Approaches and methods to study wildlife cancer.

The last few years have seen a surge of interest from field ecologists and evolutionary biologists to study neoplasia and cancer in wildlife. This contributes to the One Health Approach, which investigates health issues at the intersection of people, wild and domestic animals, together with their changing environments. Nonetheless, the emerging field of wildlife cancer is currently constrained by methodological limitations in detecting cancer using non-invasive sampling. In addition, the suspected differential susceptibility and resistance of species to cancer often make the choice of a unique model species difficult for field biologists. Here, we provide an overview of the importance of pursuing the study of cancer in non-model organisms and we review the currently available methods to detect, measure and quantify cancer in the wild, as well as the methodological limitations to be overcome to develop novel approaches inspired by diagnostic techniques used in human medicine. The methodology we propose here will help understand and hopefully fight this major disease by generating general knowledge about cancer, variation in its rates, tumour-suppressor mechanisms across species as well as its link to life history and physiological characters. Moreover, this is expected to provide key information about cancer in wildlife, which is a top priority due to the accelerated anthropogenic change in the past decades that might favour cancer progression in wild populations.

Early Onset Anorexia Nervosa in Children Aged 8 to 12 Years: a Retrospective Study.

INTRODUCTION: Early onset anorexia nervosa accounts for 8% of anorexia cases worldwide. Very few studies on this subject exist, given the difficulty of obtaining a sufficiently significant sample. The aim of this study was to supplement the literature and investigate the clinical characteristics of early onset anorexia nervosa. SUBJECTS AND METHODS: This is a monocentric retrospective study carried out on the records of patients aged 8 to 12 years with early onset anorexia nervosa, hospitalized in the child psychiatry unit at the Queen Fabiola University Children's Hospital, in Brussels, from 01/01/2000 to 01/07/2023. Descriptive statistics were then performed on the sample and compared with the data found in the literature. RESULTS: This study included 48 children aged 8 to 12 with early onset anorexia nervosa. In the sample, three children were diagnosed with autism spectrum disorder during hospitalization. The sample included 36 girls and 12 boys, with an average age of 11 years and 7 months. The patients had an average body mass index of 13.6 kg/m2 on admission. A total of 87% of the parents had a history of psychiatric illness. A large majority of the children had underlying depressive and anxiety disorders. The duration of anorexia nervosa in these children was 13.5 months from diagnosis, with an average hospital stay of 5.4 months. CONCLUSIONS: This study seems to show the difficulty for parents and caregivers in diagnosing this illness and the probable impact of genetics and maternal depression on the development of early onset anorexia nervosa.

Covariation between glucocorticoids, behaviour and immunity supports the pace-of-life syndrome hypothesis: an experimental approach.

The biomedical literature has consistently highlighted that long-term elevation of glucocorticoids might impair immune functions. However, patterns are less clear in wild animals. Here, we re-explored the stress-immunity relationship considering the potential effects of behavioural profiles. Thirteen captive roe deer (Capreolus capreolus) were monitored over an eight-week period encompassing two capture events. We assessed how changes in baseline faecal cortisol metabolite (FCM) concentrations following a standardized capture protocol and an immune challenge using anti-rabies vaccination affected changes in 13 immune parameters of innate and adaptive immunity, and whether these changes in baseline FCM levels and immune parameters related to behavioural profiles. We found that individuals with increased baseline FCM levels also exhibited increased immunity and were characterized by more reactive behavioural profiles (low activity levels, docility to manipulation and neophilia). Our results suggest that the immunity of large mammals may be influenced by glucocorticoids, but also behavioural profiles, as it is predicted by the pace-of-life syndrome hypothesis. Our results highlight the need to consider covariations between behaviour, immunity and glucocorticoids in order to improve our understanding of the among-individual variability in the stress-immunity relationships observed in wildlife, as they may be underpinned by different life-history strategies.

Pathobiology of highly pathogenic H5 avian influenza viruses in naturally infected Galliformes and Anseriformes in France during 2015-2016.

