藏獒源犬细小病毒的分离鉴定

为弄清犬细小病毒(CPV)在藏獒中的流行情况,将临床上经胶体金试纸检测为阳性的藏獒直肠棉拭子8份处理后接种猫肾传代细胞(F81细胞),分离到6株病毒,对其进行了血凝试验(HA)、半数组织培养物感染剂量(TCID50)的测定,理化性质鉴定,提取分离株DNA并通过聚合酶链式反应(PCR)扩增其一特异性片段和VP2全序列,并与GenBank上登录的CPV毒株进行比对。结果表明,接毒后第6～13天在F81细胞上出现了明显的细胞病变(CPE),表现为细胞融合、变圆或拉长;所得分离株能使猪红细胞发生凝集反应,血凝效价为27～211;能抗酸(pH3)、热(60℃)、200mL/L乙醚、480mL/L氯仿;从细胞培养物中分别扩增出与特异性片段825bp一致以及与VP2全基因1 755bp一致的条带。将扩增的VP2序列与GenBank上登录的HQ883267.1、FJ222823.1、FJ005214.1等10个国内外CPV分离株的VP2序列进行比对;结果显示,与北京地区一分离株(HQ883267.1)的同源性最高,为99.0%～99.6%。所得分离株为不同毒株,但均为CPV-2a型。

Isolation and identification of CPV strains from Tibetan mastiff

To study the epidemiology of canine parvovirus(CPV) in Tibetan mastiff,eight fecal samples were collected by cotton swab from Tibetan mastiff whose CPV test results were positive according to the immune Colloidal Gold kit.Six strains were isolated by cultivation in F81 cells.These strains were identified by HA,TCID50,physicochemical assay.The results showed that the typical cytopathogenic effect(CPE) was spotted in F81 cells from 6 to 13 days after infection.The isolates were able to agglutinate porcine erythrocytes,and the HA titer were from 27 to 211. These strains were all anti-acid(pH3),anti-heat(60℃) and insensitive to 200mL/L aether and 480mL/L chloroform.The VP2 gene(1755bp) and a 825bp fragment on it were amplified by PCR. Compared the isolates’ VP2 with 10 reference strains’(HQ883267.1,FJ222823.1,FJ005214.1,et al) from GenBank,the most similar one was HQ883267.1 with identity of 99.0% to 99.6%.All isolates were proved to be CPV-2a.