Development of an experimental model using cold stress to assess the pathogenicity of two Moroccan AI H9N2 isolates from 2016 and 2022 in commercial broiler chickens

Since 2016, low pathogenic avian influenza virus (LPAIV) H9N2 became a major issue for poultry production in Morocco. Even though the agent was classified as low pathogenic, AI H9N2 cause significant economic losses, particularly during co-infections. Experimentally, it has been difficult to reproduce the clinical picture without appealing other viral or bacterial pathogens. Our study was carried out to evaluate a new challenge model using cold stress in commercial broilers infected with two Moroccan H9N2 viruses isolated in 2016 and 2022. One hundred twenty day-old chicks were divided into four groups: A, B, and C exposed to cold stress, and D was kept as negative control. At 21 days of age, Groups A and B were challenged by oculo-nasal route with 107 EID50 of H9N2 strains, isolated respectively during 2016 and 2022. Meanwhile, chicks of group C were exposed to only cold stress. The assessment of body weight gain, clinical signs, lesions, mortality, and oropharyngeal viral shedding was monitored for 15 days post-challenge. Results showed that cold stress exacerbated H9N2 clinical signs, allowing us to establish a scoring system and to validate the challenge model without co-infections. Gross and microscopic lesions, induced by the virus primarily in the respiratory tract, peaked at 5 dpi and significantly decreased at 15 dpi. Group B harbored the highest viral loads with viral shedding persisting beyond 11 dpi in both groups. This study demonstrates a clear clinical difference among the two isolates; A/chicken/Morocco/178-2/2022(H9N2) showed a significant increase in virulence compared to the firstly isolate A/chicken/Morocco/SF1/2016(H9N2). The novel H9N2 challenge model using cold stress will contribute to a better understanding of LPAI pathogenesis and epidemiology and allow for research closer to the field.