Epidemiology of avian influenza Type A viruses with specific emphasis on H9N2 in Pakistan
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This thesis examines the epidemiology of avian influenza viruses (AIVs) in domestic poultry in Pakistan. Major aim of the current research was: to identify risk factors associated with the spread of these viruses; to quantify their prevalence in live bird retail stalls (LBRSs) and backyard poultry in Lahore district and to genetically characterize AIVs circulating in these stalls. Four independent studies were conducted which included (i) a retrospective matched case-control study in commercial poultry farms in Pakistan to identify the risk factors (ii) an estimation of the seroprevalence of AI from a cross-sectional study of backyard poultry flocks in Lahore district (iii) a cross-sectional study of LBRSs to estimate virus prevalence and identify associated risk factors and (iv) the genetic characterization of isolates collected from LBRSs. The retrospective matched case-control study identified five risk factors for AI infection. Multivariate conditional logistic regression model showed that distance of less than 0.5 kilometer of a commercial farm from the nearest case farm (OR= 145.4; 95% CI: 13.6-1553.5), followed by “previous history of infection of flock with infectious bursal disease (OR= 3.77; 95% CI: 1.18-11.97)”, selling of birds/eggs directly to live bird retail stalls from the farm premises (OR= 9.5; 95% CI: 1.7-51.9) have significant influence on spread of AI infection amongst the commercial farms. Other significant potential risk factors are “age of flock at the time of testing (OR= 1.0; 95% CI: 1.00-1.02)” and “a truck entering the farm areas (OR= 30.74; 95% CI: 1.56-604.78)”. Complete fencing of the farm was observed to be a protective factor (OR= 0.12; 95% CI: 0.02-0.63). The cross-sectional survey of backyard poultry flocks for AI (H9, H7 and H5) showed a seroprevalence of 67% (95% CI: 56.9-77.1) for H9 and 21% (95% CI: 13.8-28.1) for H5. Co-infection with both H9 and H5 was observed in 17 villages. Seroprevalence for H9 was significantly associated with the breed of bird. No samples were positive for H7. The cross-sectional survey of LBRSs in 07 towns of Lahore district showed the prevalence of H9N2 virus to be estimated at 10% (95% CI: 6.4-13.6). Subtypes H5N1 and H7N3 were not detected in any sample. Three risk factors showed a strong association with prevalence of H9N2 which are “adding new birds to the cages that already contained birds (OR= 9.2; 95% CI: 2.4-35.1)”, “purchasing birds for sale on the stall from mixed sources (other live poultry markets, auction markets, farm/individual producers) (OR= 3.4; CI 95%: 1.3-8.8)”, and “keeping birds partially inside and outside on the stalls during the day (OR= 1.7; CI 95%: 1.0-3.0)”. Phylogenetic analysis of ten H9N2 viruses isolated from LBRSs of Lahore district showed that four genes (HA, NA, M and NP) of all viruses belonged to G1-lineage and clustered with A/Quail/Hong Kong/G1/97 reference virus while the other four genes (PB2, PB1, NP and NS) from two of the viruses analysed clustered with a group of viruses from Indian subcontinent, Persian Gulf and Middle East. One recently reported H7N3 isolate from Pakistan also clustered with these genes. All H9N2 viruses examined harboured the mutation known to alter the receptor binding profile to one that preferentially binds to human receptors. The analysis shown in this study confirmed that further gene assortment has occurred since its emergence in poultry in Pakistan and Middle East, which could evolve into new genotype. Understanding the epidemiology of avian influenza has always been considered important in formulating and implementing control policies. Results from the current studies illuminate various aspects of epidemiological features of avian influenza viruses within poultry marketing systems in Asia. The current thesis has identified different risk factors and has also reported the prevalence estimates in backyard birds and LBRSs. The presence of reassortants of H9N2 with public health importance in LBRSs has also been reported in the current thesis. These results could be considered to plan future research and appropriate control and prevention strategiess for AIV by the global community. Continued surveillance and monitoring is essential to identify the viral gene pool circulating in live bird retail stalls and backyard poultry and to better understand the public-health risk posed by these viruses.