Modulation of lethal HPAIV H5N8 clade 2.3.4.4B infection in AIV pre-exposed mallards

Figures

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  Figure 1.. Clinical score and survival rate of the target group (seropositive Pekin ducks; B1; B2) and the control group (seronegative Pekin ducks; B1; B2). Of the 7 mallards, only 2 ducks appeared depressed for 2–3 days, but one died, while 3 of 4 contact ducklings died until 4 days post contact. In the seronegative Pekin duck group, all 7 ducks, including all 4 contact ducklings, developed severe clinical disease and died within 9 days post infection/contact. Three birds that were humanely killed were registered as “3” (dead) the day after.

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  Figure 2.. Oral versus cloacal shedding in virus equivalents per ml (VE/ml) per log10 derived from H5-specific RTqPCR. A: target group (seropositive mallard ducks) and contact ducklings; B: infection control group (seronegative Pekin ducks) and contact ducklings. In the infection control group, the shedding was terminated after 9 dpi due to the death of all ducks in the group. Individual results of detected RNA copy numbers are given as virus equivalents (VE), calculated using a standard curve with data from each PCR run. The lines connect the medians calculated for each day post infection. The limit of detection is at 101.43 VE/ml (dotted line).

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  Figure 3.. Results of the competitive ELISA kits showing NP- and H5-specific seroconversion of the seropositive mallards and one contact duckling that survived a challenge infection with HPAIV H5N8B (A) and of the seronegative Pekin ducks serving as infection control group with contact ducklings (B). Lines represent the median. Inhibition of less than 45% for NP and of 50% for H5 is regarded as seropositive. NP = Nucleoprotein.

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  Figure 4.. Macroscopic findings in the livers of experimentally H5N8B-infected ducks. (A) Seronegative Pekin duck, H5N8B-infected, euthanized 9 dpi due to neurological symptoms, liver. Macroscopically normal, brown-red, acutely-angled liver without immunohistochemically-detectable hepatocellular influenza A virus matrix protein. (B) Pekin contact duckling in the mallard group, died 4 dpc, liver. Swollen, brick-red-colored, friable liver with rounded edges, interpreted as severe, acute, diffuse, necrotizing hepatitis with immunohistochemically-detectable hepatocellular influenza A virus matrix protein. (C) Seropositive mallard, H5N8B-infected, clinically normal, 14 dpi, liver. Swollen, beige, greasy liver with rounded edges, interpreted as moderate, acute, diffuse hepatocellular lipidosis (background pathology) without immunohistochemically detectable hepatocellular influenza A virus matrixprotein.

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  Figure 5.. Light microscopic finding in the livers of experimentally H5N8B-infected ducks. (A, B) Seropositive mallard, H5N8B-infected, clinically normal, 34 dpi, liver. (A) No obvious findings. (B) Lack of immunohistochemically-detectable hepatocellular influenza A virus matrixprotein antigen. (C, D) Pekin duck, contact animal, died 4 days post contact, liver. (C) Marked hypereosinophilia, hepatocellular vacuolation, membraneous rupture and nuclear pyknosis, karyorrhexis and lysis interpreted as severe, acute, coalescing to diffuse necrotizing hepatitis. (D) Immunohistochemistry reveals coalescing intrahepatocytic, intracytoplasmic and intranuclear influenza A virus matrix protein. (A, C) Hematoxylin-eosin, (B, D) Immunohistochemistry using the avidin-biotin-peroxidase-complex method with a monoclonal antibody against influenza A virus matrix protein (ATCC clone HB-64), 3-amino-9-ethylcarbazol chromogen (redbrown) and hematoxylin counterstain (blue). (A, C) bars = 20 μm. (B, D) bars = 50 μm.

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  Figure 6.. Light microscopic assessment of virus tropism (A) and lesion profile (B,C) revealed a predilection of the liver for H5N8B induced lesions in contact mallards, Pekin ducks, and contact Pekin ducks. (A) The distribution of influenza virus matrixprotein was evaluated on an ordinal scale: 0 = none, 1 = focal/oligofocal, 2 = multifocal, 3 = coalescing/diffuse, and revealed a systemic virus infection in many contact mallards, Pekin ducks, and contact Pekin ducks as compared to the mallards. (B, C) The severity of parenchymal necrosis (B), or lymphocytic necrosis, apoptosis and depletion in the spleen (C) was evaluated on an ordinal scale as follows: 0 = unchanged, 1 = mild, 2 = moderate, 3 = severe. Box-and-whisker plots display the median, the quartiles as well as min and max of the semiquantitative scores.

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  Figure 7.. Survival rate and clinical score (A), viral shedding (B) and serological results (C) for homologously HPAIV H5N8 re-infected ducks (21 days after the first infection). B1+B2: Individual results of detected RNA copy numbers are given as virus equivalents (VE), calculated using a standard curve with data from each PCR run. The limit of detection is at 101.43 VE/ml (dotted line). C1+C2: Results of competitive NP- and H5-specific ELISAs. Inhibition of less than 45% for NP and of 50% for H5 is regarded as seropositive. The lines in B and C represent the medians calculated for each day post infection. NP = Nucleoprotein.