Population genetics of Toxoplasma gondii: new perspectives from parasite genotypes in wildlife

Toxoplasma gondii, a zoonotic protozoal parasite, is well-known for its global distribution and its ability to infect virtually all warm-blooded vertebrates. Nonetheless, attempts to describe the population structure of T. gondii have been primarily limited to samples isolated from humans and domesticated animals. More recent studies, however, have made efforts to characterize T. gondii isolates from a wider range of host species and geographic locales. These findings have dramatically changed our perception of the extent of genetic diversity in T. gondii and the relative roles of sexual recombination and clonal propagation in the parasite's lifecycle. In particular, identification of novel, disease-causing T. gondii strains in wildlife has raised concerns from both a conservation and public health perspective as to whether distinct domestic and sylvatic parasite gene pools exist. If so, overlap of these cycles may represent regions of high probability of disease emergence. Here, we attempt to answer these key questions by reviewing recent studies of T. gondii infections in wildlife, highlighting those which have advanced our understanding of the genetic diversity and population biology of this important zoonotic pathogen.     

弓形虫MIC3基因真核表达质粒的构建及在 IBRS-2细胞内的表达

采用PCR技术从弓形虫RH株的基因组DNA中扩增编码MIC3的基因,克隆入pMD18-T载体,转化至E.coliDH5α感受态细胞,经抗性平板筛选、小量抽提质粒进行酶切、PCR及DNA测序鉴定后,亚克隆入真核表达载体pcDNA3.1。然后筛选含有目的基因的重组质粒,并转染IBRS-2细胞,在G418压力下进行筛选,利用SDS-PAGE、Western blot和ELISA检测表达情况。结果显示,扩增的MIC3基因与GenBank上相应基因序列(AJ132530)的一致性达99.9%,构建的真核表达质粒pcMIC3能在转染的IBRS-2细胞中表达分子量约为39.2ku的MIC3,且表达的蛋白质具有良好的免疫活性,为进一步研究该质粒的动物免疫试验奠定了基础。     

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.     

弓形虫MORN2基因敲除株的构建及其表型鉴定

本研究旨在构建弓形虫（Toxoplasma gondii）Ⅰ型RH虫株的膜占位与识别联结蛋白2（membrane occupation and recognition nexus protein，MORN2）基因敲除株，探究MORN2基因的表型功能。利用CRISPR/Cas9技术在sgRNA设计网站筛选出以MORN2基因为靶标的sgRNA并设计引物，以pSAG1∷CAS9-U6∷sgUPRT质粒为模板，在Q5酶作用下构建出MORN2靶向CRISPR的质粒；在MORN2靶点两翼约500 bp设计同源臂引物，以pUPRT∷DHFR-D质粒为模板构建含有乙胺嘧啶药物抗性基因（DHFR）的同源片段；将CRISPR质粒与同源片段电转染到弓形虫速殖子中，进行乙胺嘧啶药物筛选、96孔板单克隆筛选及PCR鉴定。结果显示，试验成功获得MORN2基因敲除株，在体外噬斑试验中，该敲除株的生长速度与野生型RH虫株基本一致；在体内感染试验中，分别以100和1 000个敲除株的速殖子腹腔注射小鼠，小鼠的死亡时间与对照组基本一致，说明MORN2基因的缺失几乎不影响RH虫株在体外培养时的生长速度和感染小鼠的毒力。本研究证明了MORN2基因在RH虫株中无表型功能，为进一步探究弓形虫的功能基因奠定了基础。     

