Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fujian Animal Disease Control Technology Development Center, Fuzhou 350013, China.
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, National Research Center for Engineering and Technology of Veterinary Bio-Products, Nanjing 210014, China.
Viruses. 2023 Jun 27;15(7):1453. doi: 10.3390/v15071453.
The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5'-UTR region and the 5/2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), (), (), and IBDV-negative field samples. The method showed a linear dynamic range between 10 and 10 DNA copies/reaction, with an average R of 0.99 and an efficiency of 93% for nVarIBDV and an average R of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the 2 gene or the reverse transcription PCR (RT-PCR) for the 5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.
新型 IBDV（nVarIBDV，特别是基因型 A2dB1）主要影响中国的肉鸡。它引起的感染特征是法氏囊萎缩、淋巴细胞水平下降、滤泡周围纤维组织增殖以及滤泡严重萎缩。用活 IBDV 疫苗接种的家禽不会出现法氏囊萎缩的挑战，这是因为缺乏其他明显的临床症状，而被误诊为 nVarIBDV。本研究旨在探索实时 TaqMan 分析方法检测和区分 nVarIBDV 基因型与非 nVarIBDV，特别是活疫苗株的潜力和可靠性。该方法将有助于监测接种疫苗的家禽，以控制和管理 nVarIBDV IBDV 的感染。本研究利用 IBDV 节 A 序列 5'-UTR 区和 5/2 重叠区的核苷酸多态性，建立了一步实时 TaqMan 逆转录聚合酶链反应（RT-PCR）方法。结果表明，该方法准确地区分了 nVarIBDV 和非 nVarIBDV 株（特别是活疫苗株），与传染性支气管炎病毒（IBV）、新城疫病毒（NDV）、禽流感病毒（AIV）、传染性喉气管炎病毒（ILTV）、禽痘病毒（FPV）无交叉反应，与 IBDV 阴性田间样本也无交叉反应。该方法在 10 到 10 DNA 拷贝/反应之间表现出线性动态范围，nVarIBDV 的平均 R 为 0.99，效率为 93%，非 nVarIBDV 的平均 R 为 1.00，效率为 94%。该方法还用于检测 84 只接种活疫苗的鸡的法氏囊。结果表明，与基于 2 基因 HVR 序列分析或 5 基因 RT-PCR 的策略相比，该方法能准确地区分 nVarIBDV 和非 nVarIBDV 株（疫苗株）。这些发现表明，一步实时 TaqMan RT-PCR 方法为检测 nVarIBDV 感染提供了一种快速、敏感、特异和简单的方法，是识别和区分 nVarIBDV 与非 nVarIBDV、特别是活疫苗株的有用临床诊断工具。
