Universitas Gadjah Mada, Faculty of Veterinary Medicine, Yogyakarta, Indonesia.
Indonesian Research Center for Veterinary Science, Bogor, Indonesia.
J Virol Methods. 2017 Nov;249:181-188. doi: 10.1016/j.jviromet.2017.08.014. Epub 2017 Aug 24.
In countries where highly pathogenic avian influenza virus (HPAIV) H5N1 is endemic and controlled by vaccination, post-vaccination serological monitoring is essential to differentiate vaccinated poultry from those that are infected. The objectives of this study were to validate two experimental ELISAs that detect antibodies raised against the M2e protein of avian influenza virus that can be used for DIVA purposes. Results from the sM2e and tM2e ELISAs were compared with other conventional tests for the detection of H5N1influenza virus (virus isolation and RT-PCR) using samples collected from 16 commercial flocks in Indonesia. These comprised vaccinated layers aged between 18 and 68 weeks old that were sampled at ten-weekly intervals. A small number of sera were positive in sM2e and tM2e ELISA, 14 (0.6%) and 17 (0.7%) respectively, with low OD (0.1-0.3), but only 4 sera were positive in both tests. At the flock level, the incidence of M2e positive sera was low (4%), well below previously established minimum of 40% for an HPAIV H5N1-infected flock. Conventional M and H5 gene RT-PCRs indicated that none of 16 flocks were infected at any time during the study. No virus was isolated from any of the 480 pooled swab samples, except from one, for which the combined data analysis suggest to be the result of a laboratory cross-contamination. Clinical disease, mortalities or reduction in production performance, indicative of field H5N1 challenge, were not observed either in any of the flocks. Birds from two surveyed flocks, challenged in the laboratory with an Indonesian HPAIV H5N1 developed M2e antibodies in 50% and 55% of surviving birds with OD in the range of 0.35-1.47 in tM2e ELISA, confirming the validity of the criteria established for use of M2e ELISA for DIVA purposes. Overall these results showed that the tM2e ELISA could be a useful monitoring tool to ascertain freedom from H5N1 infections in vaccinated commercial poultry.
在高致病性禽流感病毒（HPAIV）H5N1流行并通过疫苗接种进行控制的国家，接种后血清学监测对于区分接种疫苗的家禽和感染家禽至关重要。本研究的目的是验证两种可用于区分免疫和感染（DIVA）目的的检测针对禽流感病毒 M2e 蛋白产生的抗体的实验性 ELISA。使用从印度尼西亚 16 个商业家禽养殖场采集的样本，将 sM2e 和 tM2e ELISA 的结果与其他常规检测 H5N1 流感病毒（病毒分离和 RT-PCR）的方法进行比较。这些样本包括接种疫苗的蛋鸡，年龄在 18 至 68 周之间，每隔 10 周采样一次。在 sM2e 和 tM2e ELISA 中，分别有 14（0.6%）和 17（0.7%）份血清呈弱阳性（0.1-0.3），但只有 4 份血清在两种检测中均呈阳性。在禽群水平上，M2e 阳性血清的发生率较低（4%），远低于先前建立的 HPAIV H5N1 感染禽群的最低 40%。常规的 M 和 H5 基因 RT-PCR 表明，在研究期间，16 个禽群中没有一个禽群受到感染。从 480 个混合拭子样本中没有分离到任何病毒，除了一个样本，综合数据分析表明这是实验室交叉污染的结果。在任何一个禽群中，也没有观察到临床疾病、死亡率或生产性能下降，表明受到了田间 H5N1 的挑战。在实验室中用印度尼西亚 HPAIV H5N1 对两个调查禽群进行了挑战，在存活的鸟类中，有 50%和 55%的鸟类在 tM2e ELISA 中产生了 M2e 抗体，OD 值在 0.35-1.47 之间，证实了用于 DIVA 目的的 M2e ELISA 建立的标准的有效性。总的来说，这些结果表明，tM2e ELISA 可以成为一种有用的监测工具，以确定接种商业家禽中是否存在 H5N1 感染。
