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

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.

We address the challenge of understanding the aetiology of stunting syndrome in broiler flocks. Such an understanding will lead to effective control strategies for the syndrome. Stunting diseases have a prevalence of 10-20% in affected flocks causing young broiler chickens to fail to gain weight. The pathology manifests as lesions of damaged tissue in gastrointestinal tract, which prevent nutrient absorption. As 70% of the costs of rearing broiler chickens is due to the cost of feed, syndromes that reduce nutrient availability and impact growth cause steep financial losses and veterinarians may need to cull birds on animal welfare grounds. Smaller birds that reach the abattoir may rupture during processing and then be discarded.
Our preliminary evidence suggests that co-infections of endemic, enteric viruses are the most probable candidates for the aetiology of stunting syndrome, but it has proved difficult to identify them conclusively. We aim to identify these virus species and strains that are actively replicating within the lesions, under the hypothesis that the presence of an infection of actively replicating virus at those sites is causing the lesions. We further aim to confirm the hypothesis by isolating candidate viruses and challenging healthy chicks. Specific aims are: 1) Identification of the viruses within multiple gut lesions using cutting-edge LM-RNAseq (messenger (m)RNA transcriptomics applied to laser microdissected lesion contents), which will identify not only viral mRNA transcripts but also host cell responses to infection. 2) Isolate, purify and screen selected viral candidates identified from multiple sample lesions through cell culture and embryonated egg isolation techniques and investigate their pathogenicities using cultured cell methodologies. 3) Challenge healthy young chickens with those viruses that were most pathogenic in cell culture in co-infection studies to recreate the stunting syndrome.