弓形虫疫苗的开发利用,对于提高新生儿素质、预     

刚地弓形虫基因工程疫苗

弓形虫疫苗是预防弓形虫感染的最有效手段之一。随着生物学研究技术的不断进步,弓形虫疫苗的研究也不断深入完善。本文介绍了近年来发展起来的基因工程疫苗的最新研究现状及进展,以期望在对现有研究成果总结的基础上,为大家以后的研究提供新的视角和方向。     

宁波市育龄期男女弓形虫感染状况及对不孕不育的影响

目的分析宁波市育龄期男女弓形虫感染状况及对不孕不育的影响。方法对1 890例育龄期男女用ELISA法检测弓形虫血清抗体,进行流行病学调查,并追踪有无不孕不育史。结果 1 890例宁波地区育龄期男女弓形虫抗体IgG阳性率为6.83%(129/1890),阳性与饲养宠物、爱吃生食物、砧板生熟不分、从事养殖供应肉类职业有关,差异有统计学意义(χ2值分别为106.28、85.67、84.62、7.59,P0.05)。弓形虫抗体阳性与发生不孕不育间差异有统计学意义(χ2=12.99,P0.05),与配偶抗体阳性间差异有统计学意义(χ2=5.75,P0.05)。结论感染弓形虫可能导致不孕不育,育龄期男女加强健康教育及孕前开展弓形虫抗体检测有十分重要的意义。     

完整弓形虫P35重组蛋白IgM-酶联免疫吸附测定法检测弓形虫急性感染

为利用重组的完整弓形虫表面抗原P35 GST蛋白对弓形虫感染进行血清学诊断 ,构建了可表达P35 GST的JM10 9细胞株 .采用亲和层析对融合蛋白进行分离和纯化 ,用SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)和蛋白质印迹 (Westernblot)分析所表达的蛋白质 ,并用纯化的重组蛋白进行IgM 酶联免疫吸附测定法 (IgM ELISA)检测不同病人血清中的抗 P35抗体 .SDS PAGE分析发现P35 GST重组蛋白的大小约 6 0ku ,为一亲水性蛋白 ,蛋白质印迹分析表明该蛋白与弓形虫阳性感染病人的血清有特异性反应 .利用P35 GST为抗原 ,对 6 0例血清进行IgM ELISA分析 ,发现P35 GST可明显区分近期感染和既往感染 ,在弓形虫的诊断上有很大的应用前景 .     

Effect of Eimeria falciformis infection on the development of toxoplasmosis in mice

White mice previously infected with 10(2), 10(3) or 10(4) Eimeria falciformis oocysts on days 0, 5, 10 or 30 were inoculated per os with 10(1), 10(2), 10(3) or 10(4) Toxoplasma oocysts. While the results obtained for mice with higher Toxoplasma inocula were consistent, animals with 10(1) and 10(2) oocysts previous inoculation with Eimeria showed important differences related with those infected only with Toxoplasma. For example, survival time was higher in animals infected with both parasites, especially if inoculated with Eimeria 30 days before Toxoplasma infection. Furthermore the number of T. gondii cysts found in the animals previously infected with Eimeria was lower compared with mice inoculated with Toxoplasma only. Body weight of mice infected with Toxoplasma previous infection with Eimeria was almost normal in relation to those infected only with Toxoplasma, indicating a probable pathological effect due to the parasite, more evident in "non immunized" mice.     

Real‐time imaging of Toxoplasma‐infected human monocytes under fluidic shear stress reveals rapid translocation of intracellular parasites across endothelial barriers

Peripheral blood monocytes are actively infected by Toxoplasma gondii and can function as ‘Trojan horses’ for parasite spread in the bloodstream. Using dynamic live‐cell imaging, we visualized the transendothelial migration (TEM) of T. gondii‐infected primary human monocytes during the initial minutes following contact with human endothelium. On average, infected and uninfected monocytes required only 9.8 and 4.1 min, respectively, to complete TEM. Infection increased monocyte crawling distances and velocities on endothelium, but overall TEM frequencies were comparable between infected and uninfected cells. In the vasculature, monocytes adhere to endothelium under the conditions of shear stress found in rapidly flowing blood. Remarkably, the addition of fluidic shear stress increased the TEM frequency of infected monocytes 4.5‐fold compared to static conditions (to 45.2% from 10.3%). Infection led to a modest increase in expression of the high‐affinityconformation of the monocyte integrin Mac‐1 (CD11b/CD18), and Mac‐1 accumulated near endothelial junctions during TEM. Blocking Mac‐1 inhibited the crawling and TEM of infected monocytes to a greater degree than uninfected monocytes, and blocking the Mac‐1 ligand, ICAM‐1, dramatically reduced crawling and TEM for both populations. These findings contribute to a greater understanding of parasite dissemination from the vasculature into tissues.     

