弓形虫病治疗药物的研究进展

弓形虫病是一种呈全球分布的重要的人兽共患寄生虫病,能感染包括人在内的所有温血动物。目前对该病的防控主要依赖药物。临床上联合使用磺胺嘧啶与乙胺嘧啶是目前治疗弓形虫病的黄金标准,但该疗法具有一定的毒副作用,且长期使用易产生耐药性。因此,针对弓形虫病的药物研究一直是研究热点。螺旋霉素、甲氧苄啶－磺胺甲噁唑等常作为替代药物用于弓形虫病的治疗,但均具有不同的局限性。因而,人们将研究重点转移到天然产物上。研究以植物提取物为主,通过筛选具有抗弓形虫的活性分子,并对其进行适当的化学修饰,可增强抗虫活性,降低毒副作用。在动物提取物中,某些动物的毒液具有抗弓形虫活性,可作为候选药物。随着研究手段的提升,一些原有药物的新活性不断被发现。在"旧"药新用研究方向,一方面以亲缘关系为标准,针对其他抗寄生虫药物进行抗弓形虫活性探索;另一方面以药物作用靶点为标准,对治疗其他疾病的药物进行抗弓形虫活性研究。文章对弓形虫病临床用药史、天然产物的开发及"旧"药新用这3个方面进行综述,以期为抗弓形虫病药物的研究提供新的思路。     

抗弓形虫药物的类型和作用机制

弓形虫病是一种危害严重的人畜共患病,人和动物的弓形虫感染呈全球性分布,其感染人可导致孕妇出现流产、死胎、畸胎和弱智儿,能引起免疫功能缺陷或低下病人死亡。且给畜牧业发展造成巨大的经济损失。传统抗弓形虫药物几乎都存在着不能完全杀死虫体及包囊,停药后的复发率较高以及对免疫功能缺陷患者基本无治疗效果等诸多不足。因此,研制和开发一种高效,低毒专用于动物的抗弓形虫药物迫在眉睫。笔者就不同类别抗弓形虫药物的作用机制及效果进行阐述,并对其发展前景进行展望,为新型抗弓形虫药物研制提供参考。     

抗弓形虫药物的作用机制和效果的研究进展

弓形虫病是一种危害严重的人畜共患病,人和动物的弓形虫感染呈全球性分布,其感染人可导致孕妇出现流产、死胎、畸胎和弱智儿,能引起免疫功能缺陷或低下病人死亡。且给畜牧业发展造成巨大的经济损失。传统抗弓形虫药物几乎都存在着不能完全杀死虫体及包囊,停药后的复发率较高以及对免疫功能缺陷患者基本无治疗效果等诸多不足。因此,研制和开发一种高效,低毒专用于动物的抗弓形虫药物迫在眉睫。笔者就不同类别抗弓形虫药物的作用机制及效果进行阐述,并对其发展前景进行展望,为新型抗弓形虫药物研制提供参考。     

7种药物体内外抗弓形虫效果分析

为了初步了解甘草酸、苦参碱、黄芩苷、硝唑尼特、巴龙霉素、磺胺嘧啶钠和磺胺氯吡嗪钠7种药物抗弓形虫的效果,进行体外、体内抗弓形虫试验。采用MTT法首先测定试验药物对培养巨噬细胞(Ana-1)的安全浓度,然后用弓形虫RH株的速殖子感染培养的上述细胞,加入药物继续培养48 h后观察药物体外抑制虫体在细胞内的增殖,以相对弓形虫抑制率为指标判断体外抗弓形虫效果。用弓形虫RH株速殖子按5×105/只腹腔接种昆明种小鼠,接种后4 h,药物治疗组通过饮水中给药,观察比较给药后感染弓形虫小鼠的精神状态,并记录各组小鼠死亡的时间和数目,每组随机抽选2只小鼠镜检腹腔虫体数量。体外试验结果表明,甘草酸、黄芩苷、磺胺嘧啶钠和磺胺氯吡嗪钠4种药物可显著抑制细胞内弓形虫增殖;体内试验表明,磺胺氯吡嗪钠治疗效果最佳,磺胺嘧啶钠次之,黄芩、苷草再次之,其余药物抗弓形虫作用不明显。     

