弓形虫GJS株表面抗原1基因的克隆及真核表达质粒的构建

根据GenBank数据库中的RH株弓形虫表面抗原1(SAG1)基因序列设计1对引物,应用PCR技术从弓形虫GJS株速殖子基因组DNA中扩增编码SAG1的基因片段.PCR产物经纯化、酶切后定向插入pcDNA3.1(+)真核表达载体,转化至大肠杆菌DH5α,经酶切及PCR鉴定后测序,并进行序列分析.结果表明:从弓形虫GJS株基因组DNA中扩增出1 008 bp的特异SAG1片段,大小与预测值相符,与已知的SAG1基因核苷酸序列(GenBank登录号:S76248)的ORF和氨基酸序列的同源性均为99%.     

奶山羊资源及其弓形虫感染情况的研究进展

奶山羊产业在现代化的畜牧业经济发展中占有重要的地位。弓形虫的感染严重影响奶山羊的生产繁殖性能,给奶山羊业造成了巨大的经济损失。奶山羊也是引起人感染弓形虫的重要来源因素之一,严重威胁人类的健康。文章主要对我国奶山羊资源、奶羊业及国内外奶山羊弓形虫感染情况等方面进行了综述,以期更好地了解奶山羊及其弓形虫感染的情况,为奶山羊产业的健康发展及其相关研究提供参考。     

A Brief History and Overview of Toxoplasma gondii

Toxoplasma gondii was discovered by scientists working in North Africa and Brazil around 100 years ago. The parasite has since been found to be capable of infecting all warm‐blooded animals including humans making it one of the most successful parasitic organisms worldwide. The pathogenic potential of T. gondii was recognized in the 1920s and 1930s, in congenitally infected children presenting with the classic triad of symptoms, namely hydrocephalus, retinochoroiditis and encephalitis. In addition, around the same time T. gondii parasites were found to be associated with severe intraocular inflammation. In the 1980s, T. gondii emerged as a major cause of death in patients with acquired immunodeficiency syndrome, illustrating the importance of the immune system in controlling T. gondii infection. T. gondii was reported as a major cause of abortion in sheep in New Zealand in the 1950s, which raised questions about potential new transmission routes for the parasite. The discovery of the cat as the definitive host in the 1960s was a very important finding as it helped to complete our understanding of the parasite’s life cycle, and the oocyst stage of T. gondii shed in the faeces of infected cats was found to be an important source of infection for many intermediate hosts and helped to explain infection in herbivorous animals and people with a vegetarian diet. In addition, this stage of the parasite was very robust and could survive in the environment, depending on the climatic conditions, for up to 12–18 months. Knowledge of the parasite’s lifecycle, transmission routes, risk groups and host immune responses has helped in the development of strategies to control the disease, reduce transmission of the parasite and limit environmental contamination.     

Pigeons (Columba livia) are a suitable experimental model for Neospora caninum infection in birds

Neospora caninum infections in chickens have been recently described by epidemiological and experimental approaches, and these birds may be considered natural intermediate hosts of the parasite. It has been postulated that other bird species might perform this role in wildlife as well. To better understand the sylvatic life cycle of N. caninum, further studies are required. In that sense, this work aimed to observe infection kinetics in pigeons experimentally infected with N. caninum. Experimental infections were conducted in parallel with a related protozoan, Toxoplasma gondii, which has been already described as able to infect pigeons in nature. Our results demonstrated that N. caninum disseminated through various tissues of this host and induced parasite-specific IgG seroconversion. Infection parameters were similar to that observed in the T. gondii infected group, although N. caninum-infected pigeons presented lower IgG titers during acute phase. The results herein described demonstrate that pigeons are a suitable model for N. caninum infection, considering that these data are in agreement with those observed in chickens experimentally infected with this parasite. As pigeons may be revealed as important reservoirs for N. caninum infection in nature, future studies are necessary to determine the real prevalence of this parasite in this and other birds in wildlife.     

Prevalence of Toxoplasma gondii antibodies in stray cats in Sari, northern Iran

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete environmentally resistant oocysts. T.gondii is a major zoonotic agent which infects up to one-third of the world population. Toxoplasmosis in neonates and immunocompromised patients can lead to severe disease and death. A cross- sectional parasitological and serological survey with latex agglutination test (LAT) to detect anti-T. gondii antibodies was conducted on 100 serum samples collected from stray cats in five urban areas of Sari, Northern Iran, from April to November 2004. Classification by age, sex, weight, season and region was made. Results analyzed according to specific variables. The overall prevalence of T. gondii IgG antibodies (LAT titre ≥1:1) were found in 40 of 100(40%) of stray cats, with regional variations. Overall 16 of 100(16%) of stray cats had diagnostically significant antibody titres (LAT ≥ 1:64). Prevalence was significantly higher in adult cats (1.5–3.0 kg, 54.5% of 66) than in juvenile cats and kittens (≤1.4 kg, 11.8% of 34) and higher in female stray cats (44.4% of 72) than in male stray cats (28.6% of 28). Toxoplasma seroprevalence was highest in the season of spring (22.4%). There was a significant difference in the prevalence of infection relative to host age and weight (P < 0.05). No significant difference was found between the prevalence of infection relative to host gender, urban sites and season (P > 0.05). Prevalence of T. gondii oocyst was also analyzed by a routine coprological method in 100 cats. T. gondii oocysts were not found in any faecal samples analyzed. Only 2 out of 100 smear preparations of intestinal mucosa showed trophozoites of T. gondii.     

