目的 建立牛血清中牛病毒直接免疫荧光检测方法,并进行验证.方法 建立直接免疫荧光法检测牛血清中牛病毒,对细胞接种浓度和血清浓度、病毒接种量、荧光抗体浓度、染色温度及时间进行优化;对优化的方法进行重复性、特异性、灵敏度验证,并与细胞培养法的检测结果进行比较.结果 优化的试验条件为:Vero和BT细胞的接种浓度分别为0.5×105和1.0× 105个/ml,血清浓度为5％,病毒接种量为100～300 CCID50,荧光抗体1∶10稀释,4℃染色12h以上,但不超过24 h.2名实验人员按建立的方对3批牛血清分别进行3次检测,均未检出6种牛病毒;每种病毒仅在相应抗体进行染色时出现荧光;该方法检测6种牛病毒的灵敏度较高.分别采用细胞培养法和直接免疫荧光法检测15批新生牛血清和5批胎牛血清,结果均未检出牛病毒.结论 建立的牛血清中牛病毒直接免疫荧光检测方法重复性好,特异性强,灵敏度高,操作简便,与细胞培养法的检测结果无差异,可应用于牛血清中牛病毒的检测.

Objective To develop and verify a direct immunofluorescence assay for bovine virus in bovine serum.Methods A direct immunofluorescence assay was developed for determination of bovine virus in bovine serum,of which the cell concentration for inoculation,serum concentration virus inoculation quantity,fluorescent antibody concentration as well as time duration and temperature for staining were optimized.The optimized method was verified for reproducibility,specificity and sensitivity,by which the determination result was compared with that by cell culture method.Results The test condition was optimized as follows:the concentrations of Vero and BT cells for inoculation were 0.5 × 105 and 1.0 ×105 cells / ml,the serum concentration was 5％,the virus inoculation quantity was 100 ～ 300 CCID50,the dilution of fluorescent antibody was 1 ∶ 10,while the temperature for staining was 4 ℃,and the time for staining was more than 12 h but not more than 24 h.Three batches of bovine sera

为了解新疆地区部分规模化奶牛场牛传染性鼻气管炎病毒、牛病毒性腹泻病毒、牛呼吸道合胞体病毒、牛副流感3型病毒和牛支原体的感染状况,本试验采用酶联免疫吸附试验(ELISA)检测试验区奶牛场中未接种上述疫病疫苗的奶牛血清,对自然感染状况进行评估。结果表明,牛传染性鼻气管炎病毒抗体平均阳性率为82.5%(66/80),牛病毒性腹泻病毒抗体平均阳性率为88.8%(71/80),牛呼吸道合胞体病毒抗体平均阳性率为82.5%(66/80),牛副流感3型病毒抗体平均阳性率为91.3%(73/80),牛支原体抗体平均阳性率86.3%(69/80);试验区牛场均存在2种~5种病原的混合感染现象,其中5种病原混合感染率为71.3%。结果显示在新疆地区部分规模化奶牛场中普遍存在上述5种病原的感染,且混合感染现象明显,奶牛生产中必须加强这些疫病的防控工作。

In order to find out the infection status of infectious bovine rhinotracheitis virus (IBRV) ,bovine viral di-arrhea virus(BVDV) ,and bovine respiratory syncytial virus (BRSV) ,bovine parainfluenza 3 virus(BPIV3) and Mycoplasma bovis infections in several large-scale dairy cow farms in Xinjiang ,the enzyme-linked immunosorbent assay (ELISA) was used to detect bovine sera from pre-immune dairy cows and to evaluate naturally occurred in-fections of these diseases in experimental dairy farms .The results showed that the average antibody positive rates for infectious bovine rhinotracheitis virus ,bovine viral diarrhea virus ,bovine respiratory syncytial virus ,parainflu-enza virus 3 ,and Mycoplasma bovis were 82 .5% (66/80) ,88 .8% (71/80) ,82 .5% (66/80) ,91 .3% (73/80) and 86 .3% (69/80) ,respectively .There was a mixed infection with 2 to 5 kinds of pathogens in the investigated dairy farms ,and mixed infection with these five pathogens was reached to 71 .3% .These results indicated that

