Rapid detection of the avian influenza virus H5N1 subtype in Egypt

Abstract

Influenza A virus continue to cause widespread morbidity and mortality. The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Egypt is threatening poultry and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, polyclonal antibodies (PAbs) were developed against the H5N1 avian influenza virus (AIV) and implemented an enzyme-linked immunosorbent assay (ELISA) to detect the H5 viral antigen. A group of rabbits was immunized with viral vaccine (H5N1) therefore, purifiedantibodies from rabbit serum, which secrete immunoglobulin G (IgG) was served as the detector antibody after conjugation with horse radish peroxidase and fluorescent isothiocyanate (FITC). The reactivity of the obtained peroxidase and fluorescent conjugated PAb of influenza virus revealed that they are specifically recognized H5N1 virus antigen. Specimens containing AIV subtypes collected from different Governments in Egypt yielded specific and strong signals with hemagglutination test. The detection limit of ELISA using the prepared peroxidae conjugated PAbs was 1:100000, while using fluorescent conjugated PAb was 1:10000. Reconstituted clinicalsamples consisting of H5 AIVs mixed with pharyngeal-tracheal mucus from healthy chickens also yielded positive signals in ELISA, and the results were confirmed using reference virus antigens. This investigation enhanced the usage of these PAbs in the surveillance and diagnosis of H5N1 AIV in Egypt. Key words: Avian influenza virus, H5N1, fluorescent antibody enzyme-linked immunosorbent assay (ELISA) technique, polyclonal antibodies.