Resumen de Estudis sobre la proventriculitis viral transmissible en pollastre
SUMMARY Transmissible Viral Proventriculitis (TVP) is an emergent viral infectious disease that affects mainly broiler chickens. It is characterized by impaired feed digestion, poor growth, and poor feed conversion rate, causing economic losses to the poultry industry. Enlargement, thickening, fragility, and paleness of the proventriculus, together with weakness and dilation of gastric isthmus is observed in TVP cases. Although proventricular gross lesions could be indicative of TVP, they are not specific. The disease is characterized by its histologic lesions: necrosis of oxynticopeptic cells, inflammation with a predominance of lymphocytes, and replacement of glandular epithelium by hyperplasic ductal epithelium. Therefore, the main objective of this thesis was to determine the presence of the disease and the new viral agent in Spanish poultry farms and further characterize this viral agent by ultrastructural studies and next generation sequencing techniques.
In the first study, the presence of CPNV in TVP clinical cases from Spanish poultry farms was retrospectively evaluated from 1999 to 2019 in FFPE proventricular tissue. Histopathological examination, CPNV RT-PCR, and partial sequence genome of positive cases obtained using Sanger sequence was carried out in 42 clinical cases. In adition, a new ISH technique was set up as a new method to detect the virus. The study identified the presence of CPNV in Spanish chicken farms since at least 1999. Moreover, ten proventriculi from seven different clinical cases were positive to CPNV RT-PCR and ISH, and all of them showed the characteristic histopathological features of TVP (necrosis of oxynticopeptic cells and gland interstitial inflammation). Phylogenetic studies showed that the Spanish CPNV partial sequences were very closely related to the available UK and USA CPNV sequences.
The second study of this thesis aimed to identify, visualize, and localize the causative agent of TVP by using TEM. Proventricular samples from twelve different clinical cases were used. Eight of the samples were positive by RT-PCR and ISH to CPNV, while the other three, although showing gross and microscopic lesiosn consistent with TVP, were negative to CPNV ISH and RT-PCR. Icosahedral, 70 nm, non-enveloped, intranuclear and/or intracytoplasm viruses were observed in four samples. Two of these samples were negative to CPNV by molecular methods, while the other two gave positive results to RT-PCR and ISH CPNV techniques. These results, together with the finding of virions in the nuclei of infecetd cells, a finding which is not usually seen in RNA viruses, raised the question whether TVP could also be caused by another viral agent simultaneoulsy or without the contribution fo CPNV. To further undesrtand this last hypothesis, a third study was done to further characterize the genome of the virus/es involved in the TVP cases. Eight TVP proventriculi samples were studied by NGS and partial or complete sequences of CPNV Segment B were found in all of them, furter confirming the involvement of this virus in TVP clinical cases. However, in five of the cases, partial sequences of Avian adenovirus A were found, particularly the two cases where intranuclear virions had been observed by TEM. These results confirm the hypothesis that Avian adenovirus A can be present in TVP clinical cases, although its role in the development of the disease needs further studies.