In late 2015, an epizootic of Highly Pathogenic Avian Influenza (H5Nx) was registered in Southwestern France, including more than 70 outbreaks in commercial poultry flocks. Phylogenetic analyses suggested local emergence of H5 viruses which differed from A/goose/Guangdong/1/1996 clade 2.3.4.4b lineage and shared a unique polybasic cleavage site in their hemagglutinin protein. The present work provides an overview of the pathobiological picture associated with this epizootic in naturally infected chickens, guinea fowls and ducks. Upon necropsy examination, selected tissues were sampled for histopathology, immunohistochemistry and quantitative Real Time Polymerase Chain Reaction. In Galliformes, HPAIVs infection manifested as severe acute systemic vasculitis and parenchymal necrosis and was associated with endothelial expression of viral antigen. In ducks, lesions were mild and infrequent, with sparse antigenic detection in respiratory and digestive mucosae and leukocytes. Tissue quantifications of viral antigen and RNA were higher in chickens and guinea fowls compared to duck. Subsequently, recombinant HA (rHA) was generated from a H5 HPAIV isolated from an infected duck to investigate its glycan-binding affinity for avian mucosae. Glycan-binding analysis revealed strong affinity of rHA for 3'Sialyl-LacNAc and low affinity for Sialyl-LewisX, consistent with a duck-adapted virus similar to A/Duck/Mongolia/54/2001 (H5N2). K222R and S227R mutations on rHA sequence shifted affinity towards Sialyl-LewisX and led to an increased affinity for chicken mucosa, confirming the involvement of these two mutations in the glycan-binding specificity of the HA. Interestingly, the rHA glycan binding pattern of guinea fowl appeared intermediate between duck and chicken. The present study presents a unique pathobiological description of the H5 HPAIVs outbreaks that occurred in 2015-2016 in Southwestern France.

Diversity of avipoxviruses in captive-bred Houbara bustard.

Implementation of conservation breeding programs is a key step to ensuring the sustainability of many endangered species. Infectious diseases can be serious threats for the success of such initiatives especially since knowledge on pathogens affecting those species is usually scarce. Houbara bustard species (Chlamydotis undulata and Chlamydotis macqueenii), whose populations have declined over the last decades, have been captive-bred for conservation purposes for more than 15 years. Avipoxviruses are of the highest concern for these species in captivity. Pox lesions were collected from breeding projects in North Africa, the Middle East and Central Asia for 6 years in order to study the diversity of avipoxviruses responsible for clinical infections in Houbara bustard. Molecular and phylogenetic analyses of 113 and 75 DNA sequences for P4b and fpv140 loci respectively, revealed an unexpected wide diversity of viruses affecting Houbara bustard even at a project scale: 17 genotypes equally distributed between fowlpox virus-like and canarypox virus-like have been identified in the present study. This suggests multiple and repeated introductions of virus and questions host specificity and control strategy of avipoxviruses. We also show that the observed high virus burden and co-evolution of diverse avipoxvirus strains at endemic levels may be responsible for the emergence of novel recombinant strains.

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.

Surveys on Baylisascaris procyonis in two of the three French wild raccoon populations.

Human infection by Baylisascaris procyonis can result in larva migrans syndromes, which can cause severe neurological sequelae and fatal cases. The raccoon serves as the definitive host of the nematode, harboring adult worms in its intestine and excreting millions of eggs into the environment via its feces. Transmission to paratenic hosts (such as rodents, birds and rabbits) or to humans occurs by accidental ingestion of eggs. The occurrence of B. procyonis in wild raccoons has been reported in several Western European countries. In France, raccoons have currently established three separate and expanding populations as a result of at least three independent introductions. Until now the presence of B. procyonis in these French raccoon populations has not been investigated. Between 2011 and 2021, 300 raccoons were collected from both the south-western and north-eastern populations. The core parts of the south-western and north-eastern French raccoon populations were free of B. procyonis. However, three worms (molecularly confirmed) were detected in a young raccoon found at the edge of the north-eastern French raccoon population, close to the Belgian and Luxemburg borders. Population genetic structure analysis, genetic exclusion tests and factorial correspondence analysis all confirmed that the infected raccoon originated from the local genetic population, while the same three approaches showed that the worms were genetically distinct from the two nearest known populations in Germany and the Netherlands. The detection of an infected raccoon sampled east of the northeastern population raises strong questions about the routes of introduction of the roundworms. Further studies are required to test wild raccoons for the presence of B. procyonis in the area of the index case and further east towards the border with Germany.

