Emerging Microbes & Infections 2023, VOL. 12, 2272644 (11 pages) https://doi.org/10.1080/22221751.2023.2272644 The feather epithelium contributes to the dissemination and ecology of clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in ducks Nicolas Gaidea*, Fabien Filairea,b*, Kateri Bertranc,d, Manuela Crispoa, Malorie Dirata, Aurélie Seculaa, Charlotte Foret-Lucasa, Bruno Payrée, Albert Perlasc,d, Guillermo Canteroc,d, Natàlia Majóc,f, Sébastien Soubiesa and Jean-Luc Guérina a IHAP, Université de Toulouse, INRAE, ENVT, Toulouse, France; bTHESEO France, LanXess Biosecurity, LanXess Group, Laval, France; cUnitat mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Catalonia, Spain; dIRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Catalonia, Spain; eCMEAB, University of Toulouse, Toulouse, France; fDepartament de Sanitat i Anatomia Animals, Facultat de Veterinària, Campus de la Universitat Autònoma de Barcelona (UAB), Catalonia, Spain ABSTRACT Immature feathers are known replication sites for high pathogenicity avian influenza viruses (HPAIVs) in poultry. However, it is unclear whether feathers play an active role in viral transmission. This study aims to investigate the contribution of the feather epithelium to the dissemination of clade 2.3.4.4b goose/Guangdong/1996 lineage H5 HPAIVs in the environment, based on natural and experimental infections of domestic mule and Muscovy ducks. During the 2016–2022 outbreaks, H5 HPAIVs exhibited persistent and marked feather epitheliotropism in naturally infected commercial ducks. Infection of the feather epithelium resulted in epithelial necrosis and disruption, as well as the production and environmental shedding of infectious virions. Viral and feather antigens colocalized in dust samples obtained from poultry barns housing naturally infected birds. In summary, the feather epithelium contributes to viral replication, and it is a likely source of environmental infectious material. This underestimated excretion route could greatly impact the ecology of HPAIVs, facilitating airborne and preening-related infections within a flock, and promoting prolonged viral infectivity and long-distance viral transmission between poultry farms. ARTICLE HISTORY Received 31 July 2023; Revised 9 October 2023; Accepted 15 October 2023 KEYWORDS Avian influenza; feather; viral tropism; aerosol; environment Introduction the pre-symptomatic phase, which can last several High pathogenicity avian influenza viruses (HPAIVs) days. The main shedding routes include digestive of clade 2.3.4.4b goose/Guangdong/1996 (Gs/GD) and respiratory tracts [3–5]. Therefore, to develop H5 lineage have greatly impacted both the poultry effective control measures, it is essential to properly industry and wild bird populations during the last understand the mechanisms underlying HPAIVs few years, resulting in large-scale culling of infected infection and dissemination in ducks. In commercial flocks and mass mortality of endangered species ducks, infection with clade 2.3.4.4b H5 HPAIVs leads worldwide [1,2]. Ducks, in particular, play a crucial to neurological disease and mortality. Typical patho- role in the transmission of HPAIVs due to the shed- logical findings include acute encephalitis, myocar- ding of large amounts of infectious material during ditis, and pancreatitis. However, since these viruses CONTACT Jean-Luc Guérin jean-luc.guerin@envt.fr *NG and FF contributed equally and should be both considered as 1st authors. Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2023.2272644. © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. 2 N. Gaide et al. replicate systemically, they can be detected in many 2020–2021 (n = 10), and H5N1 2021–2022 (n = 18) other tissues [3,6]. HPAIVs. All samples were obtained under the super- A key feature of viral infection is tropism, which vision of the French Official Veterinary Services and in refers to the ability of a virus to infect and replicate in compliance with the French legislation on veterinary specific cells or tissues. Viral tropism plays a critical practices and notifiable diseases. At necropsy, feath- role in viral ecology since the range of infected tissues ered skin (including wing feathers, caudal tract, and and organs determines clinical signs, mean death time capital tract) was collected and fixed in 10% neutral and viral excretion routes [6]. Growing feather follicles buffered formalin for histopathological examination have been identified as a site of H5 and H7 HPAIVs and viral in situ detection. Titration of viral infectivity replication in ducks [7–9] and other gallinaceous in feather samples was conducted on two flocks of species [10–12]. Feathers are abundant external ducks naturally infected with H5N1 2021–2022 cutaneous appendages implicated in a variety of func- HPAIV (5 ducks per flock). Additionally, feathered tions, including flight, insulation, and