桧烯抗弓形虫活性的体外评价

本研究旨在筛选具有抗弓形虫活性的萜类化合物,并进行体外活性评价。对26个萜类化合物进行体外抗弓形虫增殖作用的筛选,采用CCK-8法确定活性化合物对Vero细胞的毒性和安全浓度,Giemsa染色进一步确定对弓形虫的活性,通过细胞免疫荧光、Giemsa染色和生物化学发光法评价活性化合物对弓形虫的体外活性。结果表明,26个萜类化合物中,桧烯对弓形虫有显著的活性,桧烯对RH-2F的IC₅₀为90.76 μg·mL^-1,Giemsa染色和细胞免疫荧光的结果表明,桧烯对细胞内的两种弓形虫虫株都有显著的抗增殖作用,并且对弓形虫的入侵有极显著的抑制作用（P<0.001）。综上表明,桧烯对弓形虫的入侵和细胞内增殖具有显著的抑制作用,可作为潜在的抗弓形虫先导化合物。     

Evaluation of immune responses induced by SAG1 and MIC3 vaccine cocktails against Toxoplasma gondii

The aim of this study was to evaluate the immune responses of a SAG1 and MIC3 vaccine cocktail in BALB/c mice. Ninety-six BALB/c mice were randomly divided into eight groups, including three plasmid DNA vaccine groups (pcDNA-MIC3, pcDNA-SAG1, pcDNA-MIC3+pcDNA-SAG1), three recombinant pseudotype baculovirus vaccine groups (BV-G-MIC3, BV-G-SAG1, BV-G-SAG1+BV-G-MIC3) and two control groups (PBS and BV-G-EGFP). All groups were immunized intramuscularly twice at three-week intervals. The production of anti-Toxoplasma gondii lysate antigen (TLA) antibodies, lymphoproliferation, levels of IFN-γ, IL-4 and IL-10 and the survival time were monitored after vaccination. The results showed that immunization of BALB/c mice with MIC3 and SAG1 vaccines stimulated both the cellular and humoral immune responses with the production of anti-T. gondii TLA antibodies. The vaccine cocktails of pcDNA-MIC3+pcDNA-SAG1 or BV-G-SAG1+BV-G-MIC3 induced significantly higher immunogenicity than a single-gene vaccine (P<0.05). Splenocytes from the immunized mice significantly proliferated in response to the TLA and released interferon (IFN)-γ (P<0.05). However, the levels of IL-4 and IL-10 in the sera of the immunized mice were not significantly different from those of the controls (P>0.05). Immunization with the vaccine cocktail (BV-G-SAG1+BV-G-MIC3) in mice significantly prolonged survival (50%; P<0.05) against a lethal challenge of T. gondii (RH tachyzoites), while all mice in the other immunized groups and control groups died within 20 and 4 days post-infection, respectively. Furthermore, the recombinant pseudotype baculovirus vaccines induced better immunogenicity than the plasmid DNA vaccines (P<0.05). These results suggest that an excellent vector-mediated vaccine cocktail strategy might be used to develop a new generation of vaccines against T. gondii infection.     

几种检测弓形虫血清抗体方法的比较分析

本文对5种弓形虫血清IgG抗体检测方法进行比较评价。5种方法平行检测了80份血清,比较各检测方法的阳性检出率,敏感性,特异性,符合率及Youden指数。结果经配对2检验,ELISA-200,IHA,MAT,SPA-ELISA 4种方法在阳性检出率、平行检测结果之间无显著性差异(P<0.05)。ELISA-200敏感性、特异性和符合率均高于90%,Youden指数(0.85)显著高于其他方法(P<0.05)。IHA,MAT,SPA-ELISA 3种方法各指标处于中等水平,各有适合的检测环境。ELISA-100与其他四种方法在检测结果上差异极显著(P<0.01),特异性(38.3%)、符合率(58.75%)及Youden指数(0.383)都显著低于其他方法。     

Prevalence of antibodies against Toxoplasma gondii in roe deer from Spain

Roe deer (Capreolus capreolus) is an important game animal in Spain. Sera from 278 roe deer from eight areas in mainland Spain were assayed for antibodies to Toxoplasma gondii by modified agglutination test (MAT). Titers of 1:25 or higher were found in 109 (39.2%) of 278 deer. No significant differences in antibody prevalence were found between sex or age categories. In contrast, significant differences in seroprevalence between locations were evident. Roe deer from the Northern coastal habitats (high humidity and roe deer density) had the highest prevalence, compared with low prevalence in Central Spain (arid areas and low roe deer density). There was a positive correlation between antibody prevalence and mean annual rainfall (r(s)=0.85, n=8, P<0.01). These findings have environmental and/or public health implications because venison can be an important meat source of T. gondii infections for humans and feral cats.     

