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.

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.

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.

Detection and Typing of a Fowl Adenovirus Type 1 Agent of Pancreatitis in Guinea Fowl.

Adenoviral pancreatitis has been amply described for decades in guinea fowl. Although its pathologic picture has been characterized fairly well, its etiology still remains only partially clarified. Based on several outbreaks diagnosed on commercial guinea flocks raised in France since 2017, we performed direct whole-genome sequencing from pancreatic lesional tissue by using the Oxford Nanopore Technologies (ONT) sequencing method. We generated 4781 viral reads and assembled a whole genome of 43,509 bp, clustering within fowl adenovirus type 1 (FAdV-1). A phylogenetic analysis based on a partial sequence of the hexon and short fiber genes on viruses collected in France showed 98.7% and 99.8% nucleotide identity, respectively. Altogether, these results confirm that an FAdV-1 closely related to chicken and other avian strains is the agent of pancreatitis in guinea fowl. This study illustrates the potential of ONT sequencing method to achieve rapid whole-genome sequencing directly from pathologic material.

Detección y tipificación de un adenovirus aviar tipo 1 (FAdV-1), agente de pancreatitis en gallinas de Guinea. La pancreatitis adenoviral se ha descrito ampliamente durante décadas en gallinas de Guinea. Aunque su cuadro patológico se ha caracterizado bastante bien, su etiología todavía permanece sólo parcialmente aclarada. Sobre la base de varios brotes diagnosticados en parvadas comerciales de guineas criadas en Francia desde el año 2017, se realizó una secuenciación directa del genoma completo a partir del tejido de la lesión pancreática mediante el método de secuenciación desarrollado por Oxford Nanopore Technologies. Se generaron 4781 lecturas virales y se ensambló un genoma completo de 43,509 pb, que se agrupó dentro del adenovirus aviar tipo 1 (FAdV-1). Un análisis filogenético basado en una secuencia parcial de los genes hexón y de fibra corta de virus recolectados en Francia mostró identidades de nucleótidos de 98.7% y 99.8%, respectivamente. En conjunto, estos resultados confirman que un adenovirus aviar tipo 1 estrechamente relacionado con el pollo y otras cepas aviares es el agente de la pancreatitis en la gallina de Guinea. Este estudio ilustra el potencial de las tecnologías desarrolladas por Oxford Nanopore Thechnologies para lograr una secuenciación rápida de todo el genoma directamente a partir de material patológico.

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.

Will urbanisation affect the expression level of genes related to cancer of wild great tits?

Recent studies suggest that oncogenic processes (from precancerous lesions to metastatic cancers) are widespread in wild animal species, but their importance for ecosystem functioning is still underestimated by evolutionary biologists and animal ecologists. Similar to what has been observed in humans, environmental modifications that often place wild organisms into an evolutionary trap and/or exposes them to a cocktail of mutagenic and carcinogenic pollutants might favor cancer emergence and progression, if animals do not up-regulate their defenses against these pathologies. Here, we compared, for the first time, the expression of 59 tumor-suppressor genes in blood and liver tissues of urban and rural great tits (Parus major); urban conditions being known to favor cancer progression due to, among other things, exposure to chemical or light pollution. Contrary to earlier indications, once we aligned the transcriptome to the great tit genome, we found negligible differences in the expression of anti-cancer defenses between urban and rural birds in blood and liver. Our results indicate the higher expression of a single caretaker gene (i.e. BRCA1) in livers of rural compared to urban birds. We conclude that, while urban birds might be exposed to an environment favoring the development of oncogenic processes, they seem to not upregulate their cancer defenses accordingly and future studies should confirm this result by assessing more markers of cancer defenses. This may result in a mismatch that might predispose urban birds to higher cancer risk and future studies in urban ecology should take into account this, so far completely ignored, hazard.

Aspergillus fumigatus in Poultry.

Aspergillus fumigatus remains a major respiratory pathogen in birds. In poultry, infection by A. fumigatus may induce significant economic losses particularly in turkey production. A. fumigatus develops and sporulates easily in poor quality bedding or contaminated feedstuffs in indoor farm environments. Inadequate ventilation and dusty conditions increase the risk of bird exposure to aerosolized spores. Acute cases are seen in young animals following inhalation of spores, causing high morbidity and mortality. The chronic form affects older birds and looks more sporadic. The respiratory tract is the primary site of A. fumigatus development leading to severe respiratory distress and associated granulomatous airsacculitis and pneumonia. Treatments for infected poultry are nonexistent; therefore, prevention is the only way to protect poultry. Development of avian models of aspergillosis may improve our understanding of its pathogenesis, which remains poorly understood.

Clinical, mycological and pathological findings in turkeys experimentally infected by Aspergillus fumigatus.

Experimental aspergillosis was induced in 1-day-old turkeys by intra-air-sac inoculation of a spore suspension of a 3-day-old Aspergillus fumigatus culture (CBS 144.89) containing 10(7) spores. Ten additional poults were used as controls. Infected and non-infected animals were closely observed at least twice a day for the appearance of clinical signs and were sequentially sacrificed at days 1, 2, 3, 5 and 7 post-inoculation. In the infected group, most lung tissues and air sac swabs were culture positive from day 1 to day 5. At 1 day post-inoculation, air sac membranes were multifocally and moderately to severely thickened by an oedema and covered by an exudate. A small number of germinating conidia were present in the superficial exudate, already giving rise to small radiating hyphae. Lung lesions were mild, dominated by a diffuse congestion and a mild heterophilic infiltration. From 2 to 3 days post-inoculation, air sac membranes were more severely affected and several granulomas were observed. Both granulomas and exudates were rich in germinated conidia and hyphae. Pulmonary lesions consisted in a diffuse pneumonia. Five days post-inoculation, air sac membrane lesions progressed to a severe, multifocal, heterophilic and granulomatous inflammation. Seven days post-inoculation, a reduction of the severity of the diffuse pneumonia was detected. Concomitantly, the fungal elements were mainly observed as fragmented tubules in the cytoplasm of multinucleate giant cells. The present study demonstrated that healthy turkey poults might be able to withstand exposure to 10(7) A. fumigatus spores.