Introduction

Avian Metapneumovirus (AMPV) is a single-strand, negative-sense RNA virus belonging to Paramixoviridae family, Pneumovirinae subfamily, and Metapneumovirus genus. It causes contagious infection of the upper respiratory tract in turkey, chicken, and some poultry species with different mortality rate. In addition to economic losses to the broiler growers, AMPV infection increases feed conversion ratio, egg drop, incidence of eggshell breakage, and hatching rate (Umar et al., 2016). According to the nucleotide and deduced amino acid sequence data, AMPV is divided into 4 subtypes: A, B, C and D. A, B, and D are closely similar. The respiratory distress and reproductive disorder are the main problems of this infectious agent. Secondary bacterial infections play an important role in exacerbating disorders and wild birds could be as natural reservoirs for this virus. The  clinical diseases caused by AMPV previously referred to as avian infectious pneumoniae (APV), swollen head syndrome (SHS), turkey rhinotracheitis (TRT) and avian rhinotracheitis (ART) are the acute and highly contagious infection of the upper respiratory tract in turkey, chicken and some poultry species (Suarez et al., 2020).

AMPV was first reported in South Africa in the late 1970s, initially detected in turkeys. In 1984, the virus was isolated in France and the UK. Then, it was isolated from chickens and caused the upper respiratory infection in poultry. It was also found that the virus in some poultry species causes SHS (Brown et al., 2019; Mayahi et al., 2017). In Iran, it has been first reported by Sheikhi and Masoudnia (2011), using serological ELISA test. Then, Hoseini and Ghalyanachi (2012) conducted the first molecular epidemiology study. There are considerable differences in the clinical signs and the mortality rates between experimentally and naturally infected chickens. Rhinotracheitis is a very important manifestation in AMPV, which targeted the respiratory tissue organs. On the other hand, concurrent infection with exacerbating agents such as E. coli, Bordetella avium, Ornithobacterium rhinotracheale, Riemerella anatipestifer, Mycoplasma gallisepticum, and lentogenic Newcastle disease increase the incidence of clinical signs significantly (Miller et al., 2013; Umar et al., 2016; Brown et al., 2019). The transmission of the virus through the air and direct contact with wild birds is possible (Suarez et al., 2020). In this regard, the present study was conducted with the aim of molecular detection and subtyping of AMPV infection in broiler flocks from 2016-2020 in Semnan province, Iran, using RT-PCR method.

Materials and Methods

Sample Collection

The lesion observation and necropsy time of upper respiratory tract are very important because AMPV is present in the sinuses and turbinates only for 6–7 days.  For this study, sampling was carried out from 85 broiler flocks from the Semnan province of Iran with the respiratory clinical signs. The samples were taken from the upper part of the trachea, choana, and sinuses of dead broiler chickens. All flocks were older than 3 weeks of age, characterized by mild respiratory symptoms, SHS, and suspected to AMPV infection in autumn and winter seasons, between 2016 and 2020. For each flock, 10 swabs were taken while 5 of each were pooled and considered as one sample (total 170 samples, 2 samples from each flock). The samples were transferred to the Faculty of Veterinary Medicine, Semnan University for RNA extraction and RT-PCR amplification.

RNA Extraction

Swabs were first diluted with 0.50 mL phosphate buffer saline (PBS) and then RNA was extracted using super plus RNA extraction kit (maxcell, Iran) according to the manufacturer's instruction.

Primers

In this study, the Nd/Nx and Ga/G12 primers were used for detection of AMPV and subtype B, respectively (Table 1). These primers were previously used by Bayon-Auboyer et al. (2000).

Table 1. Primers Applied in Current Study

CDNA Synthesize

The first-strand cDNA was synthesized by SinaClon First-Strand cDNA Synthesis Kit (Cat. No. RT520100).The mixture was subjected to 12 cycles of 21°C for 30 sec, 43°C for 4 min, and 54°C for 30 min followed by a final inactivation for 5 min at 95°C. The amplification of first-strand cDNA was performed in a thermal cycler (Lab gene Scientific Co., Zurich, Switzerland).

The PCR Amplification

The PCR Mix including 10 μL of Parstous PCR Mix® (Pars-Tous, Mashhad, Iran), 2 μL of forward and reverse primers, 5 μL of DEPC treated water (CinnaGen, Karaj, Iran) and 3 μL (200 ng) of each cDNA sample was prepared. The samples were subjected to 1 cycle of initial denaturation at 94°C for 3 min, 30 cycles of 94°C for 1 min, annealing temperature for 30 sec (51°C for geneN and 54°C for geneG), and 72°C for 60 sec, followed by a final elongation step at 72°C for 5 min. The PCR products were electrophoresed on 2% agarose gel at 140 V for 40 min and then visualized by DNA safe stain (CinnaGen) (Table 2).

Table 2. Primer Set, Annealing Temperature and Expected Products Size

Results

Using the Nd/Nx primer set, from 85 flocks and 170 samples, 30 flocks (35.3%) and 51 samples (30%) were positive for AMPV. They included 3 out of 20 (15%) broiler flocks from 2016, 13 out of 30 (43%) from 2017 to 2018 and 14 out of 35 (40%) from 2019 to 2020. Overall, all positive flocks for AMPV had a history of upper respiratory distress and SHS signs during the clinical examination. Twenty-eight flocks were found infected with subtype B. Interestingly, 2 flocks were positive for another subtype, probably belonging to A, C or D subtypes. The characteristics of positive flocks for the AMPV were shown in Table 3.

Table 3. Positive flocks and their details, WINT: Winter, AUT: Autumn, NON B: not belonging to B subtype 

Discussion