Studies on standard procedures for diagnosis of avian influenza virus

The broiler chickens were grouped into A, B and C, were kept as non vaccinated group, while A and B were vaccinated with H7 N2 and H9 N2 vaccines by intramuscular route. The samples of sera were obtained from these chickens vaccinated with a local strain vaccine. The geometric mean titre (GMT) value for H7 and H9 was 7.90 and 5.95 respectively. Then all those sera were checked for AGP reactivity. The high titre sera among these samples showed positive reaction both for H7 and H9 when they kept in their respective wells, while, low titer serum gave negative results. In case of virus neutralization, the high titer sera also neutralized the virus. When the ELISA test was perform to determine antibodies in the sera of chicks, it also correlate with HI, AGP and virus neutralization, but ELISA found most sensitive because it has ability to detect very low antibody titer. The results revealed that there were no difference and a degree of positive correlation among these tests, but the AGP is a qualitative (type specific), HI only detects subtypes, only ELIA is most sensitive but needs maximum reagents and skills, difficult for standardization and purification, so the haemagglutination inhibition test for titration of antibodies by (GMT) is economical and reliable technique to measure the protective level of antibodies and to assess the efficacy of vaccines. The stability of H7 and H9 antigen with three different concentrations of glycerol at 25 degree C temperature was also examined. The results were recorded after a weak interval. The results showed that 50% glycerin was moderately superior other than 10% and 25%, at 25 degree C for 10 weeks. The rate of elution was also stable at 20-25 degree C for 60-90 minutes. The prevalence of H7 and H9 subtypes was also checked, on the basis of HA activity in the samples collected from surrounding of Korangi, Karachi. Total 27 samples found positive, for H7 and H9 subtypes. The results revealed that layer is more susceptible to H7 subtype (found 19% positive) while broilers are susceptible to H9 subtype found 33% positive. The study indicated that H7 subtype is more prevalent in layers, while H9 is common in broilers. It is concluded that there is a positive correlation between HI, AGP, ELISA and virus neutralization test. Although ELISA and HI are of equal value when testing in a population with high level of antibodies, both tests have significantly improved the ability for quickly detecting the antibody level of avian influenza virus in a vaccinated or infected flock although ELISA is more accurate in confirmation of avian influenza virus positive status in vaccinated or infected flock even at low antibody titre, but (HI) test is better to measure the protective level of antibodies for avian influenza virus due to feasible, convenient and commonly used in diagnostic labs and advantageous due to the heamagglutination property of avian influenza virus. The stability of the HA activity after treatment of antigen with glycerol simplified the handling and storage, so it was concluded from this experiment that when avian influenza virus treated with 50% glycerin, it remained stale up to months. The rate of elution has also become stable for 90 minutes. The H7 and H9 subtypes of avian influenza virus are prevalent in the surroundings of Korangi and it is a potential threat to poultry industry. The results of study revealed the H7 subtype is more common in layers, while H9 subtype in Broilers.