Molecular quantitation of H9N2 avian influenza virus in various organs of broiler chickens using TaqMan real time PCR

Abstract

During the past decade, H9N2 low pathogenic avian influenza virus (LPAI) has caused considerable economic loss due to decreased production, increased mortality and the cost of vaccination in Iranian poultry industry. Because of widespread occurrence of this disease and the virus potential to mutate to highly-pathogenic (HP) form and transmission to humans, it is, therefore, imperative to understand the pathogenesis and properties of these viruses. In this study, a two step TaqMan real time PCR assay was performed for the quantitation of A/chicken/Iran/772/1998(H(9)N(2)) virus in various organs of broiler chickens at different days post inoculation (DPI). Forty 5-week-old commercial broiler chickens were inoculated with the virus. Five chickens were randomly selected on days 1, 3, 6 and 9 PI. Their trachea, lungs, spleen, kidneys, pancreas, blood and faeces were collected for virus detection. A PCR test was performed and the positive samples were used for quantitative real time PCR assay. The result of RT-PCR assay showed the presence of the virus in trachea (40%, 33%), lungs (20%, 66.6%) and spleen (20%, 50%) of infected chickens on days 3 and 6 PI, respectively. The virus was also detected in the kidneys of inoculated chickens on 3 (40%), 6 (60%) and 9 (100%) DPI. In faecal samples the virus was only detected on day 6 PI (83.3%). The molecular quantitation of AIV showed that the AIV titre in the trachea, lungs and spleen of chickens at 3 DPI is lower than the AIV titre at 6 DPI in these organs. The highest titre was observed in the faeces. The AIV titre in all organs of the birds which died at 6 DPI was higher than those of the same organs in the other experimental birds.