The effect of age on the pathogenesis of a highly pathogenic avian influenza (HPAI) H5N1 virus in Pekin ducks (Anas platyrhynchos) infected experimentally

Abstract

Background: Highly pathogenic avian influenza (HPAI) H5N1 viruses have recently displayed increased virulence for wild waterfowl.

Objectives: To study the effect of host age on the shedding and tissue dissemination of a HPAI H5N1 virus in infected Pekin ducks.

Methods: Pekin ducks in two age-matched groups (n = 18), 8 and 12 weeks old (wo) were each infected with 10(6) EID(50)/0.1 ml of HPAI A/turkey/Turkey/1/05 (H5N1, clade 2.2). Each day for 5 days, birds were monitored clinically, and cloacal and oropharyngeal swabs collected, before three birds from each group were selected randomly for post-mortem examination. Tissue samples were collected for examination by real-time RT-PCR, histopathology and immunohistochemistry (IHC).

Results: Severe clinical signs, including incoordination and torticollis were observed in the 8 wo group resulting in 100% mortality by 4 dpi. Mild clinical signs were observed in the 12 wo group with no mortality. Real-time RT-PCR and IHC results demonstrated the systemic spread of H5N1 virus in birds of both age groups. Higher levels of virus shedding were detected in oropharyngeal swabs than in cloacal swabs, with similar levels of shedding detected in both age groups. Variations in level and temporal dissemination of virus within tissues of older ducks, and the presence of the virus in brain and heart were observed, which coincided with the appearance of clinical signs preceding death in younger birds.

Conclusions: These results are consistent with reports of natural infections of wild waterfowl and poultry possibly indicating an age-related association with dissemination and clinical outcome in ducks following infection with H5N1 HPAI virus.

Figure 1

Immunohistochemical detection of Influenza A nucleoprotein in 8‐ (A, C, E and G) and 12‐week‐old Pekin ducks (B, D, F and H) challenged with A/turkey/Turkey1/2005 HPAI H5N1. ENVISION. (A) Lung, 3 dpi, immunolabelling of air capillary epithelial cells, 20×. (B) Lung, 3 dpi, 20×. (C) Heart, 3 dpi, multifocal labelling of myocardiocytes, 10×. (D) Heart, 3 dpi, focal labelling of myocarddiocytes, 10×. (E) Spleen, 2 dpi, small groups of macrophages expressing viral antigen in their cytoplasm, 10×. (F) Spleen, 2 dpi, 10×. (G) Brain, 3 dpi, abundant neuronal and glial labeling, 10×. (H) Brain, 3 dpi, focal area of viral antigen detection, 10×.

Figure 2

Immunohistochemical detection of Influenza A nucleoprotein in 8‐ (A, C, E and G) and 12‐week‐old Pekin ducks (B, D, F and H) challenged with A/turkey/Turkey1/2005 HPAI H5N1. ENVISION. (A) Thymus, 3 dpi, abundant thymic epithelium and lymphocyte immunolabelling, 10×. (B) Thymus, 3 dpi, scarce labelling in thymic epithelial cells, 10×. (C) Skeletal muscle, 3 dpi, abundant immunolabelled myocytes, 5×. (D) Skeletal muscle, 3 dpi, focal area of immunolabelling of few myocytes, 20×. (E) Pancreas, 4 dpi, multifocal labelling in pancreatic acinary cells, 10×. (F) Pancreas, 4 dpi, focal labeling, 10×. (G) Feather follicle, 4 dpi, multiple areas of immunolabelling in feather shaft and pulp, 5×. (H) Feather follicle, 4 dpi, substantially reduced immunolabelling in feather shaft and pulp, 5×.

Figure 3

Matrix gene RRT‐PCR results for oropharyngeal and cloacal swabs taken over 5 dpi with A/turkey/Turkey/1/05 H5N1. Viral RNA was detected by RRT‐PCR (y‐axis) and quantified as relative equivalent units (REU) of RNA against a 10‐fold dilution series of RNA purified from infective allantoic fluid of a 10⁶ EID₅₀/0.1 ml dose of A/turkey/Turkey/1/2005 H5N1 and are therefore a guide to the amount of infectious virus present. Error bars indicate SE.

Figure 4

Detection of viral RNA by RRT‐PCR in tissues of lung, kidney, caecal tonsil and feathers. Viral RNA detected by RRT‐PCR (y‐axis) was measured as a relative equivalent unit (REU) of RNA against a 10‐fold dilution series of RNA purified from infective allantoic fluid of a 10⁶ EID₅₀/0.1 ml dose of A/turkey/Turkey/1/2005 H5N1. Error bars indicate SE. Note y‐axes are not presented on the same scale.

Figure 5

Detection of viral RNA by RRT‐PCR in tissues of thigh and breast muscle. Viral RNA detected by RRT‐PCR (y‐axis) was measured as a relative equivalent unit (REU) of RNA against a 10‐fold dilution series of RNA purified from infective allantoic fluid of a 10⁶ EID₅₀/0.1ml dose of A/turkey/Turkey/1/2005 H5N1. Error bars indicate SE. Note y‐axes are not presented on the same scale.

Figure 6

Detection of viral RNA by RRT‐PCR in tissues of brain and heart tissues coinciding with the appearance of clinical signs at 3–4 dpi, preceding death in younger birds. Viral RNA detected by RRT‐PCR (y‐axis) was measured as a relative equivalent unit (REU) of RNA against a 10‐fold dilution series of RNA purified from infective allantoic fluid of a 10⁶ EID₅₀/0.1 ml dose of A/turkey/Turkey/1/2005 H5N1. Error bars indicate SE.