Diagnosis of enzootic pneumonia in Danish cattle:: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle
Diagnosis of enzootic pneumonia in Danish cattle:: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle
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DOI：
10.1177/104063879901100505
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发表时间：
1999-09-01
影响因子：
1.5
通讯作者：
Uttenthal, Å
中科院分区：
文献类型：
--
作者：
Larsen, LE;Tjornehoj, K;Uttenthal, Å
建立了一种逆转录 - 聚合酶链反应（RT - PCR）检测方法，用于检测自然感染和实验感染牛肺组织中的牛呼吸道合胞体病毒（BRSV）。引物选自编码F融合蛋白的基因，该基因在BRSV分离株中相对保守。RT - PCR检测具有高度特异性，仅在对BRSV感染的细胞培养物或组织进行检测时才出现阳性反应。RT - PCR检测的检测限评估为5个半数组织培养感染剂量（TCID₅₀）。在用BRSV实验感染2 - 8天后对小牛实施安乐死，在其呼吸道组织和气管支气管淋巴结中检测到BRSV，而在所有时间点，其他组织样本以及模拟感染动物的样本均为阴性。对肺的8个不同区域的肺样本进行检查发现，尽管病毒最常出现在颅腹侧肺小叶，但也经常存在于所有肺小叶中。对1年内因诊断目的送检的所有牛的急性致命性肺炎病例（135头动物）进行微生物学检查，结果显示化脓放线菌（11%）、睡眠嗜血杆菌（10%）、巴斯德氏菌属（7%）和溶血性巴斯德氏菌（7%）是在肺中最常见的细菌病原体。使用常规抗原酶联免疫吸附测定（ELISA）在23头（17%）动物中鉴定出BRSV。已建立的BRSV特异性RT - PCR检测方法对相同的23头动物得出阳性结果，此外，ELISA检测为阴性的10头动物，RT - PCR检测为阳性。这些结果表明，RT - PCR检测可作为常规诊断程序的一种灵敏、可靠的替代方法。
A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal lymph node of calves euthanized 2-8 days after experimental infection with BRSV, whereas samples of other tissues and samples from mock-infected animals were negative at all time points. Examination of lung samples from 8 different regions of the lungs revealed that although the virus was most often found in the cranioventral lobules, it was frequently present in all lung lobules. Microbiologic examinations of all acute fatal cases of pneumonia (135 animals) in cattle submitted for diagnostic purposes during 1 year revealed that Actinomyces pyogenes (11%), Haemophilus somnus (10%). Pasteurella sp. (7%), and Pasteurella haemolytica (7%) were the most common bacterial agents found in the lungs. BRSV was identified using a conventional antigen enzyme-linked immunosorbent assay (ELISA) in 23 (17%) animals. The established BRSV-specific RT-PCR assay yielded positive results for the same 23 animals, in addition, 10 animals that were negative with the ELISA were positive with the RT-PCR assay. These results indicates that the RT-PCR assay can be a sensitive, reliable alternative to conventional diagnostic procedures.