communication skin from dead chickens (4 flocks, n = 3–5), turkeys [13]. Immature feathers are growing tubular structures (1 flock, n = 4), quails (1 flock, n = 5), geese (1 flock, budding from the skin, covered by a protective external n = 6) and wild swans (3 birds) naturally infected sheath and nourished by a vascularized dermal pulp with H5 HPAIV were collected in France between (supplementary data 1). The feather epithelium grows 2016 and 2022. Tissue samples were treated and ana- and differentiates into barbs and barbules. Upon matu- lyzed similarly to ducks. Results are provided in sup- ration, the external sheath opens and the epithelium plementary data. comes into contact with the air [14]. Experimental data show that infected feathers pulp is a site of pro- Experimental infection ductive viral replication, and feathers obtained from infected duck carcasses contain infectious material for Fifty 5-week-old domestic male mule ducks were up to 160 and 15 days post-mortem at 4 and 20°C, obtained from a commercial producer (GALLSA, Tar- respectively [12,15]. Furthermore, the stability of ragona, Spain). Ducks were challenged by intrachoa- avian influenza viruses has been reported to increase nal inoculation with 5 log10 50% egg infectious doses with preening oil, which is secreted by the uropygial (EID50) of either A/mulard duck/France/171201 g/ gland and used for preening behaviour [16]. 2017(H5N8) (H5N8/2017) reverse genetics-engin- Dust found in poultry houses is composed of eered virus (accession numbers MK859904 to diverse particles in terms of size, density, and shape, MK859911) [24] or A/Mule duck/France/20320/2020 originating from feed, faeces, skin, feather and litter (H5N8) (H5N8/2020) (accession numbers debris, microorganisms, and insect parts [17–19]. MZ166297 to MZ166304), and monitored for 14 Feathers are reported to account for 10% of dust days post-inoculation (dpi). Groups included sham found in commercial layer chicken houses [17,18]. birds (n = 3), inoculated birds (n = 16) and contact Not only is dust known to contribute to viral dispersal birds placed 24 h post-inoculation (n = 6), for each within farms, but also inhalable particles (<100 µm) viral strain. Feather pulp samples were collected on can lead to bird-to-bird airborne transmission [19– 10 inoculated and 6 contact birds at 0, 2, 4, 7, 10 23]. While dust is a recognized source of pathogens and 14 dpi for viral RNA detection. Complete necrop- [19], the specific contribution of feathers in infectious sies were performed at 3 and 5 dpi for each virus on 3 viral shedding, as well as viral environmental spread inoculated birds and on 3 sham birds. At necropsy, and ecology, remains unclear. sections of feathered skin, including capital and caudal This study aims to investigate the role of feather tracts [9], as well as detached immature wing feathers epitheliotropism in the environmental dissemination were collected for histopathology and stored in 10% of clade 2.3.4.4b H5 HPAIVs. neutral buffered formalin. All procedures involving birds were reviewed and approved by the IRTA (#258-2021) and the Catalan Government (#11467) Materials and methods Ethics and Animal Experimentation Committees, sub- ject to national and European regulations. All pro- Field cases cedures involving virus were performed in biosafety Eight flocks of commercial ducks (two flocks of Mus- level-3 (BSL-3) laboratory and animal facilities at covy ducks [Cairina moschata] and six flocks of mule IRTA-CReSA, in accordance with procedures ducks [Cairina moschata x Anas platyhrynchos]) posi- approved by IRTA Biosafety Committee (#59-2021). tive for H5 HPAIVs by official testing during the 2016–2022 HPAIVs outbreaks in France were Viral molecular detection included in the study. Pathological investigations were conducted on a total of 36 dead animals, natu- Total RNA was extracted from experimental and rally infected with H5N8 2016–2017 (n = 8), H5N8 selected field samples using the magnetic bead-based Emerging Microbes & Infections 3 ID Gene Mag Fast Extraction Kit and IDEAL 32 and post-stained with 3% uranyl acetate in 50% etha- extraction robot (Innovative Diagnostics, Grabel, nol and with 8.5% lead citrate before being examined France) according to the manufacturer’s instructions. on a HT 7700 Hitachi electron microscope at an accel- A one-step, real-time reverse transcription quantitat- erating voltage 80 KV. ive PCR (rRT-qPCR) targeting the H5 subtype was then performed using the ID GENE Influenza H5/ Dust morphological and immunofluorescent H7 Triplex kit (Innovative Diagnostics, Grabel, study France). Dust samples were collected from four H5N1 2021– 2022 HPAIV-positive farms using a dry cyclonic air Histology and viral in situ detection sampler Coriolis Compact (Bertin Technologies) for Fixed tissue samples were paraffin-embedded and cut 20 min at 50 L/min. Samples were resuspended in at 3 