间接免疫细胞化学法快速检测新城疫病毒抗原的研究

本研究旨在建立一种简单、方便、准确、特异的方法检测新城疫病毒（Newcastle disease virus, NDV)。比较间接HRP-免疫细胞化学法与间接免疫荧光法两种方法检测NDV在COS-7细胞中的定位。两种试验方法都可以检测到NDV主要定位在COS-7细胞的胞浆内。间接HRP-免疫细胞化学法检测NDV要求一抗滴度≤1∶100,间接免疫荧光法要求的滴度≤1∶100。间接HRP-免疫细胞化学法检测NDV要求二抗滴度≤1∶300,间接免疫荧光法要求的滴度≤1∶50。而且结果观察中间接HRP-免疫细胞化学法要比间接免疫荧光法方便。间接HRP-免疫细胞化学法进行NDV检测优于间接免疫荧光法,是一种简单、快捷、准确的适用于检测NDV的技术。     

湖南省新化县高风险区羊布鲁氏菌病横断面研究及活羊流通网络调查

湖南省新化县近几年动物布鲁氏菌病疫情和人间病例不断增多。为掌握新化县羊群布鲁氏菌病的群体流行率,了解饲养场户、贩运户和经销商对该病的知晓情况以及羊贩运户和经销商的一般经济行为,寻找养殖环节中导致布鲁氏菌感染的风险因素,应用流行病学方法,以新化县10个乡镇现存栏羊15只以上的养殖场户为研究对象,第一阶段以场户为单位,以估计流行率为目的,以名单为抽样框,随机抽样;第二阶段在群内选择个体,以发现疫病为目的,随机或基于风险采样。将采集的血清样品,采用虎红平板凝集和竞争酶联免疫吸附垂直试验进行检测。重点调查出现动物布鲁氏菌病疫情和人间病例乡镇的养殖场,以及活羊贩运经纪人和经销商等从业人员,掌握羊群布鲁氏菌病流行情况和活羊流通情况。调查结果显示:新化县高风险区存栏羊15只以上养殖场户的羊布鲁氏菌病流行率为0(95%CI:0～2.8%);新化县羊养殖场户以小规模自繁自养为主,一般放牧于自家附近山头;出栏羊主要在县内流通,养殖场户内生物安全防护措施不到位;养殖场户的布鲁氏菌病总体知晓率为80.6%,屠宰零售商为15.2%,贩运经纪人为25.0%,职业人群自我防护差;活羊调入以省外为主,冬季调运频繁,主要流向屠宰终端,经纪人在流通环节中充当重要角色。调查结果提示:应加强活羊流通领域的检疫监管,特别是冬季,要提高监测频次;当地需坚持实行"监测+扑杀"的综合防控策略,持续开展布鲁氏菌病相关的健康宣教和行为干预,重点针对屠宰经销商和经纪人,以提高职业人群的预防意识,培养其良好的职业行为和习惯。本研究为新化县布鲁氏菌病防控提供了依据,也为全国布鲁氏菌病防控积累了经验。     

基于SSR标记分析四川省桑树种质资源的群体结构和遗传多样性

利用14对SSR引物对四川省89份桑树种质资源的基因组DNA进行PCR,分析种质资源间的群体结构和遗传多样性。共扩增出迁移率不同的条带65条,多态性条带65条,多态性条带比率达100%;Structure群体结构分析将89份桑树种质资源划分为4个群体,4个群体的多态性信息含量在0.552 0~0.599 8之间,总体为0.657 7;基因多样度在0.554 8~0.600 3之间,总体为0.579 0;等位基因数目在66~76个之间,总体为134个;等位基因丰富度在3.327 2~3.549 1之间,总体为3.983 1;遗传多样性主要来源于品种内,属中等偏高水平;14对SSR引物位点的总基因多样度Ht在0.291~0.931之间,整体为0.699;Nei’s种群内基因多样度Hs为0.287~0.910,整体为0.662;种群间基因多样度为0.003~0.098,整体为0.037;种群间基因分化系数为0.008~0.047,整体为0.053;基因流为2.901~62.000,整体为8.934,遗传变异主要发生在群体内,群体间分化程度低,基因流大。UPGMA聚类分析与群体结构分析结果大致相同,2种分析结果都显示分群无地理迁移规律。     

IBRV重组gD蛋白的真核表达与间接ELISA检测方法的建立

通过优化牛传染性鼻气管炎病毒（IBRV）gD基因序列为Sf9细胞偏好密码子,转座形成穿梭载体,将质粒瞬时转染Sf9昆虫细胞,利用昆虫杆状病毒表达系统表达纯化重组IBRV gD蛋白;以制备的重组IBRV gD蛋白为包被抗原,建立检测IBRV血清中和抗体的间接ELISA方法,并通过比对检测已知血清中和抗体效价的牛血清,验证所建立的间接ELISA方法的敏感性、特异性与重复性等指标。结果显示：本研究建立的间接ELISA方法对相关的牛病毒血清抗体无交叉反应,敏感性可达1:512,组内和组间变异系数均低于10%;使用建立的间接ELISA检测方法结合血清中和试验,对480份临床血清样品进行检测,发现两者敏感性符合率为98.18%,特异性符合率为93.33%,总符合率为96.67%。结果表明,本研究建立的检测IBRV血清中和抗体的间接ELISA方法具有良好的特异性、敏感性和重复性,可开发为临床适应的检测试剂盒。     

