Age is not a determinant factor in susceptibility of broilers to H5N2 clade 2.3.4.4 high pathogenicity avian influenza virus

Abstract

In 2014-2015, the US experienced an unprecedented outbreak of H5 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) virus. The H5N2 HPAI virus outbreak in the Midwest in 2015 affected commercial turkey and layer farms, but not broiler farms. To assess any potential genetic resistance of broilers and/or age-related effects, we investigated the pathogenesis and transmission of A/turkey/Minnesota/12582/2015 (H5N2) (Tk/MN/15) virus in commercial 5-week-old broilers, 8-week-old broilers, and >30-week-old broiler breeders. The mean bird lethal dose (BLD₅₀) was 5.0 log₁₀ mean egg infectious dose (EID₅₀) for all age groups. The mean death time (MDT) was statistically not different among the three age groups, ranging between 3.2 and 4.8 days. All broilers that became infected shed high levels of virus with transmission to contacts and demonstrated severe pathology. Mortality and virus shedding results indicated that age is not a determinant factor in susceptibility of broilers to H5N2 clade 2.3.4.4 HPAI virus. Previously, the Tk/MN/15 virus had a BLD₅₀ of 3.6 log₁₀ EID₅₀ and MDT of 2 days in White Leghorn chickens and a BLD₅₀ of 5.0 log₁₀ EID₅₀ and MDT of 5.9 days in turkeys, suggesting that the broiler breed is less susceptible to Midwestern H5N2 virus than the layer breed but similarly susceptible to turkeys. Therefore, genetic resistance of broilers to infection may have accounted only partially for the lack of affected broiler farms in the Midwestern outbreaks, with other contributing factors such as fewer outside to on farm exposure to contacts, type of production management system or enhanced biosecurity.

Figure 1

Relaxed clock molecular phylogenetic tree for H5 clade 2.3.4.4 HPAI viruses in the US. At each node, the number indicates a posterior probability. Blue branches represent the Midwest H5N2 cluster. The virus used in this study, A/turkey/Minnesota/12582/2015 (H5N2), is colored in red.

Figure 2

Mean viral oral shed from broilers directly inoculated or contact exposed with A/turkey/Minnesota/12582/2015 (H5N2) virus. Virus detection by qRRT-PCR at 2 and 4 dpc (or 1 and 3 dpe). The limit of detection for Tk/MN/15 virus was 2.0 log₁₀ EID₅₀/mL; therefore, qRRT-PCR negative samples were given a numeric value of 1.9 log₁₀ EID₅₀/mL. Intranasally inoculated 5-week old broilers (5w), 8-week old broilers (8w), and >30-week-old broiler breeders (>30w); contact exposed 5-week old broilers (5w-c), 8-week old broilers (8w-c), and >30-week-old broiler breeders (>30w-c).

Figure 3

Histopathological findings in broilers intranasally inoculated with high dose of A/turkey/Minnesota/12582/2015 (H5N2) virus. 5-week old broilers (5w), 8-week old broilers (8w), >30-week-old broiler breeders (>30w); ×40; immunohistochemical detection of viral antigen staining in red. A Cerebellum, 5w. Vacuolation of the molecular and granular layers of the cerebellum with necrosis of the Purkinje neurons. Viral antigen present in the neuropil of both layers (inset). B Heart, 8w. Degeneration and necrosis of myocardiocytes. Extensive intranuclear and intracytoplasmic viral antigen of myocardiocytes (inset). C Lung, 5w. Severe congestion, interstitial edema, and interstitial heterophilic and monocytic infiltration. Viral antigen in epithelium of air capillaries and vascular endothelium (inset). D Spleen, 5w. Multifocal areas of necrosis and depletion of the white pulp. Viral antigen in mononuclear cells (inset). E Pancreas, >30w. Multifocal areas of degeneration of pancreatic acinar cells. Viral antigen in acinar cells (inset). F Kidney, 8w. Focal necrosis of tubular epithelium. Extensive intranuclear and intracytoplasmic viral antigen of tubular epithelial cells (inset). G Adrenal gland, 8w. Diffuse intranuclear and cytoplasmatic viral antigen in corticotropic cells. H Brain, >30w. Viral antigen in ependymal cells of the ventricles. I Skeletal muscle, 8w. Intranuclear and intracytoplasmic viral antigen of myocytes and vascular endothelium.

Figure 4

Virus detection in tissues of broilers inoculated with high dose of A/turkey/Minnesota/12582/2015 (H5N2) virus. Virus detection by qRRT-PCR. The limit of detection for Tk/MN/15 virus was 3.0 log₁₀ EID₅₀/g; therefore, qRRT-PCR negative samples were given a numeric value of 2.9 log₁₀ EID₅₀/g. Virus titers in each type of tissue were statistically similar among the three age groups (Kruskal–Wallis test, p > 0.7). Intranasally inoculated 5-week old broilers (5w), 8-week old broilers (8w), >30-week-old broiler breeders (>30w).