Highly pathogenic avian influenza management policy in domestic poultry: from reacting to preventing.

The emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza (HPAI) viruses in 2021 has led to unprecedented epidemics in poultry, changing epidemiological patterns of year-round infections in resident wild avifauna and more frequent spill-over events to mammals. Given this situation, it is important that we recognise that traditional HPAI management strategies are no longer sufficient, and policy changes are required. Poultry vaccination has emerged as a crucial intervention in the current control of HPAI, as evidenced by France's nationwide campaign targeting domestic ducks. However, due to the logistical challenges and potential trade implications of vaccination, broader structural reforms appear also necessary. These include a shift from farm-level to territorial-level biosecurity approaches, putting into practice the concept of 'regional biosecurity'. Given the role duck farm density has played in successive HPAI epidemics in France, there is a need to think about the spatial distribution of poultry farms as a structural component of regional biosecurity and to consider the reduction of farm concentration as a measure to prevent viral spread. The integration of regional biosecurity and poultry vaccination into prevention strategies should impact the way poultry are produced and traded in the future.

Making the invisible visible: Effectiveness of a training program to increase awareness and biosecurity compliance in poultry barn anterooms, using glowing substances.

Biosecurity is the cornerstone of prevention strategies to limit infectious pathogen spread between poultry farms. However, poor compliance of biosecurity measures is often observed when entering or exiting barns. The lack of knowledge, understanding, risk perception or technical skill are often described for anteroom-related biosecurity errors (i.e., not respecting anteroom zoning in relation to changing boots, clothing, and hand sanitation). In this study, we designed and assessed the efficacy of an intervention aiming at improving biosecurity compliance in anterooms, using substances glowing under ultraviolet light, which mimic microbiological contamination. Training exercises offered in a single session were proposed to 145 participants with an interest in animal farming (veterinary students, agricultural students, poultry farmers and poultry farm advisors). The exercises' efficacy was assessed by measuring the change in attitude and intentions of participants about specific biosecurity measures. The participants' attitudes and intentions were quantified using a questionnaire before and after training. The change in score was assessed in a multivariate linear regression model to measure the effect of various parameters on this change. The attitudes and intentions of participants were statistically improved (p < 0.05) and were affected by the initial attitude and intention score, the gender of the participants and the status of the participant (type of student or activity in the poultry industry). The use of this exercise material, perceived as playful and engaging, could be included in the framework of biosecurity training for all stakeholders of the poultry industry.

Viral shedding and environmental dispersion of two clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in experimentally infected mule ducks: implications for environmental sampling.

High pathogenicity avian influenza viruses (HPAIVs) have caused major epizootics in recent years, with devastating consequences for poultry and wildlife worldwide. Domestic and wild ducks can be highly susceptible to HPAIVs, and infection leads to efficient viral replication and massive shedding (i.e., high titres for an extended time), contributing to widespread viral dissemination. Importantly, ducks are known to shed high amounts of virus in the earliest phase of infection, but the dynamics and impact of environmental contamination on the epidemiology of HPAIV outbreaks are poorly understood. In this study, we monitored mule ducks experimentally infected with two H5N8 clade 2.3.4.4b goose/Guangdong HPAIVs sampled in France in 2016-2017 and 2020-2021 epizootics. We investigated viral shedding dynamics in the oropharynx, cloaca, conjunctiva, and feathers; bird-to-bird viral transmission; and the role of the environment in viral spread and as a source of samples for early detection and surveillance. Our findings showed that viral shedding started before the onset of clinical signs, i.e., as early as 1 day post-inoculation (dpi) or post-contact exposure, peaked at 4 dpi, and lasted for up to 14 dpi. The detection of viral RNA in aerosols, dust, and water samples mirrored viral shedding dynamics, and viral isolation from these environmental samples was successful throughout the experiment. Our results confirm that mule ducks can shed high HPAIV titres through the four excretion routes tested (oropharyngeal, cloacal, conjunctival, and feather) while being asymptomatic and that environmental sampling could be a non-invasive tool for early viral RNA detection in HPAIV-infected farms.

High and low pathogenicity avian influenza virus discrimination and prediction based on volatile organic compounds signature by SIFT-MS: a proof-of-concept study.