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.

Normal bacterial flora of the oral cavity in healthy pet rabbits (Oryctolagus cuniculus).

BACKGROUND: Rabbits often suffer from dental disease, including dental abscesses and periodontal/apical infections. With odontogenic infection and abscessation, a bacterial aetiology can be proven by bacterial culture and identification. Although studies exist on the bacterial flora of dental abscesses, the information available to date on the bacterial flora of the oral cavity in healthy rabbits is limited. OBJECTIVES: This study aims to evaluate the cultivable bacterial flora in the oral cavity of healthy, young, pet rabbits and to compare this flora with the pathologic flora of odontogenic abscesses described in the literature. METHODS: Samples were collected from the oral cavity of 33 healthy, young pet rabbits undergoing routine procedures. Oral cavity culture specimens were collected by rolling a sterile flocked paediatric swab in the mouth. Identification was first attempted by morphological assessment, Gram staining and mass spectrometry (MALDI-TOF). Colonies that could not be identified by mass spectrometry were identified by amplification and molecular sequencing of a part of the 16s rRNA gene. RESULTS: Bacteria were recovered from 100% of oral swabs; 220 isolates of 35 different genera of bacteria were cultured. The most frequently isolated bacteria were Streptococcus sp. (19.8%), Rothia sp. (17.9%), Enterobacter sp. (7%), Staphylococcus sp. (6.6%) and Actinomyces sp. (5.7%). Four phyla are represented: Proteobacteria (38.3%), Firmicutes (30.5%), Actinobacteria (26.9%) and Bacteroidota (4.3%). CONCLUSIONS: A wide range of commensal bacteria are present in the mouths of rabbits. Bacterial cultures taken from cases of dental abscesses often reveal bacteria. Streptococcus sp., Staphylococcus sp. and Actinomyces sp. are frequently found in cultures from dental abscesses, in contrast to Rothia and Enterobacter species. Our findings enhance the knowledge of rabbit microbial communities throughout oral cavity.

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.

Concurrent Mycobacterium genavense infection and intestinal B-cell lymphoma in a pet rabbit (Oryctolaguscuniculus).

A 6-year-old male intact pet rabbit was evaluated for chronic weight loss. A large mass was detected by palpation in the mid-abdomen and ultrasound examination suggested a jejunal location. Explorative laparotomy revealed a nodular mass within the jejunal wall. Histological examination of a biopsy revealed mycobacterial granulomatous enteritis with an atypical lymphoblastic proliferation suggestive of lymphoma. Neoplastic lymphocytes were immunopositive for Pax-5 but negative for CD3, which is diagnostic of a B-cell neoplasm. Numerous acid-fast bacteria were seen within histiocytes and identified by polymerase chain reaction as Mycobacterium genavense, which is a non-tuberculous and opportunistic mycobacterium with zoonotic potential. To the best of our knowledge, this is the first documented case of a concurrent B-cell lymphoma and M. genavense infection in a rabbit. Concomitant mycobacteriosis and lymphoma have been rarely described in animals and the coexistence of neoplasia and mycobacterial infection within the jejunum suggests a potential pathogenetic association. Interestingly, the rabbit owner worked in an anti-tuberculosis clinic, and an anthropic origin of the mycobacterial infection could not be excluded.

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.

Causes of Mortality and Pathological Findings in European Hedgehogs (Erinaceus europaeus) Admitted to a Wildlife Care Centre in Southwestern France from 2019 to 2020.