Anti-protozoal efficacy of medicinal herb extracts against Toxoplasma gondii and Neospora caninum

The purpose of this study was to determine whether alcohol extracts of herbs (Sophora flavescens Aiton, Sinomenium acutum (Thunb.) Rehder and E.H. Wilson, Pulsatilla koreana (Yabe ex Nakai) Nakai ex T. Mori, Ulmus macrocarpa Hance and Torilis japonica (Houtt.) DC.) from South Korea, possess in vitro anti-protozoal activity against cultures of Toxoplasma gondii and Neospora caninum. These herbs have been used as human anti-parasitics in Asian countries for many years. Alcohol extracts of these herbs were serially diluted to final concentrations ranging from 625 to 19.5 ng/ml in media and added to wells containing either T. gondii or N. caninum tachyzoites in equine dermal (ED) cells. Parasite growth inhibition was measured using 3H-uracil incorporation as compared to untreated controls. T. japonica inhibited T. gondii proliferation by 99.3, 95.5, 73.0 and 54.0% in the range from 156 to 19.5 ng/ml, and S. flavescens inhibited T. gondii proliferation by 98.7, 83.0 and 27.2% in the range from 156 to 39 ng/ml. T. japonica inhibited N. caninum proliferation by 97.8, 97.9, 85.3 and 46.4% in the range from 156 to 19.5 ng/ml. S. flavescens inhibited N. caninum proliferation by 98.6, 97.0, 69.5 and 14.0% in the range from 156 to 19.5 ng/ml. Toxicity to host cells was noted when concentrations of T. japonica and S. flavescens exceeded 625 ng/ml. The herb extracts from S. acutum, Pulsatilla koreana, and U. macrocarpa also showed toxicity at higher levels but did not achieve the same inhibition effects at the lower concentrations against T. gondii and N. caninum as T. japonica and S. flavescens.     

3株不同宿主源弓形虫分离株GRA7基因的克隆与序列分析

对分离于江苏地域的3株不同宿主源弓形虫YZ-1(ChineseⅠ基因型)、YZ-2(ChineseⅠ基因型)和KS(基因型Ⅰ型)分离株的GRA7基因的全长序列进行克隆与序列分析,结合GenBank中的相关序列进行遗传进化分析。结果表明:KS株的GRA7基因完整开放阅读框长711 bp,与RH参考株完全一致;YZ-1和YZ-2株GRA7基因序列均为708 bp,与KS株相比在290-292位点缺失3个碱基;氨基酸序列和抗原表位分析显示,YZ-1和YZ-2株的GRA7蛋白在97位缺失1个谷氨酸,但比KS和RH株多1个抗原表位。结果表明,GRA7基因可区分Ⅰ、Ⅱ、Ⅲ型虫株,ChineseⅠ型与Ⅱ型虫株的亲缘关系最近;ChineseⅠ型GRA7基因在290-292位点缺少3个碱基,其GRA7蛋白多1个抗原表位。     

细胞渗透肽TAT与3种弓形虫抗原的融合重组表达

为了探索新型弓形虫疫苗的传递系统,本试验分别构建了细胞渗透肽反式转录激活因子(TAT)与3种弓形虫抗原和增强型绿色荧光蛋白(EGFP)融合表达的重组质粒,即pET28a-TAT-EGFP,pET28a-TAT-SAG1,pET28a-TAT-GRA4和pET28a-TAT-AMA1质粒。重组质粒被转化到大肠杆菌中并通过IPTG诱导,成功进行了融合表达,表达产物采用Ni-NTA树脂进行纯化,然后进行SDS-PAGE分析。结果得到31 ku的TAT-EGFP融合蛋白、34 ku的TAT-SAG1融合蛋白、38 ku的TAT-GRA4融合蛋白和62 ku的TAT-AMA1融合蛋白,经过Western blotting分析,感染弓形虫的小鼠血清可特异性地识别融合TAT的SAG1、GRA4蛋白和微弱地识别TAT-AMA1蛋白。     

弓形虫疫苗研究进展

弓形虫是细胞内寄生的一种原虫,弓形虫病是由弓形虫引起的一种重要人兽共患寄生虫病,弓形虫疫苗作为防控弓形虫病的重要措施是目前研究的热点。弓形虫进入机体后以细胞免疫为主,使得疫苗可以发挥最大功用。疫苗的免疫效果与候选抗原和佐剂的使用密切相关,不同类型的抗原和佐剂用于疫苗中能够产生不同的免疫效果。目前,弓形虫疫苗的候选抗原主要以虫体特异组分蛋白和新型表位基因为主,佐剂也从传统的弗氏佐剂拓展到了霍乱毒素、透明质酸酶和CpG-ODN等。综合考虑各方面因素进行疫苗研制,将成为生产具有理想保护性弓形虫疫苗的研究方向。     

Relation between the occurrence of antibodies against Toxoplasma gondii and clinical findings in cats