抗弓形虫和新孢子虫药物体外筛选方法的研究进展

弓形虫和新孢子虫是2种在形态结构及生物学特征方面非常相似的细胞内寄生性原虫,二者引起的疾病对人和动物健康造成了严重危害。开发和筛选高效、低毒的抗弓形虫和新孢子虫药物一直是抗原虫药物研究领域的重要方向之一。药物筛选包括体外筛选和体内筛选,体外筛选的结果是进行体内筛选的依据和基础。对抗弓形虫和新孢子虫药物的体外筛选方法研究进展进行综述,以期为开发更加高效的抗弓形虫和新孢子虫药物体外筛选技术提供参考。     

弓形虫病的治疗研究进展

弓形虫病是一种世界性的人兽共患寄生虫病,不仅给畜牧业生产带来了严重危害,也严重影响人类健康。弓形虫病分为先天性弓形虫病和获得性弓形虫病,其中眼弓形虫病和脑弓形虫病是最常见的临床表现。乙胺嘧啶和磺胺嘧啶的联合用药是目前治疗弓形虫病的黄金标准。免疫力强的弓形虫病患者一般无需治疗,但对免疫功能不全或低下的患者可能会造成致命后果,需要长期监测和治疗。本文从先天性与获得性弓形虫病、弓形虫病临床用药、临床常见治疗方法以及新型抗弓形虫药物的研究进行综述。     

弓形虫病治疗：从传统药物到纳米药物

刚地弓形虫(Toxoplasma gondii)是一种呈世界性分布的机会致病原虫，几乎感染所有的温血动物，引起人兽共患弓形虫病。免疫力正常的个体感染常呈隐性感染，无明显的临床症状；但免疫功能缺陷的人或动物感染则会导致严重后果，需进行有效治疗。目前，弓形虫病已是全世界绵羊和山羊流产的常见原因，该病给人类健康和畜牧业生产都造成了严重影响。因此，控制人和动物弓形虫病对公共卫生和畜牧业经济发展极为重要。以往用于弓形虫病治疗的药物，由于其副作用和/或疾病分期的特点，治疗效果大大受限，然而纳米药物可有效克服传统药物的局限性。本文就纳米技术在弓形虫病治疗方面的新进展作一综述，以期为抗弓形虫病药物的研究提供新思路。     

四种中药体外抗弓形虫效果的比较

目的:通过黄芩、甘草、马齿苋、桦褐孔菌四种中药体外抗弓形虫效果的比较,探讨其体外抗弓形虫的效果。方法用弓形虫RH株的速殖子感染培养的非洲绿猴肾细胞(VERO),加受试药物,以蒿甲醚为阳性药物对照,在加药后的12h、24h、48h观察细胞和弓形虫的生长情况及形态。加药48h后,分离出虫体,台酚蓝染色,计数,判断药物作用效果。结果:黄芩、甘草体外可明显抑制弓形虫的生长和繁殖,作用效果与药物浓度呈正相关;而马齿苋及桦褐孔菌体外抑制弓形虫效果较弱。     

弓形虫致密颗粒蛋白的生物学功能研究进展

刚地弓形虫（Toxoplasma gondii）是一种重要的人兽共患寄生原虫,具有中间宿主广泛、生活史复杂、传播方式多样、呈全球性分布等特点,可感染几乎所有的恒温动物,导致人兽共患的弓形虫病。弓形虫慢性感染约1/3的世界人口,对免疫缺陷患者、孕妇、孕畜的健康更是造成严重威胁。致密颗粒蛋白是由致密颗粒（弓形虫的一种亚细胞分泌器官）分泌的蛋白质,它们可通过操控宿主（细胞）基因表达、信号通路进而调控宿主细胞的免疫反应和细胞周期,并在蛋白质的转运和定位、营养物质的摄取、纳米管网络（IVN）的形成与稳定性的维持、逸出、慢性感染等生理过程中发挥重要作用。本文综述弓形虫致密颗粒蛋白基本生物学功能的最新研究进展,旨在为深入研究弓形虫的致病机制、鉴定新的抗弓形虫潜在药物靶点和疫苗候选分子提供借鉴。     

