Disclosing respiratory coinfections: a broad-range panel assay for avian respiratory pathogens on a nanofluidic PCR platform

Respiratory syndromes (RS) are among the most significant pathological conditions in edible birds and are caused by complex coactions of pathogens and environmental factors. In poultry, low pathogenic avian influenza A viruses, metapneumoviruses, infectious bronchitis virus, infectious laryngotracheitis virus, <i>Mycoplasma</i> spp. <i>Escherichia coli</i> and/or <i>Ornithobacterium rhinotracheale</i> in turkeys are considered as key co-infectious agents of RS. <i>Aspergillus</i> sp., <i>Pasteurella multocida, Avibacterium paragallinarum</i> or <i>Chlamydia psittaci</i> may also be involved in respiratory outbreaks. An innovative quantitative PCR method, based on a nanofluidic technology, has the ability to screen up to 96 samples with 96 pathogen-specific PCR primers, at the same time, in one run of real-time quantitative PCR. This platform was used for the screening of avian respiratory pathogens: 15 respiratory agents, including viruses, bacteria and fungi potentially associated with respiratory infections of poultry, were targeted. Primers were designed and validated for SYBR green real-time quantitative PCR and subsequently validated on the Biomark high throughput PCR nanofluidic platform (Fluidigm©, San Francisco, CA, USA). As a clinical assessment, tracheal swabs were sampled from turkeys showing RS and submitted to this panel assay. Beside systematic detection of <i>E. coli</i>, avian metapneumovirus, <i>Mycoplasma gallisepticum</i> and <i>Mycoplasma synoviae</i> were frequently detected, with distinctive coinfection patterns between French and Moroccan flocks. This proof-of-concept study illustrates the potential of such panel assays for unveiling respiratory coinfection profiles in poultry.

呼吸道综合征（RS）是食用禽类中最为重要的病理状态之一，其发生由病原体与环境因素的复杂协同作用共同介导。在家禽中，低致病性甲型禽流感病毒、禽偏肺病毒（metapneumoviruses）、传染性支气管炎病毒、传染性喉气管炎病毒、支原体属（Mycoplasma spp.）、大肠埃希菌（Escherichia coli），以及火鸡体内的鼻气管炎鸟杆菌（Ornithobacterium rhinotracheale）均被认为是RS的关键共感染病原体。曲霉属（Aspergillus sp.）、多杀性巴氏杆菌（Pasteurella multocida）、副鸡禽杆菌（Avibacterium paragallinarum）以及鹦鹉热衣原体（Chlamydia psittaci）也可能参与呼吸道疫情的发生。
一种基于纳米流体技术的创新型实时定量PCR方法，可在单次实时定量PCR运行中，同时使用96条病原体特异性PCR引物对最多96份样本进行筛查。该平台被用于禽类呼吸道病原体的筛查：本研究靶向了15种与禽类呼吸道感染潜在相关的呼吸道病原体，涵盖病毒、细菌与真菌三大类群。引物针对SYBR Green实时定量PCR进行设计与验证，并随后在Biomark高通量PCR纳米流体平台（Fluidigm©，美国加利福尼亚州旧金山）上完成了性能验证。
作为临床评估环节，研究人员从表现出RS症状的火鸡体内采集气管拭子，并将其提交至该多重检测体系进行检测。除了系统性检测大肠埃希菌外，禽偏肺病毒、鸡毒支原体（Mycoplasma gallisepticum）以及滑液支原体（Mycoplasma synoviae）的检出率同样较高，且法国与摩洛哥的养殖禽群呈现出截然不同的共感染模式。本概念验证研究证实了此类多重检测体系在揭示禽类呼吸道共感染特征方面的应用潜力。