Serological Surveillance of Wild Waterfowl in Northern Australia for Avian Influenza Virus Shows Variations in Prevalence and a Cyclical Periodicity of Infection.

The virological surveillance of 3582 wild waterfowl in northern Australia from 2004 to 2009 for avian influenza virus (AIV) found an apparent prevalence (AP) of 1% (31 of 2989 cloacal swabs; 95% CI: 0.71%-1.47%) using a Taqman Type A real-time reverse transcription polymerase chain reaction test and no viral isolations from 593 swabs tested by the embryonating chicken egg culture method. From serological testing using a nucleoprotein competitive enzyme-linked immunosorbent assay for AIV antibody, 1131 of 3645 sera had ≥ 40% inhibition, indicating an apparent seroprevalence of 31% (95% CI: 29.5%-32.6%). This value suggests that the low AP from virological testing does not reflect the dynamics of AIV infection in these populations. Spatiotemporal and species variations in seroprevalence were found at wetland sampling sites, with consistently higher values at Kununurra in Western Australia (AP  =  39%, 95% CI: 36.9%-41.4%) compared to other locations. At Kununurra, seroprevalence values had a two-year cyclical periodicity and suggest this location is a hotspot of AIV activity. From hemagglutination inhibition (HI) testing using multiple subtype antigens, the highest AP of HI reactions were to H6 and H5 subtypes. The phenomenon of cyclic periodicity in NP seroprevalence at Kununurra is hypothesized as being related to the prevalent H6 subtype that may have either become predominant or cycled back into a mostly AIV naïve flock. The inclusion of serological testing provided insight into the dynamics of AIV infection in wild birds such as species risk profiles and spatiotemporal patterns, important epidemiological information for a risk-based approach to surveillance.

Surveillance of Charadriiformes in northern Australia shows species variations in exposure to avian influenza virus and suggests negligible virus prevalence.

The virologic surveillance of 4248 Charadriiformes since 1992 primarily from coastal northwest Australia did not detect any evidence of avian influenza virus (AIV) excretion (test prevalence = 0%; 95% confidence interval [CI]: 0%-0.09%). Past exposure to AIV was evident from serologic testing using nucleoprotein (NP) competitive-ELISA (c-ELISA) with an overall seroprevalence of 8.8% (95% CI: 8%-9.7%). The c-ELISA seroprevalence of family Scolopacidae and genus Numenius was significantly higher when compared with other families and genera, respectively. Exposure risk profiles, based on c-ELISA seroprevalence, were compiled for 40 species with the following species having significantly higher values when compared with the combined value of all other species: eastern curlew (Numenius madagascariensis), whimbrel (Numenius phaeopus), ruddy turnstone (Arenaria interpres), grey plover (Pluvialis squatarola), little curlew (Numenius minutus), red knot (Calidris canutus), sharp-tailed sandpiper (Calidris acuminata), and red-necked stint (Calidris ruficollis). From hemagglutination inhibition (HI) testing, the more prevalent HI reactions were against H2, H5, H6, and H9 subtypes, with no reactions against subtypes H11, H14, H15, and H16. Serologic testing using c-ELISA provided species risk profiles for optimizing a surveillance strategy for AIV in diverse populations of wild birds. The paucity of knowledge about the role of waders in the ecology of AIV and the overall very low to negligible virus prevalence reported globally, and in this study, suggests that waders are spillover hosts in shared ecosystems with a lesser role than previously considered.

Evaluation of avian influenza serologic and virologic diagnostic methods in wild Anseriformes and Charadriiformes.

Evaluation of avian influenza virus (AIV) diagnostic methods, including a nucleoprotein (NP) competitive enzyme-linked immunosorbent assay (c-ELISA), hemagglutination inhibition (HI) test, type A real-time reverse transcription polymerase chain reaction (RRT-PCR), and embryonating chicken egg (ECE) virus isolation (VI), suggested validity of these tests in wild birds comparable to that reported in poultry. This was determined by analyzing the results from experimental inoculation of three species of wild birds with a low-pathogenicity AIV and from field surveillance data. The NP c-ELISA in a high-AIV prevalence setting had 100% diagnostic sensitivity (Se; 95% confidence interval [CI]: 81.5%-100%) and 91% diagnostic specificity (Sp; 95% CI: 70.8%-98.9%) in negative controls compared with the RRT-PCR. In low-AIV prevalence flocks using a > 60% inhibition positivity threshold, relative to the HI test, c-ELISA performed with 90.5% Se (95% CI: 86.2%-93.8%) and 41.2% Sp (95% CI: 38.1%-44.5%). Assessment of HI suggests a titer > or = 8 is a positive test result in wild-bird sera, and using this titer had 83.3% Se (95% CI: 58.6%-96.4%) in experimentally infected birds. The RRT-PCR diagnostic performance compared with VI in cloacal swabs varied over 2-6 days postinoculation, having high Se (83.3%-100%) and Sp (94.1%-100%) with substantial agreement (kappa = 0.8). The cycle thresholds (C(t)) for the RRT-PCR of C(t) < 37 for positivity and C(t) = 37-40 as indeterminate were found to be valid for the species included in this study. In view of the interpretative diagnostic difficulties in heterogeneous populations of wild birds, this evaluation in three species of wild birds and in surveillance data should provide greater confidence in the application of these methods routinely used in poultry.

Variation in the responses of wild species of duck, gull, and wader to inoculation with a wild-bird-origin H6N2 low pathogenicity avian influenza virus.

There is poor understanding of host responses to avian influenza virus (AIV) infection in wild birds, with most experimental studies using captive-bred birds and highly pathogenic AIVs that have an early endpoint. The objective of this study was to experimentally assess antibody responses and patterns of viral excretion in wild birds challenged with a low pathogenicity AIV. Ruddy turnstones (Arenaria interpres), silver gulls (Chroicocephalus novaehollandiae), and wandering whistling ducks (Dendrocygna arcuata) were challenged with a H6N2 virus, and blood, cloacal, and oropharyngeal (OP) swabs were analyzed from each bird over 28 days, with serology conducted on the ducks for a further 7 mo. Nineteen of 22 birds showed evidence of infection, with respiratory infection prevalent in the turnstones and gulls as mostly low titer viral excretion to 4 days postinoculation (DPI) with gastrointestinal replication detected in only one turnstone. In AIV naive ducks, there was gastrointestinal tropism with moderately high titer viral excretion via the cloaca to 6 DPI and low-grade OP viral excretion to 4 DPI. The hemagglutination inhibition antibody response was poor in the ducks, declining from 19 to 56 DPI, with higher titer responses in the gulls and turnstones. All infected birds responded with elevated nucleoprotein antibodies (in competitive enzyme-linked immunosorbent assay) by 7-10 DPI, and in the ducks these waned slowly after 42 DPI and were long-lived to at least 8 mo. The interspecies variability in response was consistent with a subtype that had adapted well in ducks, while the response of the turnstones may have been influenced by preexisting immunity to AIV. These findings provide insight into AIV infection dynamics in wild birds and highlight the need for further research.