JOURNAL OF POULTRY SCIENCE AND TECHNOLOGY Journal homepage: www.jakraya.com/journal/jpst ORIGINAL ARTICLE Prevalence of Avian Influenza Virus Associated with Abiotic Components of Live Bird Markets in Gujranwala, Pakistan Toqeer Ahmad 1, Rana Haider Ali 1* and Shakil Abbas 2 1Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan. 2Faculty of Veterinary Sciences, Gomal University, Balochistan. Abstract Avian Influenza virus is of great concern for both livestock and human. Avian influenza virus has given rise to various genotypes. Live bird markets are cause of risk for poultry and human as they are potential source *Corresponding Author: of the influenza A viruses. In markets the abiotic factors are critical in the spread of virus but are somehow ignored. The water, cutting boards, cages Rana Haider Ali and knife are not regularly washed and cleaned. In the current study, 240 Email: [email protected] swab samples were tested of drinking water, fecal dropping, cage, chopping board, vehicle and sewage. The samples were inoculated in nine to eleven Received: 19/06/2020 days old embryonated eggs for the cultivation of virus. After 48 hours of Accepted: 29/06/2020 incubation, amnio-allantoic fluid was harvested and checked for the hemagglutination activity followed by haemagglutination inhibition test. The results showed 1 sample positive for H9N2 taken from cage swab sample. Present study showed detection of avian influenza virus subtype H9N2 from an abiotic factor in live bird market in Gujranwala, Pakistan. There is a strong need of check and balance on live bird markets in order to avoid any outbreak in the future. Keywords : Live bird markets, Avian influenza virus, Subtypes, Abiotic components, Hemagglutination activity. 1. Introduction commercial flocks in Pakistan (Naeem et al., 2007). Infectious diseases cause significant harm to the Influenza A virus pandemic strains ascend by genetic poultry industry (Ullah et al., 2020). Avian influenza is recombination between virus of avian and human an extremely transmissible disease produced by Avian origin. Most of the natural infection of avian influenza influenza virus type A (Fouchier et al ., 2005). Avian virus are found in free living wild birds including Influenza virus A is member of the orthomyxoviridae thirteen avian order and approximately 90 species. Most family which is negative sense single stranded RNA of these species are aquatic living (Muramoto et al ., virus with an envelope. The complete genome of this 2006). Spread of avian influenza virus through indirect virus consists of eight segments (Tan et al ., 2015). The and direct routes like fecal dropping and aerosol has virus is divided into various subtypes depending upon been well established (Alexander et al., 1986). Viruses its HA and NA gene. To the current time there are 18 H also transmit through indirect route including fomites. and 11 N subtypes that have been discovered according In live bird market setting the transmission of pathogen to hemagglutinin and neuraminidase property, making a spread is through fecal droppings. Another mean of huge hoard of virus (Le and Nguyen, 2014). In 1933, transmission is through aerosol. During the handling of Wilson Smith and his colleagues first isolated the virus birds virus spread from infected animal to healthy (Yee during epidemic of influenza at beginning of year 1933. et al., 2009). Commercial poultry markets provide huge According to an estimate influenza virus has caused density of avian host that is why it provide about 3 to 5 million intense cases and about 25 to 50 maintenance, dissemination and amplification of avian thousand deaths throughout the globe (Smith et al ., influenza virus (Offeddu et al ., 2016). It is very 2004). From year 2003 to 2004 epidemic of high necessary to do continuous surveillance of the poultry pathogenicity avian influenza of subtype H7N3 markets in order to identify the present contamination occurred in Pakistan and it affected the main broiler level so that effective measure may be applied as and layer breeder raising in the different areas. It went preventive measure of this disease (Wang et al ., 2017). along with by an outbreak of a low pathogenicity avian influenza virus H9N2 in broilers and layers, this 2. Materials and Methods infection continued during 2005. Consequently, in February 2006 for the first time avian influenza virus 2.1 Study Area subtype H5N1 was observed in two isolated Journal of Poultry Science and Technology | April-June, 2020 | Volume 08 | Issue 02 | Pages 41-44 © 2020 Jakraya Ahmad et al…Prevalence of Avian Influenza Virus Associated with Abiotic Components of Live Bird Markets in Gujranwala, Pakistan Study area was department of Microbiology, centrifuged at 3000 to 5000 revolutions per minute for University of Veterinary and Animal Sciences, Lahore. 