Interactions between Pulmonary Inflammation, Airway Hyperresponsiveness, and the Lung Bacterial Microbiome in Horses with Inflammatory Airway Disease

背景：炎症性呼吸道疾病(IAD)是马患的一种肺部炎症性疾病，与咳嗽、运动耐受和肺功能损害有关。IAD在动物福利和马业方面意义重大，因为它与价值损失和其他健康动物的提前退休有关。与其他呼吸系统疾病一样，在运动马的表现限制条件方面，它仅次于肌肉关节骨骼损伤。2007年，美国兽医内科学会的一个专家小组得出结论，谷仓中存在的空气中的粉尘颗粒很可能是导致这种疾病的原因，但没有明确的证据表明存在过敏性反应。基于将运动耐量和咳嗽与气管和支气管的高细菌负荷联系起来的研究，我们认为较低的呼吸道细菌群落有助于IAD炎症和肺功能损害的持久存在。新的证据也支持这一点，即细菌在不被认为主要是传染性的肺部疾病中发挥作用，如哮喘。传统的依赖培养的技术只能检测到三分之一或更少的细菌种群，不再被认为是研究肺部细菌群落的理想工具。分子技术的发展可以很容易地对整个细菌种群的基因组进行排序--称为微生物组--这导致了一个发现，即使是健康的肺也不是无菌的。这是重温网络成瘾潜在病因学的难得机会。**目的：本项目的主要目的是阐明肺部细菌菌群在IAD中的作用。一般的基本假设是患有IAD的马具有随时间变化的肺微生物群，并有助于持久的炎症和肺功能损害。具体目标是：1)开发有效绕过上呼吸道细菌菌群的下呼吸道采样技术；2)确定不同居住条件下正常肺细菌微生物组；3)确定健康和IAD感染马的肺细菌微生物组随时间的变化；以及4)确定抗菌剂对IAD马肺细菌微生物组和肺部炎症和功能的影响。**科学方法：将通过大量测序来研究肺细菌微生物组，使用称为16S rRNA基因的一部分细菌DNA，该基因既有高度保守的部分，也有高度可变的部分，分别用于定量和鉴定。肺微生物组将在支气管肺泡灌洗和肺活检中进行研究，并与通过脉冲振荡法和乙酰甲胆碱支气管激发法测量的呼吸道炎症和肺功能相关。将测量同一个体随时间的稳定性、住房的影响以及抗菌治疗的效果，并将其与健康和受IAD影响的马的炎症相关联。**新颖性和预期意义：据我们所知，还没有用先进的分子技术研究患有IAD的马的下呼吸道细菌种群。该程序将提供有关肺部细菌微生物群随时间的稳定性、其随居住环境的变化以及其对抗菌素干预的反应的有价值的信息。预计这项研究计划将证明患有IAD的马改变了肺部细菌种群，这有助于持久的呼吸道炎症和功能损害。调整我们的治疗干预的重点，包括恢复正常的肺部微生物群，最终可能是有益的。

Background: A pulmonary inflammatory condition suffered by horses, Inflammatory Airway Disease (IAD) is associated with cough, exercise intolerance, and lung function impairment. IAD is significant in the context of animal welfare and the horse industry, as it is associated with loss of value and the early retirement of otherwise healthy animals. Along with other respiratory diseases, it is second only to myoarthroskeletal injuries when it comes to performance-limiting conditions for sport horses. In 2007, a panel of experts from the American College of Veterinary Internal Medicine concluded that airborne dust particles present in barns were likely contributing to the disease, but there was no clear evidence of an allergic-type response. Based on studies that link exercise intolerance and cough to a high bacterial load in the trachea and bronchi, we propose that lower airway bacterial communities contribute to the perpetuation of inflammation and lung function impairment in IAD. This is also supported by new evidence that bacteria play a role in lung diseases not considered primarily infectious, such as asthma. Conventional culture-dependent techniques can only detect a third or fewer of the bacterial populations, and are no longer considered the ideal tool to investigate lung bacterial communities. The development of molecular techniques that can readily sequence the genome of entire bacterial populations-called microbiome-has led to the discovery that even healthy lungs are not sterile. This is a unique opportunity to revisit the potential aetiology of IAD. **Objectives: The main goal of this program is to elucidate the role of lung bacterial microbiome in IAD. The general underlying hypothesis is that horses with IAD have an altered lung microbiome that varies in time and contributes to perpetuating inflammation and lung function impairment. The specific objectives are 1) To develop sampling techniques of the lower airways that effectively bypass the upper airway bacterial flora; 2) To define normal lung bacterial microbiome in different housing conditions; 3) To determine the variation over time of the lung bacterial microbiome in healthy and IAD-affected horses; and 4) To determine the effect of antimicrobials on the lung bacterial microbiome and pulmonary inflammation and function in horses with IAD.**Scientific approach: The lung bacterial microbiome will be studied by mass sequencing, using a portion of bacterial DNA called the 16S rRNA gene, which has both highly conserved sections and hypervariable sections, used for quantification and identification, respectively. The lung microbiome will be studied in bronchoalveolar lavages and lung biopsies, and correlated to airway inflammation and lung function measured by impulse oscillometry and methacholine bronchoprovocation. The stability over time in the same individuals, the effects of housing, and the effects of antimicrobial treatment will be measured and correlated to inflammation in healthy and IAD-affected horses. **Novelty and expected significance: To our knowledge, the lower airway bacterial populations of horses with IAD have not been investigated with advanced molecular techniques. This program will provide valuable information on the stability of the pulmonary bacterial microbiome over time, its change with housing environment, and its response to intervention with antimicrobials. It is anticipated that this research program will demonstrate that horses with IAD have altered lung bacterial populations and that this contributes to perpetuating airway inflammation and function impairment. It may eventually be beneficial to refocus our therapeutic interventions to include the restoration of the normal lung microbiome.