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Abstract 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.

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.

The passive protection afforded by the colostrum from cattle that were vaccinated prepartum with an inactivated combination vaccine against the bovine respiratory syncytial virus (BRSV) was evaluated after an experimental challenge of calves. Pregnant cows without or with a low ELISA and neutralizing BRSV antibody titers were twice vaccinated or not vaccinated, the last immunization being at one month prior to calving. Vaccination was followed by a rapid increase in BRSV antibody titers after the second immunization. Twenty-eightnewborn calves were fed during the 6 h following birth, with 4 L of colostrum sourced from vaccinated cows (14 vaccine calves) or non-vaccinated cows (14 control calves) and were challenged with BRSV at 21 days of age. We showed that maternal immunity to BRSV provides a significant reduction in the clinical signs of BRSV in calves, especially for severe clinical forms. This protection was correlated with reduced BRSV detection in the lower respiratory tract but not in nasal swabs, indicating an absence of protection against BRSV nasal excretion. Finally, transcriptomic assays in bronchoalveolar lavages showed no statistical differences between groups for chemokine and cytokine mRNA transcriptions, with the exception of the overexpression of IL-9 at days 6 and 10 post-challenge, and a severe downregulation of CXCL-1 at day 3 post-challenge, in the vaccine group.

Abstract Background The production of foie gras involves different metabolic pathways in the liver of overfed ducks such as lipid synthesis and carbohydrates catabolism, but the establishment of these pathways has not yet been described with precision during embryogenesis. The early environment can have short- and long-term impacts on the physiology of many animal species and can be used to influence physiological responses that is called programming. This study proposes to describe the basal hepatic metabolism at the level of mRNA in mule duck embryos in order to reveal potential interesting programming windows in the context of foie gras production. To this end, a kinetic study was designed to determine the level of expression of selected genes involved in steatosis-related liver functions throughout embryogenesis. The livers of 20 mule duck embryos were collected every 4 days from the 12th day of embryogenesis (E12) until 4 days after hatching (D4), and gene expression analysis was performed. The expression levels of 50 mRNAs were quantified for these 7 sampling points and classified into 4 major cellular pathways. Results Interestingly, most mRNAs involved in lipid metabolism are overexpressed after hatching (FASN, SCD1, ACOX1), whereas genes implicated in carbohydrate metabolism (HK1, GAPDH, GLUT1) and development (HGF, IGF, FGFR2) are predominantly overexpressed from E12 to E20. Finally, regarding cellular stress, gene expression appears quite stable throughout development, contrasting with strong expression after hatching (CYP2E1, HSBP1, HSP90AA1). Conclusion For the first time we described the kinetics of hepatic ontogenesis at mRNA level in mule ducks and highlighted different expression patterns depending on the cellular pathway. These results could be particularly useful in the design of embryonic programming for the production of foie gras.

ABSTRACT Bovine respiratory disease (BRD) is a major disease of young cattle whose etiology lies in complex interactions between pathogens and environmental and host factors. Despite a high frequency of codetection of respiratory pathogens in BRD, data on the molecular mechanisms and pathogenesis associated with viral and bacterial interactions are still limited. In this study, we investigated the effects of a coinfection with influenza D virus (IDV) and Mycoplasma bovis in cattle. Naive calves were infected by aerosol with a French IDV strain and an M. bovis strain. The combined infection shortened the incubation period, worsened the disease, and led to more severe macroscopic and microscopic lesions compared to these parameters in calves infected with only one pathogen. In addition, IDV promoted colonization of the lower respiratory tract (LRT) by M. bovis and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The gamma interferon (IFN-γ) gene was shown to be the gene most statistically overexpressed after coinfection at 2 days postinfection (dpi) and at least until 7 dpi, which correlated with the high level of lymphocytes in the LRT. Downregulation of the PACE4 and TMPRSS2 endoprotease genes was also highlighted, being a possible reason for the faster clearance of IDV in the lungs of coinfected animals. Taken together, our coinfection model with two respiratory pathogens that when present alone induce moderate clinical signs of disease was shown to increase the severity of the disease in young cattle and a strong transcriptomic innate immune response in the LRT, especially for IFN-γ. IMPORTANCE Bovine respiratory disease (BRD) is among the most prevalent diseases in young cattle. BRD is due to complex interactions between viruses and/or bacteria, most of which have a moderate individual pathogenicity. In this study, we showed that coinfection with influenza D virus (IDV) and Mycoplasma bovis increased the severity of the respiratory disease in calves in comparison with IDV or M. bovis infection. IDV promoted M. bovis colonization of the lower respiratory tract and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The IFN-γ gene in particular was highly overexpressed after coinfection, correlated with the disease severity, immune response, and white cell recruitment in the lungs. In conclusion, we showed that IDV facilitates coinfections within the BRD complex by modulating the local innate immune response, providing new insights into the mechanisms involved in severe respiratory diseases.

Influenza D virus (IDV) has been identified in several continents, with serological evidence for the virus in Africa. In order to improve the sensitivity and cost−benefit of IDV surveillance in Togo, risk maps were drawn using a spatial multicriteria decision analysis (MCDA) and experts’ opinion to evaluate the relevance of sampling areas used so far. Areas at highest risk of IDV occurrence were the main cattle markets. The maps were evaluated with previous field surveillance data collected in Togo between 2017 and 2019: 1216 sera from cattle, small ruminants, and swine were screened for antibodies to IDV by hemagglutination inhibition (HI) assays. While further samples collections are needed to validate the maps, the risk maps resulting from the spatial MCDA approach generated here highlight several priority areas for IDV circulation assessment.

