Three separate experiments were conducted in birds divided into 3 groups (I–III), with 15 mallards in each group.
Group I: Twelve 6-week-old mallards were infected intraocularly and intranasally with HPAIV A/herring gull/Poland/MB082B/2016 (H5N8) at the dose of 106 EID50/bird in 0.1 mL. Three contact ducks were placed in the same room 24 h later.
Group II: Twelve 4-week-old mallards were infected intraocularly and intranasally with LPAIV A/mallard/Poland/141/2015 (H5N1) at the dose of 106 EID50/bird in 0.1 mL. Two weeks later (i.e., at 6 week of age), the ducks were infected with HPAIV A/herring gull/Poland/MB082B/2016 (H5N8) (infection route, virus dose, and volume as in group I). Three contact ducks were placed in the same room after 24 h.
Group III: Twelve 4-week-old mallards were infected intraocularly and intranasally with LPAIV A/mallard/Poland/358/2006 (H3N8) at the dose of 106 EID50/bird in 0.1 mL. Two weeks later (i.e., at 6 week of age), the ducks were infected with HPAIV A/herring gull/Poland/MB082B/2016 (H5N8) (infection route, virus dose, and volume as in group I). Three contact ducks were placed in the same room after 24 h.
Mallards in all groups were observed for 14 days post-infection (dpi) with LPAIV and/or HPAIV. At 2, 4, 7, 10, and 14 days after infection with HPAIV (in case of contact ducks: 1, 3, 6, 9, and 13 days post-contact (dpc)), oropharyngeal and cloacal swabs were collected from all birds, placed in viral transport medium (COPAN, Italy), and stored at −80 °C until further use (viral RNA quantification by real time RT-PCR). Additionally, 4 and 14 days after HPAIV infection, organ samples (brain, trachea, lung, heart, liver, spleen, kidney, pancreas, proventriculus, dudodenum, ileum) from 2 dead or euthanized ducks (4 dpi) or all birds euthanized after termination of the experiment (14 dpi) were collected for viral RNA quantification, histopathology, and immunohistochemistry. Blood samples were also collected at 14 dpi with LPAIV and HPAIV for the purpose of serological testing.
Group I: Twelve 6-week-old mallards were infected intraocularly and intranasally with HPAIV A/herring gull/Poland/MB082B/2016 (H5N8) at the dose of 106 EID50/bird in 0.1 mL. Three contact ducks were placed in the same room 24 h later.
Group II: Twelve 4-week-old mallards were infected intraocularly and intranasally with LPAIV A/mallard/Poland/141/2015 (H5N1) at the dose of 106 EID50/bird in 0.1 mL. Two weeks later (i.e., at 6 week of age), the ducks were infected with HPAIV A/herring gull/Poland/MB082B/2016 (H5N8) (infection route, virus dose, and volume as in group I). Three contact ducks were placed in the same room after 24 h.
Group III: Twelve 4-week-old mallards were infected intraocularly and intranasally with LPAIV A/mallard/Poland/358/2006 (H3N8) at the dose of 106 EID50/bird in 0.1 mL. Two weeks later (i.e., at 6 week of age), the ducks were infected with HPAIV A/herring gull/Poland/MB082B/2016 (H5N8) (infection route, virus dose, and volume as in group I). Three contact ducks were placed in the same room after 24 h.
Mallards in all groups were observed for 14 days post-infection (dpi) with LPAIV and/or HPAIV. At 2, 4, 7, 10, and 14 days after infection with HPAIV (in case of contact ducks: 1, 3, 6, 9, and 13 days post-contact (dpc)), oropharyngeal and cloacal swabs were collected from all birds, placed in viral transport medium (COPAN, Italy), and stored at −80 °C until further use (viral RNA quantification by real time RT-PCR). Additionally, 4 and 14 days after HPAIV infection, organ samples (brain, trachea, lung, heart, liver, spleen, kidney, pancreas, proventriculus, dudodenum, ileum) from 2 dead or euthanized ducks (4 dpi) or all birds euthanized after termination of the experiment (14 dpi) were collected for viral RNA quantification, histopathology, and immunohistochemistry. Blood samples were also collected at 14 dpi with LPAIV and HPAIV for the purpose of serological testing.
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