大通县城奶牛乳样中葡萄球菌的分离与鉴定

通过细菌分离培养法对大通县城奶牛乳汁样品36份进行金黄色葡萄球菌的分离，并采用金黄色葡萄球菌常规鉴定方法血浆凝固酶试验、甘露醇发酵试验和接触酶试验进行了鉴定，36份乳样中检出金黄色葡萄球菌阳性7份，阳性率为19．7％(7／36)。     

乳房炎奶牛金黄色葡萄球菌毒素基因的检测及PFGE分型研究

作者针对临床及亚临床乳房炎奶牛乳汁中金黄色葡萄球菌分离株的毒素基因进行检测和脉冲场凝胶电泳(PFGE)基因分型,比较2种类型乳房炎金黄色葡萄球菌分离株的差异.无菌法采集奶样,采用国际标准方法从中分离金黄色葡萄球菌,用多重PCR方法扩增nuc基因和mecA基因以确证金黄色葡萄球菌(SA)和耐甲氧西林金黄色葡萄球菌(MRSA).进一步用PCR方法检测SA的各种毒素基因(SEs、ETs、TSST 1和PVL基因等).利用限制性内切酶Sma Ⅰ对SA基因组DNA进行酶切和PFGE分析,最后利用BioNumerics软件进行聚类分析.结果:19.3％(23/119)的临床乳房炎奶样和14.8％(26/176)的亚临床乳房炎奶样确定为金黄色葡萄球菌阳性样品,分别从中分离鉴定出43株和26株金黄色葡萄球菌,其中临床乳房炎分离株中有5株为mecA基因阳性.临床乳房炎奶牛奶样中检测到SA的SEA、SEB、SED、SEJ和PVL毒素基因,检出率分别为3.8％(1株)、11.5％(3株)、19.2％(5株)、7.7％(2株)和31.2％(10株);亚临床乳房炎奶牛乳样中仅检测到SA的SEA和PVL毒力基因,检出率分别为7.0％(3株)和84.1％(37株).表明临床与亚临床乳房炎奶牛乳汁中SA菌株携带的毒素基因不一样,SEs可能是临床乳房炎菌株的重要致病基因,PVL可能是亚临床乳房炎菌株的重要致病基因.69株SA使用Sma Ⅰ酶切分型后,可分为7个大簇、50个基因型,来源相同的SA分型后大部分位于同一簇内.临床乳房炎奶牛乳汁中检测到MRSA菌株,PVL基因在亚临床乳房炎中的检出率为临床乳房炎的2.7倍.PFGE方法能较好的区分临床乳房炎和亚临床乳房炎的SA分离菌株.     

温度对奥尼罗非鱼受精卵孵化和仔鱼活力的影响

研究了温度对奥尼罗非鱼(Oreochromis niloticus×O.aureus)受精卵孵化和仔鱼活力的影响。在盐度为5不同温度(22、24、26、28、30、32和34℃)下,比较观察奥尼罗非鱼受精卵的培育周期、孵化周期、孵化率和畸形率。同时在以上不同温度条件下对初孵的奥尼罗非鱼仔鱼进行耐饥饿试验,测定其不投饵存活系数(Survivalactivity index,SAI)。结果表明:在温度为24~32℃,受精卵孵化率较高(84.3%~91%),温度高于34℃和低于22℃时,孵化率较低。温度与培育周期呈负相关关系,函数关系为y=0.3607x2-26.836x+553.5,R2=0.973。仔鱼生存适宜温度是24~30℃,最适的温度是24~26℃。温度为22℃时,SAI值分别为19.9,但是仔鱼发育的缓慢,部分仔鱼的身体色素一直没有出现,不适合仔鱼的培育。     

聚合酶链式反应(PCR)技术检测牛奶中金黄色葡萄球菌

根据金黄色葡萄球菌特异性STAA-Aul基因序列,设计、合成了一对引物,运用聚合酶链式反应(PCR)技术从10头患有奶牛乳房炎的奶牛奶样中扩增得到8个大小为420 bp的DNA产物;阴性样品中未扩增到产物。测定了其中2个DNA片段的核酸序列,确定为金黄色葡萄球菌的STAA-Aul基因。     

Goat horns: Platforms for viroid transmission to fruit trees?

Mechanical inoculations with contaminating tools and propagation of infected budwood were considered the main causes for the omnipresence of multiple viroid species among citrus and other Middle Eastern and Mediterranean fruit trees and grapevines. However, neither means could explain viroid infections of wild trees — scattered on terrains inaccessible to humans — nor the finding of similar viroids among graft-incompatible plants. Northern hybridization of RNA extracts made of scrapings from the surfaces of goat (Capra hircus) horns that were rubbed against etrog (Citrus medica) stems infected with a citrus viroids complex, revealed accumulation of considerable amounts ofCitrus exocortis viroids (CEVd) andHop stunt viroids (HSVd). Experimental transmission of both CEVd and HSVd was obtained by rubbing healthy citrus plants with goat horns that had been rubbed 24 h earlier on infected etrog stems. These results implicate goats as possible vectors of viroids. Transmissionvia goats could have facilitated the long-range spread of viroids among cultivated and wild plants andvice versa and also among graft-incompatible plants.     

