Biological Studies of Various Avian Influenza Viruses in Poultry

This study was conducted to (1) isolate and identify various AIV types infecting domestic chicken and wild birds in and around Lahore, (2) develop inactivated (monovalent/bivalent) AIV vaccines and compare their efficacy with the locally manufactured and imported vaccines, (3) compare the relative efficacy and economics of diagnostic tests such as HA/HI, AGPT, ELISA and PCR against various AI virus types infecting poultry. A total of 100 day old chicks, 25 from each type of broiler, layer, golden desi and quails were examined for the presence of HI maternal antibodies produced in response to H7N3 and H9N2 AIVs. Very low HI titers in the range of 0-8 were observed in the day old chicks. Isolation of AIVs from 100 tissue samples (trachea, lungs, spleen, liver, intestines etc) collected from 10 poultry farms was attempted. These samples were inoculated in developing 9 - 11 day old chicken embryos. The AF from the inoculated embryos was harvested and tested for HA activity. Of the total ten farms, 3 were positive for H7N3 virus infection and 2 were positive for H9N2 virus infection. Isolation of H7N3 virus was possible from the broiler flocks, aged 4-7 weeks which had suffered from respiratory distress signs and mortality of 15-25%. The H9N2 positive broiler flocks (age 4 weeks and 6 weeks) also suffered from respiratory signs, poor growth and weight gain with 5-11% mortality. None of those flocks had received any locally prepared or imported AIV killed vaccination. The AIV HI antibody titers in those flocks were not significant and ranged between 0-2 log 2 titers. Isolation of AIV from 200 cloacal swab samples obtained from 20 different broiler breeder flocks was also attempted. None of the samples was found positive for AIV. The AIV isolation was also attempted from desi chicken, ducks, pigeons, quails and sparrows using their 100 cloacal swab samples. All attempts to isolate AIV failed as none of the samples was positive for AIV, upon inoculation of material from the swab samples to 11-day old embryos. The attempts to isolate AIV (H5N1) from broiler, broiler breeder, goose, and layer flocks showing the respiratory illness and mortality ranging from 26-73% were also made. All the examined flocks (except one layer flock) were positive for H5N1 virus infection. Seroconversion in H7N3 and H9N2 virus (imported/local, bivalent) vaccine inoculated six broiler breeder flocks was also studied. The maximum GMT titers were observed as 27.9 and 29.9 for H7N3 virus and 26 and 55.7 for H9N2 virus in the vaccinated birds. The efficacy of three commercially available vaccines and one newly prepared vaccine was studied. The experimental findings indicated that all the three commercially available vaccines were able to generate good immune response in chicks housed under controlled hygienic conditions. The maximum titer was achieved after 12 -15 days of vaccination. The GMTs of the HI titers of chicks in groups A, B, C and D on 21st day post vaccination were recorded as 55.7, 52, 59.7 and 29.9 against H7N3 and 27.9, 68.6, 64 and 24.3 against H9N2 virus, respectively. Comparative efficacy of various diagnostic procedures for AIVs was also studied. The results indicated that ELISA and RT-PCR have good relative specificity but are costly procedures as compared to HA/HI and AGP tests. Fifty known positive field isolates along with fifty known negative samples were run each for H7N3 and H9N2 AIVs by HA/HI, AGPT, ELISA and RT-PCR. A sensitivity of 98%, 78%, 98% and 100% for H7N3 was observed each for HA/HI, AGPT, ELISA and RT-PCR, respectively. The results obtained for H9N2 subtype were 98%, 82%, 100% and 100% respectively for HA/HI, AGPT, ELISA and RT-PCR. As far as economics of AIV diagnosis is concerned, the HA/HI is recommended as the method of choice as it is highly efficient and economical. However, selection of any diagnostic procedures described depends upon the time, facilities and the financial resources available.