Immunogenicity and therapeutic effects of recombinant Ag85AB fusion protein vaccines in mice infected with Mycobacterium tuberculosis

The immune function of tuberculosis (TB) patients is disordered. By using immune regulators to assist chemotherapy for TB the curative effect might be improved. In this study, a vaccine containing Mycobacterium tuberculosis (M. tuberculosis) recombinant Ag85AB fusion protein (rAg85AB) was constructed and evaluated. The mice were immunized intramuscularly three times at two-week intervals with Ag85AB fusion protein combined with Corynebacterium parvum adjuvant (rAg85AB+CP). In comparison to control mice that received either CP alone or saline, the mice that received rAg85AB+CP had significantly higher number of T cells secreting IFN-γ and higher levels of specific antibodies of IgG, IgG1 and IgG2a isotypes in sera. The specific antibodies also had higher ratios of IgG2a to IgG1, indicating a predominant Th1 immune response. To test for immunotherapy of TB, M. tuberculosis infected mice were given three intramuscular doses of 20 μg, 40 μg or 60 μg of rAg85AB in rAg85AB+CP, or phosphate-buffered saline (PBS), or CP or Mycobacterium phlei (M. Phlei) F.U.36. Compared with the PBS group, 20 µg, 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups reduced the pulmonary bacterial loads by 0.13, 0.15, 0.42 and 0.40 log₁₀, and the liver bacterial loads by 0.64, 0.64, 0.53 and 0.61 log₁₀, respectively. Pathological changes of lungs were less, and the lesions were limited to a certain extent in 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups. These results showed that rAg85AB+CP had immunotherapeutic effect on TB, significantly increasing the cellular immune response, and inhibiting the growth of M. tuberculosis.     

Determination of relationships among non-toxigenic Vibrio cholerae O1 biotype El Tor strains from housekeeping gene sequences and ribotype patterns

Sequencing of three housekeeping genes, mdh, dnaE and recA, and ribotyping for seven non-toxigenic Vibrio cholerae O1 strains isolated from different geographic sources indicate a phylogenetic relationship among the strains. Results of MLST and ribotyping indicate a clear difference between three toxigenic strains (N16961, O395, and 569B) and three non-toxigenic strains from India (GS1, GS2, and GW87) and one Guam strain (X392), the latter of which were similar in both MLST and ribotyping, while two other non-toxigenic strains from the USA and India (2740-80 and OR69) appeared to be more closely related to toxigenic strains than to non-toxigenic strains, although this was not supported by ribotyping. These results provide clues to the emergence of toxigenic strains from a non-toxigenic progenitor by acquisition of virulence gene clusters. Results of split decomposition analysis suggest that widespread recombination occurs among the three housekeeping genes and that recombination plays an important role in the emergence of toxigenic strains of V. cholerae O1.     

Spatial spread and demographic expansion of Lyme borreliosis spirochaetes in Eurasia

The Lyme borreliosis (LB) group of spirochaetes currently comprises 18 named species that vary in their geographic distribution, host specificity and ability to cause disease in humans. In Europe three species are most abundant, Borrelia afzelii, Borrelia garinii and Borrelia valaisiana but only two of these (B. garinii and B. afzelii) are regularly found in Asia as well. A recently published study has shown that Borrelia species associated with birds, such as B. garinii, showed limited geographic structuring between European countries while, the rodent associated species, B. afzelii, showed extensive spatial structuring in Europe. Here, we use multilocus sequence analysis to show that when the wider, inter-continental, distribution is considered, there is evidence of spatial structuring even in the bird-associated species B. garinii. Furthermore, our investigations into historical LB populations provided evidence for range expansions of B. garinii and B. afzelii populations in Europe in the distant past. We propose that the expansion of B. afzelii in Europe may be linked to rodent population expansions after the last glacial maximum.     

Phylogenetic and recombination analysis of human bocavirus 2

Background  

Human bocavirus 2(HBoV2) and other human bocavirus species (HBoV, HBoV3, and HBoV4) have been discovered recently. But the precise phylogenetic relationships among these viruses are not clear yet.     

