长角血蜱和微小扇头蜱的形态与分子生物学鉴定

目的 建立鉴定长角血蜱和微小扇头蜱的形态学和分子生物学相结合的方法.方法 采集自湖北和河南省布尼亚新病毒疫区家养动物体表寄生的蜱及草丛、灌木中的蜱;先以形态学鉴定,后用PCR方法扩增得到蜱的12S rDNA,测序后进行同源性和系统进化分析.结果 形态学方法鉴定采集到两种蜱:长角血蜱(Haemaphysalis longicornis)和微小扇头蜱(Rhipicephalus microplus).蜱的12S rDNA经克隆、测序,用PAUP 4.0软件构建系统发生树,湖北、河南省两种蜱的12S rDNA序列分别与长角血蜱和微小扇头蜱聚类,与形态学鉴定结果一致.结论 在传统形态学分类的基础上结合分子生物学鉴定方法能更准确地鉴定蜱的种类.     

新疆边境地区伊宁县图兰扇头蜱形态学及分子生物学鉴定

目的 对新疆边境地区伊宁县图兰扇头蜱进行形态学和分子生物学鉴定.方法 在新疆边境地区的伊宁县随机采集畜牧体表寄生蜱324只,进行形态学初步筛选,从中选取6只形态差异较大的图兰扇头蜱,进行线粒体16S rRNA和线粒体细胞色素C氧化酶亚基Ⅰ (CO Ⅰ)基因序列扩增、测序,并与GenBank登录的图兰扇头蜱参考序列进行遗传进化分析.结果 形态学鉴定采集的伊宁县蜱类皆为图兰扇头蜱,两段序列的碱基组成均显示较高的A+T比例(16SrRNA基因为73.8％,CO Ⅰ基因为70.34％),在变异程度上16S rRNA基因较CO Ⅰ基因明显保守.结论 首次应用形态学、16S rRNA和CO Ⅰ基因测序分析表明新疆伊宁县采集蜱皆为图兰扇头蜱,并证明图兰扇头蜱具有生物多样性.     

褐黄血蜱与铃头血蜱的分子生物学鉴定

目的建立2种形态学特征非常相近的褐黄血蜱及铃头血蜱的分子生物学鉴定方法,探讨它们的系统发生关系。方法在湖北省从动物体表采集寄生蜱,形态学鉴定完毕后,采用PCR方法从2种血蜱基因组中扩增12SrDNA、16SrDNA及线粒体细胞色素氧化酶亚基Ⅰ(COⅠ)基因,测序后进行同源性分析,用PAUP4.0软件分别构建系统发生树并进行系统进化分析。结果 2种血蜱12SrDNA、16SrDNA及COⅠ基因之间的同源性分别为90.8%、90.4%和86.8%,湖北省采集的褐黄血蜱3个基因片段与已知褐黄血蜱的同源性分别为100%、99.5%和99.7%。用这2种蜱的12SrDNA、16SrDNA及COⅠ基因的核苷酸序列分别构建系统发生树,湖北省采集的褐黄血蜱与已知的褐黄血蜱聚在一起,褐黄血蜱及铃头血蜱均形成独立的分支,但用3个基因构建的系统发生树中血蜱属不同种间的亲缘关系不同。结论对于形态学特征相近蜱种的鉴定,在传统形态学分类的基础上结合分子生物学鉴定方法能更准确地鉴定蜱的种类,也能更好地了解其进化关系,为蜱传播疾病的预防控制提供依据。     

通用型基因悬浮芯片检测生物恐怖细菌的方法研究

Objective To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. Methods 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp. and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B,the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. Results After PCR amplification by 16 S rDNA primers and specific probe hybridization,the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/μl (Burkholderia pseudomallei),20 pg/μl (Brucella spp.), 7 pg/μl (Bacillus anthracis), 0.1 pg/μ1 (Francisella tularensis), and 1.1 pg/μl (Yersinia pestis),respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%,it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. Conclusion The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.     

