Aspects of the Pathogenesis, Host Immune Response and Viral Kinetics of Infectious Laryngotracheitis in Chickens

Abstract

<p>Infectious laryngotracheitis caused by infectious laryngotracheitis virus (ILTV) is a highly contagious disease of the upper respiratory tract and conjunctiva that causes huge economic losses in terms of mortality and loss of productivity. Despite their serological homogeneity (Magouz <i>et al.</i> 2018), the pathogenicity of ILTV strains varies substantially (Kirkpatrick <i>et al.</i> 2006a" Menendez <i>et al.</i> 2014). This is further complicated by the emergence of new strains that emerge from recombination of field and live attenuated vaccine strains (Lee <i>et al.</i> 2015" Agnew-Crumpton <i>et al.</i> 2016). The dominance of some of these new recombinant ILTV strains has been linked to improved fitness in terms of high virulence and transmission (Lee <i>et al.</i> 2015" AgnewCrumpton <i>et al.</i> 2016). ILTV DNA has been demonstrated in a wide range of organs other than the upper respiratory tract and conjunctiva, including feathers (Davidson <i>et al.</i> 2016) and blood fractions (Tran <i>et al.</i> 2021) . This raises the speculation of the involvement of the blood cells in the dissemination of the virus. Local cell mediated immunity is considered critical for protection against ILTV (Devlin <i>et al.</i> 2010; Vagnozzi <i>et al.</i> 2016" Beltrán <i>et al.</i> 2019" Krunkosky <i>et al.</i> 2020). Earlier in vivo studies have shown the role of splenic and blood lymphocytes against ILTV (Fahey <i>et al.</i> 1984" Honda <i>et al.</i> 1994). However, the effector immune cells in peripheral blood associated with protection have not been characterised. Furthermore, data on the influence of the inoculation route on host cellular response and the effect of repeated inoculation on replication characteristics is scarce.</p> <p>This PhD project was developed with three broad objectives, namely 1) To compare the virulence of newly isolated Class 9 and Class 14 strains to known virulent Class 10 strain in layer and meat chickens in terms of clinical signs, mortality, and viral shedding; 2) To improve our understanding of the effect of the ILTV strain and route of inoculation on the onset, severity, and duration of clinical signs as well as the level of viral replication in chickens following primary and repeated inoculation; and 3) To characterise blood cells harbouring the ILTV antigen and to investigate the dynamics of T cells in the peripheral blood of challenged or vaccinated chickens.</p> <p>The major findings from the PhD project were firstly that newly isolated Class 9 recombinant strain inoculated birds exhibited severe virulence (in terms of clinical signs and microscopic lesions) similar to that of known virulent Class 10 strain, while another newly isolated Class 14 strain inoculation resulted in subclinical infection. These differences were also evident from higher viral load in oropharyngeal swabs and higher proportion of positive oropharyngeal and cloacal swabs compared to inoculation with Class 14 (<i>P</i> < 0.0001).The second major finding was the inoculation route and bird type on the virulence. Ocular inoculation of Class 9 or SA2 resulted in a higher proportion of sick birds, earlier onset of clinical signs and fewer days to peak clinical signs than oral inoculation or vent inoculation. Ocular signs remained the prominent clinical sign type irrespective of the route of inoculation for both strains. While there was no significant difference on the viral replication in upper respiratory tract of birds inoculated with Class 9 via ocular and oral routes (P = 0.3), there was a trend (P=0.06) towards higher overall values in ocularly or orally inoculated birds than in birds inoculated by vent route. The ILTV genomic copies (GC) load of the SA2 strain in the cloaca was much greater following vent inoculation than following ocular or oral inoculation routes (P< 0.0001). Comparison of strains showed that birds inoculated with Class 9 strain exhibited a higher viral load than birds inoculated with the SA2 strain (P=0.0004). Birds after primary inoculation demonstrated marked resistance to induction of severe clinical signs and a rise in the viral load to repeated inoculation. Additionally, variation in host susceptibility was found, with Class 9 causing more severe disease in layer chickens than in meat chickens.</p> <p>With regard to the third objective, specific fluorescence for the ILTV antigen was detected in B cells and all evaluated T cell types, but not in monocytes and erythrocytes in blood samples from infected birds. Overall, the proportion of cells carrying the ILTV antigen was highest in CD4 cells (22.3 %) followed by TCRαβ/Vβ₁ cells (20.6%), CD8 cells (15.4%), TCRαβ/Vβ₂ cells or B cells (14.4) and least in TCRγδ cells (13.3%). However, attempts to determine the proportion of T cell subsets colocalised with ILTV antigen in a large number of cells at various days post inoculation and the influence of strain and route using flow cytometry was unsuccessful. Flow cytomteric analysis showed variation in their percentages of peripheral blood T cell subsets with respect to the route of inoculation and ILTV strain. Higher values of TCRγδ cells in birds inoculated by SA2 via vent than in birds inoculated by the oral route with the same strain at 3 dpi (P = 0.02). Almost a four-fold expansion in the percentage of IFN-γ positive TCRαβ/νβ₂ cells at 10 dpi and a six-fold rise in the percentage of IFN-γ positive TCRγδ cells at 5 dpi in birds inoculated by SA2 via vent route was observed. The percentages of CD4 cells, TCRαβ/νβ₂ cells, IFN-γ producing TCRγδ cells also varied with the inoculation strain type at various dpis. Overall, the alterations in the percentages of T cell subsets were more evident in birds inoculated by SA2 via the vent route.</p> <p>The outcomes of this thesis improve our understanding of the viral kinetics of field and vaccine ILTV strains as well as the factors influencing them. This thesis also provides new information about the possible role of immune cells in dissemination of the virus and further supports the idea of involvement of a systemic immune response against ILTV. These findings could assist in future work for the control of this disease.</p>