Host Responses Following Infection with Canadian-Origin Wildtype and Vaccine Revertant Infectious Laryngotracheitis Virus

Abstract

Infectious laryngotracheitis (ILT) is caused by Gallid herpesvirus-1 (GaHV-1) or infectious laryngotracheitis virus (ILTV) and was first described in Canadian poultry flocks. In Canada, ILTV infection is endemic in backyard flocks, and commercial poultry encounters ILT outbreaks sporadically. A common practice to control ILT is the use of live attenuated vaccines. However, outbreaks still occur in poultry flocks globally due to ILTV vaccine strains reverting to virulence and emergence of new ILTV strains due to recombination in addition to circulating wildtype strains. Recent studies reported that most of the ILT outbreaks in Canada were induced by the chicken-embryo-origin (CEO) live attenuated vaccine revertant strains with the involvement of a small percentage of wildtype ILTV. It is not known if the host responses induced by these two ILTV strains are different. The objective of the study was to compare the host responses elicited by CEO revertant and wildtype ILTV strains in chickens. We infected 3-week-old specific pathogen-free chickens with the two types of ILTV isolates and subsequently evaluated the severity of clinical and pathological manifestations, in addition to host responses. We observed that both of the isolates show high pathogenicity by inducing several clinical and pathological manifestations. A significant recruitment of immune cells at both 3 and 7 days post-infection (dpi) was observed in the tracheal mucosa and the lung tissues of the infected chickens with wildtype and CEO vaccine revertant ILTV isolates when compared to uninfected controls. Overall, this study provides a better understanding of the mechanism of host responses against ILTV infection.

Keywords: Canada; backyard poultry; host response; infectious laryngotracheitis virus (ILTV); pathogenesis; poultry; vaccine revertant infectious laryngotracheitis virus.

Figure 1

The clinical scores and body weights of chickens following infection with wildtype (AB-S63) and chicken-embryo-origin (CEO) vaccine revertant (AB-S45) ILTV isolates. The means of the clinical scores observed from 0–6 dpi and the body weights recorded on days 0, 3, and 7 following ILTV infection are plotted in (a,b), respectively. The error bars represent the standard error of means (SEM). The clinical signs were scored as described in the material and methods and statistically analyzed by employing the Kruskal–Wallis and Dunn’s multiple comparison tests. The differences in body weights between the groups were analyzed with the two-way ANOVA and Tukey’s multiple comparison tests. ** p < 0.01, *** p < 0.001.

Figure 2

The ILTV genome loads at 3 and 7 dpi following infection with wildtype (AB-S63) and CEO vaccine revertant (AB-S45) ILTV isolates. ILTV genome loads in (a) oropharyngeal swabs; (b) cloacal swabs; (c) lung; and (d) trachea are illustrated. The Kruskal–Wallis test, followed by Dunn’s multiple comparison test, was employed to identify group differences in ILTV genome load. The error bars indicate the SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001.

Figure 3

The immune cell recruitment in infected and control trachea following infection with wildtype (AB-S63) and CEO vaccine revertant (AB-S45) ILTV isolates and mock infection with PBS. The representative images show (a) CD4+ T cells, (b) CD8+ T cells, and (c) macrophages present in the trachea of the infected and uninfected chickens along with quantitative data. The Mann–Whitney U test was employed to compare infected and uninfected groups at 3 and 7 dpi. The error bars indicate the SEM. Significance: * p < 0.05, ** p < 0.01.

Figure 3

The immune cell recruitment in infected and control trachea following infection with wildtype (AB-S63) and CEO vaccine revertant (AB-S45) ILTV isolates and mock infection with PBS. The representative images show (a) CD4+ T cells, (b) CD8+ T cells, and (c) macrophages present in the trachea of the infected and uninfected chickens along with quantitative data. The Mann–Whitney U test was employed to compare infected and uninfected groups at 3 and 7 dpi. The error bars indicate the SEM. Significance: * p < 0.05, ** p < 0.01.

