Highly Pathogenic Avian Influenza A (H5N1) Infection in Bearded Vultures in Spain

ABSTRACT

We report mortality in bearded vultures ( Gypaetus barbatus ) associated with highly pathogenic avian influenza HPAI A H5N1 clade 2.3.4.4b, responsible for the current HPAI panzootic. Between April and May 2022, a total of five bearded vultures from Spain, four free‐ranging and one captive individual, were found dead in their nest or with acute symptoms of disease. Complete necropsies were performed and histopathological, immunohistochemistry and molecular analyses were carried out. The presence of the HPAI H5N1 virus was confirmed in different organs, oropharyngeal and cloacal swabs and feathers from the affected individuals. The complete viral genome was obtained from three of the affected bearded vultures. Phylogenetic analyses revealed that the sequences obtained from the free‐ranging individuals and the captive specimen belonged to the clade 2.3.4.4b and clustered separately. Furthermore, it supports that direct or indirect contact with other sympatric wild birds could be the most likely source of infection. This research highlights the susceptibility of the endangered bearded vulture to HPAI H5N1, thereby contributing to the broader understanding of the virus's host range.

Summary.

• We report, for the first time, mortality outbreaks caused by HPAI H5N1 clade 2.3.4.4b in the threatened bearded vulture.

• Typical HPAI macro‐ and microscopic lesions and viral RNA were confirmed in samples from deceased individuals.

• This research contributes to a deeper understanding of HPAI H5N1 epidemiology, specifically by expanding the known host range.

1. Introduction

In Europe, highly pathogenic avian influenza HPAI H5N1 clade 2.3.4.4b was first detected in October 2020 in wild birds. Since then, outbreaks have increased dramatically, leading to the largest and most devastating known worldwide HPAI panzootic posing major threats to animal health, especially in poultry and wildlife conservation (EFSA et al. ²⁰²³). To date, more than 10,900 outbreaks have been reported in wild birds in Europe, affecting more than 450 species, including waterfowl, shorebirds and vultures (EURL‐AF ²⁰²⁴). Before this concerning situation, vulture species had been only sporadically affected by HPAI viruses, with limited literature documenting cases of disease or death worldwide. Since late 2020, mortality associated with HPAI H5N1 has been reported in black vulture ( Coragyps atratus ), king vulture ( Sarcoramphus papa ) and turkey vulture ( Cathartes aura ) in the USA, as well as in hooded vultures ( Necrosyrtes monachus ) in Africa. Meanwhile, in Europe, griffon vultures ( Gyps fulvus ) have been affected by HPAI H5N1, leading to a significant indirect impact on reproduction with record‐low breeding success at French sites in 2022, attributed to a confluence of factors, including direct mortality of adult breeders, impaired parental care resulting from illness and nest immobility during recovery (Duriez et al. 2023). Transmission of the HPAI H5N1 virus in raptors (including vultures) has been scarcely studied, although evidence suggests that it may occur through direct (aerosol or droplet transmission) or indirect contact (fomite transmission or feeding on infected prey) (van den Brand et al. ²⁰¹⁵). Most of the aforementioned species are listed as endangered or critically endangered, particularly the Old‐World vultures (IUCN ²⁰²⁴). Consequently, outbreaks of HPAI affecting them may pose dramatic conservation consequences. This highlights the need to detect and report any suspicious cases or outbreaks involving vultures to better understand their susceptibility to HPAI H5N1. Here, we describe for the first time fatal HPAI H5N1 infections in free‐ranging and captive bearded vultures ( Gypaetus barbatus ).

2. Materials and Methods

Between 26 and 30 April 2022, environmental officers from the High‐Altitude Intervention Group of Navarra found three free‐ranging juvenile bearded vultures (BV1, BV2 and BV3) dead in their nests in different locations of Northern Spain (Figure 1). On 11 May 2022, environmental officers associated with the Andalusian Plan for the Recovery and Conservation of Necrophagous Birds found another free‐ranging juvenile bearded vulture dead (BV4) in its nest in Southern Spain (Figure 1). Finally, on 26 May 2022, an adult female bearded vulture (BV5) housed at the Cordoba Zoo Conservation Center (CZCC) (Southern Spain) showed ruffled feathers, dyspnoea, difficulty walking and spasmodic movements, depression, lethargy and weakness. This specimen was immediately placed in a warm environment, an intravenous line was set up and oxygen therapy was administered. Prophylactic antibiotic coverage (amoxicillin–clavulanic acid) and furosemide were also provided by CZCC veterinary services. Unfortunately, the bearded vulture died a few hours later.

