Investigating the Role of Neuraminidase Activity in the Co-pathogenesis of Mycoplasma gallisepticum and Low Pathogenic Avian Influenza A Virus

The avian pathogen Mycoplasma gallisepticum, the etiologic agent of chronic respiratory disease in chickens, exhibits enhanced pathogenesis in the presence of a co-pathogen such as low-pathogenic avian influenza virus (LPAIV). M. gallisepticum Rlow can utilize α-2,3 linked sialic acids, abundant in the avian respiratory tract, to bind host cells in vitro. To further investigate the intricacies of this co-pathogenesis, chickens were mono- or co-infected with either virulent M. gallisepticum strain Rlow, attenuated M. gallisepticum neuraminidase mutant P1C5 and Mycoplasma specific lipoprotein A mutant P1H9, or LPAIV H3N8 (A/duck/Ukraine/1963). These chickens were then assessed for tracheal histopathology, pathogen load, and transcriptomic host response to infection using RNA-sequencing. Chickens co-infected with M. gallisepticum Rlow followed by H3N8 exhibited significantly more severe tracheal lesions and mucosal thickening in response to infection than chickens infected with H3N8 alone. Viral load was also significantly increased in this group over chickens who were infected first with H3N8 and subsequently with M. gallisepticum Rlow. The attenuated M. gallisepticum mutants P1C5 and P1H9, previously shown to be cleared 14 days post-infection, were able to persist 6 to 7 days post-infection in the presence and absence of co-infection with H3N8. P1H9 was able to persist to 14 days post-infection only in the presence of H3N8. The transcriptional response to mono- and co-infection with M. gallisepticum and LPAIV highlighted the involvement of differential expression of genes such as TLR4, TLR15, TLR21, IL-1β, IRF4, MMP1, and MMP9. Pathway and gene ontology analysis of these differentially expressed genes suggests that co-infection with virulent M. gallisepticum and LPAIV induces a downregulation of ciliary activity in vivo and alters the multiple immune-related signaling cascades. Although H3N8 is susceptible to neuraminidase inhibition by oseltamivir in vitro, this antiviral treatment was not effective in vivo at reducing H3N8 load in the trachea of co-infected chickens. These data indicate that the co-pathogenesis of LPAIV and M. gallisepticum is not strictly neuraminidase-dependent and warrants further experimental understanding