µm. Sections were stained with haematoxylin and 1 mL of phosphate buffered saline (PBS). Dust micro- eosin (H&E) for histopathological analysis and anti- scopic examination was conducted following an agar- influenza A virus nucleoprotein immunohistochemis- based method, with a similar approach to the cell try (anti-IAV NP IHC) using anti-NP influenza A block technique used in cytopathology [28]. Briefly, HB65 antibodies (NP, HB65, lot 758121J1, Biozol, 100 µL were centrifuged for 10 min at 300 g. The Eching, Germany). Briefly, the IHC protocol included supernatant was removed and the pellet, consisting an antigen retrieval step with 0.05% pronase applied of dust, was resuspended in 50 µL of PBS and then for 10 min at 37°C, a peroxidase blocking step of 100 µL of Histogel® heated at 60 +/− 5°C (Thermo 5 min at room temperature (S2023; Agilent) followed Scientific Richard-Allen Scientific, Waltham, MA). by saturation of nonspecific binding sites with normal Dust blocks were formalin-fixed, paraffin-embedded goat serum (X0907; Agilent) applied for 25 min at and serially cut at 3 µm. One section was stained room temperature, and overnight incubation with with H&E for histological analysis. On a second sec- anti-IAV NP antibody (1:2000 dilution) at 4°C. Signal tion, a double immunofluorescence (IF) was per- amplification and revelation were assessed using the formed using non-commercial polyclonal rabbit EnVision FLEX system and 3,3’-diaminobenzidine primary antibodies targeting corneous beta-proteins (DAB) revelation according to the manufacturer’s rec- (CBP) and mouse monoclonal anti-NP influenza A ommendations. Avian Influenza Matrix gene (M antibodies [29]. The antigenic retrieval and saturation gene) RNAscope in situ hybridization (RNAscope steps were similar to those used in the anti-IAV NP ISH) was performed on selected samples, as previously IHC. Both antibodies were co-incubated at dilutions described [25]. Viral antigen detection was deter- of 1/2000 (IAV NP) and 1/100 (CBP) overnight at 4° mined for epithelial and mesenchymal tissues of grow- C. Anti-mouse 594 (Invitrogen, A11032) and anti-rab- ing feathers, resting feather epidermis and dermis bit 488 (Jackson ImmunoResearch, 711-546-152) sec- [26]. Results were expressed as frequency (number ondary antibodies were then used at a dilution of 1/ of positive birds/number of total birds *100) and dis- 500, incubated 1 h at room temperature. Slides were tribution was determined according to Landman mounted with Fluoromount G with DAPI (Thermo- (2021) for each flock [27]. Fischer Scientific, 00-4959-52). Images were collected using a Zeiss LSM 710 confocal microscope. Transmission electron microscopy Viral titration Ultrastructural analyzes were performed on samples obtained from an experimentally infected duck with Growing feathers collected from ten ducks belonging H5N8/2017, which was selected based on histopatho- to two flocks (five ducks per flock) that tested positive logical lesions and viral antigen detection. Growing for H5N1 during the 2021 outbreaks in France were feathers from the caudal tract stored in formalin used to determine the infectivity of the feather sub- were cut (6 transversal sections), immersed in 2% glu- compartments through viral titration. Feather tips taraldehyde in Sorensen’s phosphate buffer (0.1 mol/L, were sealed with heated paraffin, and the feather pH = 7.4) for 1 h, and washed with Sorensen’s phos- outer sheath was decontaminated by immersion in phate buffer for 12 h. Then samples were incubated 70% ethanol for 5 min at room temperature, followed with 1% OsO4 in Sorensen’s phosphate buffer (0.05 by a washing step in PBS with 0.5% bovine serum mol/L, glucose 0.25 mol/L, OsO4 1%) for 1 h, dehy- albumin (BSA). Feather outer sheath was then longi- drated in an ascending ethanol series until ethanol tudinally opened to dissect both the distal epithelial 100° and then with propylene oxide, and embedded fraction (whitish, matt, filamentous material) and the with epoxy resin (EMBed 812). After 48h of polymer- basal pulp (pink-red, soft, translucent and moist ization at 60°C, ultrathin sections (70 nm) were material). Epithelial samples were incubated in PBS- mounted on 100 mesh collodion-coated copper grids 0.5% BSA for 30 min at room temperature. 