Onset of Viral Shedding and Clinicopathological Findings in Chickens Infected with Different Strains of Infectious Laryngotracheitis Viruses

Infectious laryngotracheitis (ILT) caused by infectious laryngotracheitis virus (ILTV), is a highly contagious upper respiratory disease of poultry causing great economic losses to the chicken industry. New strains have been reported to emerge due to recombination of vaccine and field strains, some of them replacing less virulent strains because of their high virulence. The present study aimed to evaluate the viral shedding and clinic pathological findings in chickens infected with different strains of ILTV. For that, meat and layer chickens were divided into 8 groups (each group with 15-16 chickens) and inoculated with 10⁴ TCID₅₀ of ILTV class 9, 10 or a putative class 14 isolate (Nazir et al., 2020) or sham-inoculated by eye drop at 15 or 22-day age. Four chickens from each treatment were euthanized at 5 days post-infection (dpi) and the remaining birds at 9 dpi. Chickens were observed for clinical signs from 2 to 9 dpi. Viral DNA shedding was measured in oropharyngeal (OL) and cloacal swabs (CL) and in dust samples at 2, 4, 6 and 9 dpi using quantitative PCR. Microscopic lesions in trachea and conjunctiva were scored at 5 and 9 dpi. In addition, feather tissues from inoculated chicken were collected at 5 and 9 dpi and used for examination of ILTV antigen using immunohistochemistry. Class 9 and class 10 induced severe clinical signs with marked bilateral severe conjunctivitis and caused characteristic cytolytic lesions in the conjunctiva. The mortality/euthanasia induced by class 9 (41.9%) and 10 (26.7%) was significantly higher (P < 0.0001) than class 14 (0%). ILTV was detected in OL and CL and in dust samples of chickens inoculated with ILTV classes 9, 10, and 14 from 2 to 9 dpi. The overall levels of ILTV DNA in OL and CL and the proportion of positive samples in chickens infected with class 9 (detection rate of 26/26 in OL and 23/26 in CL) and 10 (OL 31/32; CL 28/32) were significantly higher (P< 0.0001) than in chickens infected with class 14 (OL 11/32; CL 10/32). Likewise, dust samples collected from isolators from chickens inoculated with class 9 or class 10 showed a trend towards higher viral load than class 14 isolator samples. The higher virulence and shedding of class 9 and 10 strain leading to better dissemination and transmission of these strains compared to class 14 could explain the disappearance of the latter from the field after its detection for a brief period in the central coast of NSW. A significant interaction of bird type and ILTV strain was observed with class 9 and 10-inoculated chickens, with class 10 causing more severe disease in broilers and class 9 in layers. This may reflect immunological differences between meat and layer chickens. None of the sections of feathers of ILTV-infected birds were positive for ILTV antigen by immunostaining. Although the molecular testing of feather tips has been used to measure ILTV vaccine uptake (Davidson et al., 2016), the absence of microscopic changes and ILTV antigen staining in feather follicles of infected chickens suggest that there is no active replication of the virus despite the presence of ILTV DNA. Further investigations are needed to determine the differences in the three strains at the genomic level and to determine the source of ILTV DNA detected in feather follicles.

ACKNOWLEDGEMENTS: Authors acknowledge the support by AgriFutures Australia (grant number PRJ-010639) and the Australian government research training program (RTP) funds.