犬细小病毒的分离鉴定与生物学特性分析  

Isolation and Biological Characterization of Canine Parvovirus Isolates

作　　者：周云朵[1] 康真玉[1] 陈月平[1] 史小娜[1] 陈建国[2] 赵雅心[3] 刘正飞[1] 

机构地区：[1]华中农业大学动物医学院农业微生物国家重点实验室,武汉430070 [2]华中农业大学动物医学院临床兽医系,武汉430070 [3]华中农业大学动物医学院实验教学中心,武汉430070

出　　处：《畜牧兽医学报》2011年第10期1402-1408,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：农业微生物学国家重点实验室教育部专项科研经费资助

摘　　要：本研究旨在获得华中地区的犬细小病毒分离株并对其基因型分型、生物学特性及致病力进行探讨。从武汉部分宠物医院采集胶体金初检阳性的14份可疑病料,用猫肾细胞(F81)进行病毒分离。根据犬细小病毒2型(CPV-2)保守的VP2,先后设计2对引物,进行PCR扩增。在细胞上对其进行空斑纯化,进行一步法生长曲线测定,并进行电镜观察。最后对这5株病毒都进行动物回归试验。细胞传代结果表明得到5株细小病毒。PCR扩增结果表明获得390和583bp 2个片段。将2个片段测序的结果与GenBank发布的CPV-2比对,表明所获得的5株病毒中,有4株属于2a型,1株属于2b型。空斑纯化及生长曲线绘制结果表明分离毒株增殖滴度可达105.5 PFU.mL-1。电镜观察结果表明,感染细胞的线粒体肿胀。动物试验结果表明犬都能表现不同程度的发病状况,大体解剖发现肠管有坏死、心肌变薄。将病变的心脏制作切片观察,结果表明心肌细胞变性、坏死。本研究结果为进一步研究CPV-2的分子生物学和分子致病机理奠定了基础。The aim of this study was to obtain Canine Parvovirus（CPV） strains in the middle of China and study their genetype,biological characterization and pathogenicity.14 feces swabs that were positive by colloidal gold test were collected from pet clinics in Wuhan.Then they were passaged in Feline kidney 81（F81）.2 pairs primers were designed according to conservative Capsid protein 2（VP2）.The strains were purified by plaque assay,and then one-step growth curves were obtained.Subsequently transmission electron microscopic（TEN） sections were made.And the animal experiment was carried out.5 CPV strains were isolated.390 bp and 583 bp nucleotide sequences were acquired.After sequencing and analyzing the gene fragments,the result shows that 4 strains were CPV-2a and 1 was CPV-2b.The curves show the growth of CPV at different stages after infection.The reproductive activity can reach or beyond 105.5 PFU·mL-1.The photographs of TEN showed that mitochondria are swelling.Finally,animal experiment shows that every dog had clinical symptoms in different degrees.The results of dissection reflect that there are necrosis in the small intestine and the myocardium becomes thinner.After pathological sections were made,observation was taken up and the result shows that myocardial cells are metamorphic and necrotic.This study provides groundwork for further study of molecular biology and pathopoiesia of CPV.

关 键 词：犬细小病毒 分离 电镜 动物回归试验 

分 类 号：S852.659.2[农业科学—基础兽医学]