4 N. Gaide et al. Subsequently, the PBS-0.5% BSA solution was were associated with epithelial disruption and loss of replaced and vortexed for 30 s. Feather outer sheath the original architecture (Supplementary data 2). Epi- (before and after decontamination), basal pulp, and thelial necrosis and signs of dermal pulpitis, such as distal epithelial fractions (with and without mechan- hyperaemia, exudation, leukocytic infiltration com- ical disruption) were subjected to viral titration. Titra- prised of lymphocytes, plasma cells and occasional tions were performed using Madin-Darby canine heterophils, were also present. In experimentally kidney (MDCK) cells, followed by an immunoperoxi- infected ducks, lesions were similar with those dase monolayer assay. Results were expressed as log10 observed in naturally infected birds. focus forming units per mL (FFU/mL) according to Matrosovitch et al. [30]. H5 HPAIVs have marked and persistent feather epitheliotropism Statistics Viral antigen was detected within lesions in both cyto- Statistical analyzes were performed using R Studio plasm and nuclei of cells (Figure 1A). Overall, in dom- software (https://www.R-project.org) to compare estic ducks the most frequently and severely affected viral titres among tissue compartments (feather tegumental compartment was the growing feather epi- outer sheath before and after decontamination, distal thelium, followed by the feather sheath (Figure 1B,C). feather epithelium before and after vortexing, and In growing feather epithelium, viral antigen was fre- basal pulp). Data for each sample were analyzed with quently and extensively detected in barbules cells, a linear model that used the bird as a random par- barb cells (ramus), rachidial cells, and supportive ameter, and the tissue compartment and flock as cells from marginal and axial plates. In contrast, fixed parameters. viral antigen detection was less frequent and widely spread in feather dermal pulp and resting feathers, Results except in ducks naturally infected with H5N1 from 2021 to 2022. In the dermal pulp, viral NP was mainly H5 HPAIVs lead to severe damage of growing detected in mesenchymal stromal cells and leukocytes. feather epithelium In resting feathers, viral antigen was inconstantly Growing feathers of domestic ducks naturally infected detected in dermal papilla and lining epithelium. with H5 HPAIVs between 2016 and 2022 exhibited Feathered skin dermis and epidermis were mostly segmental and multifocal to diffuse epithelial necrosis negative, except in some ducks naturally infected (Figure 1A). Lesions were identified in several layers with H5N1 from 2021 to 2022. Viral antigen detection exhibiting different stages of differentiation, including in other species including chicken, turkey, quail, geese, the epidermal collar and early and late barb ridges, and and wild swan is presented in Supplementary data 3. Figure 1. Feather lesions and in situ viral detection in commercial ducks naturally and experimentally infected with clade 2.3.4.4b H5 HPAIVs. (A) Growing feather from a healthy duck showing normal dermal pulp surrounded by intact feather epithelium (H&E, top figure). In contrast, severe epithelial necrosis and disruption associated with inflammation of feather pulp (H&E, middle figure) and viral antigen detection (IHC, bottom figure) is observed in a growing feather from a duck naturally and experimentally infected with H5 HPAIVs, respectively. Bars represent 100 µm. Frequency (B) and average score (C) of viral antigen detection by IHC according to tissue compartment in naturally and experimentally infected ducks. Flocks are labelled as f and are numbered for each period. Flocks included 4–6 birds per flock. Emerging Microbes & Infections 5 These results indicate that H5 HPAIVs have a marked Virion-shaped particles are observed in infected tropism for feather epithelium in domestic ducks. feather epithelium Ultrastructural analysis was performed on a total of six H5 HPAIVs are detected in growing feathers different sections of growing feathers obtained from early in the course of infection the caudal tract of a duck experimentally infected with H5N8/2017. Regions of interest were selected In experimentally infected ducks, viral RNA was based on semi-thin sections and observation of epi- detected in growing feathers as early as 2 dpi, with thelial necrosis. Filamentous particles, ranging 60- peak of detection at 4 dpi and positive detection up to 80 nm in diameter and up to 500 nm in length, were 14 dpi in the remaining ducks (Figure 2A). At 3 dpi, seen budding from the cytoplasmic membrane of the when the majority of the ducks were in the pre-symp- feather epithelium and accumulating between bar- tomatic phase, viral antigens were multifocally detected bules, extracellularly (Figure 3). within the dermal pulp and basal layer of growing epi- These results indicate that infection of the feather thelium, extending through marginal plates, and then epithelium generates production of virions released in the lower portion of barb and barbule cells (Figure extracellularly between barbules. 