毕氏肠微孢子虫基因分型及公共卫生学重要性

微孢子虫(Microsporidia)是一类细胞内专性寄生的真核生物,广泛存在于无脊椎动物如蜜蜂、蚕和几乎所有种类的脊椎动物中.在已知并被命名的1 200多个虫种中,共有8个属中的14个虫种可以感染人,其中毕氏肠微孢子虫(E.bieneusi)、兔脑炎微孢子虫(E.cuniculi)、肠脑炎微孢子虫(E.intestinalis)以及海伦脑炎微孢子虫(E.hellem)较为常见[1-3].E.bieneusi是感染人和动物最常见的病原体,能导致免疫妥协或低下者长期的致死性腹泻,也能够导致免疫功能正常者剧烈的自限性腹泻[2-3].     

蓝狐源兔脑炎微孢子虫的分离与PCR鉴定

为了对吉林省九台地区的发病蓝狐进行兔脑炎微孢子虫虫株的分离和进一步鉴定,试验从发病蓝狐采集脑组织病料,进行虫株的分离、培养及染色,同时对分离虫株进行了PCR鉴定和序列分析。结果表明:在脑组织滤液中分离得到微孢子虫,并命名为JL株。革兰氏染色镜检可观察到虫体染色呈阳性,呈明显的串珠样排列;改良马松三色染色进一步观察有明显红色、浅红色着色。PCR检测鉴定到转录间隔1区(ITS1)基因、极管蛋白(PTP)2基因和PTP4基因。分离的微孢子虫JL株的序列与GenBank上登录的兔脑炎微孢子虫的同源性在99%以上,并且ITS1基因5′-GTTT-3′重复4次,分离的虫株为兔脑炎微孢子虫Ⅲ型。说明吉林省九台地区发病蓝狐感染的是兔脑炎微孢子虫Ⅲ型。     

兔脑炎微孢子虫病的免疫学研究进展

兔脑炎微孢子虫病是由兔脑炎微孢子虫引起的一种隐性、慢性感染或临床经过的人畜共患的原虫病。近十余年来 ,出现大量有关兔脑炎微孢子虫引起艾滋病患者致死性临床感染的报道 ,此病日益引起医学界的广泛关注和重视。就兔脑炎微孢子虫病的免疫学检测方法、细胞免疫应答、感染兔脑炎微孢子虫后人及动物的免疫学比较的研究状况进行了综述。     

微孢子虫类的分子生物学研究简报

<正> 1 家蚕微孢子虫基因组DNA的PCR检测 1.1 在严格设置对照实验的情况下,设计和合成的一对引物NP1和NP2可将家蚕微孢子虫中国广东株、中国浙江株及中国四川株基因组DNA扩增出一条约317bp的特异性DNA带。供试的其余7种Nosema属微孢子虫以及一种Vairimorpha属的纳卡变形微孢子虫和一种Pleistophora属的金鱼具褶微孢子虫基因组DNA均无此单一的特异扩增带。建立的PCR方法检测家蚕微孢子     

隐孢子虫保护性单克隆抗体的制备

应用从鼠粪便中分离的小球隐孢子虫(Cryptosporidium parvum)卵囊,经阿拉伯胶梯度离心后,超声粉碎制成可溶性抗原.按常规方法免疫BALB/c小鼠,经融合、克隆筛选建立了5个单克隆抗体株(1A_8、2B_2、3C_4、1A_3和2C_3).通过免疫荧光和免疫组化检测,发现1A_8、2B_2和3C_4克隆株针对子孢子,而lA_3和2C_3针对卵囊壁.1A_8、2B_2和3C_4株单抗腹水分别以10%、20%和30%的量与小球隐孢子虫子孢子(约4×10~4个)共同孵育1h后,接种到人胎肺细胞上培养,结果观察到30_8株腹水、10+_2株腹水和20<_4株腹水对子孢子有明显细胞毒作用.进而观察了2B_2株单抗腹水对培养虫体的反应性,发现其对裂殖体期虫体亦有作用;进行了2B_2株腹水对BALB/c 小鼠的被动保护性试验,发现以1mL/只剂量分别于攻虫前后1d接种,其对5×10~5个卵囊攻击的BALB/c鼠有明显的保护作用.Western印迹结果表明,2B_2株单抗是针对小球隐孢子虫抗原的28000蛋白质分子.     

