In the current study, 83.0% of clinical blood samples from poultry farms were positive for ORT antibodies by ELISA. The ORT isolated from the infected lungs of a 32-day-old broiler grew on sheep blood agar, and the H9N2 virus was isolated from the same broiler and identified by HI and RT-PCR. Based on the sequence analysis, the Chinese ORT isolate is 98.0 to 100% homologous to other ORT isolates in GenBank. Broilers inoculated intraperitoneally with ORT/chicken/Shandong/2011 alone displayed pneumonia and typical airsacculitis, and co-infection of the broilers with ORT and H9N2 virus isolates induced higher mortality than infection with ORT or H9N2 virus alone. Therefore, these results satisfy Koch’s postulates for confirming the role of a suspected bacterial pathogen in disease. The results of this study strongly suggest that co-infection with ORT and H9N2 virus is responsible for the current severe pneumonia with high mortality in broilers of China.
In our clinical setting, ORT was associated with 20-30% and 10-20% mortality in broilers and layers, respectively. However, the birds infected with the ORT isolated in this study had a 50% mortality rate, and a co-infection with the H9N2 virus resulted in 70% death. Our findings suggest that primary infection with ORT might play a major role in the development of severe pneumonia, and secondary infection with H9N2 further increases the mortality. These findings are different from previous reports in which no pneumonia or airsacculitis was induced by aerosol, intra-tracheal or intra-thoracic inoculation with ORT alone [18
]. Only intravenous inoculation has been reported to induce clinical signs in SPF chickens, with at most 20% mortality [19
], and no airsacculitis has been previously seen in the field. Given these conflicting results, it is uncertain if ORT should be regarded as a primary pathogen [1
]. Additionally, it is generally acknowledged that ORT and Avian pneumovirusvirus
(APV) infections synergistically aggravate respiratory symptoms in turkeys. An aerosol inoculation of ORT without a viral primer did not result in lesions [20
]. Furthermore, viral agents could trigger higher mortality independent of ORT infection, such as NDV [1
] and APV [21
]. A recent report confirmed that IBV and E. coli
infection exacerbated ORT pathogenesis in adult laying hens [23
]. However, none of the above-mentioned viruses were identified in the present study. Interestingly, the birds infected with ORT alone developed an exudative pneumonia and extensive haemorrhage in the lungs and kidneys. This pattern of pathology is not consistent with previous reports. These histological results indicate that this newly isolated ORT is different from formerly reported ORT serotypes [20
]. Because the serotype of the ORT isolated in this study is unclear, further investigation is needed to identify its specific serotype and characterise ORT pathogenesis based on serotype. After inoculation of broilers with ORT and H9N2 virus together, widespread haemorrhage and fibrosis in the respiratory tract were the most notable features of the infection, which led to occlusion of the air capillaries, respiratory distress and the increased mortality. These histopathological lesions are analogous to those described in birds late during the course of avian influenza H9N2 infection alone [11
]. Notably, the severe pulmonary fibrosis was observed in the animals inoculated first with ORT followed by the H9N2 virus. The clinical signs and the respiratory lesions observed during necropsy of the birds infected with the H9N2+ORT combination and the H9N2 group confirm that ORT, not a viral agent, triggers the overt respiratory symptoms.
Our findings are also different from previous reports in which ORT could be involved in infections with E. coli O2:K1
], Bordetella avium
], and Chlamydophila psittaci
] as well as a combination of E. coli
and APV [22
]. In our pilot study (unpublished data), E. coli
and Chlamydophila psittaci
were occasionally identified in the latter phase of the ORT infection. However, co-infection of ORT and E. coli
or Chlamydophila psittaci
could not reproduce the typical pathology of the pneumonia and airsacculitis, such as clots of fibrin in the air sacs and haemorrhage in lungs. In the co-infection, ORT may dominate the primary infection, followed by secondary bacterial infections. ORT was isolated and identified in birds with clinical signs immediately after hatching in the previous reports [24
]. In current study, ORT/chicken/Liaoning/2010 was isolated from the egg yolks of the breeder’s eggs, suggesting the possibility of vertical transmission from parents to offspring.