2B). At 5 dpi, when mortality peaked, the detection of severe and diffuse viral antigens extended to barbs, bar- bules, and inter-barbular spaces (Figure 2B). Viral anti- High titres of infectious material are associated gen distribution was similar in H5N8/2017 and H5N8/ with immature feather epithelium 2020 infected ducks within feather tissue. However, immunoreactive areas tended to be more widespread Both the detection of viral RNA and proteins in the in H5N8/2017 infected ducks. feather epithelium and the observation of particles These results indicate that H5 HPAIVs rapidly evocative of filamentous influenza virions, prompted infect and diffuse into the feather epithelium over us to determine whether the infected feather epi- the course of infection. thelium produced infectious particles. To do so, Figure 2. Viral antigen dynamics in commercial ducks experimentally infected with H5 HPAIV from 2017 and 2020. (A) Viral RNA detections in growing feathers from ducks experimentally infected with H5N8/2017 (red) or H5N8/2020 (blue), represented as 40- Ct values. Means calculated on living birds at the time of sampling are represented as points, and standard deviations as hooks for both inoculated (I) and contact (C) birds. N+, N−, ND dots represent number of positive, negative and dead birds, respectively. H5N8/2017_I: H5N8/2017 inoculated birds, H5N8/2017_C: H5N8/2017 contact birds, H5N8/2020_I: H5N8/2020 inoculated birds, H5N8_C: H5N8/2020 contact birds. (B) Viral antigen detection with anti-IAV NP IHC in growing feathers of experimentally infected ducks with H5N8 2017 at 3 and 5 dpi. 6 N. Gaide et al. Figure 3. Ultrastructural viral detection in growing feather epithelium of commercial ducks experimentally infected with H5 HPAIVs. (A) Ultrastructural images of feather epithelium of a duck experimentally infected with H5N8/2017 HPAIV at 6 dpi was assessed after selecting regions of interest (ROI) on histological sections. Bar, 200 µm. (B) ROI within feather growing epithelium included barbules, inter-barbular spaces, and necrotic areas. Bar, 50 µm. (C–G) Detection of budding and free, filamentous virion- shaped particles (arrowheads) within intercellular spaces of differentiating barbules. Transmission electron microscopy. Bar, 500 nm. immature duck feathers from naturally infected flocks the pulp and sheath compartments (p < 0.05). These were carefully dissected after sheath disinfection results support the hypothesis that infectious viral (Figure 4A). The association of infectious material particles are produced by the infected immature with the various feather subcompartments was then feather epithelium. determined by viral titration in MDCK cells (Figure 4B). Although moderate levels (mean concentration Feather fragments and viral antigens colocalize of 2.16 log10 FFU/mL) of infectious material were in dust from influenza outbreak farms present on feather sheaths, ethanol incubation led to a drastic reduction of infectivity, below the assay Histologically, dust collected from four H5 HPAIV- detection limit, in 8/10 samples, ruling out sub- positive farms revealed the presence of frequent sequent contamination of internal feather elements light to dense, eosinophilic and elongated structures by external infectious material (Figure 4B). High admixed with bacteria, fungi and granular material concentrations of infectious material (mean concen- of unknown origin (Figure 5). Prior to IF, Corneous tration around 4.39 log10 FFU/mL) were detected beta-protein (CBP) antigen detection was first deter- after simple incubation, without agitation of epi- mined by IHC on normal feathered skin: CBP was thelium samples. Similar concentrations (mean con- detected in cornifying cells, including barbules and centration of 4.39 log10 FFU/mL) were detected after the feather outer sheath (Supplementary data 4). replacement of the diluent and vortexing. Finally, Subsequently, double IF, targeting both viral IAV mean concentrations of 3.53 log10 FFU/mL were NP and CBP, was conducted on sections of feathered detected in feather pulp sampled in the area closest skin of a duck experimentally infected with H5N8/ to the skin. Overall, viral titres were statistically 2017 to assess positive detection of both targets, higher in the epithelial compartment compared to and then on dust blocks obtained from H5 Emerging Microbes & Infections 7 Figure 4. Viral infectivity of growing feathers in commercial ducks naturally infected with clade 2.3.4.4b H5N1 HPAIV. (A) Wing (top left) and caudal tract (top right) growing feathers of a 40-day-old male mule duck. Detached growing feathers are comprised of an external outer sheath (sh) and a growing and differentiating epithelium (ep) supported by a central core of dermal pulp (pu) (bottom left). For viral titration, distal epithelium was collected (bottom right). (B) Viral titres in MDCK cells, expressed as log10 FFU/ mL, determined on feather outer sheath before and after decontamination, feather epithelium with and without mechanical dis- ruption, and feather pulp. Histograms and the dotted line represent the mean viral titres and the limit of detection, respectively. Each dot represents an individual subject, with blue and red colours indicating the two flocks included in the analysis. Different letters indicate significant