弓形虫两种保种方法的试验

目的寻求一个简便、适宜的弓形虫保种方法。方法采用不同温度的冷冻保存法和细胞培养保存法对弓形虫保存进行了试验研究,为深入研究弓形虫提供保证。结果弓形虫在液氮(-196℃)、-70℃能长期保存,4℃能保存14 d。通过细胞培养能获得大量的弓形虫滋养体,对培养细胞的选择性不强。结论两种保种方法保存后弓形虫毒力不变。     

北京地区犬猫弓形虫病血清学调查

比较了酶联免疫吸附试验（ELISA）和乳胶凝集试验对犬猫进行龚地弓形虫（Toxoplasma gondii）抗体检测的结果.在66只犬猫的血清中,ELISA方法检测的阳性率为24.0%,乳胶凝集试验阳性率为21.0%,二者的检测结果差异不显著（P＞0.05）.后采用ELISA方法对北京地区家养的159只犬和128只猫进行了弓形虫IGg抗体的检测,共有21只犬（13.21%）和18只猫（14.06%）检测结果为阳性.1岁以下犬（3.70%）、猫（4.35%）低于1至3岁犬（11.29%）和猫（6.98%）的感染率.3～6岁犬猫的感染率分别是27.27%和16.22%,6岁以上犬（30.0%）、猫（32.0%）的感染率最高.不同性别之间感染无明显差异（P＞0.05）.     

洱源、怒江猫源弓形虫虫株的分离与鉴定

为了分离猫源弓形虫,本试验从云南洱源、怒江两地区捕捉18只野猫,取其心脏、肝脏、肺脏、脑组织用盐酸—胃蛋白酶溶液消化处理后,腹腔接种小白鼠,将分离到的弓形虫虫株至少传3代,用特异PCR方法对所分离的虫株进行鉴定。结果表明,从18只野猫的样品中分离出3株弓形虫虫株,用特异性引物对3株虫株进行PCR鉴定,均得到弓形虫的特异性目的条带,测序结果表明所扩增出的DNA片段确为弓形虫核糖体B1基因部分序列。同源性比对分析结果显示分离株与T.gondii B1的同源性为100.0%。将动物组织用盐酸—胃蛋白酶溶液消化处理后腹腔接种小白鼠是一种分离弓形虫虫株较理想的方法,对弓形虫B1基因进行特异性扩增,可以快速地鉴定弓形虫虫株。     

Seroepidemiology of infection with Toxoplasma gondii in waste pickers and waste workers in Durango, Mexico

Municipal waste is a potential source of infection for Toxoplasma gondii as it may contain contaminated meat with parasite tissue cysts and cat excrement with parasite oocysts. Therefore, we sought to determine the prevalence of T. gondii infection and associated characteristics in two populations exposed to municipal solid waste in Durango, Mexico. Ninety waste pickers and 83 waste workers of Durango City, Mexico were examined for T. gondii infection. They were tested for anti-T. gondii IgG and IgM antibodies using enzyme-linked immunoassays. In addition, socio-demographic and behavioural characteristics from each participant were obtained. Nineteen (21.1%) of the 90 waste pickers and seven (8.4%) of the 83 waste workers were positive for anti-T. gondii IgG antibodies. The difference in prevalence among the groups was statistically significant (P =0.03). Waste pickers aged 31-50 years showed a significantly higher prevalence (40.9%) than waste workers of the same age group (2.9%, P < 0.001). Anti-T. gondii IgM antibodies were found in two (2.2%) of the waste pickers but in none of the waste workers. The seroprevalence of T. gondii was significantly higher in workers of the waste transfer station (25.0%) than in drivers or helpers of waste vehicles (2.5%) (P =0.03). Multivariate analysis showed that T. gondii infection was associated with consuming food found in the garbage [adjusted odds ratio (OR) = 4.4; 95% confidence interval (CI) 1.6-11.8] and with lack of education (adjusted OR = 3.2; 95% CI 1.1-8.8). From this study, we conclude: (i) waste pickers may represent a risk group for T. gondii infection; (ii) lack of education might be a contributing factor for T. gondii infection; (iii) the higher the exposure to garbage, the higher the seroprevalence of T. gondii infection; (iv) Eating food products from the garbage may represent an important route for T. gondii infection.     

弓形虫棒状蛋白2研究进展

棒状蛋白2(ROP2)为弓形虫生活史各期均能分泌的抗原,具有高度的保守性和免疫反应性.综述了近年来关于ROP2分子生物学特征、免疫特性、疫苗的研究进展.     

