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Recently seven isolates of avian influenza virus (AIV) serotype H9N2 recovered from an outbreak of AI were analyzed on the basis of their biological and molecular characteristics. All the isolates belonged to the low-pathogenicity group of AIV. To further evaluate their pathogenic potential in association with other organisms, an isolate was inoculated experimentally in chickens using different routes and subsequently challenged with infectious bronchitis virus, Ornithobacterium rhinotracheale or Escherichia coli. The virus isolation and seromonitoring data revealed a significant role of Escherichia coli in aggravating the clinical condition of the birds earlier infected with AIV (H9N2). The AIV-antigen was detected in lung, trachea, kidney, and cloacal bursa among the infected birds, using immunofluorescent antibody technique. In another experiment, chickens that were immunosuppressed chemically showed high mortality when challenged with AIV H9N2. The results indicated that this low pathogenicity AIV (H9N2) isolate could produce severe infection depending on the type of secondary opportunistic pathogens present under field conditions. This may explain the severity of infection with the present H9N2 outbreak in the field. A prolonged antibacterial therapy in flocks infected with AIV H9N2 and use of oil-based vaccine at an early age in new flocks has helped to control this infection and the disease.

BackgroundAvian influenza virus (AIV) infections have caused heavy economic losses to the poultry industry in Pakistan as well as numerous other regions worldwide. The first introduction of H7N3 AIV to Pakistan occurred during 1995, since then H7N3, H9N2 and H5N1 AIVs have each been sporadically isolated. This report evaluates the genetic origin of the H7N3 viruses from Pakistan collected 1995-2004 and how they disseminated within the country. To accomplish this we produced whole genome sequences for 6 H7N3 viruses and data for the HA and NA genes of an additional 7 isolates. All available sequence from H7N3 AIV from Pakistan was included in the analysis.ResultsPhylogenetic analysis revealed that there were two introductions of H7 into Pakistan and one N3 introduction. Only one of the H7 introductions appears to have become established in poultry in Pakistan, while the other was isolated from two separate outbreaks 6 years apart. The data also shows that reassortment has occurred between H7N3 and H9N2 viruses in the field, likely during co-infection of poultry. Also, with the exception of these few reassortant isolates, all 8 genes in the predominant H7N3 virus lineage have evolved to be phylogenetically distinct.ConclusionsAlthough rigorous control measures have been implemented in commercial poultry in Pakistan, AIV is sporadically transmitted to poultry and among the different poultry industry compartments (broilers, broiler breeders, table egg layers). Since there is one primary H7 lineage which persists and that has reassorted with the H9N2 AIV in poultry, it suggests that there is a reservoir with some link commercial poultry. On a general level, this offers insight into the molecular ecology of AIV in poultry where the virus has persisted despite vaccination and biosecurity. This data also illustrates the importance of sustained surveillance for AIVs in poultry.

The role of avian adenovirus isolate PARC-1 as an immunosuppressive agent was investigated using a Newcastle disease virus (NDV) vaccine immune response procedure. The immunosuppressive effect on the humoral immune response was investigated up to 21 days after inoculation with adenovirus. Infected chickens showed a serologic response to NDV that was reduced compared with that of the controls. To further investigate the effect of the virus on major lymphoid organs, the pattern of virus dissemination in various organs was studied at various time intervals after inoculation. Spleen, thymus, bursa of Fabricius, and cecal tonsils of broilers were examined using a dot-immunobinding assay. The virus was found to have a predilection for lymphoid organs, and virus from lymphoid organs was capable of producing disease when inoculated into healthy chickens. The relationship of virus predilection to its immunosuppressive effect also was studied.

From November 2003 to June 2004 an epidemic of high pathogenicity avian influenza (HPAI) virus of subtype H7N3 affected the major layer and broiler-breeder raising areas of the country. This was accompanied by an outbreak of low pathogenicity avian influenza (LPAI) virus of type H9N2 in broilers and layers, which continued during 2005. Subsequently, in February 2006 avian influenza virus (AIV) subtype H5N1 was for the first time found in two isolated commercial flocks in this country. The HPAI outbreak of 2003-2004 was eventually overcome by enforcing biosecurity measures, controlling poultry movements, using inactivated vaccines, and introducing a comprehensive AI surveillance network throughout the country. However, similar measures undertaken to control H9N2 outbreaks have not been successful in the affected areas, with continuing increased mortality and heavy production losses in broilers and layers, respectively. A similar strategy has been devised to combat the spread of newly introduced H5N1 HPAIV. The description of these outbreaks and the results of the control strategy are reported here.