Role of Terrestrial Wild Birds in Ecology of Influenza A Virus (H5N1)

Recent viruses are pathogenic for some small terrestrial bird species.

H ighly pathogenic avian infl uenza viruses of subtype H5N1 were identifi ed in Southeast Asia in 1996 and have spread in recent years across broad regions of Eurasia and Africa. These viruses have shown high lethality in chickens and other poultry species (1)(2)(3). Outbreaks of avian infl uenza, H5N1 subtype and others, have caused massive losses to commercial poultry fl ocks in recent years (4). Direct transmission of H5N1 subtype from infected poultry is thought to be responsible for virtually all of the human infl uenza (H5N1) infections since 1997. Because of the effects of infl uenza (H5N1) on human health and agriculture and its potential to mutate and cause a global pandemic, epidemiologic studies of the viruses' host range and their means of dispersal are urgently needed (5).
Highly pathogenic poultry isolates from the 1997 and 2001 infl uenza (H5N1) outbreaks typically cause few disease signs in experimentally infected ducks (6,7). These viruses' low pathogenicity in waterfowl presumably facilitated effi cient carriage to the highly susceptible hosts. Some infl uenza (H5N1) strains isolated during subsequent outbreaks are highly pathogenic in waterfowl (7,8), and some are shed by infected ducks for prolonged periods (9). Together with the commercial transportation of poultry and poultry products, migratory waterfowl are likely to have played a role in the wide dispersal of highly pathogenic infl uenza (H5N1) viruses.
Land-based wild bird populations may also be vulnerable to lethal infl uenza (H5N1) infection and could contribute to the spread and interspecies transmission of the viruses. Small terrestrial birds are potentially important hosts in infl uenza (H5N1) ecology because many of them intermingle freely with wild and domestic populations of waterfowl and poultry. However, data describing their susceptibility to infl uenza virus (H5N1) infection or their potential to transmit the viruses are limited.
A study investigating the host range of A/chicken/ Hong Kong/220/97 showed that it causes lethal infection in budgerigars and fi nches (10). In contrast, the same virus replicated poorly in sparrows, causing no deaths, and, when pigeons were inoculated, replication of this virus was not evident. A more recent chicken infl uenza (H5N1) isolate (A/chicken/Yamaguchi/7/2004), highly lethal to chickens and quail, also replicates extensively and causes high mortality rates in budgerigars (11). Since 2002, infl uenza (H5N1) viruses have been isolated from dead birds of several wild terrestrial species, including magpie, tree sparrow, pigeon, and large-billed crow (8,12,13). Viruses of a novel infl uenza (H5N1) genotype were isolated during a survey of live tree sparrows (Passer montanus); these isolates were highly pathogenic to chickens (14). Together, these reports indicate that some small, land-based bird species are susceptible to infection, sometimes fatal, with highly pathogenic infl uenza (H5N1) viruses.
We inoculated sparrows, starlings, and pigeons with several recent highly pathogenic infl uenza (H5N1) viruses isolated from a variety of avian hosts. The primary aims of the study were to test the susceptibility of different species to infection, investigate the duration and routes of viral shedding from the birds, and assess the possibility of intraspecies viral transmission in these hosts.

Infl uenza A Viruses
Four infl uenza A (H5N1) virus strains were studied, 2 from previously known susceptible hosts (duck and quail) and 2 from previously unknown hosts (common magpie and Japanese white-eye).

Animal Studies
Wild house sparrows (Passer domesticus) and European starlings (Sturnus vulgaris), both members of the order Passeriformes, were captured. Six-week-old white Carneux pigeons (Colomba spp.), members of the order Columbiformes, were purchased from Palmetto Pigeon Plant (Sumter, SC, USA) and Double T farms (Glenwood, IA, USA). Birds were housed in cages in the St Jude Children's Research Hospital Animal Biosafety Level 3+ containment facility, food and water were provided ad libitum, and general care was provided as required by the Institutional Animal Care and Use Committee. Before inoculation with virus, oropharyngeal and cloacal swabs were collected to exclude preexisting infl uenza A virus infection.
Three sparrows and pigeons were inoculated intranasally with 1 million 50% egg infectious doses (EID 50 ) in 50 μL or 500 μL phosphate-buffered saline, respectively, for each virus. Because of their limited availability, starlings were inoculated with 3 viruses (1 million EID 50 in 150 μL), and group sizes were reduced (1 bird for A/DK/TH/144/05, 3 birds for A/CM/HK/645/06, and 2 birds for A/JW/ HK/1038/06). One day after inoculation, uninfected contact birds, at a ratio of 1:1 for sparrows and starlings or 2:3 for pigeons, were housed together with inoculated animals to study intraspecies transmission. Birds were monitored daily for death and illness for a 14-day period. After inoculation, oropharyngeal and cloacal swabs were collected on days 2, 4, 6, 8, and 11 for sparrows and starlings and days 3, 5, and 7 for pigeons. Infl uenza virus was detected by using 10-day-old embryonated chicken eggs as previously described (7). EID 50 virus titers were determined in positive swabs by using the method of Reed and Muench (15). The lower limit of quantitation of the assay is 10 0.75 EID 50 /mL, and average virus titers in organs and swabs were calculated by using the log 10 value of each sample.

