棉枯萎菌血清学检测技术初步研究

以棉枯萎镰刀菌菌丝中孢子提取的蛋白质为抗原免疫家兔制备的２种抗血清Ａｓ２、Ａｓ１０中成分复杂，用琼脂双扩散法和比较对流免疫电泳法测定特异性，交叉反应明显。Ａｓ２用木贼镰刀菌的异原、Ａｓ１０用禾谷镰刀菌的异种抗原吸收排除非特异性成分后，抗血清的专化性提高，消除了交叉反应。特异性测定结果表明；利用此种经吸收后的抗血清检测棉枯萎镰刀菌可以准确地鉴定到尖孢镰刀菌种的水平，但不能区分专化型。     

Quantification and evaluation of antimicrobial drug use in group treatments for fattening pigs in Belgium

To control the emergence of antimicrobial resistance, knowledge of antimicrobial drug consumption is essential. Because consumption data are not available in Belgium, a study was conducted between March and October 2003 to investigate the antimicrobial drug consumption in pigs, using the treatment incidence based on the animal daily dose pig (ADDpig), the treatment incidence based on the used daily dose pig (UDDpig) (number of ADDpig or UDDpig/1000 pigs at risk/day), and the ratio UDDpig/ADDpig.

The sampling frame consisted of 821 pig herds that (a) used a closed or semi-closed production system, (b) were located in the most dense pig areas of Belgium, and (c) had at least 150 sows and 600 fattening pigs each. Of 50 randomly selected herds, all group treatments with antimicrobial drugs, applied to fattening pigs that were within 2 weeks of slaughter (median age 187 days), were collected retrospectively.

The treatment incidence based on ADDpig for all oral and injectable antimicrobial drugs was 178.1 per 1000 pigs at risk per day. The treatment incidence based on UDDpig shows that in reality fewer pigs were treated, namely 170.3 per 1000 pigs at risk per day. Proportionally, the most often applied oral antimicrobial drugs were: doxycycline, amoxicillin, combination trimethoprim-sulphonamides and polymyxin E. The most often applied injectable antimicrobial drugs were long-acting amoxicillin and ceftiofur. The distribution of the UDDpig/ADDpig ratio per antimicrobial drug shows that 50–75% of the oral formulations were underdosed. Injectable formulations were almost always overdosed (>90%).     

棉枯萎镰刀菌血清学检测方法和技术的研究

以棉枯萎镰刀菌公安菌株为材料,用物理法和化学法提取抗原并制备抗血清,并选有代表性的抗血清对其特异性和灵敏度分别采用4种检测方法进行了检测比较研究。结果表明:(1)采用 Richard’s培养基振荡培养菌株,化学法提取菌丝孢子抗原,按改进的 J.E.Devay 设计的免疫方案免疫,能产生效价高达1:64的抗血清。(2)琼扩、比较对流免疫电泳、间接 ELISA,SPA-ELISA 等4种方法检测其特异性具有鉴别到尖孢镰刀菌种的水平的特异性,但不能鉴别专化型和生理小种。并用上述方法检测抗血清灵敏度。     

广灵驴ADD1基因的克隆和序列分析与组织表达

运用RT–PCR方法，克隆广灵驴的ADD1基因的CDS区，对其进行序列分析，并通过qRT–PCR技术鉴定ADD1基因在广灵驴心脏、肝脏、脾脏、肺脏、肾脏和背最长肌中的相对表达水平。结果表明：广灵驴ADD1基因完整的CDS序列全长为2 223 bp，可编码740个氨基酸，序列已提交到NCBI，登录号为MN_166472，其核苷酸序列与马的核苷酸序列的同源性最高，达99.6%；生物信息学预测ADD1蛋白为结构稳定的亲水性蛋白，理论等电点为5.58，二级结构主要以α–螺旋和无规则卷曲为主，存在1个Ⅱ类醛缩酶和内收蛋白N端超家族结构域，该蛋白没有信号肽及跨膜区域，主要定位在细胞核中，序列中共存在88个磷酸化位点，69个O–糖基化位点和3个N–糖基化位点；实时荧光定量检测结果表明，ADD1基因在广灵驴的6种组织中均有表达，但存在差异，在背最长肌中表达量最丰富，在肝脏中表达量最低。     