猪弓形虫病的中西药结合疗效分析

目的:研究分析中西药结合治疗猪弓形虫病的疗效情况。方法:应用复方磺胺类药物与自拟抗弓形虫中药方剂相结合治疗感染弓形虫病的猪,同时检测猪腹水内的含虫量。然后对小白鼠接种感染猪病料,予以动物回归试验。结果:中西药结合治疗感染弓形虫病的猪达到了100%灭活弓形虫滋养体的作用;回归试验结果显示,一只小白鼠的脑组织内含有少量弓形虫。结论:中西药结合治疗猪弓形虫病获得了比较满意的疗效。     

A new type of pterocarpanquinone that affects Toxoplasma gondii tachyzoites in vitro

Toxoplasma gondii, the agent of Toxoplasmosis, is an obligate intracellular protozoan able to infect a wide range of vertebrate cells, including nonprofessional and professional phagocytes. Therefore, drugs must have intracellular activities in order to control this parasite. The most common therapy for Toxoplasmosis is the combination of sulfadiazine and pyrimethamine. This treatment is associated with adverse reactions, thus, the development of new drugs is necessary. In previous studies, naphthoquinone derivatives showed anti-cancer activity functioning as agents capable of acting on groups of DNA, preventing cancer cells duplication. These derivatives also display anti-parasitic activity against Plasmodium falciparum and Leishmania amazonensis. The derivative pterocarpanquinone tested in this work resulted from the molecular hybridization between pterocarpans and naphtoquinone that presents anti-tumoral and anti-parasitic activities of lapachol. The aim of this work was to determine if this derivative is able to change T. gondii growth within LLC-MK2 cells. The drug did not arrest host cell growth, but was able to decrease the infection index of T. gondii with an IC(50) of 2.5 μM. Scanning and transmission electron microscopy analysis showed morphological changes of parasites including membrane damage. The parasite that survived tended to encyst as seen by Dolichos biflorus lectin staining and Bag-1 expression. These results suggest that pterocarpanquinones are drugs potentially important for the killing and encystment of T. gondii.     

Enrofloxacin is able to control Toxoplasma gondii infection in both in vitro and in vivo experimental models

Currently, toxoplasmosis is treated with sulfadiazine and pyrimethamine. However, this treatment presents several adverse side effects; thus, there is a critical need for the development and evaluation of new drugs, which do not present the same problems of the standard therapy. Enrofloxacin is a fluoroquinolone antibiotic known to control infection against several bacteria in veterinary medicine. Recently, this drug has demonstrated protective effects against protozoan parasites such as Neospora caninum. The present study aimed to determine the effect of enrofloxacin in the control of Toxoplasma gondii infection. For this purpose, human foreskin fibroblast (HFF) cells were infected with T. gondii RH strain and treated with sulfadiazine, penicillin/streptomycin, pyrimethamine, or enrofloxacin. Following treatment, we analyzed the infection index, parasite intracellular proliferation and the number of plaques. Additionally, tissue parasitism and histological changes were investigated in the brain of Calomys callosus that were infected with T. gondii (ME49 strain) and treated with either sulfadiazine or enrofloxacin. Enrofloxacin was able to reduce the infection index, intracellular proliferation and the number of plaques in HFF cells infected by T. gondii in comparison with untreated or penicillin/streptomycin-treated ones. Enrofloxacin was more protective against T. gondii in HFF infected cells than sulfadiazine treatment (P<0.001). In addition, pyrimethamine, enrofloxacin or the associations of sulfadiazine plus pyrimethamine, enrofloxacin plus sulfadiazine or enrofloxacin plus pyrimethamine-treatments were able to reduce the plaque numbers in HFF cells infected by T. gondii when compared to medium, penicillin/streptomycin or sulfadiazine alone. In vivo experiments demonstrated that enrofloxacin diminished significantly the tissue parasitism as well as the inflammatory alterations in the brain of C. callosus infected with T. gondii when compared with untreated animals. Based on our findings, it can be concluded that enrofloxacin is a potential alternative drug for the treatment of toxoplasmosis.     