statistical difference at p < 0.05 determined by ANOVA analysis HPAIV-positive farms. In feathered skin, both CBP antigen colocalized with the CBP-positive elongated and IAV NP antigens colocalized in the growing structures and the granular material (Figure 6, Sup- feather epithelium, particularly in the barbule cells plementary data 5 and 6). (Figure 6). IAV NP antigen was also observed These results indicate that fragments detached from between barbules. In dust blocks, CBP antigen was the infected feather epithelium are aerosolized in detected within the elongated structures. AIV NP infected poultry houses and remain infected. Figure 5. Histological analysis of aerosols (dust) collected from H5 HPAIV-positive farms. (A) Histogel-based Coriolis Compact (Ber- tin Technologies) sampled dust blocks (arrows). (B–F) Dust blocks from H5 HPAIV-positive farms stained with H&E revealed the presence of frequent eosinophilic elongated structures (arrowheads), admixed with bacteria (asterisks), fungi and granular material of unknown origin. Bar, 50 µm (A) and 20 µm (B). 8 N. Gaide et al. Figure 6. Immunofluorescent detection of avian CBP and influenza A NP antigen in growing feather and aerosols (dust). Immu- nofluorescent detection of avian CBP and influenza A NP antigen in a growing feather of an H5N8/2017 experimentally infected mule duck, and in histogel-based dust block sampled by Coriolis Compact (Bertin Technologies) from four HPAIV-positive farms. 4’,6-diamino-2-phenilidole (DAPI, blue), Corneous beta-protein (CBP, green), Nucleoprotein influenza A (NP, red). Discussion feathers, allowing to differentiate viral titres obtained from the feather epithelium, pulp, and sheath (Figure 4). The contagiousness of avian influenza viruses and, in Our data show a marked tropism of clade 2.3.4.4b particular, of HPAIVs derived from the Gs/GD line- H5 viruses for the epithelium of growing feathers in age, is now universally recognized, especially in ducks. A similar marked epitheliotropism was ident- ducks. Experimental studies showed that both oro- ified in other waterfowl species, including commercial pharyngeal and cloacal viral shedding occur as early geese and wild mute swans naturally infected with H5 as a few hours post-infection and can last up to 14 HPAIVs (supplementary data 3). On the contrary, in days [5,31–33]. In comparison, the duration of viral gallinaceous birds naturally infected with H5 HPAIVs, excretion in infected chickens is much shorter (<2 including commercial chickens, quail, and turkeys, days) due to their short MDTs, contributing to the viral antigen detection was more frequent and widely higher excretion potential of ducks compared to galli- spread in the feather pulp and skin dermis compared naceous birds [4,5,24]. to the feather epithelium (supplementary data 3). Feather tropism of HPAIVs is now well documented, These results are consistent with experimental infec- particularly for viruses of the Gs/GD lineage [7– tions [9] and suggest rapid death of infected galli- 9,32,34]. However, virological investigation of feather formes is associated with limited viral extension tropism have mainly focused on the feather pulp, from pulp to the feather epithelium. regarded both as a marker of systemic infection and a The total number of feathers present in a single bird target for diagnostic purposes, but not on the feather is poorly known although, on average, it could account epithelium, as a potential excretion route of infectious for 3-6% of the total adult body weight [36]. In 1937, viruses. In these studies, detached feather follicles were George Andrew Ammann allegedly counted 25,216 homogenized following extraction and processed as a feathers in a swan. Phoebe Knappen reported a total whole biological matrix. As a consequence, the precise of 11,903 feathers in a female adult duck [37]. In tissue location was lost [8,12,31,35]. In our study, par- young birds, feather growth is an extremely fast and ticular attention was given to the dissection of growing relatively synchronous process that can reach up to Emerging Microbes & Infections 9 4 cm over 10 days in 40-day-old ducks [38]. For this Determining the infectious viral titre of organic reason, the feather epithelium compartment represents material released into the environment is currently a an extremely large surface area and volume in young challenge. Optimizations, including pre-treatments, individuals (Figure 4A). In contrast, adult birds have are underway in our laboratory to overcome methodo- a higher number of mature feathers that are associated logical limitations related to the study of environ- with lower viral detection [32]. However, moulting can mental matrices. In order to overcome this issue, we result in increased proportion of growing feathers in tested the infectivity of the epithelial layer in a con- adults. Different periodic and spontaneous moulting