Unravelling the aetiology of Stunting in UK Broiler Flocks through the use of novel microdissection and viral meta-transcriptomic sequencing tools

这个项目面临的挑战是解开英国肉鸡发育迟缓疾病的原因。自第二次世界大战后商业化肉鸡群工业化以来，发育迟缓综合症一直是肉鸡生产中的一个问题，但对于尼日利亚等中低收入国家的小生产者来说，这也是一个问题。在这些国家，妇女经常饲养少量鸡以补充家庭收入。发育迟缓问题有许多名称，包括传染性发育迟缓、发育迟缓综合征和吸收不良病。它指的是尽管有足够的饲料，但仍不能正常生长。鸟类通常在孵化后的第二到第三周出现发育迟缓问题，但临床症状可能更早观察到。除了体重增加减少（严重感染的鸡体重只占其年龄潜在体重的一小部分）之外，还可能出现其他迹象，如食欲改变、水样粪便、羽毛异常和肠炎。我们和其他研究小组有证据表明，发育迟缓是由传染性因子引起的：主要是怀疑病毒病原学，在AFBI分别使用特定的流行性病毒、肠道病毒、星状病毒和呼肠孤病毒菌株进行实验感染，导致发育迟缓期间观察到的一些迹象，例如体重减轻或肠道黏膜病变。其他群体也表现出一些发育迟缓的迹象，因为给鸟类接种了这些病毒和其他候选病毒的单一病毒株，例如乙肝病毒；然而，没有人完全重现了发育迟缓综合征，同时感染一种以上的病毒被认为是更可能的病因。此外，这些病毒的不同毒株正在传播，已知其致病性差异很大，其中大多数可能具有低致病性或无致病性，因此在未受影响的健康雏鸟中可能检测到高水平的这些病毒。归因疾病病原学的其他混淆因素包括存在母源抗体、传播途径（从母鸡到它们的环境）和鸟类感染的年龄，因为年龄的增加似乎赋予了抵抗力。所有这些因素也阻碍了查明导致发育迟缓疾病的病毒。该提案的具体目标是：1)确定在与发育迟缓疾病相关的病变内积极复制的病毒或在临床病例的许多类似病变中存在的病毒。组织病理学和激光显微解剖将识别和切除病变，核酸将被测序，以确定哪种病毒株存在于指示活动性感染的水平。2)确认所鉴定的病毒是发育迟缓的病原；首先，通过实验室中基于细胞和组织的方法，其次，通过使用已鉴定病毒的纯化分离物对健康鸡进行攻毒研究，这将证明典型发育迟缓疾病的标志性病变和相关临床症状的发展。这些目标将通过使用最先进的成像和基因组方法，并通过与行业（Moy Park，英国顶级肉鸡生产商之一和St David's Poultry Team，该国领先的家禽兽医实践）的合作来实现。目前没有商业疫苗来防止发育迟缓，也没有针对受感染禽群的特定治疗方法，以便扑杀受影响的鸟类。这一项目的结果将为发育迟缓的致病因素提供证据，使兽医能够改进诊断和可能的干预措施，并使疫苗公司能够开发适当的疫苗。

The challenge addressed by this project is to unravel the causes of stunting disease in UK broiler chickens. Stunting syndrome has been a production issue in commercial broiler flocks since they became industrialised after the second world war, but is also a problem for small producers in low and middle income countries, such as Nigeria, where women often raise small numbers of chickens to supplement the family income. The stunting problem has many names, including infectious stunting, runting stunting syndrome and malabsorption disease. It pertains to a failure to grow properly despite sufficient feed. Birds typically present with stunting problems around the second to third week post hatching, but clinical signs may be observable earlier. As well as the reduction in weight gain, which can be substantial with severely affected chickens only a fraction of their potential weight for age, there may be other signs such as altered appetite, watery droppings, abnormal feathering and enteritis. We and other groups have evidence that the stunting is caused by infectious agents: chiefly, viral aetiology is suspected and experimental infections at AFBI using specific strains of the endemic, enteric viruses, astroviruses and reoviruses separately have resulted in some of the signs observed during stunting, e.g. weight loss or lesions in the gut mucosa. Other groups have also demonstrated some of the signs of stunting from inoculating birds with single viral strains of these and other viral candidates, e.g. birnavirus; however, none have fully recreated the stunting syndrome, and co-infections with more than one viral agent are considered the more probable aetiology. Moreover, diverse strains of these viruses are in circulation which are known to vary widely in pathogenicity with the majority potentially of low or no pathogenicity and accordingly high levels of these virus may be detected in young, healthy, unaffected birds. Other confounding factors in attributing disease aetiology includes the presence of maternally-derived antibodies, route of transmission (from the hen versus their environment) and the age at which birds become infected, since increasing age appears to confer resistance. All these factors have also hampered the identification of the virus(es) responsible for stunting diseases.The specific aims of the proposal are to: 1) identify a virus or viruses that are actively replicating within the lesions associated with stunting disease and that are present in many similar lesions from clinical cases. Histopathology and laser microdissection will identify and excise lesions from which nucleic acids will be sequenced to determine which virus strain(s) is present at levels indicative of an active infection. 2) confirm that the virus or viruses identified are aetiological agents of stunting; firstly, by cellular and tissue-based methods in the laboratory and secondly, by challenge studies of healthy chickens using purified isolates of the identified virus(es), which will demonstrate the development of hallmark lesions and associated clinical signs typical of stunting disease. These objectives will be achieved through the use of state of the art imaging and genomic methodologies, and through collaboration with the Industry (Moy Park, which is one of UK's top broiler meat producers and St David's Poultry Team, the leading poultry veterinary practice in the country).Currently there are no commercial vaccines to prevent stunting or specific treatments for affected flocks so that affected birds are culled. The results of this project will provide evidence of the causal agents of stunting that will enable improved diagnostics and potential interventions for veterinarians, and for vaccine companies to develop appropriate vaccines.