FIGURE 1.

Location of bearded vultures ( Gypaetus barbatus ) dead by highly pathogenic avian influenza H5N1 and surrounding outbreaks in Spain.

Complete necropsies were performed on all the birds, and tissue samples were systematically collected and fixed in 10% neutral phosphate‐buffered formalin for 24 h, routinely embedded in paraffin wax and processed using standard haematoxylin and eosin procedures to analyse the histopathological lesions. In parallel, brain, feather, oropharyngeal and cloacal samples were collected in sterile bags and stored at −80°C.

When available, brain, feather, oropharyngeal and cloacal samples were tested by real‐time RT‐PCR (rRT‐PCR) for HPAI H5N1 virus using several targets with probes: matrix protein (M) as previously described (Spackman et al. 2002; Slomka et al. ²⁰¹⁰) to confirm the presence of influenza A virus and the haemagglutinin (HA) and/or neuraminidase (N) subtypes H5 and N1 genome segments, respectively (Spackman et al. 2002; Agüero et al. ²⁰⁰⁷; Hoffmann et al. ²⁰¹⁶). Each rRT‐PCR assay was performed in a final volume of 20 μL using the commercial kit known as AgPath‐ID One‐Step RT‐PCR Reagents (Applied BioSystems Waltham, MA, USA). All probes were labelled with FAM (Reporter) and Dark Quencher. When available, other co‐infections and contaminants were discarded in the samples as part of the routine diagnostic process for endangered wild species. This included bacterial pathogens such as Pasteurella spp., Yersinia spp. and Salmonella spp.; viral pathogens (West Nile virus, Bagaza virus and Newcastle disease virus), as well as fungal pathogens (Aspergillus spp. and Candida spp.). Additionally, potential contaminants (lead or toxic substances such as carbamates, organophosphates, organochlorines, strychnine, antimicrobials or anti‐inflammatory drugs) were discarded in the samples.

When HPAI H5N1 was confirmed by rRT‐PCR, and it was possible, its presence was further corroborated by immunohistochemistry (Bertran et al. 2023). Moreover, to further characterise the HPAI H5N1, samples from positive animals with a lower cycle threshold (Ct) (< 30) were sent to the EU Reference Laboratory for Avian Influenza and Newcastle Disease (EURL, Italy), to sequence the complete viral genome on an Illumina MiSeq platform. HA sequences were compared with those deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database. Phylogenetic analysis of the sequences was performed by the maximum‐likelihood method, using IQtree v1.6.6 software (Trifinopoulos et al. 2016), and the best‐fit model of nucleotide substitution was selected by ModelFinder (included in IQtree).

3. Results and Discussion

Following external inspection, cachexia was observed in BV1 and BV2 during post‐mortem examination, whereas the cloacae of BV5 and BV6 exhibited yellowish diarrhoea staining. No external signs were observed in BV3 during post‐mortem examination. Upon examination of the internal organs, BV1 exhibited serous atrophy of epicardial fat and congestion in the kidneys and intestines, while BV2 showed congestion in the lungs, kidneys and intestines. BV3 displayed haemorrhages in the lungs. Gross lesions in BV4 included generalised congestion and necrotising hepatitis. Additionally, the captive bearded vulture (BV5) exhibited generalised congestion, multifocal haemorrhages in the lungs, fibrinous tracheitis and moderate splenomegaly with pallor. Microscopic examination revealed the presence of moderate mononuclear inflammatory infiltrates in the brain (BV1), lungs (BV1 and BV2) and kidneys (BV2), alongside renal tubule degeneration and vacuolar degeneration of hepatocytes (BV1 and BV2). Moreover, BV4 and BV5 displayed widespread moderate mononuclear inflammatory infiltrates and necrosis in multiple organs, including the brain, lung, heart, liver, spleen and pancreas, accompanied by marked gliosis and moderate lymphoid depletion in the spleen (Figure 2A,B). Vascular lesions, including congestion, haemorrhages and oedema, were evident in the lungs, kidneys and brain of BV4 and BV5.