High and low pathogenicity avian influenza viruses (HPAIV, LPAIV) are the primary causes of poultry diseases worldwide. HPAIV and LPAIV constitute a major threat to the global poultry industry. Therefore, early detection and well-adapted surveillance strategies are of the utmost importance to control the spread of these viruses. Volatile Organic Compounds (VOCs) released from living organisms have been investigated over the last decades as a diagnostic strategy. Mass spectrometry instruments can analyze VOCs emitted upon viral infection. Selected ion flow tube mass spectrometry (SIFT-MS) enables direct analysis of cell headspace in less than 20 min. As a proof-of-concept study, we investigated the ability of a SIFT-MS coupled sparse Partial Least Square-Discriminant Analysis analytical workflow to discriminate IAV-infected cells. Supernatants of HPAIV, LPAIV, and control cells were collected from 1 to 72 h post-infection and analyzed using our analytical workflow. At each collection point, VOCs' signatures were first identified based on four independent experiments and then used to discriminate the infectious status of external samples. Our results indicate that the identified VOCs signatures successfully discriminate, as early as 1-h post-infection, infected cells from the control cells and differentiated the HPAIV from the LPAIV infection. These results suggest a virus-dependent VOCs signature. Overall, the external samples' status was identified with 96.67% sensitivity, 100% specificity, and 97.78% general accuracy.

Infection dynamics of subtype H9N2 low pathogenic avian influenza a virus in turkeys.

While mammals can be infected by influenza A virus either sporadically or with well adapted lineages, aquatic birds are the natural reservoir of the pathogen. So far most of the knowledge on influenza virus dynamics was however gained on mammalian models. In this study, we infected turkeys using a low pathogenic avian influenza virus and determined the infection dynamics with a target-cell limited model. Results showed that turkeys had a different set of infection characteristics, compared with humans and ponies. The viral clearance rates were similar between turkeys and ponies but higher than that in humans. The cell death rates and cell to cell transmission rates were similar between turkeys and humans but higher than those in ponies. Overall, this study indicated the variations of within-host dynamics of influenza infection in avian, humans, and other mammalian systems.

Pathological investigation of high pathogenicity avian influenza H5N8 in captive houbara bustards (Chlamydotis undulata), the United Arab Emirates 2020.

At the end of 2020, an outbreak of HPAI H5N8 was registered in captive African houbara bustards (Chlamydotis undulata) in the United Arab Emirates. In order to better understand the pathobiology of this viral infection in bustards, a comprehensive pathological characterization was performed. A total of six birds were selected for necropsy, histopathology, immunohistochemistry, RNAscope in situ hybridization and RT-qPCR and nanopore sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue blocks. Gross lesions included mottled and/or hemorrhagic pancreas, spleen and liver and fibrinous deposits on air sacs and intestine. Necrotizing pancreatitis, splenitis and concurrent vasculitis, hepatitis and fibrino-heterophilic peritonitis were identified, microscopically. Viral antigens (nucleoprotein) and RNAs (matrix gene) were both detected within necro-inflammatory foci, parenchymal cells, stromal cells and endothelial cells of affected organs, including the myenteric plexus. Molecular analysis of FFPE blocks successfully detected HPAI H5N8, further confirming its involvement in the lesions observed. In conclusion, HPAI H5N8 in African houbara bustards results in hyperacute/acute forms exhibiting marked pantropism, endotheliotropism and neurotropism. In addition, our findings support the use of FFPE tissues for molecular studies of poorly characterized pathogens in exotic and endangered species, when availability of samples is limited.

High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear (Ursus thibetanus): investigations based on paraffin-embedded tissues, France, 2022.

High pathogenicity avian influenza viruses (HPAIVs) H5Nx of clade 2.3.4.4b have been circulating increasingly in both wild and domestic birds in recent years. In turn, this has led to an increase in the number of spillover events affecting mammals. In November 2022, an HPAIV H5N1 caused an outbreak in a zoological park in the south of France, resulting in the death of a Tibetan black bear (Ursus thibetanus) and several captive and wild bird species. We detected the virus in various tissues of the bear and a wild black-headed gull (Chroicocephalus ridibundus) found dead in its enclosure using histopathology, two different in situ detection techniques, and next-generation sequencing, all performed on formalin-fixed paraffin-embedded tissues. Phylogenetic analysis performed on the hemagglutinin gene segment showed that bear and gull strains shared 99.998% genetic identity, making the bird strain the closest related strain. We detected the PB2 E627K mutation in minute quantities in the gull, whereas it predominated in the bear, which suggests that this mammalian adaptation marker was selected during the bear infection. Our results provide the first molecular and histopathological characterization of an H5N1 virus infection in this bear species. IMPORTANCE: Avian influenza viruses are able to cross the species barrier between birds and mammals because of their high genetic diversity and mutation rate. Using formalin-fixed paraffin-embedded tissues, we were able to investigate a Tibetan black bear's infection by a high pathogenicity H5N1 avian influenza virus at the molecular, phylogenetic, and histological levels. Our results highlight the importance of virological surveillance programs in mammals and the importance of raising awareness among veterinarians and zookeepers of the clinical presentations associated with H5Nx virus infection in mammals.