弓形虫急性感染与小鼠妊娠失败相关性研究

研究弓形虫感染对小鼠妊娠的影响。用免疫组织化学方法测定子宫部分免疫细胞,用酶联免疫吸附法测定子宫匀浆中肿瘤坏死因子 α(TNF α)和白介素 10(IL 10)的含量。发现孕0天和孕6天接种弓形虫,小鼠全部流产。感染组子宫内膜中仅见少量CD8+T淋巴细胞。对照组未见阳性细胞。F4/80+巨噬细胞在感染组小鼠子宫内膜中大量存在。对照组仅见少量F4/80+细胞。细胞因子测定结果表明,TNF α弓形虫感染组子宫匀浆中含量高达76 862±30 354pg/mg蛋白,显著高于正常妊娠7天时小鼠子宫中TNF α的含量(P<0 01)。结果提示,已妊娠母鼠感染弓形虫可引发流产,并与子宫胎儿界面的TNF α分泌增多有关。孕0天感染,孕鼠子宫匀浆上清液中IL 10含量平均值为19 696±9 811pg/mg蛋白,显著低于对照组(48 856±16 518pg/mg,P<0 05)。孕6天感染,子宫匀浆IL 10含量与对照组比较差异不显著(P>0 05)。     

免疫学技术在弓形虫检测方面的研究概况

弓形虫病是世界分布最广泛的一种人兽共患病,此病在温暖潮湿的地区感染率极高,所以建立高效敏感的检测方法是非常必要的。目前,国内检测弓形虫病的技术有多种,其中以免疫学技术为主。虽然病原学检测方法的敏感性高,特异性强,但其成本高,对人体会产生严重的副作用,所以并不适宜推广。近年来,通过对弓形虫表面抗原的大量研究,检测弓形虫病的免疫学技术也有了显著提高。从敏感性差、操作复杂到现在的特异性强、快速简便,从单一抗原的检测到现在的多表面抗原的检测,无疑为检测弓形虫带来了新的思路。论文主要对检测弓形虫的ELISA、改良凝集试验以及化学发光免疫分析的最新进展进行了综述。     

Seroprevalence of Toxoplasma gondii in mainland and sub-Antarctic New Zealand sea lion (Phocarctos hookeri) populations

AIMS: To investigate the seroprevalence of antibodies to Toxoplasma gondii in New Zealand sea lions (Phocarctos hookeri), as a potential contributor to reproductive failure.

METHODS: Archived sera were sourced from New Zealand sea lions from two recolonising mainland populations in the Otago Peninsula (n=15) and Stewart Island (n=12), as well as a declining population at Enderby Island (n=28) in the New Zealand sub-Antarctic. Sera were tested for antibodies to T. gondii using a commercially available ELISA (with samples considered positive if the sample to positive ratio was?>30%), and latex agglutination test (LAT; with titres ≥1:32 considered positive). Western blot analysis was used to validate the results of a subset of 14 samples.

RESULTS: Five samples from sea lions in mainland locations were confirmed positive for antibodies to T. gondii. Two adult females exhibited high LAT antibody titres (min 1:2048, max 1:4096) on both occasions when sampled 1 and 2 years apart, respectively. No animals from Enderby Island were seropositive.

CONCLUSIONS: Toxoplasma gondii infection is unlikely to be a major contributor to poor reproductive success in New Zealand sea lions. However, continued surveillance is pertinent to assess subclinical and clinical impacts of the parasite on these threatened populations. The commercial tests evaluated here, with further species-specific threshold refinement could provide a fast, inexpensive and reliable indicator of T. gondii exposure in New Zealand sea lions.     

Construction and Identification of Cell Penetrating Peptides VP22 Gene Fused with Enhanced Green Fluorescent Protein and Three Antigens of Toxoplasma gondii

To study a novel vaccine of Toxoplasma gondii,the recombinant plasmids that cell penetrating peptides VP22 gene fused with one enhanced green fluorescent protein（EGFP)(pcDNA-VP22-EGFP)and three antigens of T.gondii（pcDNA-VP22-SAG1,pcDNA-VP22-GRA4 and pcDNA-VP22-AMA1）were constructed,respectively.The recombinant plasmids were identified by PCR,restriction endonuclease digestion and DNA sequencing.The pcDNA-VP22-EGFP plasmid was transfected into COS7 cells,and the expression of VP22-EGFP in COS7 cells was confirmed by fluorescence microscope and RT-PCR.The results showed that all recombinant plasmid were constructed correctly.Green fluorescence was observed successfully under the fluorescence microscope in transfected cells after 72 h.The gene fragment of 1 449 bp was obtained by RT-PCR.The results indicated that the recombinant plasmids were constructed successfully.