Serology
Fourteen days after inoculation with virus, serum specimens were collected from inoculated and contact birds, and hemagglutination-inhibition (HI) titers were determined according to standard methods (16,17) by using chicken erythrocytes and 4 hemagglutinating units of virus. An HI titer >10 suggested a recent infl uenza virus infection; an HI titer <10 was considered negative.

Infection of Different Bird Species with Infl uenza (H5N1) Virus
The ability of 4 different infl uenza A (H5N1) viruses to infect and cause disease in house sparrows, European starlings, and white Carneux pigeons was determined. Infection of sparrows caused death in 66%-100% of the infected animals, depending on the inoculated virus ( Table 1). The average time to death varied from 4.2 days for A/DK/TH/144/05 to 6.3 days for A/Q/TH/551/05 virus (data not shown). High viral loads were detected in brain and lung tissues of deceased sparrows (Figure, panel C). In contrast, none of the starlings or pigeons died after inoculation with these viruses.
Re-isolation of virus from oropharyngeal and cloacal swabs obtained at various time points after inoculation indicated that all the sparrows and starlings were infected by all viruses tested. In contrast, the frequency of virus re-isolation from inoculated pigeons varied widely among viruses. Of the 4 different H5N1 subytpes, A/CM/HK/645/06 demonstrated the broadest host range, infecting not only spar-rows and starlings but also all of the inoculated pigeons. The A/DK/TH/144/05 virus, which caused 100% mortality in sparrows within 4.2 days after inoculation, was not reisolated from inoculated pigeons.
Quantifi cation of the virus titer in the swabs demonstrated that sparrows and starlings shed similar amounts of virus in oropharyngeal swabs. However, virus titers in the cloacal swabs of sparrows were higher than in those obtained from infected starlings ( Table 1). Comparison of peak virus titers in oropharyngeal swabs confi rmed the similarity in oral shedding between sparrows and starlings. In contrast, peak virus titers in the cloacal swabs of starlings were lower ( Figure, panels A and B). The 2005-2006 infl uenza (H5N1) viruses replicated relatively poorly in pigeons, as shown by average oropharyngeal and cloacal shedding on days 3 and 5 (Table 1) and by peak virus titers in oropharyngeal and cloacal swabs (Figure, panels A and B).

Intraspecies Transmission of Infl uenza (H5N1) Viruses
The capacity of current infl uenza (H5N1) viruses to transmit from infected birds to same-species uninfected birds was assessed for these 4 viruses. No evidence of transmission in sparrows and pigeons was found, as attempts to isolate the virus from contact birds failed (Table 2). Also, no virus-specifi c antibodies were detected by HI in the contact birds (data not shown). In starlings, transmission of virus to contact birds was observed once for A/CM/HK/645/06 virus, but this was not seen in 2 further experiments.

Discussion
The susceptibility of 3 species of wild terrestrial birds to infl uenza A (H5N1) virus and their ability to transmit to contact birds were assessed. Our studies show that major differences in susceptibility to infl uenza (H5N1) virus infection exist among these bird species and that, under  our conditions, transmission occurred infrequently. Pigeons, starlings, and sparrows were more susceptible to experimental infection with the recent (H5N1) isolates than they were to A/chicken/Hong Kong/220/97 (H5N1) virus (6,10,18). Although drawing conclusions on the basis of a single 1997 isolate is inappropriate, these data are consistent with studies that have demonstrated increased virulence or host range for recent infl uenza (H5N1) viruses in mammalian species, including mice, ferrets, and domestic and wild cats (19)(20)(21)(22). Whereas a previous study showed that a 2003 chicken infl uenza (H5N1) isolate can cause severe neurologic disease in pigeons, we observed no signs of disease in infl uenza (H5N1)-infected pigeons (23). Such a difference in pathogenicity between our study and others may be due to subspecies differences or a change in inoculum size. A critical question concerning these small avian species is whether they can serve as intermediate hosts or reservoirs for infl uenza (H5N1) viruses and transmit them to poultry and mammals. Sparrows were highly susceptible to infl uenza (H5N1) infection; however, they did not transmit to sentinel contact birds, despite a relatively low infectious dose (≈500 EID 50 for A/DK/TH/144/05 virus, data not shown) and the fact that virus was common in drinking water and fecal samples. Although it is possible that the high pathogenicity of these viruses prevented bird-to-bird transmission, the data suggest that this species can act as an intermediate host and potentially transmit to both poultry and mammals but not serve as a reservoir for prolonged shedding of highly pathogenic infl uenza (H5N1) viruses. In contrast, the characteristics of infl uenza (H5N1) infection in starlings, i.e., nonfatal with longer-term shedding, suggest that starlings could act as an intermediate host and a reservoir for infl uenza (H5N1) virus. However, evidence of transmission to contact starlings was limited, which implies that these strains are unsustainable in a starling population. Because pigeons shed only low amounts of virus upon infection and they did not transmit to contact birds, their role in the ecology of infl uenza (H5N1) virus may be minor.
Our results indicate that there are considerable differences in susceptibility to infl uenza (H5N1) virus among various small terrestrial wild bird species. The high virulence of several recent isolates in sparrows suggests that this and other populations of small terrestrial birds may have substantial losses during current and future outbreaks. Further mutation of circulating infl uenza (H5N1) viruses might enhance their adaptation to hosts such as starlings and sparrows, further increasing virulence or allowing these species to become effi cient intermediate hosts in the ecology of infl uenza (H5N1) viruses.