Molecular and Serological Detection of Avian Influenza H9N2 Virus in Asymptomatic Commercial Layers in Faisalabad District, Punjab

A total of 120 blood samples (10 samples from each flock) and 24 tissue samples (Trachea, lungs, and Intestine) were collected randomly from various layer flocks reared in Faisalabad district of Pakistan. For serum collection, 1 ml blood was taken from the wing vein by using appropriate syringe. Serum was isolated from clots by centrifuging at 4000 rpm/m for 10 min. For tissue collection, birds were slaughtered and then trachea, Intestines and lungs were aseptically removed. Rinsed in tap water and immediately transported to the lab of in cold conditions for further processing.

To test the presence of H9N2 specific antibodies in the serum, Hemagglutination Inhibition (HI) test was performed in V-bottom shaped 96-well microtiter plates. The NOBILIS Influenza H9N2 Vaccine virus (Novus International® USA) was used in the study along with 1% of chicken erythrocytes according to standard protocol (Swayne, 1997). The antibody titer was calculated according to the last well reading which showed the complete inhibition of haemaggutinin activity induced by the H9N2 subtype used in the study.

For RT-PCR analysis trachea, lung, and intestine samples were processed for RNA extraction according to Mannhalter et al. (2000). The RNA pellet was solubilized in 50µl of DEPC treated water. Its quality and quantity was measured by using NanoDrop®.

The RT-PCR was performed to amplify H9 specific 808-bp fragment of avian influenza virus using Forward 5’- AGCAAAAGCAGGGGAACTCC-3’Reverse 3’- CCATACCATGGGGCAATTAG-5’ primers as describe by Dybkaer et al. (2004). The 50µl reaction mixture was prepared by adding 25µl QuickTM Master Mix, 1µl (5u) of AMV Reverse Transcriptase (Sigma, USA), 5µl RNA template, 1µl of 1.0µM upstream and downstream primers. The conditions used in one step RT-PCR were include reverse transcription with the Incubation at 45°C for 45 min, initial denaturation at 95°C for 5 min, and 40 cycle each of 95°C for 40 sec, annealing at 56°C for 1 min, primer extension at 72°C for 30 sec followed by final extension at 72°C for 10 min. The amplified PCR products were visualized on 0.8% agarose gel, stained with ethidium bromide.

The results of the investigation revealed that overall seroprevelance was 60% (72/120) and geometric mean titer of 5.37 (Table I) indicating the natural exposure of the flocks to the H9N2 subtype of avian influenza Virus. The positive results showed the amplification of 808 bp fragment of H9 from H9N2 subtype (Fig. 1). Out of 24 tissue samples each from trachea, lung, and intestine samples tested, 92% (22/24), 88% (21/24), 75% (18/24) were found positive, respectively. The results reveal the presence of live virus in the asymptomatic birds. Moreover, results show that the respiratory tract is the preferred path of virus multiplication.

Table I.- Seroprevelance and antibody titer against H9N2 AIV in asymptomatic layer flock. The table shows seroprevelance and antibody titer against H9N2 AIV in asymptomatic layer flock tested by HI. The results are presented as geometric mean titer GMT of log 2 titers.

In Asia, prevalence of H9N2 has been established since 1990 (Guan et al., 1999). This virus belongs to LPAI group and is associated with mild infection in poultry. On the other hand, in the presence of opportunistic pathogens it may cause severe morbidity and sometimes leads to death (Huang et al., 2015). The pproposed study was conducted with the objectives of the estimation of antibodies against H9N2 and confirmation of the virus in asymptomatic commercial layers of various flocks located in district Faisalabad, Punjab by using RT-PCR.

In the current study, we collected samples from randomly selected 12 layer flocks with the age of 35-50 weeks. These flocks were apparently healthy and had no signs and symptoms of avian influenza. Results of HI revealed 60% seroprevalence with a GMT of 5.37. It indicates the birds were naturally exposed to H9N2 AIV. Natural exposure to the virus may be due to the risk factors associated with the environment which include the rearing of the domestic poultry in the vicinity of layer farms, because the prevalence of AI in domestic poultry and its transmission through aerosol has been established (Alexander, 2007).

Presence of H9N2 was further verified by RT-PCR based molecular detection of viral RNA in various organs of birds. RT-PCR results showed a surprisingly high prevalence of live virus; 92 % trachea, 88% lungs and 75% intestine samples were found positive for H9N2 RNA. Kwon et al. (2008) reported the detection of AIV A/Chicken/HS/K5/01(H9N2) in various organs including bursa, spleen, kidneys, lungs and trachea. It has also been observed that H9N2 has the ability to stay in various tissues like spleen and kidneys of the commercial layers (Lee et al., 2007; Mosleh et al., 2009). Results of the present study are also in accordance with a study conducted by Shamseddini et al. where the researchers detected H9N2 of Iranian origin in numerous tissues include lungs, trachea and kidneys (Shamseddini et al., 2002).

In conclusion, results of the present study show that the healthy layer flocks can be naturally exposed to AIV H9N2. Furthermore, our findings affirm the fact that AIV is disseminated to various body organs. In continuation of this study prospective studies should be planned with the objective of screening of all poultry population and live poultry markets (LPMs) as well with special reference to AIV at national level.

We appreciate the National Agriculture Research Council for the permission to use their laboratory facilities.

Authors have declared no conflict of interest.