Veterinary antimicrobial-usage statistics based on standardized measures of dosage

In human medicine, the defined daily dose is used as a technical measure of drug usage, which is independent of the variations in the potency of the active compound and the formulation of the pharmaceutical product—therefore providing a measure of the relative importance of different drugs. A national system of animal defined daily doses (ADD) for each age-group and species has been defined in VetStat (the Danish national system monitoring veterinary therapeutic drug use). The usage is further standardized according to the number of animals in the target population, acquired from production data on the national level or on herd size by species and age in the Danish central husbandry register (CHR). Statistics based on standardized measures of VetStat data can be used for comparison of drug usage between different herds, veterinary practices, or geographic regions (allowing subdivision by animal species and animal production class, route of administration, disease categories, season and geographic location). Individual statistics are available as interactive reports to the control authorities, farmers and veterinary practitioners by a secure access to the database. The ADD also is used in pharmaco-epidemiogical research and to assist in the interpretation of resistance-surveillance data.     

山羊ADD1基因部分序列的克隆与分析

提取湘东黑山羊基因组总DNA，用所设计的3对引物以聚合酶链式反应扩增山羊ADD1（Adipocyte Determination and Differentiation factor-1）基因，并进行克隆测序。序列分析表明所克隆的山羊ADD1基因包含exon 2～8、intron 2～7序列，将序列提交GenBank，获2个登录号：DQ480338、DQ487874；对编码序列（exon 2～8）与牛、人同区域进行Blast对比，同源性分别达到了96．14％和83．82％，所翻译氨基酸序列与牛属同源性更高达97．9％；此外，通过不同引物的测序结果比较，分别在exon6的第42处和intron6的第82处发现了一个碱基突变（C→T）和碱基插入／缺失（G／-），前者属于沉默突变，未引起氨基酸的变化。     

DHA对C2C12细胞成脂分化过程中相关基因表达的影响

采用胰岛素、地塞米松和3-异丁基-1-甲基黄嘌呤联合诱导C2C12细胞分化为脂肪细胞,诱导分化后加入100 mmol/L二十二碳六烯酸(Docosahexaenoic acid,DHA).DHA处理8d后,收集细胞,提取总RNA,采用实时荧光定量PCR方法检测脂肪分化相关基因PPARγ、C/EBPα和ADD1转录表达水平.结果表明,C2C12细胞成脂分化时添加DHA能显著上调PPARγ、C/EBPα和ADD1基因的表达,并且联合添加维生素E后效果更显著.     

山羊ADD1基因 exon13～18、intron 13～17序列的克隆分析

提取湘东黑山羊基因组总DNA,用所设计的引物以聚合酶链式反应扩增山羊ADD1基因,并进行克隆测序。通过对克隆所得片段的测序结果分析,得到了山羊ADD1基因外显子(exon)13~18、内含子(intron)13~17序列,并将序列提交GenBank,获两个序列号:DQ483057、DQ455606;对编码序列(exon 13~18)与牛、人同区域进行Blast对比,同源性分别达到了97.43%和86.12%。聚类分析结果表明,在6个物种中,牛与山羊ADD1基因的同源性最高,其它几个物种间同源系数相差不大,在83%至89%之间。     

马铃薯疮痂病菌(Streptomyces scabies)的血清学检测

以马铃薯疮痂病菌RB5(Streptomyces scabies)的菌丝-孢子为抗原免疫家兔,制备了抗血清AsRB5,并采用琼脂双扩散和酶联免疫吸附试验对抗血清进行特异性测定,结果表明,供试的10 株马铃薯疮痂病菌,1 株玫瑰黄链霉菌和1株玫瑰暗黄链霉菌与AsRB5 反应为阳性,其他15 株疮痂链霉菌与AsRB5 反应为阴性,作为对照的棉花黄萎病菌、立枯病菌、炭疽病菌与AsRB5 反应为阴性。以上结果表明,使用抗血清AsRB5 检测马铃薯疮痂病菌,基本上可以将其鉴定到种的水平上。但不能对同种内菌株进一步区分或进行多样性评价,并且这种鉴定与其地理来源及寄主无关。