番鸭小鹅瘟病免疫荧光诊断方法的建立及应用
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摘要
小鹅瘟病是由鹅细小病毒(Goose Parvovirus,GPV)感染1月龄以内雏鹅和雏番鸭,引起拉稀和小肠形成栓塞为特征的一种传播快、死亡率高的高度接触性传染病。该病常与番鸭细小病毒病混合感染,根据流行病学、临床症状和病理变化难以确诊,因此,研制简便快速的诊断方法对本病确诊具有重要意义。
采用透析法将抗GPV单抗标记上异硫氰酸荧光素(FITC),研制出GPV直接荧光抗体试剂,建立了检测GPV抗原的直接免疫荧光检测方法(DFA)。该方法特异性强(仅与GPV反应,对MPV、MDRV、PMV等均无交叉反应)、敏感性高(最佳工作浓度为1:150,1:450倍稀释仍对抗原敏感)、快速检测(60分钟内结果)和重复性好等特点。该方法与病毒分离法和间接荧光法(IFA)比较,符合率分别为100%和95.%,三者总符合率为95.%。应用DFA检测GPV在人工感染番鸭的第3天,第5天,第7天和第10天各器官中病毒的动态分布结果表明,感染早期在心、肝、脾、肾、胰腺、盲肠扁桃体均为强阳性;感染后期在心、肝、脾呈弱阳性,在胰腺和盲肠扁桃体呈强阳性。进一步证实了DFA方法特异性好,可用于番鸭小鹅瘟病的临床快速诊断和GPV抗原在组织中的定位。
The goose plague, caused by Goose Parvovirus (GPV) in gosling and Muscovy duckling within 30 days old, is an epidemic contagious infection with diarrhea, embolism in small intestine and high mortality. It’s necessary to establish a simple and rapid diagnostic method of the disease for outbreak cases are often infected contemporaneously with muscovy ducks parvovirus and difficult to diagnose by epidemiology, clinical symptoms and postmortem. In this study, direct immunofluorescence assay (DFA) was developed to detect GPV antigen, with dialysis of GPV monoclonal antibody labeled by fluorescein isothiocyanate (FITC). The results showed that positive reaction to GPV is only observed after DFA reacts to MDRV, MPV and PMV for 60 minutes. Optimum concentration of the reagent of DFA is 1:150, it is also sensitive until dilution by 1:450. Compared to pathogen isolation and indirect immunofluorescence assay (IFA ), coincident rate between them was 100%, was 95%, respectively and average coincident rate of DFA, IFA, and pathogen isolation was 95%. With DFA, the distributions of the virus in various tissues were determined at the third day, the 5th day, the 7th day and 10th day after muscovy ducklings were challenged by GPV,showing that GPV is detected obviously from heart, liver, spleen, kidney, lung, pancreas and caecum at the early stages of infection and the virus is detected obviously from pancreas and caecum at the later stages of infection. Sensitivity of DFA to GPV was further confirmed. The results indicate that DFA is specific, sensitive, repeatabe to determine GPV, it can be used for the rapid diagnosis and epidemiological investigation of Muscovy duckling infection of GPV.
采用透析法将抗GPV单抗标记上异硫氰酸荧光素(FITC),研制出GPV直接荧光抗体试剂,建立了检测GPV抗原的直接免疫荧光检测方法(DFA)。该方法特异性强(仅与GPV反应,对MPV、MDRV、PMV等均无交叉反应)、敏感性高(最佳工作浓度为1:150,1:450倍稀释仍对抗原敏感)、快速检测(60分钟内结果)和重复性好等特点。该方法与病毒分离法和间接荧光法(IFA)比较,符合率分别为100%和95.%,三者总符合率为95.%。应用DFA检测GPV在人工感染番鸭的第3天,第5天,第7天和第10天各器官中病毒的动态分布结果表明,感染早期在心、肝、脾、肾、胰腺、盲肠扁桃体均为强阳性;感染后期在心、肝、脾呈弱阳性,在胰腺和盲肠扁桃体呈强阳性。进一步证实了DFA方法特异性好,可用于番鸭小鹅瘟病的临床快速诊断和GPV抗原在组织中的定位。
The goose plague, caused by Goose Parvovirus (GPV) in gosling and Muscovy duckling within 30 days old, is an epidemic contagious infection with diarrhea, embolism in small intestine and high mortality. It’s necessary to establish a simple and rapid diagnostic method of the disease for outbreak cases are often infected contemporaneously with muscovy ducks parvovirus and difficult to diagnose by epidemiology, clinical symptoms and postmortem. In this study, direct immunofluorescence assay (DFA) was developed to detect GPV antigen, with dialysis of GPV monoclonal antibody labeled by fluorescein isothiocyanate (FITC). The results showed that positive reaction to GPV is only observed after DFA reacts to MDRV, MPV and PMV for 60 minutes. Optimum concentration of the reagent of DFA is 1:150, it is also sensitive until dilution by 1:450. Compared to pathogen isolation and indirect immunofluorescence assay (IFA ), coincident rate between them was 100%, was 95%, respectively and average coincident rate of DFA, IFA, and pathogen isolation was 95%. With DFA, the distributions of the virus in various tissues were determined at the third day, the 5th day, the 7th day and 10th day after muscovy ducklings were challenged by GPV,showing that GPV is detected obviously from heart, liver, spleen, kidney, lung, pancreas and caecum at the early stages of infection and the virus is detected obviously from pancreas and caecum at the later stages of infection. Sensitivity of DFA to GPV was further confirmed. The results indicate that DFA is specific, sensitive, repeatabe to determine GPV, it can be used for the rapid diagnosis and epidemiological investigation of Muscovy duckling infection of GPV.
引文
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3 Hlinak A,Muller T,Kramer M, Muhle Ru, Liebherr H, Ziedler K.Secological survey of viral pathogens in bean and white-fronted geese from Germany.[J].Wildl Dis.1998 Jul: 34 (3):479-486.
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5 Sirivan P, Obayashi M, Nakamura M, Tantaswasdi U, Takehara K.Detection of goose and Muscovy duck es using polymerase chain reaction-restriction enzyme fragment length polymorphism analysis[J].Avian Dis.1998 Jan-Mar;42(_1):133-139.
6殷震,刘景华主编.动物病毒学.第2版[M].北京:科学出版社,1997,1165-1168.
7章金刚,向华,杜华茂,禽类细小病毒的分子生物学[J].中国兽医学报,1999,19(4)412.
8 Qiu J, Cheng F, Yoto Y, Zadori Z, Pintel D. The expression strategy of goose exhibits features of both the Dependvirus and general[J].Virol,2005 Sep;79(17): 1035-1044.
9 Kisary J, Valose B, Miller Faurs A, etal. The genome structure of a new chicken virus identifies it as a [J].Journal of General Virology, 1985, 66: 2259-2263.
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11 Brown K E, Green S W, Yong N S. Goose Parvovirus an Autonomous Member of the depend virus Gene [M] Virology, 1995, 210:283-291.
12 Zadori Z,Erdei J,Nagy J, etal,Characteristies of the genome of goose [M].Avian pathol,1994,23:359-364.
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