Isolation of Avian Influenza Virus A Subtype H5N1 from Internal Contents (Albumen and Allantoic Fluid) of Japanese Quail (Coturnix coturnix japonica) Eggs and Oviduct during a Natural Outbreak

Abstract

Avian influenza virus (AIV) was recovered from the internal contents of eggs, including mixture of albumen and allantoic fluid, and from the oviduct of naturally infected Japanese quail (Coturnix coturnix japonica) flocks in the southern part of Thailand. The virus titers of 10(4.6)-10(6.2) ELD(50)/mL were directly measured from the internal content of infected eggs. The virus was isolated by chorioallantoic sac inoculation of embryonating chicken eggs. Infected allantoic fluid was identified as hemagglutinating virus and then was indicated the presence of H5 hemagglutinin. The virus was confirmed to be H5N1 subtype influenza A virus by reverse transcriptase-polymerase chain reaction. Additionally, real-time reverse transcriptase-polymerase chain reaction assay could specifically detect influenza virus subtype H5. Furthermore, indirect fluorescent antibody (IFA) test by using specific anti-influenza A monoclonal antibody indicated that virus antigens were detected in the parenchyma of multiple tissues. Systemic localization of viral antigen detected was certainly considered to be viremic stage. In addition, influenza virus antigen was also detected by IFA in allantoic fluid sediments isolated from internal content of egg or oviduct. The conclusion of isolated AIV type A subtype H5N1 from these two infected materials was correlated to the viremic stage of infection because the virus antigens could be observed in almost all tissues. Conclusively, the need for adequate safeguards to prevent contamination and spread of the virus to the environment during movement of eggs--including hatching eggs, cracked eggs, and other relevant infected materials-- or egg consumption from area of outbreak is emphasized and must not be ignored for the reasons of animal, public, and environmental health.