trolled setting, as this sub-tissular compartment is in processes can be observed in wild birds, which can be direct contact with the environment, compared to influenced by photoperiod, nutritional deficiency, the feather pulp. This experiment showed that the migration timing constraints, and global warming infectious viral load was higher in the epithelial layer [39,40]. Therefore, attention should not only be given compared to the pulp and feather sheath, further sup- to the study of host species but also to host age, breed- ported by the observation of filamentous virion- ing practices, plumage moult, and their potential shaped particles by electron microscopy in infected impact on viral dissemination. feather epithelium. Filamentous influenza viral par- Normal and pathological behaviours may also lead ticles are indeed preferentially observed after infection to increased viral transmission from birds with of epithelial cells and their genesis appears to depend infected feathers to other individuals. Ducks are well on both host and virus-derived factors [44]. The rela- known for their preening behaviour, which is very tive frequency of filamentous versus spherical viral specific of waterfowl and to which they devote several particles produced by infected feather epithelium is hours a day. This behaviour can involve only one bird currently under investigation. Altogether, these data (self-preening) or several congeners (allo-preening) support the notion that infected duck feather debris [33]. Additionally, feather pecking is a pathological are infectious. Persistence of infectivity over time injurious behaviour resulting in removal and eating and dispersion of such infectious debris in the of feather’s congeners [41]. Consequently, feathers environment remains to be assessed, in particular for represent an external organ strongly involved in nor- long-distance contamination and between-farm mal and pathological ethograms of wild and domestic dissemination. birds. This suggests possible direct transmission and Active viral shedding occurs along the time course ingestion of viral particles from feathers. of infection from pre-clinical to clinical phases [4]. The quantitative importance of the feather However, attention should also be given to the pas- excretion route, compared to the respiratory and sive post-mortem viral shedding from infected car- digestive shedding routes, still needs to be assessed casses littering the ground. Stability in detached [33]. Nevertheless, although a definitive conclusion infected feathers has been demonstrated for both is still premature, the intensity of the viral signal H5 and H7 HPAIVs in experimental conditions detected in the feather fraction identified in dust [12,15]. In birds, feather loss and disruption take samples is remarkable. Current data available in place throughout the decomposition process [45], chicken suggest that feather particles make up as underlying the importance of rapid disposal of much as 10% of the total mass of the dust present in infected carcasses and complete environmental disin- poultry houses [17], underlying the quantitative fection, considering also the risk of exposing cadave- importance of animal exposure to this type of sub- ric fauna (insects, arthropods etc.), scavenger birds, strate. A clear parallel can be drawn between AIVs and mammals to infectious tegument. Dispersion of and the aetiological agent of Marek’s disease in chick- feather debris may also occur during transportation ens; Gallid alphaherpesvirus 2 replicates to high titres of ducks from the farm to the slaughterhouse, during in the epithelium of the feather follicles, and mature processing, or in live poultry markets, which play a virions are released in association with debris originat- critical role in the epidemiology and evolution ing from the epithelial layer. In this form, herpesvirus dynamics of HPAIVs in many low- and middle- can survive for several months in the poultry house, income countries [46]. which is much longer than expected for a herpesvirus By identifying another route of viral shedding for [42]. A similar excretion route was found for the ducks, these results open the way to a paradigm shift chicken infectious anaemia virus [43]. in the epidemiology of avian influenza. Transmissibil- Staining experiments on dust samples collected ity is influenced by viral shedding and environmental from the environment of infected duck farms stability [6]. Infectious virions shed by the feather epi- confirmed that viral antigens are mostly co-detected thelium may be protected from physical factors or with avian CBPs. In feathered skin, CBP antigens even disinfectants, resulting in an unexpected persist- could be detected in barbs, barbules, feather sheath, ence of infectivity in the environment. However, and the outermost layer of epidermis, but viral antigen whether any particular resistance properties are detection was mostly positive in feather epithelium. associated with this route of excretion, thanks to the 10 N. Gaide et al. protection provided by cornified cells, endogenous or (H5N8 and H5N2) United States index viruses in uropygial lipids, still needs to be clarified [16,47,48]. Pekin ducks and Chinese geese. Vet Res. 2017;48 Combining natural and experimental infections, (1):33. doi:10.1186/s13567-017-0435-4 [4] Germeraad EA, Sanders P, Hagenaars TJ, et al. Virus our findings support the environmental shedding shedding of avian influenza in poultry: A systematic and dissemination of H5 HPAIVs through the review and meta-analysis [Internet]. Viruses. 