孕中期弓形虫感染对孕鼠血清孕酮及雌三醇含量的影响

目的探讨孕中期弓形虫感染对孕鼠血清孕酮(progesterone)及雌三醇(estriol)含量的影响。方法孕中期BALB/c孕鼠随机分为染虫组和健康对照组,染虫组经腹腔无菌接种纯化的RH强毒株弓形虫速殖子,健康对照组注射等量0.01mol/LPBS。分别于妊娠第10、12、14、16、18d,摘孕鼠眼球取血,经放射免疫法测定血清孕酮和雌三醇含量。结果在孕第10、12、14d,孕鼠血清孕酮和雌三醇含量染虫组与对照组比较差异无统计学意义(P>0.05);在孕第16、18d,两组比较差异有统计学意义(P<0.01)。结论孕中期弓形虫感染可引起孕鼠血清孕酮和雌三醇含量下降,这可能是弓形虫感染致胚胎发育迟缓、胎盘组织炎性病变及滋养层细胞凋亡增加的结果,也可能是胚胎发育迟缓、滋养层细胞凋亡增加的原因。     

弓形虫致密颗粒蛋白GRA2真核表达质粒的构建与体外表达

目的构建弓形虫致密颗粒蛋白GRA2的真核表达重组质粒。方法设计合成GRA2引物，运用PCR方法扩增其基因片段，经克隆至pMDl8-T载体后，亚克隆至真核表达质粒pcDNA3．1（-）而构建重组表达质粒pcDNA3．1-GRA2。脂质体法将构建的重组质粒转染HFF细胞，RT—PCR法检测转染细胞中GRA2的表达情况。结果PCR扩增GRA2基因序列正确，构建的重组表达质粒pcDNA3．1-GRA2经PCR、EcoRⅠ／HindⅢ双酶切和测序鉴定正确；转染GRA2基因的细胞，RT—PCR可见目的条带。结论成功获得真核表达重组质粒pcDNA3．1-GRA2，为进一步研究弓形虫疫苗的免疫保护性奠定基础。     

Different effect of Toxoplasma gondii infection on adhesion capacity of fibroblasts and monocytes

This study investigated the effect of infection with the apicomplexan parasite Toxoplasma gondii , in combination with the concomitant cytokine environment (IFN-γ/TNF-α), on adhesion of THP-1 monocytic cells to MRC-5 fibroblasts. Surprisingly, infection of THP-1 cells decreased their adhesion to the MRC-5 cell monolayer. This decrease was compensated by IFN-γ/TNF-α stimulation. In contrast, infection of MRC-5 cells significantly increased adhesion, which was synergistically augmented by cytokine stimulation. Levels of ICAM-1 (CD54) on MRC-5 cells, as well as LFA-1 (CD11a) on THP-1 cells, were not changed by infection, neither in resting, nor in cytokine stimulated cells. These results show that T. gondii infection alters adhesion properties and reactivity to cytokine stimulation in a cell-specific way.     

两种免疫调节剂增强弓形虫W2b2a表位疫苗的小鼠保护作用

目的:探讨匹多莫德、沙利度胺增强弓形虫W2b2a表位疫苗刺激机体产生免疫应答和保护免疫的效果。方法:将匹多莫德、沙利度胺分别与弓形虫W2b2a表位疫苗混合肌注免疫小鼠,检测其细胞免疫和体液免疫,并观察其受到弓形虫攻击感染后的生存时间。结果:pc DNA3-W2b2a加匹多莫德组、pc DNA3-W2b2a加弗氏佐剂组、pc DNA3-W2b2a加沙利度胺组小鼠血清IFN-γ水平显著高于pc DNA3-W2b2a组(P0.05);pc DNA3-W2b2a加匹多莫德组、pc DNA3-W2b2a加弗氏佐剂组Ig G抗体水平、CD4+/CD8+比值、T细胞增殖活性显著高于pc DNA3-W2b2a组和pc DNA3-W2b2a加沙利度胺组(P0.05);小鼠攻击试验表明,免疫组小鼠存活时间明显长于对照组(P0.05)。结论:匹多莫德、沙利度胺可增强弓形虫W2b2a表位疫苗免疫小鼠的保护作用。     

刚地弓形虫RH株GRA6分子基因克隆与序列分析

目的　克隆刚地弓形虫 RH株致密颗粒抗原 (Dense granule antigen,GRA6 )分子基因 ,为研究使用重组的GRA6分子作为抗原进行弓形虫病诊断奠定基础。　方法　根据己知的 GRA6序列合成一对引物 ,抽提弓形虫速殖子m RNA,以速殖子 m RNA为模板 ,通过反转录 PCR(RT- PCR)扩增出 GRA6的 c DNA条带。目的基因 DNA条带被克隆到质粒 p UC19中形成重组质粒。对重组质粒 DNA进行纯化 ,并对目的基因片段进行核苷酸序列分析。　结果　通过RT- PCR从 m RNA中扩增出一条分子量约 70 0 bp的 DNA条带。核苷酸序列分析表明 ,GRA6分子基因的开放的阅读框全长为 6 93bp,编码 2 30个氨基酸分子 ,与已报道的 RH株 GRA6分子具有 10 0 %的同源性。　结论　本研究获得了刚地弓形虫 RH株的 GRA6分子基因 ,为今后应用此分子作为抗原诊断弓形虫病奠定了基础     

Moving towards improved vaccines for Toxoplasma gondii

Introduction: Toxoplasma gondii is an intracellular parasitic protozoan that infects almost all warm-blooded animals and humans, resulting in threats to public health and economic losses. Despite continuous research efforts, there are still very few effective strategies against toxoplasmosis. In the past few years, numerous vaccination experiments have been performed to control T. gondii infection.