东方蜜蜂微孢子虫孢子冷冻保存后对意蜂工蜂的侵染性

东方蜜蜂微孢子虫是成年蜜蜂的重要传染性病原真菌。试验对10%的甘油、乙二醇、二甲亚砜及水在-80℃低温下冷冻保存东方蜜蜂微孢子虫孢子5个月后,孢子对意蜂工蜂的侵染性进行了考查。每只初出房工蜂接种3×104个孢子,结果发现,接种后15天,各组的孢子量无显著差异(P>0.05),产孢量为3.05⁴.13×107个/只。10%甘油和10%二甲亚砜2种保存孢子感染工蜂的22 d累计死亡率分别高达74%和68%,而水保存的累计死亡率仅12.5%。所以得出结论,10%甘油和10%二甲亚砜是比水更好的孢子冷冻保护剂。     

微孢子虫的侵染机制

蜂微孢子虫病是蜜蜂的检疫性病害,该病严重阻碍了蜂群的生产繁殖.微孢子虫孢子侵入寄主细胞有3种方式:发芽侵染、自发感染、细胞内吞感染,又以发芽侵染为主.微孢子虫孢子表面蛋白与孢子发芽密切有关,找到孢子表面发芽关键蛋白将有助于揭示微孢子虫的侵染机制.     

两种微孢子虫孢子表面蛋白及对家蚕侵染性的比较研究

对来自家蚕的内网虫属样微孢子虫和家蚕微孢子虫的形态、表面蛋白及侵染性进行了比较研究。内网虫属样微孢子虫孢子显著小于家蚕微孢子虫 ,孢囊中孢子数为 8～ 32个 ,只侵染中肠组织 ,对家蚕无胚胎传染性。SDS UreaPAGE和 2D PAGE图谱显示两种孢子表面蛋白有明显差异。在SDS UreaPAGE中 ,内网虫属样微孢子虫的主要表面蛋白为 2 2kD ,而家蚕微孢子虫为 4 5kD。 2D PAGE显示 ,当上样量为 12 0 μg时 ,内网虫属样微孢子虫孢子表面蛋白斑点有 330多个 ,而家蚕微孢子虫孢子只有 16 0多个 ,其中仅有 10多个相同或疑似蛋白点。家蚕微孢子虫的主要表面蛋白点约有 2 0个 ,以中性偏酸的小分子居多 (<18kD ,pI 5 .4～ 7.2 ) ;内网虫属样微孢子虫有 30多个 ,分布较分散。根据两种孢子都能感染家蚕的特点 ,认为相同或疑似蛋白与是否能够感染有关 ,而大量差异蛋白与对家蚕的感染程度有关。两孢子表面蛋白中的主要蛋白均为差异蛋白 ,可能是孢子表面的结构蛋白。     

家蚕微孢子虫研究

<正>微孢子虫是在细胞内专性寄生的、原始的、形成孢子的、缺乏线粒体的真核生物,可广泛寄生于动物.近年来,也有爱滋病患者被微孢子虫感染的报道.自然界,微孢子虫是控制昆虫种群数量的一个重要因子,人们希望通过用微孢子替代化学农药进行害虫的生物防治;在蚕业生产中,由于微孢子虫寄生所引起的疾病,有部分能够胚种传染,给蚕种生产带来了极大的威胁.本文想就感染家蚕的微孢子虫的种类、增殖、鉴别、分子生物学以及该病的药物治疗等方面的研究概况作一综合.     

家蚕微孢子虫原位杂交诊断技术研究

根据家蚕微孢子虫rRNA基因高度保守的特点,设计合成了1对PCR引物,从家蚕微孢子虫基因组DNA上扩增出1个12kb片段,用同位素标记后作为检测家蚕微孢子虫的特异性探针。采用原位杂交的方法,对家蚕卵及感染家蚕微孢子虫4、6、10d的幼虫进行原位杂交检测。实验结果表明,该方法可以从家蚕单粒卵内检测到296粒孢子,对感染家蚕微孢子虫4、6、10d的幼虫均有阳性杂交信号出现。     

Encephalitozoon cuniculi infection in rabbits

The microsporidian parasite Encephalitozoon cuniculi commonly infects rabbits. Most infections are initially asymptomatic, but for reasons yet to be explained, many rabbits subsequently develop disease as a result of infection with this organism. Three common forms of this disease are recognized, and they can occur individually or in combination. The ocular form is associated with cataracts and when there is extensive damage to the lens, uveitis. The neurological form can vary from a mild change in the rabbit’s behavior to severe vestibular disease. The signs associated with the renal form of the disease are those of chronic progressive renal disease. Definitive diagnosis of encephalitozoonosis in the rabbit is difficult. Animals with encephalitozoonosis are expected to be seropositive, but many apparently healthy rabbits are also seropositive, so this assay is not specific and its results must be considered in the light of other diagnostic findings. The absence of antibody, however, should cause the practitioner to consider other differentials. Drugs proven to be efficacious for E. cuniculi infections include albendazole and fenbendazole. Supportive care and treatment with antiinflammatory medications may also be necessary in some forms of encephalitozoonosis. Lens removal or removal of the lens contents is indicated in some rabbits with E. cuniculi-induced ocular disease.     

