Comprehensive pathological, immunohistochemical, and apoptotic analysis of the highly pathogenic avian influenza a virus H5N1 clade 2.3.4.4b in poultry in Taiwan.

Since 2021, the highly pathogenic avian influenza (HPAI) virus H5N1 clade 2.3.4.4b has become the predominant circulating strain in Taiwan and has caused substantial losses in the poultry industry. However, its pathogenicity and tissue tropism across different poultry species remain insufficiently characterized. This study investigated gross lesions, histopathological changes, viral antigen distribution, and apoptosis-associated signals in chickens, ducks, geese, and quails naturally infected with HPAI virus H5N1 clade 2.3.4.4b in Taiwan. Samples were collected from 41 laboratory-confirmed H5N1-positive farms between October 2023 and February 2025, including 23 chicken farms, 4 duck farms, 10 goose farms, and 4 quail farms. Gross lesions were systematically evaluated. Furthermore, tissues including the brain, heart, liver, spleen, lung, kidney, and pancreas were examined using histopathology, immunohistochemistry for influenza A virus nucleoprotein and cleaved caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Semiquantitative scoring systems were applied to compare the severity of histopathological lesions, viral antigen distribution, and apoptotic activity across tissues and species. Results showed that chickens exhibited systemic infection with widespread necrosis and viral antigen distribution. Ducks displayed milder lesions but a more coordinated apoptotic response. Geese showed intermediate pathological features, with moderate correlation between lesion severity and viral antigen distribution. Quails displayed a unique host response, including prominent splenic lesions and distinct associations among viral antigen distribution, apoptosis, and tissue damage. Interspecies comparisons further identified significant differences in splenic and renal lesion scores, renal viral antigen scores, and apoptosis-associated scores in selected organs. Overall, these findings indicate that HPAI virus H5N1 clade 2.3.4.4b induces species-dependent pathological patterns and apoptotic responses in poultry. This field-based study provides pathological evidence supporting host-specific differences in tissue tropism and disease outcome under natural infection conditions.