Limited or No Serologic Evidence for Reunion Harrier (Circus maillardi) Exposure to Avian Influenza Virus, West Nile Virus, and Infectious Bronchitis Virus Open Access

The Reunion Harrier (Circus maillardi) is one of the world’s rarest raptors. Several threats affect this endemic species to Reunion Island, in particular exposure to rodenticides (Coeurdassier et al. 2019) but also road and cable collisions and loss and fragmentation of breeding habitats (Augiron 2022). Population size has significantly decreased in recent decades, to ∼200 pairs (Bourgeois et al. 2024). This species also has a high inbreeding rate (Bourgeois et al. 2024) and could become extinct within the next 40 yr without targeted conservation measures (Fay et al. 2023).

The Reunion Island bird community has been highly impacted since human settlement in the 17th century, with the extinction of 51% of native bird species (Mourer-Chauviré et al. 2006). Introduction of exotic birds and mammals has modified predator–prey interactions and may have cascading effects on the transmission of parasites and pathogens. Indeed, Reunion Harriers currently mostly feed on rodents (e.g., Rattus rattus, Mus musculus) but also on reptiles (e.g., Furcifer pardalis, Calotes versicolor), free-living exotic bird species (e.g. Columba livia, Pycnonotus jocosus), and occasionally poultry (Probst 2008). Both commercial farms and backyard flocks of poultry are kept across the island, and poultry is the most commonly consumed type of meat locally. Knowledge on exposure of Reunion Harrier to viruses with pathogenic or zoonotic potential, in particular associated with exotic species and poultry, is currently lacking, although essential for the conservation of this critically endangered species.

Low pathogenic avian influenza virus (LPAIV) has previously been detected in seabirds on Reunion Island (Lebarbenchon et al. 2015). Although LPAIV and infectious bronchitis virus (IBV) have not been reported in poultry on the island, this may be associated with limited surveillance rather than absence of the viruses. Two highly pathogenic (HP) H5N1 epizootics were reported in backyard poultry on Reunion Island in October 2022 and July 2023. Viruses were unlikely to have been introduced by wild birds, given the absence of migratory ducks and the small number of vagrant shorebirds on Reunion Island, and might rather have occurred through the importation of poultry or ornamental species. West Nile virus (WNV) has been documented in horses (Equus cabellus) on Reunion Island (Cardinale et al. 2017), but no information is available on any potential avian host reservoir locally.

Biological samples were obtained from Reunion Harriers between January 2015 and August 2023 captured 1) in reproduction sites during brood monitoring (adults and chicks); 2) in hunting areas (adults and immature); and 3) from a rescue center at the Société d’Études Ornithologiques de La Réunion, Saint-André, Réunion. The combination of birds sampled in the field and in the care center was set up to maximize the number of tested samples and increase the probability of virus and antibody detection.

Capture, handling, and collection of biologic material was approved by the Center for Research on Bird Population Biology (National Museum of Natural History, Paris; Personal Program no. 557) and conducted under the permit delivered by the “Direction de l’Environnement, de l’Aménagement et du Logement” (no. DEAL/SEB/UBIO/2021-36). Wild birds were caught with clapnets. The netting trap had 40 × 40-mm mesh arranged in a circular frame. It was mounted on a tripod 60 cm above the ground, with an upper semicircular support made of the same netting that closes the ring when the bird touches the lower net in an attempt to capture a decoy (Gosler 2004). Age and sex were determined for each bird based on plumage color as well as morphometric measurements for young birds (Augiron 2022).

Both cloacal and oropharyngeal swabs were collected from 92 birds (including 3 birds sampled twice), with sterile rayon-tipped applicators (Puritan, Guilford, Maine, USA). For each bird, both swabs were placed in the same tube that contained 1.5 mL of virus transport medium. Swabs were maintained at 4 C in the field then held at −80 C until tested; blood was collected from 76 birds (including 2 birds sampled twice). After centrifugation, plasma was obtained for 73 samples and stored at −20 C.

Swabs were centrifuged at 1500 × G for 15 min and then RNA was extracted from 140 µL of supernatant by using the QIAamp Viral RNA mini kit (QIAGEN, Valencia, California, USA). For red blood cells, nucleic acids were extracted from 200 µL of each sample with the QIAamp cador pathogen kit (QIAGEN). Reverse transcription was performed as described previously (Lebarbenchon et al. 2017). Molecular detection of AIV, coronaviruses, and flavivirus was conducted following Spackman et al. (2002), Moureau et al. (2007), and Muradrasoli et al. (2009). The PCRs were carried out with the QuantiNova Probe PCR master mix (QIAGEN) for influenza A viruses and coronavirus and the GoTaq G2 Hot Start green master mix (Promega, Madison, Wisconsin, USA) for flaviviruses.

Antibodies detection against AIV, IBV, and WNV was performed with the ID Screen influenza A antibody competition multi-species, the West Nile competition multi-species, and the Infectious bronchitis competition ELISAs (all IDvet, Grabels, France). An optimized protocol for the detection of antibodies against influenza viruses was applied (Lebarbenchon et al. 2012). For this assay, a sample-to-negative control (S/N) ratio value <0.40 was considered positive for the presence of antibodies against influenza and tested twice to confirm positivity; incubation periods were kept to <4 h to minimize the risk of false positives (Lebarbenchon et al. 2012). Because of limited amount of plasma, samples were tested first for influenza A virus (n = 73) and then for WNV (n = 72) and IBV (n = 62).

We did not detect AIV, coronavirus, and flavivirus in swabs or red blood cells from any birds, suggesting that these viruses were not circulating in the Reunion Harrier population at the time of sampling (Table 1). Serologic analyses supported that tested birds had not previously been infected by IBV or WNV (Table 2). Two Reunion Harriers however tested positive for AIV antibodies (Table 2; Supplementary Table S1). Both samples were tested twice to confirm positivity (S/N ratios of 0.31 and 0.33 for DA282341 and 0.40 and 0.39 for DA282332). Sample DA282332 S/N ratios were close to the positivity threshold applied with our optimized protocol (Lebarbenchon et al. 2012), but below the S/N ratio cutoff recommended by the manufacturer (0.45). The AIV subtype was not investigated because of limited amount of plasma available. Although transmission pathway could not be determined in our study, virus origin could either be from other wild birds or from poultry. Among wild birds, Brown Noddies (Anous stolidus) and Lesser Noddies (Anous tenuirostris) might represent AIV reservoir hosts on Reunion Island (Lebarbenchon et al. 2015), given the absence of resident and migratory ducks and the limited number of vagrants shorebirds. Noddies and Reunion Harriers however have very different ecologies, thereby limiting contact between these species and transmission opportunities.

Although poultry consumption may only be occasional, this might represent a source of AIV infection for Reunion Harriers. To validate this hypothesis, a precise assessment of poultry predation occurrence would be needed as well as epidemiologic surveillance of LPAIV circulating among farms and backyard flocks on the island. Given the high susceptibility of raptors to H5 HPAIV (Shearn-Bochsler et al. 2019), investigation of this transmission route is critical, because it could represent an additional threat to the conservation of the Reunion Harrier.

We thank François-Xavier Couzy, Yohan Meuraillon, Marie-Alice Simbi, Julie Tourmetz, Magali Turpin, Colombe Valette, and Zéba Vally for assistance in sample collection or analysis. This research was financially supported by DEAL Reunion and the European Union through a FEDER grant (no. SYNERGIE: RE0027109 and RE0031680).

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-24-00167.