对牛病毒性腹泻-粘膜病的病毒分离、琼脂扩散试验、微量中和试验、免疫荧光技术、酶联免疫吸附试验、核酸杂交技术、聚合酶链反应等检测技术进行了综述，对7种技术运用于牛病毒性腹泻-粘膜病的检测作了简要介绍，为进一步研究牛病毒性腹泻病毒提供参考。

This paper gave a review of the diagnostic methods including virus isolation, agar gel immunodiffusion, virus micro-neutralization test, fluorescent antibody test, enzyme-linked immunosorbent assay, nucleic acid hybridization, polymerase chain reaction, introduced seven technology to detect bovine viral diarrhea virus, and provided references for the further research of bovine viral diarrhea virus.

牛呼吸道合胞体病毒是引起牛呼吸道疾病的主要病原之一。进行牛呼吸道合胞体病诊断时,首先通过临床症状观察以及病理剖检变化进行初诊,然后再进行实验室诊断。其实验室检测主要依赖于病原学诊断和血清学诊断,病原学诊断方法主要包括细胞分离培养鉴定、聚合酶链反应。血清学方法包括中和试验、免疫荧光试验、酶联免疫吸附试验等。近年来聚合酶链反应﹑酶联免疫吸附试验等方法得到快速发展,凭借其高效、快速、灵敏性高的特点成为牛呼吸道合胞体病毒检测的常用方法。牛呼吸道合胞体病在全球范围内流行,对各国养牛业造成极大危害。论文综述了牛呼吸道合胞体病毒检测方法的研究进展,为牛呼吸道合胞体病的诊断和预防提供参考。

Bovine respiratory syncytial virus is recognized as one of the crucial causes of bovine respiratory disease,which has a marked impact on the cattle industry and the dairy industry.Bovine respiratory syncy-tial virus is preliminarily diagnosed based on the clinical symptoms and pathological anatomy changes,and then through the laboratory tests.The laboratory tests of bovine respiratory syncytial virus mainly rely on etiology diagnosis and serological diagnosis.The methods for etiology diagnosis consists of cell-culture iso-lation techniques,polymerase chain reaction.And the serological methods consists of neutralization tests, immunofluorescence method,enzyme linked immunosorbent assay.For the past few years,the experimen-tal methods,such as polymerase chain reaction and enzyme-linked immunosorbent assay were developed rapidly,and became the main methods for the diagnosis of bovine respiratory syncytial virus due to their high efficiency,rapidness and high sensitivity.The bovine respira

目的对1株蝙蝠来源病毒进行分离鉴定,进行简单的基因分析。方法从云南沧源县8个居民地附近捕获50只棕果蝠并制作组织标本,采用细胞培养分离病毒,采用cDNA-RAPD方法扩增序列,采用分子克隆进一步扩增序列,将序列在NCBI上进行BLAST比对,采用DNAMAN5.0进行同源性分析,采用MEGA 5建序列进化树。结果 50份接毒细胞中,6份细胞出现细胞病变,cDNA-RAPD扩增得到1份阳性片段,测序大小为614 bp,经比对该病毒株是细小病毒科细小病毒亚科博卡病毒属牛细小病毒种的一个毒株。结论成功分离并鉴定该毒株,确定该病毒是牛细小病毒的一个新的基因型。

Objective To isolate and identify a virus from bats and to make simple gene analysis. Methods 50 rousettus leschenaultias were captured from the surrounding areas of 8 residences in Cangyuan County,Yunnan Province and some of their organs were collected;cell culture was adopted to isolate the virus;the sequence of the virus was first amplified by cDNA-RAPD and then further amplified by molecular cloning;the sequence was compared in the NCBI;the homology analysis of the virus was carried out by DNAMAN5. 0 and the phylogenetic analysis was executed by MEGA 5. Results Fifty cells were infected and six cells turned to be CPE and a positive gene fragment was obtained by cDNA-RAPD. The sequencing result indicated that the fragment was 614 bp long. The virus was identified to be one of the Bovine parvovirus of Bocavirus of Parvovirinae of Parvoviridae. Conclusions The isolation and identification of the virus was successful and the virus was confirmed to be a new genotype of Bovine parvovirus