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Broilers of Live Bird Markets at Mymensingh City Carrying H9n2 Avian Influenza Virus in Their Respiratory Tract

Sanzida Rahman
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Afroja Yasmin
Department of Pathobiology, Faculty of Veterinary Medicine and Animals Science, Bangabandhu Sheikh Mujibor Rahman Agricultural University, Gazipur- 1706, Bangladesh

Tahmina Ruba
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Mohammad A H N A Khan
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Avian influenza (AI) caused by Type A influenza virus is a global zoonosis, infecting vast majority of mammalian and avian species. Broilers are meat type birds and randomly reared and sold by the farmers in Bangladesh with poor biosecurity. This study was aimed to identify the Type and subtypes of AI viruses in the broilers of two live bird markets, Mymensingh. A total of 10 birds from each of the market were randomly selected, investigated by clinical, pathological, reverse transcriptase polymerase chain reactions (RT-PCR), sequencing and sequence analysis. Out of 20 birds investigated, 06 were sick, 02 were dead and 12 were apparently healthy. Clinically, the sick/dead birds did not reveal any changes typical to AI. During necropsy, the sick/dead birds showed congested lungs and moderate hemorrhages in the trachea. Such lesions was absent in the lungs of apparently healthy birds. Following histopathological examination interstitial pneumonia with bronchitis was seen in sick/dead birds. The RT-PCR protocol was adapted to identify matrix protein gene of Type A influenza virus and amplified 430bp fragment is even cases. To identify the sub types of AI viruses involved, hemagglutinin (HA) and neuraminidase (NA) gene specific RT-PCR was carried out. 1475bp and 1089bp amplicons specific to HA and NA genes of AI viruses were generated in 07 cases. The cDNAs of HA and NA genes were sequenced, edited and revealed that the AI virus circulated in the live bird market of Mymensingh city is H9N2 subtype. Two sick, one dead and four apparently healthy birds found to carry H9N2 AI virus. The H9N2 virus is naturally low pathogenic for poultry, has got public health significance, and may donate partial or even whole cassette of internal genes to generate novel human-lethal reassortants of AI viruses; this was main concern for AI viral outbreak investigation in this study. It needs to examine large number of samples from wider sources to trace the rate of mutation and subsequent reemergence of pandemic AI viruses.

Keywords: Live bird market, Avian influenza, Broilers, RT-PCR, Sequencing, H9N2

Location: KR market, BAU, Mymensingh and Kewatkhali local market, Mymensingh, Bangladesh

Start Date: 00-03-2016

End Date: 00-05-2016

Program Area: Animal Health and Management

Commodity: Broiler

Objectives:

To identify the clinicopathological investigation of AI in broilers at live bird markets and detection of the subtypes of AI virus existed in the upper respiratory system by using RT-PCR and sequencing.

Methodology:

Broilers of two live bird markets of Mymensingh city were investigated (n=20) and necropsised in the Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University (BAU), Mymensingh during March to May, 2016. A total of 06 sick, 02 dead and 12 apparently healthy broilers were collected from KR market, Bangladesh Agricultural University (BAU) and Kewatkhali live bird market, Mymensingh city. 10 birds (four sick/dead and six apparently healthy) from each of the market were investigated. During live bird market investigations, the sick birds (N=06) showed mild gasping, reduced feed and water intake, slightly loose feces and dizziness. The sick/ dead (N=08) and apparently healthy (N=12) broilers were brought to the Department of Pathology, necropsised and gross changes observed were recorded. Following necropsy, portion of tracheas was snap frozen. Portion of lungs, liver, heart, pancreas, skeletal muscle, duodenum, trachea and spleen were collected and fixed in 10% neutral buffered formalin for histopathological studies. Formalin fixed tissue samples were processed and stained with H&E stain. Briefly the tissues were embedded in paraffin, 4-5µm thick sections were float on lukewarm water (42ºC) containing gelatin and the floating tissues were pick up onto clean slides. The tissues on to slides were stained with hematoxylin and eosin (H&E) and were mounted using DPX. The sections on to the slides were examined at low and high power microscopic field. The images were captured and labelled for better understanding the changes. Portion of trachea (N=20) of the broilers were used in viral RNA extraction and detection of matrix protein genes of AI virus by using RT-PCR. Briefly 50mg of frozen tracheal tissues were crushed in liquid nitrogen and extracted viral RNA using QIA amp Viral RNA Mini Kit (QIAGEN, Germany). The quality and quantity of the extracted RNA was measured by using agarose gel electrophoresis and spectrophotometry (A260/A280). The matrix (M) protein gene of AI viruses was targeted to amplify in an RT-PCR setting. The RT-PCR was carried out using One-Step RT-PCR System (SuperScript® III One-Step RT-PCR System, USA). The reaction was carried out in 50μl volume consisting of 2x master Mix, 1μl of each primer (20pmol/μl), 1μl of SuperScript III RT/Platinum Taq Mix, 1μl Rnase inhibitor, 5μl template RNA (50ng) and 17μl Nuclease free water. As negative control 5μl nuclease free H₂O was used instead of template RNA. The amplification reaction was carried out in a thermal cycler (Master Cycler Gradient, Eppendorf, Germany) as standardized (Ruba et al., 2015), the final reaction was held at 4°C, the amplicons was analyzed by electrophoresis in 1.5% agarose gel containing ethidium bromide (0.5 μg/ml) and images were captured using a transilluminator (Alpha imager, USA). Viral RNA from the trachea of matrix gene positive birds were used in RT-PCR amplification of HA and NA genes of AI viruses. Published primer sequences of the HA and NA genes of AI viruses were used in RT-PCR amplification of the viral genomes (Ruba et al., 2015). The cDNAs of HA and NA genes as obtained through RT-PCR were gel cleaned and sequenced from commercial source (1st Base, Malaysia). The retrieved raw sequence data were first checked for its quality and then edited. Following sequence edition a total of 1122 nucleotide sequences of HA gene and 972 nucleotide sequences for NA gene were stand for genetic analysis. The HA and NA nucleotide sequences in the GenBank database provided by the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/genbank) were downloaded and used for phylogenetic analysis. Multiple alignments were done with Clustal W algorithm and Neighbour-Joining phylogenetic tree was constructed with MEGA6 programme. The stability of the nodes in the phylogenetic tree was tested by bootstrapping value with 1000 replications. For comparative study, available sequences of other Bangladeshi isolates of AI were downloaded from GenBank and used in phylogeny.

Source of Information: Res. Agric. Livest. Fish. Vol. 5, No. 2, August 2018 : 225-233

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Achievement/Findings:

This study investigated 20 broilers from KR market, BAU, Mymensingh and Kewatkhali local market, Mymensingh and 07 birds found to carry Type A AI viruses in their upper respiratory tracts. Clinical and pathological investigation was unable to trace infectivity of broilers due to AI viruses. The RT-PCR protocols used appeared successful in terms of detecting M, HA and NA genes of Type A influenza virus and identify H9N2 viruses in the dead, sick and apparently healthy broilers. The occurrence of H9N2 AI viruses in the broilers of live bird market possess a substantial threat to public health and the carrier birds may prone to co- infection with other infectious agents having affinity to upper respiratory tract. It requires extensive surveillance on to the distribution of H9N2 AI virus and other subtypes, identifying the effect of mutation on receptor binding domains (study in progress) and predicting possible generation of pandemic AI viruses.

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