Vaccination Technology in Poultry: Principles of Vaccine Administration.

Poultry vaccines are very important tools for disease prevention and may be administered collectively by drinking water or spray or individually by injection or oculonasal drop, whereas inactivated vaccines are administered by injection only. Poultry vaccines are increasingly delivered at the hatchery to day-old chicks or in ovo, because mass vaccination is much more efficiently implemented and controlled at the hatchery than on the farm. Mass administration on the farm by drinking water or spray requires strict compliance with guidelines regarding water quality, preparation of vaccines, and application, so as to cover the whole flock. Vaccination at the hatchery uses integrated machines to deliver vaccines to day-old chicks or, increasingly, in ovo at transfer from setters to hatchers. Regardless of the route and technology, a high quality of monitoring is critically important to maintain strict compliance and best practices from the vaccine vial to the bird, to guarantee efficient administration and intake of the vaccine by the whole flock and to secure the integrity of the vaccine itself. Major recent technical innovations in poultry vaccination covering both biology and technology open a very exciting era.

Estudio recapitulativo- Tecnología de vacunación en la avicultura: principios de administración de vacunas. Las vacunas para la avicultura son herramientas muy importantes para la prevención de enfermedades y pueden administrarse colectivamente mediante agua de bebida y por aerosol, también individualmente mediante inyección o por gota oculonasal, mientras que las vacunas inactivadas se administran únicamente mediante inyección. Las vacunas avícolas se administran cada vez más en la planta de incubación a pollitos de un día o in ovo, porque la vacunación masiva se implementa y controla mucho más eficientemente en la planta de incubación que en las granjas. La administración masiva en granja mediante agua de bebida o aspersión requiere el cumplimiento estricto de las pautas relativas a la calidad del agua, preparación de vacunas y aplicación, de manera de cubrir a toda la parvada. La vacunación en la planta de incubación utiliza máquinas integradas para administrar vacunas a los pollitos de un día o, de manera más frecuente, in ovo en el momento del traslado de las incubadoras a las nacedoras. Independientemente de la ruta y la tecnología, una alta calidad del monitoreo es de vital importancia para mantener un cumplimiento estricto y las mejores prácticas desde el vial de la vacuna hasta el ave, para garantizar la administración y la captación eficiente de la vacuna por toda la parvada y para asegurar la integridad de la vacuna. Las importantes innovaciones técnicas recientes en la vacunación en avicultura que abarcan tanto la biología como la tecnología están iniciando una era fascinante.

An amplicon-based nanopore sequencing workflow for rapid tracking of avian influenza outbreaks, France, 2020-2022.

During the recent avian influenza epizootics that occurred in France in 2020/21 and 2021/22, the virus was so contagiousness that it was impossible to control its spread between farms. The preventive slaughter of millions of birds consequently was the only solution available. In an effort to better understand the spread of avian influenza viruses (AIVs) in a rapid and innovative manner, we established an amplicon-based MinION sequencing workflow for the rapid genetic typing of circulating AIV strains. An amplicon-based MinION sequencing workflow based on a set of PCR primers targeting primarily the hemagglutinin gene but also the entire influenza virus genome was developed. Thirty field samples from H5 HPAIV outbreaks in France, including environmental samples, were sequenced using the MinION MK1C. A real-time alignment of the sequences with MinKNOW software allowed the sequencing run to be stopped as soon as enough data were generated. The consensus sequences were then generated and a phylogenetic analysis was conducted to establish links between the outbreaks. The whole sequence of the hemagglutinin gene was obtained for the 30 clinical samples of H5Nx HPAIV belonging to clade 2.3.4.4b. The consensus sequences comparison and the phylogenetic analysis demonstrated links between some outbreaks. While several studies have shown the advantages of MinION for avian influenza virus sequencing, this workflow has been applied exclusively to clinical field samples, without any amplification step on cell cultures or embryonated eggs. As this type of testing pipeline requires only a short amount of time to link outbreaks or demonstrate a new introduction, it could be applied to the real-time management of viral epizootics.