The European hedgehog (Erinaceus europaeus) is a nocturnal, solitary and non-territorial mammal endemic across Europe and central Asia. Due to population decline in recent decades in Europe, this species was declared as protected and registered as 'Least Concern' on the International Union for Conservation of Nature Red List of Threatened Species. Previous studies pointed to the possible contribution of human activities to hedgehog mortality but few reports provide a complete overview of the pathological processes associated with mortality of the European hedgehog. The aim of this study was to identify the causes of mortality and pathological findings in 35 wild adult European hedgehogs that died spontaneously or were euthanized at the Wildlife Care Centre of the Ecole Nationale Vétérinaire de Toulouse between 2019 and 2020. The main causes of mortality were trauma (41%) including collision (9%), predation (9%), trapping (6%) and trauma of unknown origin (17%). Infectious diseases associated with systemic and local bacterial infections accounted for 34% of the cases and included cutaneous wounds (23%), mandibular abscesses and botryomycosis (23%), exudative bronchopneumonia (9%), suppurative rhinitis (7%) and otitis (3%). One hedgehog (3%) had a large subcutaneous tumour consistent with a malignant peripheral nerve sheath tumour. Parasitism was associated with mortality in 11% of cases with severe debilitation, massive infestation and active larval migration. Incidental findings included splenic and hepatic extramedullary haematopoiesis (100% and 69%, respectively), pulmonary crenosomiasis (91%), gastrointestinal capillariasis (61%), renal lipofuscinosis (59%), chronic renal infarction and interstitial nephritis (50%). These data emphasize the need for further study of hedgehog mortality and of the genetic, behavioural and environmental factors that may contribute to population decline.

Exposure assessment for avian influenza and Newcastle disease viruses from peridomestic wild birds in a conservation breeding site in the United Arab Emirates.

Worldwide, wild birds are frequently suspected to be involved in the occurrence of outbreaks of different diseases in captive-bred birds although proofs are lacking and most of the dedicated studies are insufficiently conclusive to confirm or characterize the roles of wild birds in such outbreaks. The aim of this study was to assess and compare, for the most abundant peridomestic wild birds, the different exposure routes for avian influenza and Newcastle disease viruses in conservation breeding sites of Houbara bustards in the United Arab Emirates. To do so, we considered all of the potential pathways by which captive bustards could be exposed to avian influenza and Newcastle disease viruses by wild birds, and ran a comparative study of the likelihood of exposure via each of the pathways considered. We merged data from an ecological study dedicated to local wild bird communities with an analysis of the contacts between wild birds and captive bustards and with a prevalence survey of avian influenza and Newcastle disease viruses in wild bird populations. We also extracted data from an extensive review of the scientific literature and by the elicitation of expert opinion. Overall, this analysis highlighted those captive bustards had a high risk of being exposed to pathogens by wild birds. This risk was higher for Newcastle disease virus than avian influenza virus, and House sparrows represented the riskiest species for the transmission of both viruses through direct exposure from direct contact with an infectious bird that got inside the aviary and indirect exposure from consumption of water contaminated from the faeces of an infected bird that got inside the aviary for Newcastle disease virus and avian influenza virus, respectively. These results also reaffirm the need to implement biosecurity measures to limit contacts between wild and captive birds and highlight priority targets for a thoughtful and efficient sanitary management strategy.

Quantification and characterisation of commensal wild birds and their interactions with domestic ducks on a free-range farm in southwest France.

The role of commensal birds in the epidemiology of pathogens in poultry farms remains unclear. Our study aimed to identify potential key species for interactions with domestic ducks on one free-range duck farm in southwest France. Methods combined direct individual observations on duck outdoor foraging areas, network analysis, and general linear mixed models of abundances. Results showed a wide diversity of wild bird species visiting foraging areas, heavily dominated in frequency by White wagtails (Motacilla alba) and Sparrows (Passer domesticus and Passer montanus). These also were the only species seen entering duck premises or perching on drinkers in the presence of ducks. Moreover, White wagtails were the species most frequently observed on the ground and in close proximity to ducks. Network analysis suggested the role of White wagtails and Sparrows in linking ducks to other wild birds on the farm. The abundance of White wagtails was positively associated with open vegetation, with the presence of ducks and particularly in the afternoon, while the abundance of Sparrows was positively associated only with the fall-winter season. By precisely characterising interactions, the study was able to identify few wild bird species which should be prioritized in infectious investigations at the interface with poultry.

Detection of a novel enterotropic Mycoplasma gallisepticum-like in European starling (Sturnus vulgaris) around poultry farms in France.

