Performing Department
Population Health & Reproduction
Non Technical Summary
This project is meant to understand the innate immune responses to a viral infection caused by infectious bronchitis virus. The main goal is to understand these responses in vitro and using the information gathered come up with new strategies to prevent infectiopus bronchitis infection.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
OBJECTIVESModify the experimental design to eliminate the TOC processing and allantoic fluid effect in cytokine productionInvestigate the IL-6 and IL-8 cytokine production and associate it with the pathology of tracheas from relatively susceptible and resistant lines challenged with IBV.
Project Methods
PROJECT DETAILSCentrifugation of the viral stock using sucrose gradient will be performed for viral purification. In order to test viral viability, we will titrate the stock in 10-days-old embryonated specific pathogen free (SPF) eggs. (2) Ten B2 and ten B19 haplotype birds will be raised until 3 weeks of age. At that time birds will be euthanized, tracheas collected in media and processed into 2 mm tracheal rings. Rings will be cultured for 24 hours and dead or contaminated rings will be eliminated. Tracheas and media will be collected at 24, 48, 72, 96, 120, 144 and 168 hours to determine IL-6 and IL-8 mRNA expression (RT-qPCR) and protein production (ELISA) in the absence of IBV challenge, respectively. This will be done to understand the cytokine response dynamics of the tracheal cut. The challenge time for the next experiment will be selected based on the time in which cytokines IL-6 and IL-8 decrease at a reasonable level for a non-injured tissue. (3) A second experiment will be performed avoiding the cytokine response from the tracheal cut. Ten B2 and ten B19 birds will be raised until 3 weeks of age. Tracheas will be collected and cut into 2 mm tracheal rings. Rings will be distributed and divided into two groups per chicken line. The challenge, at the time determined by the previously performed experiment, will be performed with the previously purified IBV M41 with a titer of 103.5EID50%. Samples (tracheas and media) will be collected at 18, 24, 36, 48, 72, 96 and 120 hours post-infection. From tracheal tissue we will assess viral load and mRNA for IL-6 and IL-8. Media will be used for IL-6 and IL-8 ELISA's. We will also create slides for histopathology and histomorphometry evaluation of the tracheas. In addition, we will perform immunohistochemistry (IHC) using polyclonal antibodies for IBV and monoclonal antibodies for IL-6 and IL-8, adding specificity for cytokine detection in tissues