Encephalitozoon cuniculi in pet rabbits

The results of a serological test for Encephalitozoon cuniculi in 125 pet rabbits are reviewed, together with follow-up studies of clinical cases. Blood samples were taken from 38 asymptomatic rabbits and 87 rabbits showing neurological, renal or ocular signs suggestive of encephalitozoonosis. In the asymptomatic group, six of 26 (23 per cent) apparently healthy rabbits, sampled as part of a health screen, were seropositive; of the remaining 12 asymptomatic rabbits, sampled because they lived with seropositive companions, eight (66 per cent) were seropositive. Fifty-eight of the rabbits with clinical disease showed neurological signs, including head tilt, seizures, ataxia and swaying; three of them also showed renal signs and two showed ocular signs, and these five rabbits were all seropositive. Head tilt was the most common neurological sign in 21 of 23 (91 per cent) of the seropositive cases. All nine rabbits with ocular lesions were seropositive. In follow-up studies of clinical cases, the rabbits showed variable responses to treatment with albendazole, fenbendazole, antibiotics or corticosteroids, and some cases recovered without treatment.     

兔脑炎原虫的超微形态与发育

用电镜连续７代动态地观察了培养细胞中兔脑炎原虫的超微结构与发育状态。脑炎原虫含有细胞核，但无线粒体、内质网和高尔基复合体等细胞器，其表面有几根极丝。脑炎原虫在培养细胞中除二分裂增殖外，还有裂体增殖和配体增殖。由于脑炎原虫有３种增殖方式，故在光学显微镜下呈多形态结构。     

兔脑炎原虫的分离与鉴定

3只临床表现斜颈、麻痹和打滚的病兔,经病理组织学检查诊断为脑炎原虫病.取其脑组织制成乳剂,接种于兔肾细胞、Vero细胞、猫肾细胞和兔脉络丛细胞.用前3种细胞均分离出原虫.该虫体表现为多种形态,呈杆状、圆形或卵圆形;Gram染色呈阳性,Goodpasture氏染色呈红色.用20%H_2O_2处理虫体,在相差显微镜下见到虫体极丝的伸展.通过虫体的形态学、染色特征和极丝的突出实验,鉴定该虫体为脑炎原虫.兔肾细胞、Vero细胞和猫肾细胞的感染率分别为40%、40%和5%,而在兔脉络丛细胞原代培养物中未检查到虫体.     

Immune response to Encephalitozoon infection review

The microsporidia are emerging agents of infectious disease in both immunocompromised and immunocompetent mammals. Recently, there has been an increased interest in studying the immunobiology of microsporidiosis. This paper discusses the humoral and cell-mediated immune responses to Encephalitozoon spp. The T-cell-mediated responses appear to be most important in conferring resistance. This has become evident by the lethal effects of microsporidiosis in T-cell-deficient hosts. However, much still needs to be learned about the immunobiology of microsporidiosis regarding the specific T-cell responses and the cytokines that provide protective immunity and facilitate the macrophage-mediated killing of microsporidia. Such information will become important in developing immunotherapeutic strategies to control microsporidiosis in the future.     

Direct agglutination test for Encephalitozoon cuniculi

Encephalitozoon cuniculi is a small protozoan parasite in the phylum Microspora. It has been shown to naturally infect several host species, including humans. Infection with microsporidia is usually asymptomatic, except in young or immunocompromised hosts. Currently, serological diagnosis of infection is made using the indirect immunofluorescent antibody assay (IFA) or enzyme-linked immunosorbent assay (ELISA). Although these methods are sensitive and reliable, there are several drawbacks to the IFA and ELISA tests. Cross-reactivity between other Encephalitozoon species is common, and specialized equipment is required to conduct these tests. This paper reports the development of a direct agglutination test for detecting IgG antibodies to E. cuniculi. The utility of the agglutination test was examined in CD-1 and C3H/He mice infected with E. cuniculi or one of 2 other Encephalitozoon species. Test sera were incubated overnight with eosin-stained microsporidia spores in round-bottom microtiter plates. In positive samples, agglutination of spores with antibodies in test sera resulted in an opaque mat spread across the well. The results indicate that the agglutination test is 86% sensitive and 98% specific for E. cuniculi, with limited cross-reactivity to Encephalitozoon intestinalis. No cross-reactivity to Encephalitozoon hellem was observed. The test is fast and easy to conduct, and species-specific antibodies are not required.     

獭兔脑原虫病的诊断

对发生脑原虫病的獭兔进行尸体剖检和组织病理学检查，尸体剖检发现肾脏有2-4mm灰白色凹陷区，组织病理学检查可见典型的肉芽肿性脑炎及间质性肾炎的变化，这是该病特征性的病理形态学的变化，并在大脑胶质结节内和肾脏髓质见到虫体的剖面。