PRRSV间接免疫荧光检测法的建立和应用

以猪生殖与呼吸综合征病毒(PRRSV) ATCC VR 2332 株人工单独感染3 头3 周龄仔猪,并用PRRSV ATCC VR 2332株和猪圆环病毒2型(PCV2)联合感染2 头3 周龄仔猪,每日检查试验猪体温、采食情况及临床表现,并用HerdChek IDEXX 试剂盒测其血清抗体。PRRSV PCV2联合感染猪分别在感染后1、3周剖杀,PRRSV单独感染猪和健康对照猪分别在感染后1、2和3周剖杀,对全部试验猪的肺组织进行灌洗处理以获得肺泡巨噬细胞(PAM),用PAM进行抹片,同时用肺组织直接进行触片做间接免疫荧光(IIF)检测。结果表明,用所获得的3 株单克隆抗体均检测到人工感染猪肺组织中的PRRSV抗原。另外,用IIF检测7 头临床疑似PRRS病猪的PAM抹片,其中沭阳县的2头猪呈现阳性反应;对这2头猪的肺组织进行病毒分离,在盲传到第4代时Marc 145细胞上产生明显的细胞病变,进一步证实用所获得的3株单克隆抗体检测临床样品的结果准确而可靠,且获得的3株单克隆抗体均可通过IIF用于检测猪肺组织中的PRRSV抗原。

Development of indirect immunofluorescent assay to detect porcine reproductive and respiratory syndrome virus

Three 3-week-old pigs were experimentally infected with PRRSV ATCC VR-2332 strain and two pigs with both PRRSV ATCC VR-2332 and PCV2. Sera were collected before and after inoculation. One of 3 PRRSV infected pigs and 1 control pig were necropsied on week 1, 2 and 3 post-infection(PI), respectively, while two pigs infected with both PRRSV and PCV2 were necropsied on week 1 and 3 PI, respectively. The PRRSV-specific antibody response was monitored with HerdChek IDEXX Kit. The serum antibody monitoring indicated that all serum samples were negative before inoculation and positive on week 2 PI. Slight pathological changes were observed in lung. The results of indirect immunofluorescent assay (IIF) attempts on lung showed that all samples were positive except for the samples from control pigs. In IIF test, PRRSV was monitored by 3 Mabs against nucleocapsid protein of PRRSV and detected in the lung. 7 PRRS-suspected pigs were also detected and the results showed that only 2 pigs from Shuyang were PRRSV positive in IIF test. Virus isolation was proceeded and the presence of PRRSV in cell lysates was confirmed by IIF test. The virus isolation in lung tissue showed that CPE was observed at the fourth passage and the fluorescence occurred in cytoplasm of infected Marc-145 cells. All results demonstrated that the 3 MAbs against nucleocapsid protein of PRRSV based on IIF test could be used to detect PRRSV in lungs of clinical cases.