The surveillance and risk assessment of wild birds in northern Australia for highly pathogenic avian influenza H5N1 virus

Highly pathogenic avian influenza (HPAI), caused by infection with H5N1 virus, is a transboundary disease which has had a significant socio-economic impact on the poultry production systems of Eurasia, and spillover events with mortality in humans and wild birds. In northern Australia, prior to the current study there was poor understanding of the ecology of avian influenza viruses (AIV) and the risks of H5N1 transmission by wild birds. In this study, the biological pathways of risk for HPAI H5N1 by migratory birds were estimated as a negligible to very low risk to the wild birds of northern Australia. Following stochastic modelling the highest mean frequency of outbreaks was 1 year in 36 years (range 1 in 25-53 years; annual incidence of 0.028) for the Little Curlew (Numenius minutus), followed by the Sharp-tailed Sandpiper (Calidris acuminata) (1 in 56 years, range 36 to 91 years). Three species of wild birds were challenged with a H6N2 low pathogenicity AIV (LPAIV). There was poor viral replication in the Ruddy Turnstones (Arenaria interpres) and Silver Gulls (Chroicocephalus novaehollandiae) with mostly low titre oropharyngeal (OP) excretion [median titre at 4 days post inoculation (DPI) of 101.43 and 102.09 50% embryo infectious dose (EID50)/0.1 mL respectively], with the exception of an OP sample from one Silver Gull (104.26 EID50/0.1 mL at 2 DPI), and one cloacal sample from a Ruddy Turnstone (103.14 EID50/0.1 mL at 10 DPI). In the Wandering Whistling Ducks (Dendrocygna arcuata), there was gastro-intestinal tropism with moderately high titre viral excretion to 6 DPI (highest median titre of 104.58 EID50/0.1 mL in cloacal swabs at 4 DPI). The anti-haemagglutinin (HA) antibody response was poor in the ducks and declined from 19-56 DPI [highest haemagglutination inhibition (HI) test reciprocal geometric mean titre (GMT) of 16.1 at 19 DPI to a GMT of 3.7 at 56 DPI]. In the ducks after 42 DPI, nucleoprotein (NP) c-ELISA antibodies waned slowly from a median of 81% inhibition, and were long-lived to at least 8 months with a 57% median inhibition value. The evaluation of a commercial NP c-ELISA, HI test, Taqman Type A RRT-PCR and embryonating chicken egg (ECE) virus isolation methods suggests high validity of these tests in wild birds, comparable to that reported in poultry. The NP c-ELISA in high AIV prevalence situations had a 100% diagnostic sensitivity (95% CI 81.5, 100) and in controls had 91% diagnostic specificity (95% CI 70.8, 98.9). In low AIV prevalence situations using a ≥60% inhibition threshold for positivity relative to the HI test, c-ELISA performed with 90.5% diagnostic sensitivity (95% CI 86.2, 93.8) and 41.2% diagnostic specificity (95% CI 38.1, 44.5). Assessment of the HI test suggests that a titre of ≥8 is a significant result in wild birds, and using this titre the HI test had 83.3% diagnostic sensitivity (95% CI 58.6, 96.4) in the challenged birds. The Type A RRT-PCR test performance for cloacal swabs had high diagnostic sensitivity that varied between 83.3-100% and diagnostic specificity that varied between 94.1-100% over 2-6 DPI when evaluated against ECE virus isolation, with substantial to outstanding agreement (Kappa statistic=0.8) and significant positive correlation (rs=0.82). The recommended thresholds for the Type A RRT-PCR at the Australian Animal Health Laboratory (AAHL) in poultry of CT <37 for positivity with an intermediate threshold (CT 37-40) were found to be valid in wild birds. The ECE virus isolation method performed well with 89% of virus positive birds positive on the first passage.