Signaling during the invasion of host cells by Toxoplasma gondii

Invasion of host cells is essential for the pathogenicity of Toxoplasma gondii. This review examines the signal transduction pathways that lead to the internalization of T. gondii. We demonstrate that extra- and intracellular Ca(2+) mobilization, Ca(2+)-calmodulin complex and phospholipase A(2) activities are required for T. gondii entry. T. gondii also causes the activation of mitogen-activated protein kinase in infected cells and modifies its ionic environment during its intracellular state. Thus, many of the signaling systems found in other eukaryotes are operative in Toxoplasma invasion.     

Critical analysis of the topology and rooting of the parabasalian 16S rRNA tree

Between 20% and 60% of the population of most countries are infected with the protozoan Toxoplasma gondii . Subjects with clinically asymptomatic life-long latent toxoplasmosis differ from those who are Toxoplasma free in several behavioral parameters. Case–control studies cannot decide whether these differences already existed before infection or whether they were induced by the presence of Toxoplasma in the brain of infected hosts. Here, we searched for such morphological differences between Toxoxoplasma -infected and Toxoplasma -free subjects that could be induced by the parasite (body weight, body height, body mass index, waist–hip ratio), or could rather correlate with their natural resistance to parasitic infection (fluctuating asymmetry, 2D : 4D ratio). We found Toxoplasma -infected men to be taller and Toxoplasma -infected men and women to have lower 2D : 4D ratios previously reported to be associated with higher prenatal testosterone levels. The 2D : 4D ratio negatively correlated with the level of specific anti- Toxoplasma antibodies in Toxoplasma -free subjects. These results suggest that some of the observed differences between infected and noninfected subjects may have existed before infection and could be caused by the lower natural resistance to Toxoplasma infection in subjects with higher prenatal testosterone levels.     

Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

Importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis in mice

The importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis was studied in mice chronically infected with Toxoplasma gondii by using a mAb to this lymphokine. Control mice chronically infected with the ME49 strain that received saline or normal IgG had slight inflammation in their brains whereas those that received the mAb developed severe encephalitis. In contrast to control mice, the mAb-treated mice had many areas of acute focal inflammation and infiltration of large numbers of inflammatory cells in the meninges and parenchyma of their brains. In the areas of acute focal inflammation, tachyzoites and Toxoplasma Ag were demonstrated by immunoperoxidase staining with the use of rabbit anti-Toxoplasma antibody, suggesting that the focal inflammation was induced by Toxoplasma organisms. Acute inflammation was also observed around cysts of Toxoplasma. Immunohistologic staining revealed tachyzoites and Toxoplasma Ag surrounding the periphery of these cysts suggesting cyst disruption had occurred. Mice treated with mAb against IFN-gamma had five times the numbers of cysts in their brains as did control mice. These results clearly indicate that endogenous IFN-gamma plays a significant and important role in prevention of encephalitis in mice chronically infected with Toxoplasma. The mAb-treated mice had the same Toxoplasma antibody titers and the same degree of macrophage killing of Toxoplasma as did untreated controls. These results suggest that IFN-gamma may have a direct role in preventing cyst rupture and toxoplasmic encephalitis.     

Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation

European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-κB, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are?protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate?macrophage activation influence the ability of Toxoplasma to establish a chronic infection.     

The immunocytochemical demonstration of Toxoplasma antigen in the brains of congenitally infected mice

The peroxidase anti-peroxidase immunocytochemical staining method was used to identify Toxoplasma antigen in paraffin embedded sections of the brains of 22 mice congenitally infected with the parasite. Intact Toxoplasma tissue cysts were readily demonstrated in the brain in all cases. In 4 of the 22 infected mice there was evidence of rupture of the cyst wall and/or presence of extra-cystic Toxoplasma antigen. Further support for the extra-cystic location of Toxoplasma antigen was obtained by electron microscopy of reprocessed tissue which revealed endozoites in the area immediately surrounding a ruptured cyst. The possible implications of these findings in relation to the pathogenesis of congenital toxoplasmic meningo-encephalitis are discussed.     

Detection of Acute Toxoplasmosis in Pigs Using Loop-Mediated Isothermal Amplification and Quantitative PCR

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.