Comparison of the pathogenesis and immune responses following avian Metapneumovirus subtype A and B infection in broiler-type chickens

Avian Metapneumovirus (aMPV) causes TurkeyRhinotracheitis (TRT), an acute respiratory tract infection in turkeys of all ages. The virus is also associated with swollen head syndrome (SHS) in broilers and broiler breeders. Four different subtypes (A –D) of aMPV have been differentiated by nucleotide sequence analysis on the basis of the attachment (G) protein. The pathogenesis of different subtypes of aMPV had been investigated in turkeys intensively. SHS associated with avian Metapneumovirus (aMPV) subtype A or B in broilers and broiler breeders has been reported worldwide. However, data about the pathogenesis of aMPV subtype A and B in broilers are scarce and it is still unknown whether there are any differences in pathogenicity of aMPV A and B in broilers. Since aMPV causes swelling of periorbital sinuses in chickens, aMPV may also affect paraocular glands in this species. Most of the studies on immune mechanisms against aMPV have been conducted in turkeys but not in broilers. Therefore, two experiments were carried out to compare the pathogenesis of aMPV subtype A and B of turkey origin in broilers and to investigate selected parameters of the immune response of broilers to aMPVs. Sixteen-day-old and 14-day-old commercial Ross-type broilers free of aMPV maternal antibodies were used in the first and second experiment, respectively. Two groups of broilers were inoculated oculonasally with 104 median ciliostatic dose (CD50) of turkey isolates of aMPV subtype A or aMPV subtype B. The third group received virus free trachea organ culture (TOC) supernatant. Clinical signs such as depression, coughing, clear nasal exudates and frothy eyes appeared at 4 days post inoculation (dpi), followed by swelling of infraorbital sinuses at 5 dpi. Relatively higher numbers of broilers showed clinical signs in subtype B inoculated groups than in subtype A inoculated groups. Cumulative clinical score indices of subtype B-inoculated groups were significantly higher (P<0.05) than of subtype A-inoculated groups. At 15 and 17 dpi, subtype A and B-infected broilers had recovered from respiratory signs, respectively. Virus free TOC media inoculated groups were free of clinical signs in both experiments. Histopathological lesions such as thickening and destructions of the ciliated epithelium of infraorbital sinuses, nasal turbinate, and trachea due to infiltration of lymphoid cells, heterophils and formation of lymphoid follicles were observed between 3 and 11 dpi in aMPV-inoculated broilers. Histopathological lesion scores of nasal turbinate and trachea were not significantly different between subtype A and B inoculated broilers. Formation of lymphoid follicles in the submucosa of primary and secondary bronchi of the lung and in the interstitial tissues of paraocular glands was also detected in the virus infected broilers at 6 to 11 dpi. No histopathological changes were detected in spleen and bursa cloacalis. The distribution of the aMPV genome in different tissues was investigated by RT-nested PCR. The subtype B genome showed a considerably wider tissue-distribution and longer persistence than the subtype A genome. In addition to the respiratory tract tissues, the subtype B genome was also detected in the spleen, caecal tonsils, bursa cloacalis and cloacal swabs. In both experiments, aMPV specific serum VN-antibody titres increased significantly (P<0.05) at 6 dpi and reached peak levels at 10 to 11 dpi in both subtypes-inoculated groups. Serum aMPV-ELISA antibodies were detected at 10 to 11-dpi in aMPV-inoculated broilers and continued to increase until the end of experiment. The appearance of detectable ELISA aMPV-antibodies coincided with the clearance of the aMPV genome from different tissues after infection. Both subtypes stimulated not only a systemic humoral responses but also mucosal antibodies in the respiratory tract and bile. In contrast to the development of serum-ELISA-antibodies, the VN-antibody titres in tracheal washes declined when the aMPV genome was cleared from the upper respiratory tract. The infiltration of CD4+ and CD8+ T cells in the Harderian gland was observed at 6 dpi. These cells may also be involved in the local antiviral response to aMPV infection in broilers. Overall, this is the first study, which reproduced clinical disease with swelling of infraorbital sinuses in commercial broilers by experimental infection with aMPV subtype A and B of turkey origin. Both aMPVs affected all ciliated columnar epithelium of the respiratory tract tissues in broilers. Strong lymphoid cell infiltrations were detected in all respiratory mucosae following infection with both subtypes. These cells may participate in local immune responses. Infiltration of inflammatory cells was not only observed in the ciliated epithelium of the respiratory tract but also in paraocular glands, such as Harderian glands and lachrymal glands. Our study clearly suggests the role of aMPV as a primary pathogen of the respiratory tract for commercial broilers.