Study on Expression and Preliminary Immunocompetence of CIC Protein Containing Molecular Adjuvants

To express CTB-IsdBid-Clfais(CIC) protein and evaluate its immunogenicity, CTB (as a molecular adjuvant) could be tandem linked with IsdBid-Clfais gene by the overlapping PCR method, then CTB-IsdBid-Clfais(CIC) was inserted into pET-32a(+) vector to construct recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais. The recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais were transformed into Escherichia coli (E.coli) BL21 to express the CTB-IsdBid-Clfa(CIC) protein, the CIC expression protein and its immune activity were detected by Western blotting and ELISA,respectively. The results showed that the length of CIC gene were 2 072 bp, and CIC was correctly inserted into the pET-32a(+) plasmids, the pET-32a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed. Western blotting result confirmed that the molecular weight of CIC proteins was 95.9 ku, which were correctly expressed by E.coli BL21 with pET-32a (+)-CTB-IsdBid-Clfais plasmids. ELISA results showed that there was no significant difference among the CIC, IsdBid and Clfais protein groups (P>0.05), and there was extremely significant difference between CIC and BSA protein groups (P<0.01). In conclusion, the pET-32a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed, CIC proteins were correctly expressed, and were able to react with serum from mice immunized with IsdBid and Clfais,respectively,therefore, CIC proteins had strong immune activity.     

我国牛源金黄色葡萄球菌耐药现状及药敏检测方法探讨

为调查我国牛源金黄色葡萄球菌的临床耐药现状,对2009年以来新疆、浙江、山东、内蒙古和上海五省区不同地方分离的120株金黄色葡萄球菌用纸片扩散法进行临床药敏试验。结果显示,新疆分离株对红霉素、克林霉素、青霉素、复方新诺明、多西环素、四环素耐药,22%的菌株对头孢西丁耐药,43%的对氯霉素耐药,64%的对环丙沙星耐药,58%的对庆大霉素耐药;其它4地区分离株耐药情况更为严重,除头孢西丁以外,对其它9种抗生素均耐药。对菌株的多重耐药情况分析发现,新疆分离株中对5种以上抗生素耐药的占86.1%,对10种抗生素完全耐药的占17.4%;而内地分离株的数据为100%和38.2%。此外,在试验中鉴定出33株耐甲氧西林金黄色葡萄球菌,新疆19株,占新疆菌株的22.1%,内地四地区14株,占内地分离株的41.2%。结合2005~2009年间发表的相关耐药数据的分析结果,研究显示,我国牛源金黄色葡萄球菌呈现多重耐药,且出现了耐甲氧西林金黄色葡萄球菌,并在不同地区分离株中占有一定的比例;研究还发现,新疆分离株与浙江、山东、内蒙古和上海四地分离株的耐药情况有较大差异。此外,在整理药敏试验资料时,发现在金黄色葡萄球菌药敏试验中,药物的选择没有遵循选药规则,试验操作不规范和试验结果的报告不规范等问题,对此提出一些探讨性建议,供试验人员参考。     

北京地区奶牛隐性乳房炎金黄色葡萄球菌的分离鉴定及药敏试验

为了解北京地区奶牛隐性乳房炎中金黄色葡萄球菌的感染情况及对常用药物的敏感性,用科玛嘉显色培养基和16SrRNA PCR检测方法对5个奶牛场的100份隐性乳房炎奶样进行金黄色葡萄球菌的分离鉴定,采用K-B纸片扩增法进行药敏试验。结果表明,显色培养基分离到24株疑似金黄色葡萄球菌,经16SrRNA PCR鉴定,15株为金黄色葡萄球菌;药敏试验结果显示,15株金黄色葡萄球菌均对β-内酰胺类中的氨苄西林产生普遍耐药性,对克林霉素和环丙沙星的耐药率较高,分别为53.33%和40%;对氧氟沙星、庆大霉素、头孢噻肟和头孢曲松的耐药性较低,耐药率为6.67%。说明15株金黄色葡萄球菌分离株对7类9种抗菌药物都有不同程度的耐药性。     

橙皮素对金黄葡萄球菌α-溶血素表达的抑制作用

α-溶血素在金黄葡萄球菌的致病过程中发挥着不可或缺的作用。本研究采用最小抑茵浓度测定、茵液上清溶血活性以及α-溶血素含量测定、荧光定量PCR及细胞毒性的测定等相关试验验证了橙皮素对金黄葡萄球菌α-溶血素表达的影响。结果显示,无抗茵活性的橙皮素在较低质量浓度下即可抑制金黄葡萄球菌α-溶血素的表达及其编码基因的转录,是-种潜在的以毒力因子为靶标的前导化合物,并可进一步开发用于抗金黄葡萄球茵感染。     

奶牛金黄色葡萄球菌性乳房炎研究进展

奶牛乳房炎是对世界奶牛业造成经济损失最大的疾病之一,它不仅直接导致乳品工业的经济损失,而且由于患乳房炎的奶牛其乳汁中经常含有大量的毒素、病原微生物以及治疗后残留的抗生素,使得该病日益引起公众的关注。金黄色葡萄球菌是奶牛乳房炎的主要病原,由于其极易产生耐药性,使得治疗变得相当困难,因此使用预防或治疗性疫苗防治乳房炎就成为首选。金黄色葡萄球菌对上皮细胞的黏附和侵袭力被认为是奶牛金黄色葡萄球菌性乳房炎的主要致病机制,金黄色葡萄球菌的致病力涉及到许多不同的毒力因子,如黏附素、荚膜、各种毒素和酶类等。研究表明,以这些毒力因子为靶位进行疫苗研究,可能是预防奶牛金黄色葡萄球菌性乳房炎的有效途径。