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Viral shedding and antibody response of mallard ducks to avian influenza viruses




Muth, Jack P., author
Bowen, Richard, advisor
Landolt, Gabriele, committee member
Mason, Gary, committee member
Zabel, Mark, committee member

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Wild ducks are a key reservoir for avian influenza (AI) viruses. Their long distance migrations, coupled to frequent contact with domestic poultry enhances risk for spread of highly pathogenic avian influenza (HPAI) viruses. Despite years of study, our understanding of how AI viruses are maintained and transmitted in nature remains poorly understood. The work described here examines several aspects of avian influenza virus infections that play a role in perpetuation and spread of this disease, including persistence of virus in duck feces, effect of prior exposure to AI viruses on subsequent infections and the passage of maternal antibodies between hen and duckling. In recent years, the emergence of H5N1 HPAI virus stimulated establishment of massive international surveillance programs to detect that virus in wild waterfowl. One deficit in these efforts was a lack of data on the stability of AI virus and AI virus RNA in bird feces under different environmental conditions. Consequently, an experiment was designed to address this knowledge gap. Feces were collected from mallards infected with a low pathogenic avian influenza (LPAI) virus (H5N2) on days 3 and 4 post infection and kept in environmental chambers for 21 days under the following conditions: 32°C/20% relative humidity (RH), 32°C/50%RH, 32°C/90%RH, 4.5°C/50%RH, 4.5°C/90%RH, and 0°C/50%RH. Sensitivity of detection of infectious virus in fresh fecal material was equivalent to that from cloacal swab samples, while time and environmental conditions did not significantly affect detection of AI virus RNA by PCR. Infectious virus was isolated from feces for considerably shorter intervals than RNA could be detected and was isolated for longer periods of time when feces were maintained under cold conditions. High relative humidity also had a negative effect on virus isolation at 4.5°C. Use of quantitative reverse transcriptase PCR to detect AI virus in fecal samples is as a valuable tool in limiting the labor involved in surveying wild ducks for AI virus. Few prior studies have examined virus shedding over the course of short interval, sequential infections of ducks with LPAI viruses, as likely occurs in natural settings such as breeding grounds. We characterized such infections by sequential inoculation of ducks with homosubtypic versus heterosubtypic with H5N2 and H3N8 LPAI viruses. We found that prior infection with either virus reduced the duration of viral shedding during a subsequent infection initiated 14 or 28 days later. Further, shedding was significantly shorter when the secondary infection occurred 28 days following the initial infection compared to 14 days. No difference in rate of shedding for the secondary infection were noted based on the viral subtype causing the initial infection, suggesting induction of some degree of heterosubtypic immunity. As reported from previous studies, some ducks shed virus but did not develop detectable antibody titers. There was no evidence of subtype cross-reactivity by antibodies as demonstrated by hemagglutination inhibition testing. The antibody response to a heterosubtypic virus was not improved by a prior infection while a second infection with the same virus was capable of boosting the antibody response to that virus. This information should be useful in parameterizing models examining the ecology of avian influenza infection. Another factor of significance in understanding transmission of AI viruses among wild ducks is the influence of passive immunity. A third study was performed to evaluate the magnitude of passive transfer of anti-influenza virus antibodies in mallard ducks and to determine their rate of decay in ducklings. Since not all ducks develop antibodies following natural infection with AI virus and the antibody titers are typically low, a vaccine was used to induce consistent seroconversion. Four, 11 month-old mallard hens were inoculated with a recombinant H5 protein in adjuvant. Specifically, hens received a single injection of 20 μg of hemagglutinin protein derived from A/Vietnam/1203/2004 emulsified in Freund's incomplete adjuvant. Beginning two weeks post-vaccination, eggs were collected daily. Yolk was harvested from eggs laid at one-week intervals and the remainder of the eggs incubated for hatching. All hens developed detectable antibody titers with an average log 2 hemagglutination inhibition titer (HI) of 6.4. Maternal-origin antibodies were detected in the yolk of eggs laid by all hens. Antibody titers peaked in yolks three weeks post vaccination for two hens and were still rising four weeks post vaccination for the other two hens. The highest yolk HI antibody titer was 32. Serum samples from the ducklings hatched from vaccinated hens were collected between days 0 and 22 post-hatch. The calculated mean half-life of maternal antibody in ducklings was 2.3 days with a range of 1.6 to 4.0 days. The short duration of passive immunity in ducks is similar to what has been reported for other species of birds and suggests that maternal antibodies may not play a major role in modulating protection against AI virus infection in natural populations. The strong immune response elicited by the H5 protein suggested that further evaluation should be performed to determine the viability of this vaccine for ducks.


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avian influenza
maternal antibodies
mallard duck


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