鸡痘病毒282E4株2.2kbTK基因序列分析

将鸡痘病毒２８２Ｅ４弱毒株基因组中的３．７ｋｂＨｉｎｄⅢ片段克隆到ＫＳ（－）质粒中，亚克隆后，获得含ＴＫ基因正反方向插入的２．２ｋｂＨｉｎｄⅢ－ＣｌａＩ片段的质粒ｐＫＦＡ和ｐＫＦＢ，进而构建出正反方向的系列嵌套缺失质粒。用Ｔ７、Ｔ３引物所做的核苷酸序列分析表明，２．２ｋｂＨｉｎｄⅢ－ＣｌａＩ ＴＫ基因序列长为２１５９ｂｐ，含有两个完整的读码框架（ＯＲＥ）Ｘ和ＴＫ，并确证了ＴＫ基因内存在单一酶切点Ｎ     

Novel avian orthoavulavirus 13 in wild migratory waterfowl: biological and genetic considerations

Avian orthoavulavirus 13 (AOAV-13), formerly known as Avian paramyxovirus 13 (APMV-13), is found scatteredly in wild birds around the world. Although four complete genome sequences of AOAV-13 had been identified since the first discovery in Japan in 2003, the information available on the genetic variation and biological characteristics of AOAV-13 is still limited. In the present study, we isolated six AOAV-13 strains from fecal samples of wild migratory waterfowls during annual (2014–2018) viral surveillance of wild bird populations from wetland and domestic poultry of live bird markets (LBMs) in China. The phylogenetic analyses based on the HN and F genes showed that they had very close relationship and the molecular clock estimations showed a low evolutionary rate of AOAV-13. However, Bean goose/Hubei/V97–1/2015 is 1953 nt in size (ORF, 1, 776 nt), which is a unique size and longer than other reported AOAV-13 strains. Additionally, four repeats of conserved sequences “AAAAAT” was presented in the 5′-end trailer region of Swan goose/Hubei/VI49–1/2016, which is unprecedented in the AOAV-13. These findings highlight the importance of continuous monitoring the specific species of APMVs.

     

应用套入式聚合酶链反应(Nested-PCR)检测蓝舌病病毒的研究

本实验建立了一种Ｎｅｓｔｅｄ－ＰＣＲ方法。应用方法检测５株标准株蓝舌病病毒（Ｔ４、Ｔ１０、Ｔ１１、Ｔ１６、Ｔ２０）和５株国内蓝舌病病毒分离株（ＣＦ４、ＡＦ６、Ｚ１、Ｈ１、Ｇ１４）,检测结果均为阳性,而检测相关环状病毒（ＥＨＤ２、ＥＨＤ６、Ｉｂｒａｋｉ）,检测结果均生。研究证明,此方法可检测到３．５ｆｇ的ＢＴＶ－ＲＮＡ,灵敏度较高。该方法成功区分了蓝病病毒和相关环状病毒,比血清学方法更加优势,在临床     

基于16srDNA序列分析冬樱花花期蜜囊细菌多样性

对昆明冬樱花花期东、西方蜜蜂蜜囊细菌进行分离、纯培养;并对分离菌株的16srDNA基因片段进行扩增和测序,序列比对后进行系统发育分析。结果表明:东方蜜蜂蜜囊细菌包括3个纲中的10个种,西方蜜蜂蜜囊细菌包括3个纲中的13个种,花蜜细菌包括4个纲中的6个种,所采集样品除Bacillus subtili相似以外,其余菌种差异较大。     

Pharmacokinetics of cefquinome in tilapia (Oreochromis niloticus) after a single intramuscular or intraperitoneal administration

The pharmacokinetics of cefquinome was studied in plasma after a single dose (10 mg/kg) of intramuscular (i.m.) or intraperitoneal (i.p.) administration to tilapia (Oreochromis niloticus) in freshwater at 30 °C. Ten fish per sampling point were examined after treatment. The data were fitted to two‐compartment open models following both routes of administration. The estimates of total body clearance (CL/F), volume of distribution (Vd/F), and absorption half‐life (T_1/2ka) were 0.049 and 0.037 L/h/kg, 0.41 and 0.33 L/kg, and 0.028 and 0.035 h following i.m. and i.p. administration, respectively. After i.m. injection, the elimination half‐life (T_1?2β) was calculated to be 5.81 h, the maximum plasma concentration (C_max) to be 49.40 μg/mL, the time to peak plasma cefquinome concentration (T_max) to be 0.14 h, and the area under the plasma concentration–time curve (AUC) to be 204.6 μg h/mL. Following i.p. administration, the corresponding estimates were 6.05 h, 44.39 μg/mL, 0.17 h and 267.8 μg h/mL. The minimum inhibitory concentrations of cefquinome, determined for 30 strains of Streptococcus agalactiae isolated from diseased tilapia, ranged from 0.015 to 0.12 μg/mL. Results from these studies support that 10 mg cefquinome/kg body weight daily could be expected to control tilapia bacterial pathogens inhibited in vitro by a minimal inhibitory concentration value of ≤2 μg/mL.