The species diversity of ducks may also play an important role in the pathogenicity of influenza viruses. Mallard duck embryos inoculated with LPAI virus have significantly lower mortality rates than inoculated Muscovy duck embryos; however, in regards to replication, the LPAI viruses behave in a different way. Viral antigens were found in the internal organs (nasal sinuses, pharynx, trachea, bronchi, lung, and air sacs) of the mallard duck embryos, but not in those of the Muscovy duck embryos. The reason for this mortality ⁄ virus replication paradox in mallard ducks is unclear but is in keeping with the evidence that mallard ducks are considered to be the main reservoirs of LPAI viruses in nature. 25 Although several studies have examined the serum antibody response in both naturally and experimentally infected ducks, knowledge of the avian immune response to influenza viruses is very limited. 26 White Pekin ducks inoculated with an H7N2 LPAI virus developed negligible serum hemagglutination inhibition (HI) antibody titers despite fecal shedding of virus until day 7 post-inoculation. Animals reinoculated 46 days later with the same virus strain had a marked antibody response, but virus could not be isolated from any of the organs. These results and the lack of a secondary immune response after inoculation with formalin-inactivated virus suggested that the rapid immune response in re-infected birds may restrict influenza infection to short time scales. 3 It is noteworthy that prior infection does not protect ducks against subsequent infection with other virus subtypes. For example, ducks infected with an H4N6 subtype are protected from reinfection with the same virus, but they shed virions for 8 days after challenge with an H11N3 isolate. 27 These data have applications in the field, where isolation of influenza virus from migratory waterfowl is infrequent during the winter, potentially indicating the existence of a significant level of immunity in wintering ducks acquired from previous influenza infections. Further illustration of this is seen from a study of wild waterfowl in Italy over six winter seasons, in which 17 of the 20 viruses isolated were of the H1N1 subtype, suggesting that the wintering ducks had some degree of immunity to the other subtypes of circulating influenza strains. 28 Highly pathogenic avian influenza Several experimental studies have investigated the pathogenicity of H5N1 HPAI viruses (isolated since 2002) in ducks. Cherry Valley Pekin ducks inoculated with a 2003 HPAI H5N1 strain isolated from duck meat at a quarantine inspection station in China developed neurologic signs, including blindness and head shaking, although none died. High virus titers were detected in the respiratory organs (lung and trachea), brain, liver, kidneys, and colon, and microscopic lesions were observed in the brain (viral encephalitis), heart (myocarditis with degeneration and necrosis of myocytes), and bursa (mild lymphoid follicular hyperplasia). 29 Viral neurotropism and pancreatotropism have been observed in multiple other studies of recent HPAI virus isolates. Ducks lethally challenged with these H5N1 HPAI viruses showed severe neurologic signs, including torticollis, incoordination, tremors, and seizures. 30, 31 Immunohistochemistry positivity was recorded in the cerebrum and brain stem, and in situ hybridization detected virus in the neurons and glial cells of the cerebral gray matter, further confirming the strong neurotropism of post-2002 isolates. 30, 31 Although the route of entry of virus into the central nervous system has not been determined, at least two different hypotheses have been proposed, including ascending transmission of virus via vagal, olfactory, and trigeminal nerve fibers, and penetration of the blood-brain barrier. 32, 33 Another recurring characteristic of recent H5N1 HPAI viruses in ducks is that virus titers are frequently higher in oropharyngeal swabs than in cloacal swabs. 30, 34 Pharyngeal excretion of H5N1 HPAI viruses has been suggested to originate from the lung and ⁄ or air sac, as only these tissues have shown immunohistochemical evidence of virus replication. Preferential pharyngeal excretion suggests that pharyngeal swabs, as well as the customary cloacal swabs, should be taken when conducting surveillance of avian influenza viruses in wild ducks. 34 Otherwise, the prevalence of H5N1 HPAI may be underestimated. Additional studies of the role and rates of respiratory transmission of H5N1 HPAI viruses in ducks are needed, especially as they relate to cloacal excretion.