Though not natural hosts, different species of domestic birds can be infected with influenza viruses from wild bird sources or contaminated environment from these reservoirs. In order to gain detailed understanding of the biological properties of low pathogenic influenza viruses in domestic birds, we studied the replication, pathogenicity and intraspecies transmission characteristics of 20 low pathogenic influenza viruses of different origins. Our results indicated that most of these isolates can replicate and transmit within poultry without clinical disease. However, distinct virus and species specific differences in transmission to contact control birds were noted emphasizing the importance of having contact control cage mates in biological characterization experiments. Ducks supported the replication of wild bird viruses in their respiratory and digestive tracts equally well, though in contact control birds, higher tendency for viral detection from cloacal swabs was observed. The wild bird viruses did not effectively transmit between chickens. However, the wild bird isolates as well as domestic bird viruses from live bird markets and commercial poultry operations tested, replicated and transmitted in turkeys to comparatively higher titers than in chickens or ducks. Further, determination of minimal infectious doses of 3 selected viruses for the three bird species revealed lower dose requirement for infection of turkeys suggesting that turkeys could be easily infected following a low dose exposure. Our data elucidate the important role of turkeys as being more susceptible hosts for wild and domestic bird low pathogenic avian influenza (LPAI) viruses in comparison to domestic ducks and chickens. Further, we were interested in looking at the host factors, especially the presence, distribution and type of receptors that influence susceptibility to influenza viral infections. It is commonly understood that avian influenza viruses preferentially bind to α2,3sialic acid(SA)-galactose (gal) linked receptors and correspondingly avian tissues predominantly express these receptors. We undertook detailed studies on the distribution of α2,3SA-gal (avian type) and α2,6SA-gal (human type) receptors on different tissues of chickens, ducks and turkeys in day old, 2-4 week old and layer birds using specific plant lectins, MAA and SNA respectively. On the tracheal epithelium, the 3 bird species expressed almost 80-100% high intensity positive staining for the α2,3SA-gal receptors in 3 different age groups. However, the distribution of the α2,6SA-gal receptors varied with species and age. Ducks expressed high positive staining (more than 80%) for human type receptors while turkeys and chickens showed approximately 30% and 60% positive staining, respectively, with slight differences in distribution with age. The sections of large intestine consistently showed 40-50% positive staining for the avian receptors with low (less than 5%) or no staining for the human receptors. However, small and large intestinal sections from chickens showed positive staining for avian (60-80%) and human (20-50%) receptors. We also demonstrated viral binding to the receptors observed using tracheal membrane preparations from chickens and turkeys. Our results indicated that like other terrestrial domestic poultry, like chickens and quail that have been implicated as potential intermediate hosts for influenza viruses, turkeys could also support infection, replication of influenza viruses from avian and mammalian sources.
Further, we expanded our studies to determine whether turkeys supported infection, replication and transmission of swine lineage viruses equally well. We employed swine lineage triple reassortant (TR) H3N2 influenza viruses that derive their gene segments from avian, human and swine lineage viruses. Phylogenetically, all turkey origin TR H3N2 viruses isolated from different geographical locations as well as different time points shared high genetic similarity and grouped together with the swine origin isolates. Antigenically, all turkey isolates were similar, showed lesser cross-reactivity to swine origin viruses, and no cross reactivity to avian origin H3 subtype viruses that are not triple reassortants. Biologically, turkey origin TR H3N2 viruses replicated efficiently in 3-week-old turkeys and transmitted to the contact control cage mates. In 4-week-old chickens and 2-week-old ducks, these viruses replicated poorly and did not transmit. In 26-week-old layer turkeys, one of the TR H3N2 strains tested caused complete cessation of egg production within 13 days post infection. We confirmed high levels of virus replication and abundant distribution of avian specific α2,3 sialic acid-galactose receptors in the oviduct of these turkeys. Our studies indicated that influenza infections alone, without any concurrent infections, can result in drastic declines in egg production in breeder turkeys.
Further, our interests were to determine if the high viral titers observed in the oviduct of breeder turkeys could be passed into eggs laid by infected turkeys. Following experimental inoculation of the turkey origin virus, A/turkey/Ohio/313053/04, in breeder turkeys, we detected the virus in albumin of eggs using real time RT-PCR and virus isolation in embryonated chicken eggs. Swabs from egg shells were also found positive by real time RT-PCR. This is the first report of detection of low pathogenic influenza viruses from internal egg contents following experimental infection. Although, there is no available evidence for vertical transmission of influenza viruses, the possibility of hatchery contamination by egg-borne influenza viruses and spread of virus during movement of contaminated cracked eggs and egg flats pose serious food safety and public health concerns.
In summary, our biological characterization experiments indicate that turkeys support infection with influenza viruses from different sources efficiently. Also, the receptor profile, especially in the tracheal epithelium, indicates the suitability of turkeys as potential intermediate hosts for influenza viruses. The ability of influenza viruses to cause drastic declines in egg production and the transmission of influenza viruses into egg contents of infected turkeys raise serious economic and public health concerns.