新疆北疆部分地区硬蜱伯氏疏螺旋体基因型研究

目的调查和鉴定新疆北疆石河子市、沙湾县、伊宁县和察布查尔县媒介蜱,并确定其携带的莱姆病螺旋体的基因型。方法选择4县市6个牧区为调查点,采集羊源寄生硬蜱,结合形态学与16S rRNA进行蜱种鉴定;采用BSK-H培养基对莱姆病螺旋体分离培养,并用硝酸银染色和巢式PCR法进行病原检测,阳性产物测序结果比对,并与11种GenBank参考序列比对,确定莱姆病螺旋体的基因型。结果6个调查点共采集寄生硬蜱900多只,经形态学与16S rRNA鉴定分别为图兰扇头蜱、刻点血蜱、亚洲璃眼蜱和边缘革蜱;从中分别选取样本硬蜱共102只,分24管,分离培养后结合巢式PCR和硝酸银染色共获得16管阳性培养物。5S～23S rRNA基因问隔区测序比对分析表明,所有菌株为伯氏疏螺旋体与国际报道的B.burgdoq''eri Sensu Stricto(B31)基因型具有高度同源性,同源性为98.6%～99.5%;OspC基因型分析与上述结果一致。结论4县市分离获得伯氏疏螺旋体,基因型为B.burgdooCeri Sensu Stricto,且存在生物多样性。从图兰扇头蜱中分离到伯氏疏螺旋体为我国首次报道。     

驻山东省部队野外驻训地域蜱种群调查分析

目的 摸清山东省野外部队驻训地域蜱分布和种群密度.方法 驻训地域游离蜱采用人工小时布旗法;动物宿主寄生蜱采用动物体表捡蜱法.结果 4个驻训地域均有蜱活动,蜱活动高峰期共捕获蜱虫12 808只,经鉴定均为长角血蜱.不同驻训地域的蜱密度存在显著差异性(F=6.791,P＜0.05),以淄博市沂源县(199.73只/h)最高...     

从实验室孵化的幼蜱中分离到一株玫瑰单胞菌

目的:从实验室孵化的幼蜱中分离到一粉红色菌株XTD622,确定其分类学位置。方法:使用16S rDNA序列分析和同源性检索方法及细菌的形态学鉴定方法。结果:菌株XTD622为革兰氏染色阴性、严格需氧的小球杆菌,在LB平板培养基上形成的菌落较小,圆形、光滑、湿润、有光泽,颜色为浅粉红色。16S rDNA序列分析和同源性检索发现XTD622和Roseomonas(玫瑰单胞菌)一些种的同源性较高,可达99%。结论:XTD622菌株在分类学上属于Roseomonas(玫瑰单胞菌)。     

一组长角血蜱中检出莱姆病螺旋体和斑点热群立克次体复合感染

目的 了解浙江省山区野生动物和蜱中莱姆病、斑点热、埃立克体病(无形体病)的感染情况.方法 采用巢式PCR对采集的鼠、蜱标本进行莱姆病伯氏疏螺旋体、斑点热群立克次体、埃立克体(无形体)特异性核酸片段检测分析.结果 从121份鼠标本和105组蜱标本中检出阳性结果 14份.鼠标本中检出伯氏疏螺旋体5S～23S rDNA间隔区片段1份和埃立克体(无形体)16SrDNA 5'端片段2份.蜱标本中检出阳性11份,包括伯氏疏螺旋体5S～23S rDNA间隔区片段3份和斑点热群立克次体外膜蛋白OmpA基因5'端片段8份.其中1组长角血蜱成虫标本为伯氏疏螺旋体和斑点热群立克次体复合感染,5S～23S rRNA基因间隔区和ompA基因片段均阳性,分别与伯氏疏螺旋体法雷氏基因型和马赛立克次体株等关系较近.结论 在同一组长角血蜱成虫中同时检出莱姆病疏螺旋体和斑点热群立克次体复合感染.     

软骨样副脊索瘤（附病例报告）

To study the clinical pathological characteristics and differentiation diagnosis of the chondroid parachordoma from primary tumors or metastatic to ulna, femur and radius. Methods Three cases of primary chondroid parachordoma of the ulma, femur and radius were studied by histopathological observation and immunohisto chemical staining and by pathological consultation. Results The histological features of tumor were composed of two the chordoma ceils subtotal and the chondrosareoma ceils of small - portio. The tumor was arranged vary in sizes of lobular and brink of lobular were seplum small girder of chondroid, or the tissue fiber. The tumor cells were distributed sheet, trabecula with or island -like in the intralobular, and the tumor ceils were vary in size and the karyon heterotypic were no patent and the cytoplasm of rich shows large vacuole. The chondroidosarcomas intra - lobular were viewed ceils both nucleus and uninuclear of chondrolacunae and the stroma were hyaling cartilage with or blennoid in the mesenchyma, and that picture of typical chondrosarcoma. EMA, CK, Vimentin, S - 100 protein, NSE with immunohistochemical method (ABC) staining were positive and Desmin, CD34 negative in the all of tumor cells. The chondroid parachodoma was diagnosed in the pathological read- section- conference and consulation. Conclusion Primary ulna, femur and radius chondroid parachordoma is a rare in rate of neoplasm and is diagnosed diflqculdy. Immunohistochemical method would be helpful to diagnosis.     

