The Houghton Trust - Promoting research into poultry diseases
The Houghton Trust - Promoting research into poultry diseases
The Houghton Trust - Promoting research into poultry diseases
The Houghton Trust - Promoting research into poultry diseases
The Houghton Trust - Promoting research into poultry diseases
The Houghton Trust - Promoting research into poultry diseases
The Houghton Trust - Promoting research into poultry diseases
The Houghton Trust - Promoting research into poultry diseases

Pilot investigation into use of novel 3D enteroids as an in vitro model for in ovo vaccination of HVT-eGFP
Bayley Martin Towers (student), and Dr. Kate Sutton and Emma Armstrong (supervisors), The Roslin Institute

Chicken 3D intestinal enteroids consist of multiple differentiated cell types, from embryonic day (ED) 18 intestinal villi. Notably, avian enteroids contain an ‘inside-out’ conformation and inner core lamina propria, containing functional immune cells. Previous work has indicated that the primary route of uptake for in ovo inoculated HVT is potentially through ingestion into the duodenum. Therefore, this project aimed to investigate if the chicken 3D enteroids could be used as an in vitro model to investigate the initial stages of vaccine uptake. This project had two main aims:

  1. Determine the role of actin-dependent HVT uptake in chicken 3D enteroids
  2. Investigate the intestinal epithelial barrier integrity of 3D enteroids following HVT inoculation

The full results of this research are confidential.

Research Highlights

  • Chicken 3D enteroids can be infected with the HVT vaccine strain.
  • HVT can be detected in the lamina propria of chicken 3D enteroids.
  • Investigation into the role of actin during HVT uptake in the chicken 3D enteroids.
  • Investigation into the role of tight junction proteins in HVT infection.

Challenges, and lessons learned

  • Incorrectly labelling slides: Several slides were used for each technical replicate, so it was easy to label them wrong. These slides were then discarded from the analysis meaning further repetitions were needed.
  • Initial staining of 3D enteroids with ZO-1 antibody were unsuccessful but troubleshooting this allowed experience with antibody and staining optimisation.
  • When analysing the virus at different time points, the same enteroid could not be compared at each time-point, so we were unable to determine the difference in HVT uptake between different time-points by taking confocal images. Further work will use qPCR quantification of HVT DNA to address this.
  • Variation in extraction efficiency each week meant enteroid numbers were variable but a minimum of 500 enteroids per well were plated each week to allow visualisation.

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