A real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of highly pathogenic H5 clade 2.3.4.4b avian influenza viruses.

Highly pathogenic avian influenza viruses (HPAIV) are a major threat to the global poultry industry and public health due to their zoonotic potential. Since 2016, Europe and France have faced major epizootics caused by clade 2.3.4.4b H5 HPAIV. To reduce sample-to-result times, point-of-care testing is urgently needed to help prevent further outbreaks and the propagation of the virus. This study presents the design of a novel real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of clade 2.3.4.4b H5 HPAIV. A clinical validation of this RT-LAMP assay was performed on 198 pools of clinical swabs sampled in 52 poultry flocks during the H5 HPAI 2020-2022 epizootics in France. This RT-LAMP assay allowed the specific detection of HPAIV H5Nx clade 2.3.4.4b within 30 min with a sensitivity of 86.11%. This rapid, easy-to-perform, inexpensive, molecular detection assay could be included in the HPAIV surveillance toolbox.

Optimizing environmental viral surveillance: bovine serum albumin increases RT-qPCR sensitivity for high pathogenicity avian influenza H5Nx virus detection from dust samples.

IMPORTANCE: With the circulation of high pathogenicity avian influenza viruses having intensified considerably in recent years, the European Union is considering the vaccination of farmed birds. A prerequisite for this vaccination is the implementation of drastic surveillance protocols. Environmental sampling is a relevant alternative to animal sampling. However, environmental samples often contain inhibitory compounds in large enough quantities to inhibit RT-qPCR reactions. As bovine serum albumin is a molecule used in many fields to overcome this inhibitory effect, we tested its use on dust samples from poultry farms in areas heavily affected by HPAIV epizootics. Our results show that its use significantly increases the sensitivity of the method.

The feather epithelium contributes to the dissemination and ecology of clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in ducks.

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.

Predominance of low pathogenic avian influenza virus H9N2 in the respiratory co-infections in broilers in Tunisia: a longitudinal field study, 2018-2020.

Respiratory diseases are a health and economic concern for poultry production worldwide. Given global economic exchanges and migratory bird flyways, respiratory viruses are likely to emerge continuously in new territories. The primary aim of this study was to investigate the major pathogens involved in respiratory disease in Tunisian broiler poultry and their epidemiology. Between 2018 and 2020, broilers farms in northeastern Tunisia were monitored, and 39 clinically diseased flocks were sampled. Samples were screened for five viral and three bacterial respiratory pathogens using a panel of real-time PCR assays. The reemergence of H9N2 low pathogenic avian influenza virus (LPAIV) in commercial poultry was reported, and the Northern and Western African GI lineage strain was typed. The infectious bronchitis virus (IBV) GI-23 lineage and the avian metapneumovirus (aMPV) subtype B also were detected for the first time in broilers in Tunisia. H9N2 LPAIV was the most detected pathogen in the flocks tested, but rarely alone, as 15 of the 16 H9N2 positive flocks were co-infected. Except for infectious laryngotracheitis virus (ILTV), all of the targeted pathogens were detected, and in 61% of the respiratory disease cases, a combination of pathogens was identified. The major combinations were H9N2 + aMPV (8/39) and H9N2 + IBV (6/39), showing the high contribution of H9N2 LPAIV to the multifactorial respiratory diseases. This field survey provided evidence of the emergence of new respiratory viruses and the complexity of respiratory disease in Tunisia. A comprehensive and continuous surveillance strategy therefore is needed to better control respiratory pathogens in Tunisia.

Highly pathogenic avian influenza affects vultures' movements and breeding output.

An exceptional highly pathogenic avian influenza (HPAI) outbreak due to H5N1 virus genotypes belonging to clade 2.3.4.4.b has been affecting birds worldwide since autumn 2021.1,2,3 Mortality caused by viral infection has been well documented in poultry and more recently in wild birds, especially in seabird-breeding colonies.4,5,6 However, there is a critical lack of knowledge about how terrestrial birds deal with HPAI virus infections in terms of behavior and space use, especially during the breeding season.7,8,9 Understanding how birds move when they are infected could help evaluate the risk of spreading the virus at a distance among other populations of wild or domestic birds, this latter risk being especially important for commensal bird species. Through long-term GPS tracking, we described the changes in daily movement patterns of 31 adult griffon vultures Gyps fulvus in two French sites in 2022 compared with 3 previous years. In spring 2022, 21 vultures at both sites showed periods of immobility at the nest, during 5.6 days on average. Positive serological status of 2 individuals confirmed that they had been infected by HPAI viruses. Death was recorded for 3 of the 31 tracked individuals, whereas all others recovered and returned quickly to their foraging routine, although at least 9 birds failed breeding. Such immobility patterns and death rates were never observed in previous years and were not related to weather conditions. The high immobility behavior of infected birds could reduce the risks of transmission. The observed vulnerability to HPAI viruses questions the resistance of endangered vulture species worldwide if infected.

