CHARACTERIZATION AND COMPARATIVE SUSCEPTIBILITY OF HIGHLY PATHOGENIC AVIAN INFLUENZA H5N1 VIRUS INFECTIONS IN CHICKENS AND DUCKS

Abstract

Nigeria experienced outbreaks of Highly Pathogenic Avian Influenza (HPAI) H5N1 virus in poultry between 2006 and 2008. Mixed poultry rearing have been postulated as factors responsible for easy spread, while inadequate diagnostic specimens and procedures have militated against prompt diagnosis of the disease. This study was designed to elucidate the comparative susceptibility and diagnosis of H5N1 virus infection in chickens and ducks.

Clinical and pathological examinations, agar gel immunodiffusion and viral isolation were used to confirm 468 suspected chickens, ducks, turkey and geese from six (6) geopolitical zones submitted to the reference laboratory at National Veterinary Research Institute, Vom. Spatial data were mapped with Arc-view GIS. Fifty-six of the 80 confirmed H5N1-positive backyard poultry cases were compared for proportions (single and mixed species); the association was calculated by odds ratio using MedCalc Software. Immunohistochemistry (IHC), Real-time Reverse Transcriptase-Polymerase Chain Reaction (RRT-PCR), sequencing and alignment using the haemagglutinin cleavage site were used to detect and characterize H5N1 in Formalin-Fixed Paraffin-Embedded (FFPE) chicken tissues from ten of the outbreak cases. Comparative pathology; tissue virus predilection and titre were carried out using IHC and RRT-PCR in 10 Muscovy, 10 Pekin and eight Mallard 3-weeks old specific pathogen-free ducks each separately experimentally infected with clade 1 or 2.2 H5N1 virus genotypes. Twelve age-matched chickens served as in-contact sentinels.

Clinical signs, lesions and mortalities were severe in older birds, while younger and free-range chickens showed minimal clinical signs and lesions. Lesions were multi-systemic and characterized by severe haemorrhages and necrosis. Mortalities in birds were: 20.6% (north-central), 16.5% (north-east), 15.9% (north-west) and 6.0% (south-west). Wetland areas in north-west and north-east had more positive cases. There were higher risks (OR=3.02) of infection and mortalities in mixed than in single species farms. H5N1 RNA virus detection in FFPE tissues was successful in 7 of 10 while gene sequencing was possible only in four. All the viral RNA characterized belonged to the sub-clade 2.2 with >96% homology to similar virus of European origin. Along the 154 nucleotides sequenced, amino acid exchange (mutation; Ala →Thr) occurred at position 544. Clade 1-infected Muscovy ducks shed more viruses, showed more severe nervous signs and mortality than Pekin and Mallard. Pekin ducks were moderately susceptible to clade 1 but insusceptible to clade 2.2. Mallard ducks were resistant to clinical disease from both viruses. Chickens exposed to infected ducks had 100% mortality four days post-exposure. Eyelids, combs, wattles, thymus, spinal cord, pancreas, cerebrum and bursa of Fabricius had higher RRT-PCR detection than the heart, lung, trachea, liver, spleen and intestine traditionally harvested for HPAI- H5N1 antigen detection.

Co-rearing of Muscovy ducks with chicken posed greater risk of transmission of Highly Pathogenic Avian Influenza to the latter. Detection of H5N1 virus in formalin-fixed paraffin-embedded chicken tissues was an important finding useful in retrospective diagnosis of HPAI.