Abstract

In a retrospective study, histopathology and immunohistochemistry (IHC) were performed on formalin-fixed paraffin embedded (FFPE) archival tissues from chickens obtained during outbreaks of highly pathogenic avian influenza (HPAI) H5N1 that occurred in Nigeria in 2006 and 2007. Ten samples as representative of 10 outbreaks were selected, and following the detection of HPAI viral antigen in different chicken tissues using IHC, RNA was extracted from each sample and molecular analysis was performed using real-time reverse transcription-polymerase chain reaction (rRT-PCR) targeting matrix protein. Seven rRT-PCR positive samples were then subjected to conventional and rRT-PCR assays for the amplification of hemagglutinin (HA) gene. Four of them were further characterized by sequence analysis of a short HA2-part of the H5 gene. Along the 154 nucleotides sequenced, differences at 4 positions were detected in one sample. One of these mutations led to an amino acid exchange at position 544 (Ala>Thr) whereas the others were silent. The study suggests the potential application for retrospective IHC and PCR analysis of FFPE tissues from chickens involved in the AI for pathologic studies and providing short fragment sequences which may help in the characterization of viral strains and tracing the outbreaks. This is important as archived poultry tissues can be re-examined for possibility of earlier introduction of the virus.

Key words: Avian influenza; FFPE; H5N1; Nigeria; Immunohistochemistry; real-time RT-PCR