Areas covered: In this review, the authors summarize the development of T. gondii vaccines with proper adjuvants, ranging from live or live-attenuated vaccines to protein vaccines, DNA vaccines, epitope vaccines and novel vaccines. They also highlight the challenges involved in the development of T. gondii vaccines, including specific impediments and shortcomings.

Expert opinion: Moving towards the development of effective vaccines against T. gondii is not only a tedious mission but also a difficult challenge. Future studies should consider new approaches and strategies for vaccine development, particularly novel vaccines and genetic adjuvants, as well as optimizing immunization protocols and evaluation criteria.     

Seroprevalence and Risk Factors of Toxoplasma gondii Infection in Buffaloes,Sheep and Goats in Yunnan Province,Southwestern China

Background:

The seroprevalence of Toxoplasma gondii infection in buffaloes, sheep and goats in Yunnan Province, southwestern China was conducted between May 2012 and December 2013.

Methods:

A total of 973 (427 buffaloes, 154 sheep and 392 goats) serum samples were collected from seven administrative regions of Yunnan Province, and examined for T. gondii antibodies by indirect hemagglutination (IHA) test. Some risk factors related to species, age, gender and geographical origin were determined using a multinomial logistic regression.

Results:

The overall seroprevalence of T. gondii in ruminant species was estimated at 11.9%. The final logistic regression model demonstrated that host species and geographical origin were the main risk factors associated with T. gondii infection (P<0.05).

Conclusion:

Taken together, the results of the present study revealed a high exposure to T. gondii in ruminant species in Yunnan Province, which has an important implication for public health.     

Genetic Characterization of Toxoplasma gondii from Zoo Wildlife and Pet Birds in Fujian,China

Background:

Toxoplasmosis, a worldwide zoonotic disease, is caused by Toxoplasma gondii. The distribution of genetic diversity of T. gondii in wild animals is of great importance to understand the transmission of the parasite in the environment. However, little is known about T. gondii prevalence in wild animals and birds in China.

Methods:

We conducted the genetic characterization of T. gondii isolated from Zoo Wild Animals and Pet Birds in Fujian Province, Southeastern China. Heart tissues were collected from 45 zoo animals and 140 pet birds. After identified using B1 gene, the genetic diversity of T. gondii isolates were typed at 11 genetic markers, including SAG1, 5’ and 3’-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3.

Results:

Seven of 45 zoo animals and 3 of 140 pet birds were positive by PCR amplification using T. gondii B1 gene specific primers. Of these positive isolates, 3 isolates from Black-capped (Cebus apella), Peacock (Peafowl) and Budgerigar (Melopsittacus undulatus) were successfully genotyped at 11 genetic loci, and grouped to three distinct genotypes: ToxoDB Genotype #9, #2 and #10, respectively.

Conclusion:

This is the first genotyping of T. gondii isolated from zoo wild animals and pet birds in Fujian, China. There is a potential risk for the transmission of this parasite through zoo wild animals and pet birds in this region.     

孕早期妇女弓形虫感染对妊娠结局的影响分析

目的：分析孕早期妇女弓形虫感染对妊娠结局的影响。方法回顾性分析该院2014年2月-2015年2月收治的1960例孕妇,根据不同感染情况分为三组,研究A组(活动性感染)、研究B组(既往感染)、研究C组(急性感染),另选取同期120例健康孕妇作为对照组,比较各组妊娠结局情况。结果研究B组与对照组不良结局妊娠发生率比较差异无统计学意义(P>0.05),但研究A组与研究C组比对照组高差异有统计学意义(P<0.05)。结论弓形虫活动性感染与急性感染和孕早期妇女的妊娠结局密切相关,可将检测弓形虫IgM抗体作为孕妇孕前的检查项目之一。     