Figure 3

The immune cell recruitment in infected and control trachea following infection with wildtype (AB-S63) and CEO vaccine revertant (AB-S45) ILTV isolates and mock infection with PBS. The representative images show (a) CD4+ T cells, (b) CD8+ T cells, and (c) macrophages present in the trachea of the infected and uninfected chickens along with quantitative data. The Mann–Whitney U test was employed to compare infected and uninfected groups at 3 and 7 dpi. The error bars indicate the SEM. Significance: * p < 0.05, ** p < 0.01.

Figure 4

The immune cell recruitment in infected and control lungs following infection with wildtype (AB-S63) and CEO vaccine revertant (AB-S45) ILTV isolates and mock infection with PBS. The representative images show (a) CD4+ T cells, (b) CD8+ T cells, and (c) macrophages present in the lung tissue of the infected and uninfected chickens, along with quantitative data. The Mann–Whitney U test was employed to compare the infected and uninfected groups at 3 and 7 dpi. The error bars indicate the SEM. Significance: * p < 0.05, ** p < 0.01.

Figure 4

The immune cell recruitment in infected and control lungs following infection with wildtype (AB-S63) and CEO vaccine revertant (AB-S45) ILTV isolates and mock infection with PBS. The representative images show (a) CD4+ T cells, (b) CD8+ T cells, and (c) macrophages present in the lung tissue of the infected and uninfected chickens, along with quantitative data. The Mann–Whitney U test was employed to compare the infected and uninfected groups at 3 and 7 dpi. The error bars indicate the SEM. Significance: * p < 0.05, ** p < 0.01.

Figure 4

The immune cell recruitment in infected and control lungs following infection with wildtype (AB-S63) and CEO vaccine revertant (AB-S45) ILTV isolates and mock infection with PBS. The representative images show (a) CD4+ T cells, (b) CD8+ T cells, and (c) macrophages present in the lung tissue of the infected and uninfected chickens, along with quantitative data. The Mann–Whitney U test was employed to compare the infected and uninfected groups at 3 and 7 dpi. The error bars indicate the SEM. Significance: * p < 0.05, ** p < 0.01.

Figure 5

The immune gene mRNA expressions after infection with the wildtype (AB-S63) and CEO vaccine revertant (AB-S45) ILTV isolates. IFN-γ, IL-1β, and iNOS mRNA expressions in the trachea at 3 and 7 dpi is shown in (a–c), respectively, and in the lungs at 3 and 7 dpi are illustrated in (d–f), respectively. The Mann–Whitney U test was used to analyze the differences between time points and between groups. The error bars represent the SEM. Significance: * p < 0.05, ** p < 0.01.

Figure 6

The representative images of the histology of the trachea from ILTV-infected chicken and controls. (A) A normal trachea with pseudostratified ciliated columnar epithelium and normal distribution of mucous glands, score 0; (B) minimal changes with normal epithelium plus mild to moderate infiltration of lymphocytes, rare heterophils, normal mucous glands, without syncytia or intranuclear inclusions, score 1; (C) mild changes with thickened mucosa with mild to moderate cell infiltration and/or normal epithelium, except for foci of syncytia, intranuclear inclusions, and hyperemia, occasionally with cell cuffs, score 2; (D) moderate changes with thickened mucosa with moderate to marked cell infiltration, numerous syncytia, intranuclear inclusions, patches of separating or sloughing epithelium from the lamina propria, the mucosal surface well covered by normal or affected epithelium, mucous glands reduced, marked hyperemia, and mononuclear cell infiltration around blood vessels, score 3; (E) severe changes with thickened mucosa, edema, proteinaceous fluid, cellular exudate, or adherent fibrinohemorrhagic to cellular pseudomembrane on the surface, normal epithelium absent, the mucosal surface covered by a thin layer of basal cells, and syncytia with intranuclear inclusions sometimes present, score 4.