FIGURE 2.

Microscopic analysis of tissue samples from bearded vultures ( Gypaetus barbatus ) with highly pathogenic avian influenza H5N1 virus infection in Spain. (A) Brain section showing necrotising encephalitis (grey asterisk) with perivascular and glial cuffs (black arrowhead), neuronal necrosis (white arrowhead), perivascular and neuropil oedema (grey arrowhead) and congestion (blue asterisk). (B) Lung section showing mononuclear inflammatory infiltrate (white asterisks), areas of congestion (blue asterisk) and haemorrhages (black arrow). Immunohistochemical labelling for HPAI H5N1 was observed in necrotic cells, such as neurons (white arrow), glial cells (grey arrow) (C), as well as glandular cells of the exocrine pancreas (black asterisks) (D).

The HPAI H5N1 virus was detected in the brain, heart and pancreas (Figure 2C,D) of bearded vultures BV4 and BV5 by immunohistochemistry. RNA of the virus was also confirmed in brain (cycle threshold (Ct) values 11.7–12.4), feather (Ct 30.3–32.3), oropharyngeal (Ct 20.5–28.2) and cloacal (Ct 21.7–35.4) swabs in all the specimens analysed using rRT‐PCR. No additional co‐morbidities or potential contaminants (lead or toxic substances) were identified in the deceased animals. It is noteworthy that all the specimens showed detectable viral loads in feathers. In this respect, previous evidence indicates viral feather epitheliotropism, replication and survivability, potentially facilitated by the preen oil naturally produced by some bird species. Additionally, HPAI virus concentration resulting from preening activity is particularly notable in growing feathers, feather pulp and skin dermis during the early stages of HPAI infection in other wild and domestic bird species (Karunakaran et al. 2019; Uno et al. ²⁰²⁰; Gaide et al. ²⁰²³). Therefore, our results, in conjunction with the previously cited evidence, indicate that feathers, above all those growing with pulp, could be a valuable and non‐invasive sampling method for detecting HPAI infection in vulture species.

The complete viral genome was obtained from samples of four of the affected bearded vultures (BV1, BV3, BV4 and BV5) (GISAID accession numbers: EPI_ISL_18075816, EPI_ISL_15234656, EPI_ISL_13990717 and EPI_ISL_13990718) confirming the presence of HPAI H5N1 clade 2.3.4.4b RNA in these individuals by comparing their haemagglutinin sequences with those deposited in the GISAID database. HPAI H5N1 clade 2.3.4.4b is the most recent dominant clade worldwide, with numerous spillover events in domestic animals, including poultry and minks, leading to massive culling events and into wild bird populations and mammals (Good et al. 2024). The topology of the phylogenetic tree of the haemagglutinin gene suggested that the sequences obtained from the free‐ranging bearded vultures (BV1—A_bearded_vulture_Spain_2116–1‐2022_23VIR6502‐1_2023_H5N1_2022‐06‐09, BV3—A_bearded_vulture_Spain_2116‐3‐22_VIR8632‐8_2022_H5N1_2022‐06‐09 and BV4—A_Gypaetus_barbatus_Spain_1878‐9_22VIR6312‐18_2022_H5N1_2022‐05‐11) were closely related to other HPAI H5N1 viruses obtained from other raptor species in France and Spain in May–June 2022 (Duriez et al. 2023), indicating a potential regional spread. Furthermore, the sequence obtained from the captive bearded vulture (BV5—A/Gypaetus_barbatus/Spain/1956‐25_22VIR6312‐19/2022_H5N1_2022‐05‐26) was closely related to other HPAI H5N1 viruses collected from rheas, ducks and swans at the same zoo in May and June 2022 (Figure 3 unpublished data).

FIGURE 3.

Maximum‐likelihood phylogenetic tree for the haemagglutinin gene (segment 4) of highly pathogenic avian influenza virus isolates from three bearded vultures ( Gypaetus barbatus , in red) and the most closely related viruses obtained from the BLAST analysis in GISAID (grey boxes). Ultrafast bootstrap values (1000 replicates) are displayed at the branch nodes. The scale bar indicates the number of nucleotide substitutions per site.