Within a three-year period, 178 clinically healthy and 442 sick cats (patients of the Clinic for Small Animal Diseases of the University of Veterinary Medicine, Brno) were examined for the presence of antibodies to Toxoplasma gondii. Antibodies to T. gondii were detected in 34.8% of the animals without clinical signs of the disease and in 54.3% of the sick cats (P less than 0.01). Compared with the group of clinically healthy cats, the specific antibodies occurred significantly more frequently in the cats suffering from diseases of the digestive tract (P less than 0.01), particularly in those with acute gastroenteritis, and in those having liver disorders (P less than 0.01; P less than 0.05). A statistically significantly higher occurrence of antibodies to T. gondii was also recorded in the cats with disorders of the nervous system (P less than 0.05; P less than 0.01), particularly in those with symptoms of extraordinary aggressivity at the age span from four months to three years. Enlarged lymph nodes were found out in 44% of the cats having antibodies to T. gondii. This is 15% more than the average for the investigated set of animals, which is a statistically significant dependence (P less than 0.01).     

Prevalence of antibodies against Toxoplasma gondii among Brazilian white-eared opossums (Didelphis albiventris)

Considering that Toxoplasma gondii is a parasite of global importance which affects several animal species including humans, the current study aimed to investigate the prevalence of antibodies against T. gondii among 72 white-eared opossums (Didelphis albiventris) from Botucatu Municipality (22°53'S 48°26'W), S?o Paulo State, Brazil. The investigation was carried out from January 2008 to December 2009, when the animals had their blood samples collected and subjected to the modified agglutination test (MAT); 12 specimens had brain samples bioassayed in mice. Seroprevalence was 5.5% (n=4) and bioassays were negative. Older animals had higher prevalence of antibodies against T. gondii. Opossums in closer contact with the urban environment are likely more exposed to T. gondii than animals from the sylvatic environment.     

ELISA和IHA检测弓形虫抗体的比较试验研究

目的:分析ELISA方法和IHA方法检测弓形虫病的可靠性。方法:本文应用酶联免疫吸附试验(ELISA)和间接血凝试验(IHA)两种方法,平行检测来自龙岩市某个猪场的32份猪血清中的抗弓形虫特异性抗体。结果:ELISA和IHA对猪弓形虫抗体阳性检出率分别为62.5%(20/32)、53.13%(17/32),这两种方法的阳性检出符合率较高,为71.88%(23/32)。其中,ELISA方法的敏感性(93.33%)、检测效率(81.25%)以及Youden指数(63.92%)都在IHA方法之上,但ELISA方法的特异性(12/17,70.59%)略低于I-HA方法(13/17,76.47%)。结论:ELISA和IHA两种方法均可用于猪弓形虫病的诊断和血清学调查,但ELISA方法更适用于猪弓形虫病的血清学调查。     

ELISA和LAT检测猪血清弓形虫抗体的比较

应用弓形虫重组蛋白rMIC3为抗原建立的酶联免疫吸附试验(ELISA)和乳胶凝集试验(LAT)2种方法,平行检测来自36个猪场的471份猪血清中的抗弓形虫特异性抗体。结果显示,ELISA和LAT对母猪弓形虫抗体的阳性检出率分别为42.86%(21/49)和44.90%(22/49),2种方法的阳性检出符合率为95.45%(21/22);对育肥猪的阳性检出率分别为42.06%(135/321)和43.61%(140/321),2种方法的阳性检出符合率为93.57%(131/140);对仔猪的阳性检出率分别为29.07%(30/101)和45.54%(46/101),2种方法的阳性检出符合率为63.04%(29/46)。结果表明,ELISA和LAT可用于猪弓形虫病的诊断和血清学调查,LAT更适合于仔猪弓形虫病的检测。     

The prevalence of antibodies against Toxoplasma gondii among hospitalized animals and stray dogs.

Hospitalized animals and stray dogs were serologically tested for antibodies against Toxoplasma gondii. In addition, the data were examined for the possibility of toxoplasmosis infection being associated with the clinical diagnosis and with the discharge status (alive vs. dead). Among 1056 hospitalized animals, 17 (20%) of 86 cats, 112 (14%) of 804 dogs, 34 (26%) of 133 horses and 6 (18%) of 33 cattle had serological evidence of infection with T. gondii. Only 22 (6%) of 342 young (median age = one year) stray dogs were seropositive. The difference in antibody prevalence between hospitalized and stray dogs was thought to be due to age and dietary factors. Of 249 dogs grouped by clinical diagnosis, there was significantly (p less than 0.01) higher prevalence of seropositives among dogs with diseases of the kidney or with adrenocortical hyperfunction than among dogs hospitalized for other diseases. Of 19 dogs with diseases of the kidney and 12 with adrenocortical hyperfunction 37% and 42%, respectively, were seropositive.. There was higher risk of being discharged from the hospital dead among seropositive dogs, cattle and horses than among seronegative animals of the same species. The exception was cats, where of 69 seronegative cats 29% were dead at discharge and where of 17 seropositive cats 18% were dead at discharge. The possible effects of stress due to hospitalization need further research.