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. 2014 Jan 2;9(1):e83417.
doi: 10.1371/journal.pone.0083417. eCollection 2014.

Experimentally infected domestic ducks show efficient transmission of Indonesian H5N1 highly pathogenic avian influenza virus, but lack persistent viral shedding

Hendra Wibawa  1 , John Bingham  2 , Harimurti Nuradji  3 , Sue Lowther  2 , Jean Payne  2 , Jenni Harper  2 , Akhmad Junaidi  4 , Deborah Middleton  2 , Joanne Meers  5
Affiliations

Experimentally infected domestic ducks show efficient transmission of Indonesian H5N1 highly pathogenic avian influenza virus, but lack persistent viral shedding

Hendra Wibawa et al. PLoS One. .

Abstract

Ducks are important maintenance hosts for avian influenza, including H5N1 highly pathogenic avian influenza viruses. A previous study indicated that persistence of H5N1 viruses in ducks after the development of humoral immunity may drive viral evolution following immune selection. As H5N1 HPAI is endemic in Indonesia, this mechanism may be important in understanding H5N1 evolution in that region. To determine the capability of domestic ducks to maintain prolonged shedding of Indonesian clade 2.1 H5N1 virus, two groups of Pekin ducks were inoculated through the eyes, nostrils and oropharynx and viral shedding and transmission investigated. Inoculated ducks (n = 15), which were mostly asymptomatic, shed infectious virus from the oral route from 1 to 8 days post inoculation, and from the cloacal route from 2-8 dpi. Viral ribonucleic acid was detected from 1-15 days post inoculation from the oral route and 1-24 days post inoculation from the cloacal route (cycle threshold <40). Most ducks seroconverted in a range of serological tests by 15 days post inoculation. Virus was efficiently transmitted during acute infection (5 inoculation-infected to all 5 contact ducks). However, no evidence for transmission, as determined by seroconversion and viral shedding, was found between an inoculation-infected group (n = 10) and contact ducks (n = 9) when the two groups only had contact after 10 days post inoculation. Clinical disease was more frequent and more severe in contact-infected (2 of 5) than inoculation-infected ducks (1 of 15). We conclude that Indonesian clade 2.1 H5N1 highly pathogenic avian influenza virus does not persist in individual ducks after acute infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Virus isolation and H5 RNA Ct values for oral and cloacal swabs from the inoculated ducks (group 1) and the acute stage-contact ducks (group 2).
Five contact ducks of group 2 (charts at right) were mixed with five inoculated ducks of group 1 (charts at left) at one day post inoculation (1 dpi). Swabs were obtained from all ducks in group 1 for 1–15 dpi, while those for group 2 were collected from 5 ducks from 1–5 day post-contact (dpc), 4 ducks from 6–9 dpc and 3 ducks from 10–15 dpc as 2 ducks were euthanized at 5 and 9 dpc. Vertical column bars indicate the number of ducks positive for virus isolation in embryonated chicken eggs, while white circles indicate the H5 RNA Ct values from rRT-PCR. Circles for Ct values have been staggered to avoid overlap. The means of Ct values from each time point are connected (solid black line); undetectable Ct values were given the value of 45 for calculation of the means.
Figure 2
Figure 2. Virus isolation and H5 RNA Ct values for oral and cloacal swabs from the inoculated ducks (group 3) and the post-acute stage contact ducks (group 4).
Swabs from ducks of group 3 (n = 10) were collected 1–15 day post inoculation (dpi) and at 17, 20, 24 and 29 dpi. Nine contact ducks (group 4) were mixed with the inoculated ducks of group 3 at 10 dpi (arrow). Virus isolation and rRT-PCR for group 4 was performed on oral and cloacal swabs collected from all the contact ducks at 1, 5 and 7 days post contact (dpc) (equivalent to 11, 15, 17 dpi); none of these swabs were virus isolation positive and only five swabs from different contact ducks had detectable Ct values (black circles). The mean Ct values for group 3 from each time point are connected (solid black line); undetectable Ct values were given the value of 45 for calculation of the means.
Figure 3
Figure 3. Histopathology and immunohistochemistry of H5N1 HPAI virus infection in acute stage contact-infected ducks #94 and #85.
Ducks #94 (left, A–C) and #85 (right, D–F) were euthanized at 5 and 9 days post contact (dpc) following disease signs. A. Infraorbital sinus, showing influenza viral nucleoprotein antigen in epithelium and mononuclear cell inflammation (*). B. Air sac, showing abundant viral antigen in the epithelial membranes and in fibrinous exudates, and mononuclear cell infiltration (*); C. Feather, with abundant antigen staining associated with mixed cell inflammation (*) in the pulp; D. Heart, showing severe, sub-acute, diffuse mononuclear cell myocarditis (*); no antigen was detected in myocardium E. Brain, showing antigen in neuronal tissue associated with mononuclear cell encephalitis; F. Feather, with mononuclear cell pterilitis (*) observed in the pulp, and antigen staining in the epidermis. Haematoxylin and eosin stain (Fig. D) and immunohistochemistry stain (brown color) for influenza A virus nucleoprotein (Fig. A, B, C, E, F). All scale bars = 100 µm.
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Grants and funding

The study was jointly funded by CSIRO and the Australian Centre for International Agriculture Research (ACIAR) grant number AH/2004/040. Hendra Wibawa was supported by an ACIAR John Allwright Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.