Protection against Toxoplasma gondii brain cyst formation in mice immunized with Toxoplasma gondii cytoskeleton proteins and Lactobacillus casei as adjuvant

The aim of the study was to evaluate the protection generated in mice against Toxoplasma gondii brain cyst burden by vaccination with T. gondii cytoskeleton proteins using Lactobacillus casei as adjuvant. One hundred and sixty-eight NIH mice were randomly allocated into eight groups of 21 mice each. Animals were immunized as follows: in group 1 with Toxoplasma lysate antigen (TLA) in Freund's modified adjuvant, containing L. casei (FMA), in group 2 with Toxoplasma cytoskeleton proteins (TCPs) in FMA, in group 3 with FMA, in group 4 with phosphate buffered saline (PBS), in group 5 with L. casei dead by heath (Lc), in group 6 with Freund's complete adjuvant (FCA), in group 7 with TLA in FCA, and in group 8 with TCP in FCA. Mean brain cyst burden (±S.E.M.) was assessed in mice 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). The percentages of reduction in cyst burden per brain (P < 0.01) as compared with the group 4 (control: mean 3181 ± 97.5) were 77.25% (724 ± 98) in group 1, 88.02% (381 ± 97.5) in group 2, 38.92% (1943 ± 130.3) in group 3, 44.31% (1771.4 ± 102) in group 5, 59.28% (1295.2 ± 99.1) in group 7 and 55.69% (1409.5 ± 89.9) in group 8. In order of importance, the best protection was obtained in groups 2, 1, 7, 8, 5 and 3. Noticeably the mice inoculated with L. casei alone showed a significant reduction in T. gondii brain cysts (P < 0.01), while those animals treated with FCA alone did not. Additionally, IgM anti-T. gondii antibody levels, as determined by ELISA 2 weeks after challenge, were highest in group 2 (P < 0.01) than in the other seven groups. Results suggest that T. gondii cytoskeleton proteins with L. casei as adjuvant constitute a good anti-toxoplasmosis vaccine candidate.     

Prevalence of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> and <Emphasis Type="Italic">Neospora caninum</Emphasis> infections in sheep from Federal District,central region of Brazil

Serum samples from 1028 sheep were collected from 32 herds within Federal District, in the central region of Brazil. The samples were examined by indirect fluorescent antibody test (IFAT) using sera diluted 1:64 and 1:50 as cut-off values for the detection of antibodies against Toxoplasma gondii and Neospora caninum, respectively. The observed prevalence for T. gondii infection was 38.22% (26.81%<CI 0.95<49.62%), and the titers ranged from 64 to 65536. The observed prevalence for N. caninum infection was 8.81% (7.08%<CI 0.95<10.53%). The titers ranged from 50 to 51200. The reactant sera to both pathogens corresponded to 4.67% of the samples. The risk factors were not determined because of the absence of negative herds for T. gondii and the high proportion of positive herds for N. caninum (87.50%). The prevalence for T. gondii infection was significantly higher among males than in females. The present work is the first report on seroprevalence of T. gondii and N. caninum in sheep from Federal District and shows that infection by both parasites is widespread in the ovine population from this region.     

弓形虫GJS株pcDNA3-MIC3核酸疫苗的研究

根据GenBank中MIC3基因序列设计1对引物,采用PCR技术从弓形虫GJS株基因组DNA中扩增微线体蛋白3(MIC3)基因片段,克隆到pMD18-T载体,经PCR、酶切及测序鉴定后,阳性重组质粒酶切并亚克隆到真核表达载体pcDNA3.1(+)后进行PCR、酶切及测序鉴定.重组质粒pcDNA3-MIC3肌肉注射免疫BALB/c小鼠,通过ELISA检测血清特异抗体;经腹腔攻击感染弓形虫GJS株速殖子,观察小鼠的生存时间.结果成功构建了pcD-NA3-MIC3质粒;免疫组小鼠血清检测到特异性抗体;攻击感染后免疫组小鼠平均存活时间较对照组明显延长.表明该核酸疫苗具有较好的免疫原性,能诱导小鼠产生良好的免疫保护作用.     

中国4个弓形虫虫株GRA6基因的比较分析

 通过对国内来源于不同宿主的ZS人株、SH人株、CN猪株、QH绵羊株4个弓形虫虫株，以及国际标准强毒株RH株的致密颗粒抗原（GRA6）基因进行PCR-RFLP分析，首次对中国弓形虫虫株进行了基因分型研究，旨在了解中国弓形虫基因型的分布情况，从而为进一步的分子遗传学研究以及弓形虫病的防制提供资料。PCR-RFLP分析显示两种带型：RH、SH、CN 3株弓形虫为一种带型，QH和ZS 2株弓形虫为另一种电泳带型。GRA6序列分析结果显示：RH、SH、CN 3株弓形虫的GRA6基因序列上的MseΙ酶切位点与国外报道的RH株一致，同属于基因型Ι，为强毒株；QH和ZS 2株弓形虫的MseΙ酶切位点与国外报道的弱毒株BEVERLEY株和ME49株一致，同属于基因型Ⅱ，与PCR-RFLP分析结果一致。除MseΙ酶切位点外，中国的几个虫株GRA6序列与GenBankTM注册的RH株及BEVERLEY和ME49两个弱毒株有个别碱基的差异。结果证明中国人源弓形虫包含了Ι、Ⅱ两种基因型，而动物源性弓形虫则因宿主和来源地不同亦有Ι、Ⅱ两个基因型。