叔丁基对苯二酚对百草枯神经细胞毒性和氧化应激的拮抗作用

Objective To investigate the protective effects of the tert-butylhydroquinone (tBHQ)pretreatment on neurotoxicity and oxidative stress induced by paraquat (PQ) in PC12 cells. Methods Cytoyoxicity of PC12 cells was measured by MTT assay, following the PC12 cells treatment with different concentrations of 100, 300 μmol/L PQ for 24 h and 48 h. PC12 cells were pretreated with or without 40 μmol/L tBHQ for 4 h, PC 12 cells were exposed to PQ at the doses of 0, 100, 300 μmol/L for 24 h and 48 h, respectively.The viability of PC 12 cells was measured by MTT assay, the apoptosis rates of PC 12 cells were detected by flow cytometry (FCM) and the malondialdehyde (MDA) levels of PC 12 cells were examine by thiobarbituric acid (TBA) method. Results When the exposure doses of PQ were 100 and 300 μmol/L for 24 h, the viability of PC 12 cells pretreated with tBHQ was significantly higher than that of PC 12 cells only exposed to PQ (P＜0.05 or P＜0.01). When the exposure dose of PQ was 100μmol/L for 48 h, the viability of PC12 cells pretreated with tBHQ was significantly higher than that of PC12 cells only exposed to PQ (P＜0.01). When the exposure doses of PQ were 100 and 300 μmol/L for 24 h, the apoptosis rates and MDA levels of PC12 cells pretreated with tBHQ were significantly lower than those of PC12 cells only exposed to PQ (P＜0.05 or P＜0.01). Conclusions tBHQ preteatment can reduce the cytotoxicity, apoptosis and oxidative stress induced by PQ in PC12 cells.     

Rhipicephalus ticks infected with Rickettsia and Coxiella in Southern Switzerland (Canton Ticino).

Ticks of the Rhipicephalus sanguineus species complex may be vector of various pathogens including Rickettsia conorii (the etiological agent of the Mediterranean spotted fever) and Coxiella burnetii (cause of the Query (Q) fever). R. sanguineus ticks have been imported in several parts of central and northern Europe, especially in environments such as kennels and houses providing the appropriate microclimatic conditions and the blood source necessary for their survival. Since 1940 these ticks have occasionally been recorded in Switzerland. In Ticino (the southern part of Switzerland), they have been reported since 1980 and their probable establishment in this area has been suggested in the '90s. By means of PCR and direct sequencing, we tested the identity of these ticks (using 12S rDNA gene) and the occurrence of Rickettsia spp. (using 16S rDNA, gltA and OmpA genes) as well as Coxiella sp. (using 16S rDNA). The results indicated that in Ticino, two different tick species coexist, i.e. R. sanguineus sensu stricto and Rhipicephalus turanicus. A few individuals of R. sanguineus sensu stricto are infected with Rickettsia massiliae/Bar29, which are strains of unknown pathogenicity. Coxiella sp., an endosymbiont of Rhipicephalus ticks, has also been identified in both tick species. Due to climatic changes towards global warming, imported tick species may therefore adapt to new area and might be considered as epidemiological markers for a number of infectious agents transmitted by them.     

New Rickettsiae in ticks collected in territories of the former soviet union

Dermacentor nuttallii from Siberia, Rhipicephalus sanguineus from Crimea, and Rh. pumilio from the Astrakhan region were infected with Rickettsia sibirica (12%), R. conorii (8%), and the Astrakhan fever agent (3%), respectively. Three new Rickettsiae of the R. massiliae genogroup were identified in ticks by 16S rDNA, gltA, and ompA sequencing.     

