Epidemiology and molecular characterization of avian influenza virus in chickens and ducks at backyard farms in Chattogram, Bangladesh

Abstract

Bangladesh is an agriculture-based country where large portions of rural households have backyard poultry, which play an important role in their dietary protein needs. Besides that, selling the meat and egg to the local markets to support the additional family expenses. Avian influenza virus has a catastrophic impact on household poultry next to commercial poultry industries by causing high mortality or reducing egg production. Over the past two decades, highly pathogenic avian influenza (HPAI) has triggered serious outbreaks in poultry and has affected humans with causing mortality across the world, including Bangladesh. As the ducks are believed as natural reservoir of avian influenza virus, it can act as reassortment vessel in the transmission of HPAI virus and low pathogenic avian influenza (LPAI) virus among the other domesticated, wild bird species and humans. Most of the epidemiological research on avian influenza have been previously been limited into commercial poultry and live bird markets and to date, studies on apparently healthy poultry at household level are not yet available. The present cross-sectional study was carried out to reveal the epidemiological traits of avian influenza of backyard poultry in coastal (Anowara) and plain land (Rangunia) areas to find out prevalence, associated factors and molecular characterization of the avian influenza virus (AIV). A total of 300 households’ poultry (having both chicken and duck) were randomly selected and cloacal swabs of one bird per household were sampled. Structured pre-tested questionnaires were used to collect the information related to risk factors at household level by direct interview of farmers and recorded. Cloacal samples were pooled in small groups and tested first for the matrix gene (M gene) presence by real time reverse transcriptase polymerase chain reaction (rRT-PCR) with reference primers and probes, and then M gene positive swabs pooled were further tested for H5 and H9 subtypes using specific primers and probes by rRT-PCR. All AIV positive samples were subjected to sequencing for the four gene segments (M, PB1, HA and NA gene). We were able to amplified Eight (8) M genes, four (4) for each HA, NA, PB1gene segments and then performed phylogenetic analysis. We detected overall viral RNA, Influenza A (M-gene) prevalence at household level was 6% (95% CI: 3.6 – 9.3; N=300) where this prevalence was 3.6% (95% CI: 1.7 – 6.4; N = 281) in household duck and 3.2% (95% CI: 1.4 – 6.2; N= 251) in household chicken. During the winter season the prevalence was estimated 8.2% (95% CI: 4.5 – 13.3; N= 171 whereas in x the summer it was 3.1% (95% CI: 0.8 – 7.7; N= 129). According to subtype, the prevalence of H5 and H9 in backyard poultry was 2.7% (95% CI: 1.1 – 5.2; N= 300) and 3.3% (95% CI: 1.6 – 6; N= 300), respectively. The phylogenetic analysis of eight partial M gene sequences suggested that the M gene sequences detected in backyard poultry were almost similar to each other and closely related to the previously reported M gene sequences of HPAI and LPAI subtypes in poultry in Bangladesh as well as Southeast Asia. Besides, the phylogenetic analysis of HA, NA and PB1 gene also showed the similarity in sequences with each other and closely related to the gene sequences of previously reported HPAI in different poultry sectors in Bangladesh. Overall results reflect that both H5 and H9 subtypes of avian influenza virus are circulating in the household poultry with or without showing any clinical symptoms. Besides regular surveillance and early detection of avian influenza virus in this area, molecular identification of AIV’s subtypes in the study area helps to get clear idea of circulating subtypes of AIV virus in the backyard poultry rearing system of the study areas and take effective control measure to prevent the infection and control the zoonotic transmission.