Serologic prevalence of Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses in Brazil.

OBJECTIVE: To determine serologic prevalence of Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses in Brazil. DESIGN: Prevalence survey. ANIMALS: 101 Thoroughbreds in Brazil. PROCEDURE: Blood samples were obtained from horses and tested for serum antibodies against S neurona by use of an immunoblot procedure with culture-derived S neurona merozoites as antigen, and for serum antibodies against T gondii and N caninum by use of a modified agglutination test with formalin-preserved tachyzoites and mercaptoethanol. RESULTS: Antibodies against S neurona and T gondii were detected in 36 and 16 of 101 horses, respectively. Cross-reactivity between antibodies against T gondii and S neurona was not detected. Antibodies against N caninum were not detected in any samples. CONCLUSIONS AND CLINICAL RELEVANCE: The high prevalence of antibodies against S neurona detected in clinically normal horses emphasizes the importance of examining CSF for antibodies when establishing a diagnosis of equine protozoal myeloencephalitis.     

磺胺氯吡嗪处理弓形虫速殖子抑制消减文库的构建和初步分析

为了研究和探索磺胺氯吡嗪抗弓形虫的作用机理,构建磺胺氯吡嗪处理弓形虫的抑制性消减文库.将刚地弓形虫(Toxoplasma gondii)RH株速殖子腹腔注射(10000个/只鼠)感染昆明系小鼠,大量收集并纯化速殖子.用250 mg/mL磺胺氯吡嗪PBS溶液和PBS分别浸泡速殖子,37℃恒温箱孵育2h,分别提取虫体的总RNA和mRNA,以PBS处理的正常虫体cDNA为驱动组,以药物处理虫体cDNA为实验组,构建药物处理前后弓形虫速殖子的抑制性消减文库,对部分阳性克隆进行测序和序列分析.结果显示,提取的虫体总RNA纯度高,合成的双链cDNA经RsaⅠ酶切反应较完全;从消减文库中随机选择100个阳性克隆进行PCR扩增,其插入片段长度在200～750 bp之间,文库的重组率达93％以上;随机挑取30个阳性克隆进行测序,获得11个单一序列,Blast分析显示有7个单一有效EST序列与已知蛋白质相似性较高.结果表明,已成功构建磺胺氯吡嗪处理弓形虫速殖子的抑制性消减cDNA文库,并对部分阳性克隆进行了初步分析,为深入研究磺胺氯吡嗪的抗弓形虫机制和寻找药物作用的关键靶分子奠定了基础.     

Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-γ, IL-2, IL-4, and IL-10, as well as the percentage of CD4(+) cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice.     

喜马拉雅旱獭弓形虫病血清学检测

用弓形虫的重组蛋白GST-SAG1作为ELISA诊断抗原,采用ELISA检测方法,对青海省祁连县喜马拉雅旱獭血清进行了弓形虫病的血清学检测。结果表明,被检92份喜马拉雅旱獭共检出阳性血清25份,阳性率约为27.17%。结果提示,青海省祁连县的喜马拉雅旱獭中存在弓形虫病的感染。     