Efficacy of Rhipicephalus (Boophilus) microplus Bm86 against Hyalomma dromedarii and Amblyomma cajennense tick infestations in camels and cattle

The recombinant Bm86-based tick vaccines have shown their efficacy for the control of cattle ticks, Rhipicephalus (Boophilus) microplus and R. annulatus infestations. However, cattle ticks often co-exist with multi-host ticks such as Hyalomma and Amblyomma species, thus requiring the control of multiple tick infestations for cattle and other hosts. Vaccination trials using a R. microplus recombinant Bm86-based vaccine were conducted in cattle and camels against Hyalomma dromedarii and in cattle against Amblyomma cajennense immature and adult ticks. The results showed an 89% reduction in the number of H. dromedarii nymphs engorging on vaccinated cattle, and a further 32% reduction in the weight of the surviving adult ticks. In vaccinated camels, a reduction of 27% and 31% of tick engorgement and egg mass weight, respectively was shown, while egg hatching was reduced by 39%. However, cattle vaccination with Bm86 did not have an effect on A. cajennense tick infestations. These results showed that Bm86 vaccines are effective against R. microplus and other tick species but improved vaccines containing new antigens are required to control multiple tick infestations.     

海南省琼中地区家畜体表寄生蜱的调查分析

目的了解家畜体表寄生蜱物种多样性及群落结构特点，并分析其与疾病的关系。方法采用逆毛式检蜱法，定义家畜体表寄生蜱为一个群落，运用生态学中相对优势度（组成比率）、优势指数、多样性指数和均匀度等方法进行测定。结果从放养的5群东山羊（20头）、4群黄牛（10头）和水牛（5头）中共检获蜱2614只，分属4个种属（血红扇头蜱、微小牛蜱、粒形硬蜱和钝刺血蜱），前两者为优势种；其中东山羊的优势度指数、多样性指数和均匀度等均高于黄牛和水牛。结论海南琼中地区家畜体表寄生蜱有4种，优势种为血红扇头蜱和微小牛蜱；其中东山羊的群落结构最丰富。应警惕斑点热群立克次体自然疫源性疾病的流行。     

GroEL gene typing and genetic diversity of Anaplasma bovis in ticks in Shaanxi,China

Anaplasma bovis, causative agent of bovine anaplasmosis, is usually identified by nested-PCR amplifying the rrs gene. However, it is difficult to determine the genetic relationship among different variants within A. bovis using this gene because of high conservation. In this study, two tick species, identified as Rhipicephalus microplus and Haemaphysalis longicornis based on morphological and molecular methods by analyzing COI gene, were collected from cattle, goat or sheep. Subsequently, A. bovis was initially detected by PCR amplifying the rrs gene in ticks in Shaanxi Province, China. The sequencing and Blast results showed that some false positive samples were found when only based on the amplification of partial rrs gene, presenting these sequences resembled those of other Alphaproteobacteria rather than A. bovis. Although major surface proteins genes were proposed and used successfully to identify members within Anaplasmataceae, these genes were unavailable for A. bovis. Hence, primers targeting the groEL gene were designed and a PCR assay was developed. The PCR products were sequenced and similarity and phylogenetic analysis suggested all these sequences are the groEL gene of A. bovis. In addition, phylogenetic analysis based on the groEL gene also revealed the genetic diversity of A. bovis worldwide, as well as in Shaanxi Province of China, which wasn't reflected by analyzing the rrs gene. In sum, groEL gene is important for molecular detection and phylogenetic analysis of A. bovis.     

野兔寄生蜱自然感染土拉弗氏菌的初步研究

目的调查研究陕西省野兔寄生蜱对土拉弗氏菌的自然感染情况。方法查阅文献资料,布旗拖蜱法采集游离蜱、逆毛拣蜱法采集寄生蜱和巢式-PCR法进行病原分子生物学检测。结果调查获得陕西省蜱类有15种,其中野兔寄生蜱有7种,占已知蜱种46.67%。应用巢式-PCR法对蜱体内携带土拉弗氏菌进行了检测,野兔寄生蜱仅有边缘革蜱和森林革蜱自然感染土拉弗氏菌;游离蜱仅有达吉斯坦革蜱、日本血蜱和嗜群血蜱自然感染土拉弗氏菌。结论陕西省蜱类是土拉弗氏菌病的潜在媒介。