2019;11 infected plumage of domestic ducks, which may con- (9):812. doi:10.3390/v11090812. Available from: stitute an underestimated route of transmission. https://www.mdpi.com/1999-4915/11/9/812. Further investigations are needed to establish the [5] Beerens N, Germeraad EA, Venema S, et al. Comparative pathogenicity and environmental trans- importance of this alternative route compared to the mission of recent highly pathogenic avian influenza fecal-oral and the respiratory-aerosol routes, and to H5 viruses. Emerg Microbes Infect. 2021;10(1):97– define its impact in the implementation of biosecurity 108. doi:10.1080/22221751.2020.1868274 measures. [6] Pantin-Jackwood MJ, Swayne DE. Pathogenesis and pathobiology of avian influenza virus infection in birds. OIE Rev Sci Tech. 2009;28(1):113–136. doi:10. Acknowledgments 20506/rst.28.1.1869 [7] Yamamoto Y, Nakamura K, Kitagawa K, et al. The authors would like to thank Manel Vinyes (GALLSA) Pathogenesis in call ducks inoculated intranasally for providing the ducks for the experimental infections, with H5N1 highly pathogenic avian influenza virus and Miquel Nofrarías, Maria José Valdez, and Rosa Valle and transmission by oral inoculation of infective for their technical assistance. We thank Thomas Figueroa, feathers from an infected call duck. Avian Dis. Pierre Bessière and Romain Volmer for providing viral 2007;51(3):744–749. doi:10.1637/0005-2086(2007)51 stock of reverse genetics-engineered virus. We would like [744:PICDII]2.0.CO;2 to express our sincere gratitude to Professor Lorenzo Ali- [8] Yamamoto Y, Nakamura K, Okamatsu M, et al. bardi from the University of Bologna for providing primary Detecting avian influenza virus (H5N1) in domestic antibody aliquots for the detection of corneous beta pro- duck feathers. Emerg Infect Dis. 2008;14(10):1671– teins. We gratefully acknowledge Cell Imaging Facility of 1672. doi:10.3201/eid1410.080415 INFINITy for access and assistance to the confocal micro- [9] Nuradji H, Bingham J, Payne J, et al. Highly pathogenic scope. The authors are thankful to Ms Aurore Mathon avian influenza (H5N1) virus in feathers. Vet Pathol. and Ms Grace Delobel for the design of the graphical 2017;54(2):226–233. doi:10.1177/0300985816666608 Abstract and the English proofreading, respectively. [10] Bertran K, Dolz R, Busquets N, et al. Pathobiology and transmission of highly and low pathogenic avian influenza viruses in European quail (Coturnix Disclosure statement c. coturnix). Vet Res. 2013;44(1):1–11. doi:10.1186/ No potential conflict of interest was reported by the author 1297-9716-44-23 (s). [11] Bertran K, Pérez-Ramírez E, Busquets N, et al. Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus Funding infection in red-legged partridge (Alectoris rufa). Vet Res. 2011;42(1):1–9. doi:10.1186/1297-9716-42-24 This study was performed in the framework of the “Chaire [12] Busquets N, Abad FX, Alba A, et al. Persistence of de Biosécurité et Santé Aviaires”, hosted by the National highly pathogenic avian influenza virus (H7N1) in Veterinary College of Toulouse (ENVT) and funded by infected chickens: feather as a suitable sample for diag- the Direction Generale de l’Alimentation, Ministère de nosis. J Gen Virol. 2010;91(9):2307–2313. doi:10.1099/ l’Agriculture et de la Souveraineté Alimentaire, France. vir.0.021592-0 The animal experiment was partially funded by the Veterin- [13] Terrill RS, Shultz AJ. Feather function and the evol- ary Biocontained facility Network (VetBioNet) [EU Grant ution of birds. Biol Rev. 2023;98(2):540–566. doi:10. Agreement INFRA-2016-1 N°731014]. 1111/brv.12918 [14] Alibardi L. Review: cornification, morphogenesis and evolution of feathers. Protoplasma. 2017;254 Data availability statement (3):1259–1281. doi:10.1007/s00709-016-1019-2 The data supporting the findings of this study are available [15] Yamamoto Y, Nakamura K, Yamada M, et al. within the article and its supplementary materials. Persistence of avian influenza virus (H5N1) in feathers detached from bodies of infected domestic ducks. Appl Environ Microbiol. 2010;76(16):5496–5499. References doi:10.1128/AEM.00563-10 [16] Karunakaran AC, Murugkar HV, Kumar M, et al. 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