Laboratory results confirmed a fatal HPAIH5N1 infection in the five bearded vultures analysed. Histopathological findings, although not homogeneous across all specimens, were consistent with those previously reported in other bird species (Nooruzzaman et al. 2019; Schreuder et al. ²⁰²²). It should be noted that neuroinflammation, together with the high viral loads detected in the bearded vultures, has previously been associated with other bird species highly susceptible to HPAI H5N1 (Morris et al. 2023; van den Brand et al. ²⁰¹⁵).

Phylogenetic analysis confirmed that the sequences obtained from the free‐ranging individuals (BV1–BV4) and the captive specimen (BV5) clustered separately. Despite their geographical dispersion, sequences obtained from BV1‐BV4 were intimately related among them as well as to viruses from other raptor species from Spain and France during 2022 (Figure 3). Direct or indirect contact, through infected individuals or fomites (as described above), coming from adult bearded vultures or other sympatric wild birds could be the most likely source of infection. In this regard, the home range of the bearded vulture can reach 10,000 km² (Margalida et al. 2016), and bearded and griffon vultures have regular contacts at the network of supplementary feeding sites (Margalida et al. 2016; Spina et al. ²⁰²²; Duriez et al. ²⁰²³). A different picture emerged for the BV5 sequence, which was closely related to viruses previously detected in other wild bird species in Spain during 2022 and in other captive birds from the same zoo and year (Figure 3). Considering the biosecurity measures at the BV5 facility, like compartmentation, cleaning and disinfection protocols or dedicated facility clothing, virus‐contaminated fomites seem to be the most plausible route of HPAI H5N1 infection; however, direct contact with sympatric infected free‐ranging small birds that could gain access to the facility cannot be completely ruled out. Confirmation of HPAI H5N1 infection in BV5 at the CZCC, located in the city of Cordoba, is of public health concern and highlights the importance of zoos to monitor this emerging virus in anthropised areas.

It is also worth noting that another free‐ranging juvenile bearded vulture was discovered deceased in 2022, at a location geographically and temporally close (same period) to the cases detailed in this study. This specimen, located in Southern Spain (Figure 1), was confirmed to be infected by the HPAI H5N1 virus by rRT‐PCR (EURL‐AF ²⁰²⁴) but could not be histopathologically analysed due to its advanced state of decay. Also, deaths of hundreds of chicks and several adult griffon vultures, among which 35 were confirmed to be infected by the H5N1 HPAI virus in Spain and France, were reported during this period (Duriez et al. 2023). All these cases, together with those previously described worldwide in black, white‐backed ( Gyps africanus ), turkey and hooded vultures, indicate that certain vulture species, particularly juvenile and chick individuals as described here, seem to be highly susceptible to HPAI H5N1 infection, which is of conservation concern. In fact, the bearded vulture is listed as an endangered species in Spain (IUCN ²⁰²⁴) with a total population estimated between 1200 and 2000 individuals and fewer than 215 breeding pairs across the Pyrenees (France, Andorra and Spain) and other regions of Spain. The bearded vulture and the other vulture species play a key role in the food chain, rapidly and efficiently consuming large amounts of carrion, which may help in disease prevention by reducing the availability of decomposing organic material that could harbour pathogens (Blanco and Díaz de Tuesta ²⁰²¹).

4. Conclusions

To the best of the authors' knowledge, this is the first study reporting fatal HPAI H5N1 infection in bearded vultures. Our findings underline the high susceptibility of this species to HPAI H5N1 and further contribute to knowledge of the epidemiology of this emerging virus, increasing its host range. Further studies, monitoring the breeding success of this species as well as the seroprevalence of HPAI H5N1 in the bearded vulture populationsare warranted to assess the impact of HPAI H5N1 on threatened vulture species worldwide and their potential role in transmission to other bird or mammal species, including humans.

Ethics Statement

This study did not involve the purposeful killing of animals. All samples were collected from animals found dead by the authorities. Thus, no ethical approval was necessary.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.