几种药物抗小鼠弓形虫感染的初步效果

为寻找有效的抗弓形虫药物,选择了临床上使用较多的6种药物进行抗小鼠弓形虫感染初步实验。用刚地弓形虫（Toxoplasma gondii）长宁株（CN株）速殖子,按1×104/鼠腹腔接种昆明系雄性小白鼠,接种后4 h用药组通过灌服或饮水用药,每鼠剂量分别为阿奇霉素（Azithromycin）150、120、90 mg/（kg·d）,泰妙菌素（Tiamulin）100 mg/（kg·d）,克拉霉素（Clarithromycin）15 mg/（kg·d）,磺胺氯吡嗪钠（Sulfachloropyrazine sodium）150、120、90 mg/（kg·d）,甲硝唑（Metronidazole）150 mg/（kg·d）,甲氧苄胺嘧啶（Trimethoprim）10 mg/（kg·d）,连续用药5~6 d,接种后10 d结束实验。结果阿奇霉素、磺胺氯吡嗪钠组的平均存活天数明显高于感染不用药组（P〈0.05）,泰妙菌素、克拉霉素、甲硝唑、甲氧苄胺嘧啶组在所用剂量下的存活天数与感染不用药组无明显差异（P〉0.05）;用药后4－7 d,取腹腔液镜检速殖子,与感染不用药组相比,阿奇霉素组减少70%以上,磺胺氯吡嗪钠组减少99%以上。研究结果提示,磺胺氯吡嗪钠具有良好的抗弓形虫效果,其推荐剂量与用药疗程还有待进一步研究。     

Toxoplasmosis in sheep III. Further evaluation of the ability of a live Toxoplasma gondii vaccine to prevent lamb losses and reduce congenital infection following experimental oral challenge

The aim of this study was to compare the ability of a live incomplete strain (Strain 48) and a live complete strain (Strain 89) of Toxoplasma gondii to protect against abortion and congenital infection following an oral challenge of T. gondii oocysts. Sixty-nine two-tooth ewes were immunised pre-tupping with live Strain 48 of T. gondii tachyzoites and seventy ewes were immunised with Strain 89. Eighty-two serologically negative ewes served as controls. At mid-pregnancy half of the ewes were challenged orally with T. gondii oocysts (2x10(5)/ewe). The ewes vaccinated with Strain 48 were significantly (p<0.05) protected against the effects of experimental challenge and the rate of congenital infection was also significantly (p<0.15) reduced. The ewes vaccinated with Strain 89 were also significantly (p<0.05) protected. The serological response to challenge as measured by both the Dye test and the Indirect Haemagglutination test varied considerably between the two vaccinated groups.     

Prevalence of antibodies against Toxoplasma gondii and Neospora caninum in Hungarian red foxes (Vulpes vulpes)

In the present study we have investigated the seroprevalence to the protozoan parasites Toxoplasma gondii and Neospora caninum in 337 red foxes (Vulpes vulpes) from 16 out of 19 counties in Hungary. The foxes were originally collected within a National vaccination program against rabies. Antibodies to T. gondii were detected in as many as 228 (68%) of the foxes using a commercial direct agglutination test (DAT). In an indirect iscom ELISA, five foxes (1.5%) were positive for antibodies against N. caninum. The high prevalence of foxes positive for T. gondii might be explained by the widespread occurrence of the parasite in the diet of foxes. As a contrast, latent infections of N. caninum among red foxes in Hungary are much less common.     

内蒙古部分地区放牧牛羊弓形虫血清学调查

[目的]了解内蒙古部分地区放牧牛羊弓形虫病感染情况。[方法]采用间接血凝试验(IHA)对阿拉善盟及呼伦贝尔市随机采集的286份放牧牛羊血清样本进行弓形虫抗体检测。[结果]绵羊血清弓形虫抗体总阳性率为1.68%,其中,阿拉善盟阳性率为4.84%,呼伦贝尔市阳性率为0.56%;经统计学分析,两个地区的绵羊血清弓形虫抗体阳性率差异不显著(P>0.05);牛血清弓形虫抗体总阳性率为0。[结论]内蒙古主要牧区放牧绵羊存在弓形虫感染,应引起足够的重视,并制定相应的弓形虫病防治措施。