Serosurvey ofAvian metapneumovirus, Orithob

Seasonal outbreaks of airsacculitis in China’s poultry cause great economic losses annually. This study tried to unveil the potential role of Avian metapneumovirus(AMPV), Ornithobacterium rhinotracheale (ORT) and Chlamydia psittaci (CPS) in avian airsacculitis. A serological investigation of 673 breeder chickens and a case-controlled study of 430 birds were showed that infection with AMPV, ORT, and CPS was highly associated with the disease. The correlation between AMPV and CPS were positively robust in both layers and broilers. Finally, we determined the co-infection with AMPV, ORT, and CPS was prevalent in the sampled poultry farms suffering from respiratory diseases and the outbreak of airsacculitis was closely related to simultaneous exposure to all three agents.

Airsacculitis is the inflammation of the mucous membrane of birds’ air sacs. It has been prevalent in most areas in the Chinese poultry since 2012. Chickens of all ages could be infected, even newborn chicks. Numerous microorganisms are responsible for air sac disease and more than one infectious agent is often identified in an outbreak. In general, it is reported that bacteria such as Escherichia coli (E.coli), Mycoplasma gallisepticum (MG)or virusessuch as Infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and Avian influenza virus are most relevant. A series of treatments which targeted at the pathogens mentioned above have been applied, but without significant success, which implies another sources of infection. AMPV infection might contribute to avian airsacculitis[1] and it might co-infectwith other pathogens. In a recent survey, both AMPV subtype B and C were identified and seropositivity ranged from 52.4% to 74.1% in Chinese poultry farms[2]. In China ORT was identified in diseased birds from Guangxi, Shandong, Henan, Hubei and Jiangsu from2004 to 2016[3]. ORT infection usually aggravated respiratory difficulty induced by AMPV andCPS inoculation[4]. Guo et al. using a RT-PCR assay[5] indicated that 24.7% (442/1,791) of chickens were infected with CPS, and Specific pathogen free (SPF) chickens inoculated with CPS and then AIV H9N2 had severe respiratory difficulty and exhibited 35% mortality[6]. The understanding of the association between avian airsaculitis and these three agents will shed light on the etiology of airsacculitis in poultry.

During the period December 2014 to May 2015, we surveyed 14 poultry farms with outbreaks of seasonal airsacculits in seven provinces of China (Table S1, available in ). A total of 673 blood samples of breeders with a typical respiratory conditions were collected (248 broilers and 425 layers). For the unmatched case-control study, 210 commercial broilers and 220 commercial layers, which added up to 430 birds, were selected (from a farm we investigated in Hebei) (Table S2, available in ). Blood sera were centrifuged (3500 rpm for 10 min) and stored at -80 °C until assayed. Blood samples were tested with commercial ELISA kits (IDEXX, Beijing, China) to exclude the infections of E.coli (99-08701), MG (99-06729), IBV (99-09262), NDV (99-09263), and Avian influenza (99-09262). Detailed numbers of case and control birds were exhibited in Table S2. The experiments were approved by an ethical board at the China Agricultural University in full accordance with ethical principles and all birds were handled following humane protocols that minimized pain in the animals.

Antibodies against AMPV (99-) and ORT (99-) were also analyzed with commercial ELISA kits (IDEXX, Beijing, China) while CPS-specific IgG antibodies were detected using a MOMP-based ELISA kit (supplied by Professor Daisy Vanorompay, Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University).

The AMPV, ORT, and CPS positive rate between control and diseased birds in both broilers and layers was summarized using number and frequency with percentage. Spearman correlation analysis was performed to detect the correlation among these three pathogens. The individual effect of pathogen with disease status was analyzed using Chi-square test. Logistic regression model was constructed to analyze the combined effect of these three pathogens, including the two terms and three terms interactions. If possible, adjusted if there was any other virus infection. Infection status was defined into 7 levels: negative, AMPV only, ORT only, CPS only, AMPV and ORT, AMPV and CPS, ORT and CPS, all positive. The infection status difference between broilers and layers was analyzed using Chi-square test. P < 0.05 was considered as significant statistical findings. SPSS 22.0 software was used for all analyses.

To define the infection patterns, we analyzed the seropositivity status of the three pathogens in each case (Table 1). In broilers, AMPV-CPS coinfection was the primary infection pattern. There were no significant differences among all the co- infection patterns. In layers, AMPV-CPS coinfections were the most frequently encountered, followed by AMPV-ORT-CPS co-infections. There was no significant difference between the two patterns.

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