刚地弓形虫亲环蛋白基因TgCyP真核表达质粒的构建及其在Hela细胞内的表达

亲环蛋白（cyclophilin,CyP）是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫（Toxoplasmngondii）速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用。本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHI和EcoRI酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因。PCR产物连接到pMD18-T克隆载体。用限制性内切酶BamHI和EcoRI双酶切克隆载体,将TgCyP目的基因克隆到载体pVAXI,构建真核表达质粒pVAXl-TgCyP。将真核表达质粒转染Hela细胞,利用问接免疫荧光检测其在Hela细胞内的表达情况。结果表明扩增的TgCyP基因与GenBank上相应基因序列（U04633．1）的一致性达100％,构建的真核表达质粒pVAX1-TgCyP能在转染的Hela细胞中表达,其表达产物与刚地弓形虫阳性血清具有免疫反应性。本研究表明Tg-CyP有望作为弓形虫疫苗的候选抗原,将为进一步研究该质粒的动物免疫试验奠定基础。     

弓形虫基因分型研究进展

弓形虫可感染多种动物引起严重的弓形虫病,为了对不同宿主感染的弓形虫株基因型差异及不同基因型弓形虫虫株的致病力差异进行进一步研究,弓形虫基因型的分析多采用限制性片段长度多态性PCR（PCR-RFLP）、随机扩增多态性DNA、多位点PCR-RFLP分析、DNA序列分析和微卫星DNA序列分析等技术,扩增位点多选择SAG1、SAG2、SAG3、BTUB和GRA6等遗传标记。传统的弓形虫基因型主要有3个,随着基因型研究的不断深入,越来越多的基因型被发现。本文就弓形虫基因分型研究进展进行讨论分析。     

弓形虫ROP2基因在大肠杆菌内高效表达条件的优化

采用SDS-PAGE方法检测弓形虫(Toxoplasma gondii)棒状体蛋白(Rhoptry protein)ROP2基因原核表达质粒在大肠杆菌BL21Codon Plus中的表达量。通过改变菌体密度、诱导剂浓度和诱导时间进行高效表达条件的优化。结果显示,在菌体密度D600nm为0·6、IPTG浓度为0·4mmol·L-1和诱导表达3.5h时,融合蛋白的表达量最大,达到159mg·L-1培养液。这为利用重组ROP2研究和制备弓形虫病的诊断抗原奠定基础。     

几种不同免疫程序制备抗弓形虫高免血清的比较

用弓形虫血清抗体阴性的健康家兔,以4种不同的免疫程序皮下接种弓形虫速殖子裂解抗原制备高免血清.研究结果表明,先以完全弗氏佐剂和不完全弗氏佐剂分别进行一免和二免,然后再以抗原进行三免或四免制备高免血清,其抗体效价高达1:4096(IHA),与每次都使用抗原的传统免疫程序制备的抗体效价相当,但所用抗原剂量减半,该法优于另外2种免疫程序.     

Toxoplasma gondii in Switzerland: a serosurvey based on meat juice analysis of slaughtered pigs, wild boar, sheep and cattle

Toxoplasmosis is one of the most important zoonotic diseases worldwide and is caused by the protozoan Toxoplasma gondii. Besides vertical infection during pregnancy, humans can get infected post-natally either by peroral uptake of sporulated Toxoplasma oocysts or by ingestion of tissue cysts upon consumption of raw or undercooked meat. The aim of this study was to approximate the risk of human infection via meat consumption by estimating the seroprevalence of T. gondii in slaughtered animals in Switzerland and to compare data with prevalences assessed 10 years ago. The study included pigs, cattle, sheep and wild boar of different age groups and housing conditions whenever possible and applicable. A P-30-ELISA was used to detect T. gondii-specific antibodies and to determine seroprevalences in meat juice of slaughtered animals. A total of 270 domestic pigs (120 adults, 50 finishing, 100 free-ranging animals), 150 wild boars, 250 sheep (150 adults, 100 lambs) and 406 cattle (47 calves, 129 heifers, 100 bulls, 130 adult cows) were tested. Seropositivity increased with the age of the assessed animals. Independent of the age-group, the overall seroprevalence was lowest in wild boars (6.7%), followed by pigs (23.3%), cattle (45.6%) and sheep (61.6%), respectively. Conventional fattening pigs and free-ranging pigs surprisingly had comparable seroprevalences (14.0% and 13.0%, respectively). Unlike in other European countries, where generally a decrease in the number of seropositive animals had been observed, we found that the prevalence of seropositive animals, when compared with that of 10 years ago, had increased for most species/age groups. Conclusively, the results demonstrated a high seroprevalence of T. gondii in animals slaughtered for meat production and revealed that increasing age of the animals is a more important risk factor than housing conditions in Switzerland.