10 to 15 minutes. Supernatent was discarded and normal saline was added to make volume of 10 ml and 2.2 Sample Collection and Processing then again tube was centrifuged and supernatent was A total of 240 samples were collected on discarded. Steps were repeated three times until the suitable basis from live bird markets of Gujranwala supernatant became clear. From washed red blood cells, from July 2019 to September 2019. Sampling areas and 370 µl was taken and then added in 50 ml normal saline number of samples collected from each place is to make 0.735% washed red blood cells. described in Table 1. Samples include swabs from poultry cage, scale, fecal droppings, chopping board, 2.3 Hemagglutination Assay vehicle carrying the birds, drinking water and sewage Test was performed in abbexa ® 96 well micro sample. Swabs were kept in suitable virus transport titration plate. The hemagglutination test was performed medium or Brain heart infusion media. Media was to check the hemagglutination activity of the harvested prepared from SIGMA-ALDRICH ® Brain Heart amnio-allantoic fluid. Test was performed as per the Infusion Broth. Samples were transported to protocol described by Alexander and Chettle (1977). Department of Microbiology, UVAS Lahore and were stored at - 18°C. Sample processing was done as 2.4 Hemagglutination Inhibition Assay explained according to Spackman et al. (2013). The hemagglutination inhibition test was performed to check the subtype of virus by using the Table 1: Sampling areas and number of samples specific sera for H5, H7and H9. This assay was done in collected from each place accordance with the method explained by Alexander and Chettle (1977). Sr # Sampling Areas Number of samples 3. Results and Discussion 1 Naushehra Virkan town 20 Broiler chicken meat is cheap source of animal 2 Kamonki town 20 3 Wazirabad town 20 protein (Ali, 2020). In developing countries like 4 Qila Didar Singh town 20 Pakistan live bird markets hold a very significant place 5 Nandipur town 20 in supplying food and fulfilling the nutritional 6 Aroop town 20 requirements (Organization, 2006). On the other side 7 Khiali Shahpure town 20 these market also deliver best conditions for sheltering 8 Ali Pur Chatta 20 and transfer of zoonotic diseases as in these markets 9 Sodhra 20 interaction between live animal and human is very 10 Ramnagar 20 frequent (Webster, 2004). Infectious diseases trigger 11 Tariq Abad 20 12 Qasim town 20 significant damage to the poultry industry (Ali et al., 2020). In present study, out of 240 abiotic samples, 1.2 % (3 samples) were HA positive and only 0.4 % were 2.2.1 Inoculation and Harvesting In laboratory each sample was inoculated in 9 to H9 (n=1 sample) positive. The H9 positive sample was 11 days old embryonated eggs through allantoic sac isolated from cage sample. The HA titer of cage sample route using insulin syringes (Khalili et al., 2013). Eggs number 145 was 10 and the HA titer of drinking water were incubated at 37ºC for 48 hours after the sample number 188 and 211 were 8. The HA titer of inoculation. After 48 hours of inoculation, eggs were three positive sample along with their origin and type is shown in Fig 1. Haemagglutination inhibition titre of chilled overnight at -4ºC and then candling was done to check mortality of embryo. After overnight incubation samples is shown in Fig 2. The both drinking water at -4°C amnio-allantoic fluid was harvested from samples showed negative results for HI against H9 sera. Cage sample showed positive result against H9 embryonated chicken eggs in Class 2 biosafety cabinet. th 70% ethanol was used for disinfection of egg shells and sera. Titre of positive sample was upto 8 well (1: 256). None of the three positive HA samples showed activity then with help of sharp scissor, shell over the sac was against H5 and H7 sera. In the current study, there was removed. Membrane of air sac was removed and head of embryo was pressed with forceps and amnioallantoic lower detection of H9 and no sample was positive for fluid using 5 ml disposable syringes was harvested the H5 and H7. This may be because most of the markets fluid was collected in 15 ml falcon tubes that were in Gujranwala are not enclosed. As sampling in correctly labelled (Hitchner et al., 1980). summer season was done between July to September when temperature of Gujranwala is normally above 40ºC. In other studies, there was relatively high level of 2.2.2 Washing of Chicken Red Blood Cells For washing of red blood cells, 50 ml of fresh finding of H5 and H7 subtype (Chaudhry et al., 2018). blood of chicken was collected in the beaker that already The markets included in present study that are not contained 5 ml of anticoagulant EDTA. Then 10 ml contaminated with avian influenza virus were open. blood was transferred to 15 ml falcon tube and then it was The market from where the positive sample of H9 was Journal of Poultry Science and Technology | April-June, 2020 | Volume 08 | Issue 02 | Pages 41-44 © 2020 Jakraya 42 Ahmad et al…Prevalence of Avian Influenza Virus Associated with Abiotic Components of Live Bird Markets in Gujranwala, Pakistan found was an enclosed market with a very heavy in and showed positive result against H9 sera.