In situ Hybridization to Study the Pathogenicity of North American Avian Metapneumovirus Subtypes A and B in Turkeys

Avian metapneumovirus (aMPV) is a highly contagious upper respiratory pathogen that causes significant economic losses in both chickens and turkeys. Globally, there are four antigenically distinct subgroups, A, B, C, and D, based on the sequence of glycoprotein G. Subgroups A and B emerged recently in the US, causing several outbreaks in chickens and turkeys, resulting in high morbidity and mortality rates, as well as a decline in egg production. aMPV primarily infects the upper respiratory tract and eyelids/conjunctiva. <i>In-situ</i> Hybridization (ISH) is a molecular tool that localizes specific nucleic acid sequences within cells or tissues. ISH probes were designed to target the highly conserved N-gene of aMPV-A and B localizing the nucleic acid of aMPV at the cellular level in the respiratory tract, thereby allowing for the visualization of viral attachment and replication sites. Different tissues from rRT-PCR-positive turkeys were subjected to ISH. The affected turkeys displayed increased morbidity and mortality that coincided with clinical signs and lesions of catarrhal sinusitis, rhinitis, and conjunctivitis. The infraorbital sinuses, nasal turbinate, and conjunctiva were the most affected tissues. In addition to the detection of aMPV, secondary bacterial infections were detected. The designed probes identified the spread of aMPV throughout the upper respiratory mucosa including the nasal/infraorbital sinuses, trachea, and secondary bronchi. It also showed the presence of the virus in luminal secretions of the upper respiratory tract. This work describes viral distribution and shedding in the respiratory organs of commercial meat-type turkeys naturally infected with aMPV under field conditions. These results advance the understanding of aMPV pathogenesis and confirm the best diagnostic sampling.