Serological Evidence of Avian Metapneumovirus Infection in Layer Chicken Farms of Madhya Pradesh, India

Avian metapneumovirus (aMPV) can blow heavy economic losses to the poultry industry by causing respiratory and reproductive tract malfunctions in turkeys, chickens and ducks (Jones and Rautenschlein, 2013). This virus was first time reported from South Africa in 1978 (Buys et al., 1989)  and was named as turkey rhinotracheitis virus (TRT) and since then has spread to different continents (Jones, 2010). Based on their genetic and antigenic differences four distinct subtypes of aMPVA, B, C and D have been reported from various parts of the globe (Cook and Cavanagh, 2002). Out of four, two subtypes A and B have wide geographical distribution in Asia, Europe, Africa and South America (Jones, 2010; Kwon et al., 2010; Owoade et al., 2008). The predominant clinical signs in chickens include swelling of the periorbital sinuses and infraorbital sinuses, known as a swollen head syndrome. The virus may also adversely affect egg production in breeder chickens and commercial layers (Cook et al., 2000, Jones et al., 1991). In India, avian metapneumovirus antibodies have been detected in broiler breeder flocks of seven States where in, 32.12-34.02% of tested birds were positive to aMPV antibodies (Eswaran et al., 2014, Surajit et al., 2018). In India data regarding aMPV status in layers is scarce. Under field conditions egg production losses of unknown etiology are often present and are most commonly attributed to aMPV infection on the basis of serological evidence. Virus isolation is difficult from chickens than from turkeys and requires several blind passages in cell cultures (OIE, 2016). Reverse-transcription PCR (RT-PCR) is relatively a sensitive and rapid method for diagnosis of aMPV than virus isolation (Cook and Cavanagh, 2002; Gough, 2003). Serological detection of aMPV is the commonly used method for diagnosis of aMPV infections, especially in unvaccinated flocks. Enzyme-linkedimmunosorbent assay (ELISA) is the most commonly deployed test for aMPV diagnosis (OIE, 2016).
       
In this study, we have performed a survey on layer farms in selected areas of Madhya Pradesh State, India to ascertain the aMPV status in laying birds, choanal cleft swabs and sera samples were collected for ELISA and RT-PCR assay.

Study area
 
The surveillance of aMPV was conducted from October to December, 2019 in five districts (Indore, Bhopal, Sagar, Guna and Bhind) of Madhya Pradesh, India (Fig 1). A total of 263 sera and 169 choanal cleft swabs were randomly collected from layer flocks of single farm of each district. The average flock size was of 1000-5000 birds. The samples were collected from the peak (20-48 week) and late laying (50-72 week) stage birds.
 

 
Diagnostic specimens
 
About 2.0 mL blood was collected by brachial venipuncture of each chicken individually using disposable syringes. The blood specimens were kept in a slanting position for an hour to allow clotting. The sera were collected into a sterile tube and centrifuged at 1500×g for 10 min at 4°C temperature to obtain the clear sera which were stored at -80°C temperature until further analysis. Choanal cleft swabs were also collected in viral transport media and transported to the laboratory in a cold chain. The blood and choanal cleft samples were collected from apparently healthy birds, avoiding any sign of respiratory illness and any clinical signs.
 
Vaccination
 
Since vaccination against aMPV is not followed in India therefore all the flocks were reared without aMPV vaccination. Flocks were vaccinated against other diseases, such as, Mareks’s Disease, Newcastle disease, infectious bronchitis and fowl pox.
 
Indirect enzyme-linked immunosorbent assay
 
The indirect ELISA was performed for each serum sample to detect the antibodies against serotypes A and B of aMPV using indirect ELISA (ID Screen® Avian Metapneumovirus Indirect, IDVet, Grabels, France) following manufacturer’s instructions. The results were analyzed by calculating sample/positive (S/P) ratio. (S/P) ratios >0.2 and were considered positive indicating exposure to aMPV.
 
Molecular identification
 
To detect genome of aMPV a Reverse-transcription polymerase chain reaction (RT-PCR) assay was carried out on choanal cleft swab samples using published primers directed against conserved regions of the N gene to detect A, B, C and D subtypes were used in the study (Bäyon-Auboyer et al., 1999). The synthetic gene construct of N gene was synthesized commercially to produce in vitro transcribed RNA for use in RT-PCR assay as positive control.

Avian metapneumovirus diagnosis in both turkeys and chickens is mostly carried out on the basis of detecting specific antibodies by ELISA in unvaccinated birds with a history of respiratory tract infections, or by detecting avian metapneumovirus genome by RT-PCR assay in acute respiratory infection (OIE, 2016 and Cook and Cavanagh, 2002). Swollen head syndrome (SHS) in broiler and broilers breeders (Gough et al., 1994) and egg production losses in layers (Sugiyama et al., 2006) have been attributed to the predisposition of aMPV.         
       
In the present study, overall 9.88% (26/263) of the chickens harbored antibodies against aMPV in their serum. Among five districts sampled, Indore revealed the significantly (P<0.001) highest 73.33% (22/30) and Guna and Bhind lowest 0% (0/50 and 0/94) seroprevalence, respectively (Table 1). Remaining two districts Bhopal and Sagar showed 4.08% (2/49) and 5.00% (2/40), respectively (Table 1). According to the stage of laying, the highest seroprevalence 73.33% (22/30) was found in late laying (approximately 72 weeks) birds of Indore.
 
  
       
In Korean, chicken, Park et al., (2011) reported that 73.1% chicken were positive for aMPV antibodies. The incidence of aMPV antibodies was higher in broilers (97.3%) than in layers (67.5%). In a recent study in Bangladesh, Ali et al., (2019) observed that 53.29% chicken harbored antibodies against aMPV and incidence was higher in broiler breeders more than 41 week of age. In India an earlier study in Tamil Nadu State (Eswaran et al., 2014) observed 34.02% antibodies in broiler breeder chicken. Our findings are also in concurrence with these findings however, the 9.88% positivity observed in our study is lower as compared to earlier studies this could be due to difference in biosecurity measures followed in the farms sampled. RT-PCR assay was carried out in 169 choanal cleft swabs from four districts viz., Indore (30), Bhopal (49), Sagar (40) and Guna (50). All the samples were tested negative for aMPV genome. For detection of aMPV genome it is importnat to collect specimens when clinical signs are first exhibited it has been shown that maximum amount of virus is present in the nasal turbinates and trachea at 3 days post inoculation and viral RNA could be detected up to 9 days and 14 days in trachea and nasal turbinates, respectively (Velayudhan et al., 2005). It has been observed that aMPV can be detected in samples collected within 7-10 days post-exposure, however the viral concentration decreases considerably by reducing success of detection (Alkhalaf et al., 2002; Padersen et al., 2001). In our study none of the birds were showing any clinical signs during collection of specimens this could be reason of absence of aMPV genome in the samples. Since, in India chickens are not vaccinated for aMPV the seropositivity against aMPV indicates that chickens are exposed to this important poultry pathogen. aMPV may pose serious economic concerns in turkeys and chickens especially in the presence of concurrent bacterial and viral infections.

In conclusion this study highlights the presence of antibodies to aMPV in layers of Madhya Pradesh State which warrants further surveillance of aMPV in wider geographic area and isolation, characterization of aMPV to design effective control strategies for aMPV infection.

Authors are thankful to Indian Council of Agricultural Research, Director, ICAR-NIHSAD for providing funding and facilities to carry out this work.