Monitoring biosecurity in poultry production: an overview of databases reporting biosecurity compliance from seven European countries.

Compliance with required on-farm biosecurity practices reduces the risk of contamination and spread of zoonotic and economically important diseases. With repeating avian influenza epidemics in the poultry industry, the need to monitor and improve the overall level of biosecurity is increasing. In practice, biosecurity compliance is assessed by various actors (e.g., academic, private and public institutions), and the results of such assessments may be recorded and gathered in databases which are seldom shared or thoroughly analyzed. This study aimed to provide an inventory of databases related to the assessment of biosecurity in poultry farms in seven major poultry-producing European countries to highlight challenges and opportunities associated with biosecurity data collection, sharing, and use. The institutions in charge of these databases were contacted and interviewed using a structured questionnaire to gather information on the main characteristics of the databases and the context of their implementation. A total of 20 databases were identified, covering the gamut of poultry species and production types. Most databases were linked to veterinary health authorities or academia, and to a lesser extent interbranch organizations. Depending on the institutions in charge, the databases serve various purposes, from providing advice to enforcing regulations. The quality of the biosecurity data collected is believed to be quite reliable, as biosecurity is mostly assessed by trained farm advisors or official veterinarians and during a farm visit. Some of the databases are difficult to analyze and/or do not offer information concerning which biosecurity measures are most or least respected. Moreover, some key biosecurity practices are sometimes absent from certain databases. Although the databases serve a variety of purposes and cover different production types, each with specific biosecurity features, their analysis should help to improve the surveillance of biosecurity in the poultry sector and provide evidence on the benefits of biosecurity.

Validation of an RNAscope assay for the detection of avian influenza A virus.

Highly pathogenic avian influenza (HPAI) is an acute viral disease associated with high mortality and great economic losses. Immunohistochemistry (IHC) is a common diagnostic and research tool for the demonstration of avian influenza A virus (AIAV) antigens within affected tissues, supporting etiologic diagnosis and assessing viral distribution in both naturally and experimentally infected birds. RNAscope in situ hybridization (ISH) has been used successfully for the identification of a variety of viral nucleic acids within histologic samples. We validated RNAscope ISH for the detection of AIAV in formalin-fixed, paraffin-embedded (FFPE) tissues. RNAscope ISH targeting the AIAV matrix gene and anti-IAV nucleoprotein IHC were performed on 61 FFPE tissue sections obtained from 3 AIAV-negative, 16 H5 HPAIAV, and 1 low pathogenicity AIAV naturally infected birds, including 7 species sampled between 2009 and 2022. All AIAV-negative birds were confirmed negative by both techniques. All AIAVs were detected successfully by both techniques in all selected tissues and species. Subsequently, H-score comparison was assessed through computer-assisted quantitative analysis on a tissue microarray comprised of 132 tissue cores from 9 HPAIAV-infected domestic ducks. Pearson correlation of r = 0.95 (0.94-0.97), Lin concordance coefficient of ρc = 0.91 (0.88-0.93), and Bland-Altman analysis indicated high correlation and moderate concordance between the 2 techniques. H-score values were significantly higher with RNAscope ISH compared to IHC for brain, lung, and pancreatic tissues (p ≤ 0.05). Overall, our results indicate that RNAscope ISH is a suitable and sensitive tool for in situ detection of AIAV in FFPE tissues.

In Vitro and In Vivo Characterization of H5N8 High-Pathogenicity Avian Influenza Virus Neurotropism in Ducks and Chickens.