弓形虫感染对小鼠和人子宫自然杀伤细胞的影响

目的　观察弓形虫感染对小鼠和人子宫自然杀伤细胞（uterine natural killer cells, uNK cells）比例、数量、分化和功能的影响,探讨uNK细胞在弓形虫感染致早孕流产中的作用。方法　妊娠第6.5天给予孕鼠腹腔注射弓形虫速殖子,建立孕早期弓形虫感染致流产小鼠模型,妊娠第9.5天分离小鼠子宫淋巴细胞。取人正常妊娠孕早期流产新鲜蜕膜组织,分离人子宫淋巴细胞,与弓形虫RH株速殖子体外共培养48 h（弓形虫与子宫淋巴细胞比例分别为0.5∶1、1∶1、2∶1）。采用流式细胞术检测小鼠uNK细胞表型（CD122、NK1.1、DX5）、人uNK细胞表型（CD3、CD56、CD11b、CD27）及胞内细胞因子γ⁃干扰素（interferon⁃γ, IFN⁃γ）和肿瘤坏死因子⁃α（tumor necrosis factor⁃α, TNF⁃α）表达水平。磁珠分选小鼠和人uNK细胞,以小鼠YAC⁃1或人K562细胞作为靶细胞、以小鼠或人uNK细胞作为效应细胞,采用乳酸脱氢酶（lactate dehydrogenase, LDH）释放法检测效应细胞/靶细胞比例分别为1∶1、5∶1、10∶1、20∶1时uNK细胞的杀伤功能。结果　妊娠第9.5天,弓形虫感染组小鼠流产率较对照组显著上升（83.02% vs. 3.51%;[χ2] = 71.359,P < 0.001）。与对照组比较,弓形虫感染组小鼠uNK细胞绝对数量显著降低[（4 547 ± 1 610）个/只 vs.（8 978 ± 3 339）个/只;U = 2.000,P < 0.05]、细胞表面NK1.1表达水平显著下降[（74.53 ± 8.37）% vs.（93.00 ± 1.11）%;U = 0.000,P < 0.05]、NK1.1⁃DX5⁃细胞比例显著上升[（20.10 ± 8.03）% vs.（5.04 ± 0.68）%;U = 0.000,P < 0.05]、NK1.1+DX5+细胞比例显著下降[（21.70 ± 12.48）% vs.（45.75 ± 2.26）%;U = 0.000,P < 0.05]、IFN⁃γ表达水平显著升高[（16.74 ± 1.36）% vs.（8.13 ± 1.90）%;U = 0.000,P < 0.05],但TNF⁃α表达水平无显著改变[（67.98 ± 9.20）% vs.（52.93 ± 10.42）%;U = 2.000,P > 0.05]。弓形虫感染组小鼠uNK细胞表现出较强杀伤能力,且杀伤作用随着效应细胞/靶细胞比例升高逐渐增强;效应细胞/靶细胞比例为1∶1、5∶1、10∶1、20∶1时,感染组uNK细胞对YAC⁃1细胞的杀伤活性分别为2.30%、4.32%、8.12%和12.65%,对照组分别为1.21%、1.63%、2.51%和3.22%。将人子宫淋巴细胞与弓形虫RH株速殖子共培养48 h后,弓形虫感染组人uNK细胞在子宫淋巴细胞中所占比例较对照组显著降低[弓形虫2∶1组 vs. 对照组：（6.61 ± 1.75）% vs.（17.48 ± 4.81）%;F = 7.307,P < 0.01]、绝对数量显著减少[弓形虫2∶1组vs. 对照组：（12 104 ± 5 726）个/孔 vs.（65 285 ± 21 810）个/孔;H =11.540,P < 0.01]。弓形虫感染组uNK细胞中,CD56brightCD16⁃调节型NK细胞亚群比例较对照组减少（弓形虫2∶1组vs. 对照组：（25.25 ± 5.90）% vs.（36.03 ± 4.51）%;F = 3.213,P > 0.05]、CD56dimCD16+杀伤型NK细胞亚群比例增加[弓形虫2∶1组vs. 对照组：（11.15 ± 2.15）% vs.（7.09 ± 2.24）%,F = 2.992,P > 0.05],两者比值显著降低[弓形虫2∶1组vs. 对照组：（2.37 ± 0.92）vs.（5.58 ± 2.39）;H = 8.228,P < 0.05];CD11b+CD27⁃细胞比例显著上升[弓形虫2∶1组vs. 对照组：（30.28 ± 6.91）% vs.（17.48 ± 4.67）%;H = 6.556,P < 0.05],IFN⁃γ [弓形虫2∶1组vs. 对照组：（14.13 ± 1.28）% vs.（15.19 ± 1.64）%;F = 1.639,P > 0.05]、TNF⁃α表达水平无显著改变[弓形虫2∶1组vs. 对照组：（54.76 ± 10.02）% vs.（50.33 ±3.67）%;F = 0.415,P > 0.05]。弓形虫感染组人uNK细胞具有较强杀伤能力,且杀伤作用随着效应细胞/靶细胞比例升高逐渐增强;效应细胞/靶细胞比例为1∶1、5∶1、10∶1、20∶1时,感染组uNK细胞对K562细胞的杀伤活性分别为11.90%、28.11%、49.91%和73.35%,对照组分别为11.21%、21.63%、33.51%和48.22%。结论　弓形虫感染对小鼠和人uNK细胞分化和分泌功能产生了不同影响,但均可引起小鼠和人uNK细胞绝对数量减少、杀伤功能增强。