Abstract 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.

Abstract Background A fine balance of feto-maternal resource allocation is required to support pregnancy, which depends on interactions between maternal and fetal genetic potential, maternal nutrition and environment, endometrial and placental functions. In particular, some imprinted genes have a role in regulating maternal-fetal nutrient exchange, but few have been documented in the endometrium. The aim of this study is to describe the expression of 42 genes, with parental expression, in the endometrium comparing two extreme breeds: Large White (LW); Meishan (MS) with contrasting neonatal mortality and maturity at two days of gestation (D90-D110). We investigated their potential contribution to fetal maturation exploring genes-fetal phenotypes relationships. Last, we hypothesized that the fetal genome and sex influence their endometrial expression. For this purpose, pure and reciprocally crossbred fetuses were produced using LW and MS breeds. Thus, in the same uterus, endometrial samples were associated with its purebred or crossbred fetuses. Results Among the 22 differentially expressed genes (DEGs), 14 DEGs were differentially regulated between the two days of gestation. More gestational changes were described in LW (11 DEGs) than in MS (2 DEGs). Nine DEGs were differentially regulated between the two extreme breeds, highlighting differences in the regulation of endometrial angiogenesis, nutrient transport and energy metabolism. We identified DEGs that showed high correlations with indicators of fetal maturation, such as ponderal index at D90 and fetal blood fructose level and placental weight at D110. We pointed out for the first time the influence of fetal sex and genome on endometrial expression at D90, highlighting AMPD3, CITED1 and H19 genes. We demonstrated that fetal sex affects the expression of five imprinted genes in LW endometrium. Fetal genome influenced the expression of four genes in LW endometrium but not in MS endometrium. Interestingly, both fetal sex and fetal genome interact to influence endometrial gene expression. Conclusions These data provide evidence for some sexual dimorphism in the pregnant endometrium and for the contribution of the fetal genome to feto-maternal interactions at the end of gestation. They suggest that the paternal genome may contribute significantly to piglet survival, especially in crossbreeding production systems.

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.

Abstract Background Embryonic and fetal development is very susceptible to the availability of nutrients that can interfere with the setting of epigenomes, thus modifying the main metabolic pathways and impacting the health and phenotypes of the future individual. We have previously reported that a 38% reduction of the methyl donor methionine in the diet of 30 female ducks reduced the body weight of their 180 mule ducklings compared to that of 190 ducklings from 30 control females. The maternal methionine-restricted diet also altered plasmatic parameters in 30 of their ducklings when compared to that of 30 ducklings from the control group. Thus, their plasma glucose and triglyceride concentrations were higher while their free fatty acid level and alanine transaminase activity were decreased. Moreover, the hepatic transcript level of 16 genes involved in pathways related to energy metabolism was significantly different between the two groups of ducklings. In the present work, we continued studying the liver of these newly hatched ducklings to explore the impact of the maternal dietary methionine restriction on the hepatic transcript level of 70 genes mostly involved in one-carbon metabolism and epigenetic mechanisms. Results Among the 12 genes (SHMT1, GART, ATIC, FTCD, MSRA, CBS, CTH, AHCYL1, HSBP1, DNMT3, HDAC9 and EZH2) identified as differentially expressed between the two maternal diet groups (p-value

Abstract Background In mammals, the nutritional status experienced during embryonic development shapes key metabolic pathways and influences the health and phenotype of the future individual, a phenomenon known as nutritional programming. In farmed birds as well, the quantity and quality of feed offered to the dam can impact the phenotype of the offspring. We have previously reported that a 38% reduction in the intake of the methyl donor methionine in the diet of 30 female ducks during the growing and laying periods - from 10 to 51 weeks of age - reduced the body weight of their 180 mule ducklings compared to that of 190 ducklings from 30 control females. The maternal dietary methionine restriction also altered the hepatic energy metabolism studied in 30 of their ducklings. Thus, their plasma glucose and triglyceride concentrations were higher while their plasma free fatty acid level was lower than those measured in the plasma of 30 ducklings from the control group. The objective of this new study was to better understand how maternal dietary methionine restriction affected the livers of their newly hatched male and female ducklings by investigating the hepatic expression levels of 100 genes primarily targeting energy metabolism, amino acid transport, oxidative stress, apoptotic activity and susceptibility to liver injury. Results Sixteen of the genes studied were differentially expressed between the ducklings from the two groups. Maternal dietary methionine restriction affected the mRNA levels of genes involved in different pathways related to energy metabolism such as glycolysis, lipogenesis or electron transport. Moreover, the mRNA levels of the nuclear receptors PPARGC1B, PPARG and RXRA were also affected. Conclusions Our results show that the 38% reduction in methionine intake in the diet of female ducks during the growing and egg-laying periods impacted the liver transcriptome of their offspring, which may explain the previously observed differences in their liver energy metabolism. These changes in mRNA levels, together with the observed phenotypic data, suggest an early modulation in the establishment of metabolic pathways.