Location: Endemic Poultry Viral Diseases Research
Project Number: 6040-32000-083-019-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2023
End Date: Sep 30, 2027
To determine the virulence of the recombinant ILTV containing a deletion in the gene encoding virus interleukin 4 (vIL4) and investigate it protective nature against virulent challenge in vaccination/challenge experiments in birds.
The infectious laryngotracheitis virus (ILTV) recombinant containing a deletion in the gene encoding vIL4 will be propagated on chicken kidney cells to generate a viral stock. The stock will be titrated according to the tissue culture infectious dose 50 (TCID50) method. For the virulence study, 108 birds will be raised to 24 days of age. The birds will be inoculated with the vIL4 deletion recombinant and the parent virus (1874C5) via two separate routes: ocular (OC) and intratracheally (IT). The birds will be divided into groups of six. Six birds will be used to collect conjunctiva and tracheal swabs at 3 and 5 days post-inoculation to evaluate viral replication by real-time PCR. Six birds will be sacrificed at 4 days post-inoculation to evaluate the level of lytic replication in the conjunctiva and trachea (some tissues can be collected for RNA extraction).The remaining six birds will serve solely for collection of clinical signs and weight gain during the experiment. For the protection study, 85 chickens from the susceptible line B*13 will be used. Blood samples will be collected prior to vaccination and prior to challenge to determine the antibody titers. Thirty-five birds (vaccinated/challenge, Vx/Ch) will be vaccinated at 2 weeks of age with recombinant strain vIL4 at a 103.16 TCID50 dose per chicken. Fifty chickens will be sham-vaccinated, and half (n=25) will serve as the non-vaccinated/challenged (NVx/Ch) control. At 17 days post-vaccination, the vIL4- and sham-vaccinated groups will be challenged with virulent ILTV. Each chicken will receive the challenge virus (1874C5) at a dose of 103.8 TCID50 in a total volume of 200 µl, 50 µl in each eye, and 100 µl delivered intratracheally. A group of sham-vaccinated chickens (n=25) will receive a mock challenge with tissue culture media and serve as non-vaccinated/non-challenged (NVx/NCh) control groups. Clinical signs will be scored at 3–9 days post-challenge (DPC). At 3, 5, 7, and 9 DPC, tracheal swabs will be collected to quantify the challenge virus genome load by real-time quantitative polymerase chain reaction (RT-qPCR). At 4 days post-challenge, seven chickens from each group will be sacrificed, bled, and tracheae will be collected for histopathologic examination. Chickens will be monitored daily for morbidity and mortality, and any reaching the humane endpoint during the experiment will be culled. Surviving birds will be humanely euthanized at 45 days.