H5N8 high-pathogenicity avian influenza virus (HPAIV) of clade 2.3.4.4B, which circulated during the 2016 epizootics in Europe, was notable for causing different clinical signs in ducks and chickens. The clinical signs preceding death were predominantly neurological in ducks versus respiratory in chickens. To investigate the determinants for the predominant neurological signs observed in ducks, we infected duck and chicken primary cortical neurons. Viral replication was identical in neuronal cultures from both species. In addition, we did not detect any major difference in the immune and inflammatory responses. These results suggest that the predominant neurological involvement of H5N8 HPAIV infection in ducks could not be recapitulated in primary neuronal cultures. In vivo, H5N8 HPAIV replication in ducks peaked soon after infection and led to an early colonization of the central nervous system. In contrast, viral replication was delayed in chickens but ultimately burst in the lungs of chickens, and the chickens died of respiratory distress before brain damage became significant. Consequently, the immune and inflammatory responses in the brain were significantly higher in duck brains than those in chickens. Our study thus suggests that early colonization of the central nervous system associated with prolonged survival after the onset of virus replication is the likely primary cause of the sustained inflammatory response and subsequent neurological disorders observed in H5N8 HPAIV-infected ducks. IMPORTANCE The severity of high-pathogenicity avian influenza virus (HPAIV) infection has been linked to its ability to replicate systemically and cause lesions in a variety of tissues. However, the symptomatology depends on the host species. The H5N8 virus of clade 2.3.4.4B had a pronounced neurotropism in ducks, leading to severe neurological disorders. In contrast, neurological signs were rarely observed in chickens, which suffered mostly from respiratory distress. Here, we investigated the determinants of H5N8 HPAIV neurotropism. We provide evidence that the difference in clinical signs was not due to a difference in neurotropism. Our results rather indicate that chickens died of respiratory distress due to intense viral replication in the lungs before viral replication in the brain could produce significant lesions. In contrast, ducks better controlled virus replication in the lungs, thus allowing the virus to replicate for a sufficient duration in the brain, to reach high levels, and to cause significant lesions.

Two major epidemics of highly pathogenic avian influenza virus H5N8 and H5N1 in domestic poultry in France, 2020-2022.

The spread of highly pathogenic avian influenza (HPAI) viruses worldwide has serious consequences for animal health and a major economic impact on the poultry production sector. Since 2014, Europe has been severely hit by several HPAI epidemics, with France being the most affected country. Most recently, France was again affected by two devastating HPAI epidemics in 2020-21 and 2021-22. We conducted a descriptive analysis of the 2020-21 and 2021-22 epidemics, as a first step towards identifying the poultry sector's remaining vulnerabilities regarding HPAI viruses in France. We examined the spatio-temporal distribution of outbreaks that occurred in France in 2020-21 and 2021-22, and we assessed the outbreaks' spatial distribution in relation to the 2016-17 epidemic and to the two 'high-risk zones' recently incorporated into French legislation to strengthen HPAI prevention and control. There were 468 reported outbreaks during the 2020-21 epidemic and 1375 outbreaks during the 2021-22 epidemic. In both epidemics, the outbreaks' distribution matched extremely well that of 2016-17, and most outbreaks (80.6% and 68.4%) were located in the two high-risk zones. The southwestern high-risk zone was affected in both epidemics, while the western high-risk zone was affected for the first time in 2021-22, explaining the extremely high number of outbreaks reported. As soon as the virus reached the high-risk zones, it started to spread between farms at very high rates, with each infected farm infecting between two and three other farms at the peaks of transmission. We showed that the spatial distribution model used to create the two high-risk zones was able to predict the location of outbreaks for the 2020-21 and 2021-22 epidemics. These zones were characterized by high poultry farm densities; future efforts should, therefore, focus on reducing the density of susceptible poultry in highly dense areas.

SARS-CoV-2 Infection in Companion Animals: Prospective Serological Survey and Risk Factor Analysis in France.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for COVID-19 in people, has been detected in companion animals on rare occasions. A limited number of large-scale studies have investigated the exposure of companion animals to SARS-CoV-2. The objective of this prospective study was to estimate seroprevalence in privately owned dogs and cats presented in veterinary clinics in different French regions and to test the hypothesis that the occurrence of an episode of COVID-19 in the household and close contact with the owner would increase the chances of the animals being seropositive. One hundred and sixty-five dogs and 143 cats were blood-sampled between March 2020 and December 2021. Neutralizing SARS-CoV-2 antibodies were detected in 8.4% of cats (12/143) and 5.4% of dogs (9/165). Seven animals (three dogs and four cats) were seropositive in the absence of an episode of COVID-19 in the household. Despite not being statistically significant (chi-square test, p-value = 0.55), our data may suggest that the occurrence of an episode of COVID-19 in the household could increase the risk of animal seropositivity (odds ratio = 1.38; 95% confidence interval = 0.55-3.77). This survey indirectly shows that SARS-CoV-2 circulates in canine and feline populations, but its circulation appears to be too low for pets to act as a significant viral reservoir.