扩增片段长度多态性分析用于霍乱弧菌分子分型的方法建立和应用评价

目的建立霍乱弧菌的基于红外荧光标记引物的扩增片段长度多态性(AFLP)分型方法,评价脉冲场凝胶电泳(PFGE)和AFLP对霍乱弧菌的分型能力。方法选择PFGE带型均不相同的47株霍乱弧菌作为评价菌株,建立AFLP对于霍乱弧菌的分型方法;确定操作程序后选择83株分离自1962-2005年11个省的霍乱弧菌,进行AFLP和PFGE对于霍乱弧菌分型能力的比较。AFLP分型方法采用了红外荧光标记PCR引物,利用LI-COR4300自动测序仪完成电泳、SagaMX软件对电泳图谱进行编辑。PFGE采用PulseNet提供的标准化程序。结果AFLP用于霍乱弧菌分型时选择性引物携带碱基数为1,最优引物组合为EcoRⅠ-G／MseⅠ-T,AFLP分型实验的重复性达到99．2％。AFLP将83株霍乱弧菌分为52个型别,D值为0．9545;PFGE将此83株菌分为44种带型,D值为0．9251。结论优化固定了AFLP对于霍乱弧菌的分型程序,重复性好;AFLP比PFGE具有更好的分型能力,能够用于分离菌株的分子分型。结合已成为实验室网络监测标准分析方法的PFGE,可对分离株做更细致的分型比较。     

中国八省市食品来源单核细胞增生李斯特菌的脉冲场凝胶电泳分析

目的 采用国际标准化的脉冲场凝胶电泳（PFGE）分析方法对单核细胞增生李斯特菌（Lm）进行基因分型,初步建立中国Lm的基因分型数据库。方法 按照标准化的Lm-PFGE方法对来自国内8个省市、多种食品来源的118株Lm进行了PFGE分型。结果 118株Lm分成了39种带型,其中GX6A160004型有37株菌,占分析菌株的31．36%,是主要型别。结论 初步建立了中国单核细胞增生李斯特菌用于网络监测分析（PulseNet China）的基础数据库,发现同型菌株在全国多个地区广泛存在。     

实时聚合酶链反应检测O1群和O139群霍乱弧菌方法的建立及应用

目的建立检测O1群和O139群霍乱弧菌的实时荧光聚合酶链反应（RT—PCR）方法并进行水体标本的检测评价。方法根据O1群和O139群霍乱弧菌O抗原编码基因rfb序列设计引物，利用SYBRGreen染料，建立同时检测霍乱弧菌O1群和O139群的RT—PCR方法，对所建立的方法分别进行实验室内的灵敏度、特异性及重复性评价。采集河口水标本增菌后进行RT—PCR检测，与分离培养方法比较评价实际应用价值。结果建立了检测O1群和O139群霍乱弧菌的双重RT—PCR方法，根据扩增产物的溶解温度能有效区分O1群和O139群霍乱弧菌两种目标片段的扩增；对其他10种弧菌染色体DNA没有扩增；RT—PCR检测524份河口水体标本的增菌液，与常规分离方法相比显示了明显的灵敏性，并且所有常规分离方法阳性标本其荧光PCR检测亦为阳性。结论以O1群和O139群rfb基因为目标检测片段建立的霍乱弧菌RT—PCR方法可用于环境水体样本中霍乱弧菌常规分离前的快速筛查。     

猪链球菌脉冲场凝胶电泳分析方法的建立

目的 建立一种可共享的猪链球菌脉冲场凝胶电泳分析(PFGE)方法.方法 利用PulseNet技术优化猪链球菌PFGE分子分型方法,提出新的分析方案,包括胶块的制备、内切酶的选择、电泳条件等.利用DNAStar的Mapdraw软件对猪链球菌基因组序列进行分析,发现在242种内切酶中,Swa Ⅰ、Sma Ⅰ、Apa Ⅰ种酶比较适合.结果 通过对34个血清型共100株菌的分析,发现使用Swa Ⅰ可获得59种带型,使用Sma Ⅰ可获得53种带型,使用Apa Ⅰ可获得43种带型.其中以Swa Ⅰ的分辨率最好.结论 提出猪链球菌的Swa Ⅰ PFGE分析方法,并对条件进行优化,较原方法所需时间短、图像清晰,更具有可重复性,基本具备推广的条件.