Despite the widespread use of live attenuated vaccines, infectious laryngotracheitis (ILT) remains a persistent global threat to the poultry industry. Challenges in controlling this disease in Australia are partially related to the use of vaccines that provide imperfect immunity. The overarching aim of this thesis was to advance the understanding of ILTV vaccine-mediated protection in chickens. Specific objectives of this dissertation were to 1) optimise cryopreservation protocols for systemic and mucosal lymphoid tissues in chickens to enable reproducible immunophenotyping of cryopreserved samples by flow cytometry, 2) compare immune responses and protective efficacy induced by a ILTV vaccine when administered via eye drop (ED) or vent brush (VB) route to the cloaca, and 3) evaluate the durability of ILTV vaccine-mediated immunity in commercial laying hens.
To achieve the first objective, the study presented in Chapter Two evaluated the effect of cryopreservation on chicken lymphoid tissues, including peripheral blood lymphocytes (PBLs), spleen, Harderian gland, conjunctiva, caecal tonsil, and larynx-trachea. Fresh and cryopreserved samples were analysed by flow cytometry using major immune cell markers to assess cell viability and marker stability. For the second objective, outlined in Chapter Three, a challenge model was used to compare vaccine-mediated protection conferred by an ILTV vaccine, administered via ED or VB to 4-week-old layer chicks, against virulent Class 9 challenge 14 days after vaccination. The third objective, which is addressed in Chapter Four, evaluated the duration of clinical protection of commercial layer hens in three age groups (22, 49, and 62 weeks) that had received multiple doses of ILTV vaccine, in response to challenge with virulent class 9 ILTV.
The main findings of the studies in this thesis were: Objective 1) Cryopreservation of chicken lymphoid tissues showed that lymphocytes extracted from cryopreserved systemic tissues such as PBLs and spleen maintained acceptable cell viability and stable immune marker expression, demonstrating reliable preservation for later immunophenotyping. In contrast, lymphocyte extraction from cryopreserved mucosal tissues was inconsistent with loss of several cell markers, indicating the need for further optimisation. To ensure reliable immunophenotyping, fresh processing of mucosal tissues is recommended.
Objective 2) VB and ED vaccination provided equivalent clinical protection reflected by high protection indices against clinical signs after challenge (VB, 99%; ED, 90%) and pathological lesion scores in conjunctiva (VB, 83%; ED, 74%) and trachea (VB, 78%; ED, 69%). Both routes induced immune responses and gene expression profiles following challenge similar to vaccinated, non-challenged chickens, indicating comparable immunological efficacy. Findings indicated that vaccine-mediated protection was achieved through efficient recall and innate antiviral responses independent of vaccination route. Rapid activation of antiviral mechanisms, facilitated by the transient over-expression of MX1 and STAT1 in the absence of inflammatory markers of vaccinated and challenged chickens, suggests an efficient recall response with rapid viral clearance. The negative association of TLR3 expression with ILTV genome copies and clinical signs, with no increase in CD8A or CD4, further suggests that immunity relied on mucosal effector recall responses rather than T cell proliferation.
Objective 3) Substantial viral load was detected in 82% of hens on arrival. Surprisingly, a vaccine-like recombinant strain (class 7b) associated with recent outbreaks in Australia was detected in 41.7% of 49 weeks old (wo) and 25% of 62 wo hens. The silent circulation of field ILTV strains in commercial flocks is of epidemiological significance for the potential emergence of strains with increased virulence. Following challenge with class 9 ILTV, chickens showed no clinical signs, although 53.8%, 15.4%, and 0% of choanal swab samples tested positive in the 22, 49, and 62 wo groups respectively. Collectively, the results highlight the inability of the current Australian industry ILT vaccination protocols to prevent infection with field strains as early as 12 weeks post-vaccination.
This thesis advances understanding of avian immune responses and ILTV vaccine efficacy by revealing novel insights into mucosal immunity, identifying mucosal recall responses as a key mechanism of protection while demonstrating that existing vaccination protocols fail to prevent infection and circulation of field ILTV strains. It further illustrates that effective cryopreservation of mucosal tissues requires approaches beyond rapid freezing and warrants methodological refinement.