Recent outbreaks of highly pathogenic avian influenza in southwest France have raised questions regarding the role of commensal wild birds in the introduction and dissemination of pathogens between poultry farms. To assess possible infectious contacts at the wild-domestic bird interface, the presence of Mycoplasma gallisepticum (MG) was studied in the two sympatric compartments in southwest France. Among various peridomestic wild birds (n = 385), standard PCR primers targeting the 16S rRNA of MG showed a high apparent prevalence (up to 45%) in cloacal swabs of European starlings (Sturnus vulgaris, n = 108), while the MG-specific mgc2 gene was not detected. No tracheal swab of these birds tested positive, and no clinical sign was observed in positive birds, suggesting commensalism in the digestive tract of starlings. A mycoplasma strain was then isolated from a starling swab and its whole genome was sequenced using both Illumina and Nanopore technologies. Phylogenetic analysis showed that it was closely related to MG and M. tullyi, although it was a distinct species. A pair of specific PCR primers targeting the mgc2-like gene of this MG-like strain was designed and used to screen again the same avian populations and a wintering urban population of starlings (n = 50). Previous PCR results obtained in starlings were confirmed to be mostly due to this strain (20/22 positive pools). In contrast, the strain was not detected in fresh faeces of urban starlings. Furthermore, it was detected in one cloacal pool of white wagtails, suggesting infectious transmissions between synanthropic birds with similar feeding behaviour. As the new Starling mycoplasma was not detected in free-range ducks (n = 80) in close contact with positive starlings, nor in backyard (n = 320) and free-range commercial (n = 720) chickens of the area, it might not infect poultry. However, it could be involved in mycoplasma gene transfer in such multi-species contexts.

Newcastle disease virus transmission dynamics in wild peridomestic birds in the United Arab Emirates.

To understand the dynamics of a pathogen in an animal population, one must assess how the infection status of individuals changes over time. With wild animals, this can be very challenging because individuals can be difficult to trap and sample, even more so since they are tested with imperfect diagnostic techniques. Multi-event capture-recapture models allow analysing longitudinal capture data of individuals whose infection status is assessed using imperfect tests. In this study, we used a two-year dataset from a longitudinal field study of peridomestic wild bird populations in the United Arab Emirates during which thousands of birds from various species were captured, sampled and tested for Newcastle disease virus exposure using a serological test. We developed a multi-event capture-recapture model to estimate important demographic and epidemiological parameters of the disease. The modelling outputs provided important insights into the understanding of Newcastle disease dynamics in peridomestics birds, which varies according to ecological and epidemiological parameters, and useful information in terms of surveillance strategies. To our knowledge, this study is the first attempt to model the dynamics of Newcastle disease in wild bird populations by combining longitudinal capture data and serological test results. Overall, it showcased that multi-event capture-recapture models represent a suitable method to analyse imperfect capture data and make reliable inferences on infectious disease dynamics in wild populations.

Risk of Anticoagulant Rodenticide Exposure for Mammals and Birds in Parc National des Pyrénées, France.

The extensive use of anticoagulant rodenticides (ARs) to control rodent populations poses intoxication risks for wildlife: persistence of ARs in rodents can cause secondary exposure and poisoning of predators or scavengers. We investigated risk factors for wildlife exposure to ARs in the Parc National des Pyrénées (PNP), France, using a multivariable logistic regression analysis. A total of 157 liver samples were collected from carcasses of 10 mammal and three bird species found in the PNP between 2010 and 2018 and screened for presence of AR residues. First- and second-generation ARs were detected in more than 60% of red fox (Vulpes vulpes) and stone marten (Martes foina) samples and in around 40% of wild cat (Felis silvestris), European pine marten (Martes martes), American mink (Neovison vison), and Eurasian Buzzard (Buteo buteo) samples. Wildlife exposure to ARs was significantly associated with species having a regular consumption of small mammals (odds ratio [OR]: 2.5, 95% confidence interval [CI]: 1.1-5.8) being collected in the Ossau valley (OR: 2.5, 95% CI: 1.1-6.1) and between 2013 and 2015 (OR: 4.8, 95% CI: 2.0-11.7). We identified wild species that could be targeted for risk-based surveillance program for AR secondary exposure and determined high risk areas in which alternative measures should be applied for rodent control.

Backyard poultry flocks in France: A diversity of owners and biosecurity practices.

Over the past few years, the number of backyard poultry flocks has been increasing in France. A mandatory step to improve backyard poultry management is to assess health risks by characterizing the flocks and understanding the owners' motivations for keeping poultry and their husbandry practices. A survey of backyard poultry owners was conducted in France to gather information about their motivations for owning poultry, flock characteristics, and breeding and biosecurity practices. The survey was completed by 1,160 owners. The major motivations for owning poultry flocks were egg consumption (93.3 %), recycling (72.4 %) and having pet animals (53.2 %). Most owners had already heard about avian influenza (96.7 %), but were less aware about other diseases such as Newcastle Disease (41.6 %), salmonellosis (79.1 %), or campylobacteriosis (18.6 %). Owners mainly kept only egg-layers (78.4 %), and the median size flock was five egg-layers. Owners gave eggs to their relatives, occasionally or regularly, in 86.6 % of the cases. Contacts with other family poultry owners were frequent (68.9 %) and biosecurity practices were poorly implemented: 50 % of owners did not wash their hands systematically after visiting the flock and more than 60 % of owners did not wear specific shoes. Drawing from the survey data, five profiles of family poultry flocks were identified with multiple correspondence analysis and hierarchical cluster analysis. The profiles, based on flock characteristics and owners' practices and motivations, illustrate the heterogeneity of the backyard poultry sector: 1) urban poultry, 2) traditional poultry, 3) student poultry, 4) pet poultry and 5) hobby poultry. Urban poultry consisted of recently constituted (< 2 years old), small (< 3 birds) flocks of layers, and traditional poultry of older, medium-sized flocks belonging to retired and older people. These two profiles were characterized by limited contacts (direct or indirect) with other flocks and owners. Student poultry consisted of younger owners (<30 years old) with flocks over 5 years old. Pet poultry consisted of recently established, medium-size flocks of layers located in both rural or urban environments. Hobby poultry consisted of dedicated owners who breed and sell poultry and participate in exhibitions and poultry shows. Pet and hobby poultry profiles were characterized by greater knowledge of diseases and biosecurity practices, more bird movements, and reported more frequent clinical signs. The observation of different profiles can help target veterinary and public health education messages to prevent disease transmission in backyard poultry flocks in France.

Viral tropism and detection of clade 2.3.4.4b H5N8 highly pathogenic avian influenza viruses in feathers of ducks and geese.

Highly Pathogenic Avian Influenza viruses (HPAIVs) display a tissue pantropism, which implies a possible spread in feathers. HPAIV detection from feathers had been evaluated for H5N1 or H7N1 HPAIVs. It was suggested that viral RNA loads could be equivalent or higher in samples of immature feather compared to tracheal (TS) or cloacal swabs (CS). We investigated the suitability of feathers for the detection of clade 2.3.4.4b H5N8 HPAIV in ducks and geese field samples. In the six H5N8 positive flocks that were included in this study, TS, CS and immature wing feathers were taken from at least 10 birds. Molecular loads were then estimated using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) targetting H5 and M genes. In all flocks, viral loads were at least equivalent between feather and swab samples and in most cases up to 103 higher in feathers. Bayesian modelling confirmed that, in infected poultry, RT-qPCR was much more likely to be positive when applied on a feather sample only (estimated sensitivity between 0.89 and 0.96 depending on the positivity threshold) than on a combination of a tracheal and a cloacal swab (estimated sensitivity between 0.45 and 0.68 depending on the positivity threshold). Viral tropism and lesions in feathers were evaluated by histopathology and immunohistochemistry. Epithelial necrosis of immature feathers and follicles was observed concurrently with positive viral antigen detection and leukocytic infiltration of pulp. Accurate detection of clade 2.3.4.4b HPAIVs in feather samples were finally confirmed with experimental H5N8 infection on 10-week-old mule ducks, as viral loads at 3, 5 and 7 days post-infection were higher in feathers than in tracheal or cloacal swabs. However, feather samples were associated with lower viral loads than tracheal swabs at day 1, suggesting better detectability of the virus in feathers in the later course of infection. These results, based on both field cases and experimental infections, suggest that feather samples should be included in the toolbox of samples for detection of clade 2.3.4.4b HPAI viruses, at least in ducks and geese.