Highly pathogenic avian influenza subtype H5Nx clade 2.3.4.4 outbreaks in Dutch poultry farms, 2014-2018: Clinical signs and mortality.

In recent years, different subtypes of highly pathogenic avian influenza (HPAI) viruses caused outbreaks in several poultry types worldwide. Early detection of HPAI virus infection is crucial to reduce virus spread. Previously, the use of a mortality ratio threshold to expedite notification of suspicion in layer farms was proposed. The purpose of this study was to describe the clinical signs reported in the early stages of HPAI H5N8 and H5N6 outbreaks on chicken and Pekin duck farms between 2014 and 2018 in the Netherlands and compare them with the onset of an increased mortality ratio (MR). Data on daily mortality and clinical signs from nine egg-producing chicken farms and seven Pekin duck farms infected with HPAI H5N8 (2014 and 2016) and H5N6 (2017-2018) in the Netherlands were analysed. In 12 out of 15 outbreaks for which a MR was available, MR increase preceded or coincided with the first observation of clinical signs by the farmer. In one chicken and two Pekin duck outbreaks, clinical signs were observed prior to MR increase. On all farms, veterinarians observed clinical signs of general disease. Nervous or locomotor signs were reported in all Pekin duck outbreaks, but only in two chicken outbreaks. Other clinical signs were observed less frequently in both chickens and Pekin ducks. Compared to veterinarians, farmers observed and reported clinical signs, especially respiratory and gastrointestinal signs, less frequently. This case series suggests that a MR with a set threshold could be an objective parameter to detect HPAI infection on chicken and Pekin duck farms at an early stage. Observation of clinical signs may provide additional indication for farmers and veterinarians for notifying a clinical suspicion of HPAI infection. Further assessment and validation of a MR threshold in Pekin ducks are important as it could serve as an important tool in HPAI surveillance programs.

Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans.

Animal experiments have shown that nonhuman primates, cats, ferrets, hamsters, rabbits, and bats can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition, SARS-CoV-2 RNA has been detected in felids, mink, and dogs in the field. Here, we describe an in-depth investigation using whole-genome sequencing of outbreaks on 16 mink farms and the humans living or working on these farms. We conclude that the virus was initially introduced by humans and has since evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period, several weeks before detection. Despite enhanced biosecurity, early warning surveillance, and immediate culling of animals in affected farms, transmission occurred between mink farms in three large transmission clusters with unknown modes of transmission. Of the tested mink farm residents, employees, and/or individuals with whom they had been in contact, 68% had evidence of SARS-CoV-2 infection. Individuals for which whole genomes were available were shown to have been infected with strains with an animal sequence signature, providing evidence of animal-to-human transmission of SARS-CoV-2 within mink farms.

An observational field study of the cloacal microbiota in adult laying hens with and without access to an outdoor range.

BACKGROUND: Laying hens with access to outdoor ranges are exposed to additional environmental factors and microorganisms, including potential pathogens. Differences in composition of the cloacal microbial community between indoor- and outdoor-housed layers may serve as an indicator for exposure to the outdoor environment, including its pathogens, and may yield insights into factors affecting the chickens' microbiota community dynamics. However, little is known about the influence of outdoor housing on microbiota community composition in commercial layer flocks. We performed a cross-sectional field study to evaluate differences in the cloacal microbiota of indoor- vs outdoor-layers across farms. Eight layer flocks (four indoor, four outdoor) from five commercial poultry farms were sampled. Indoor and outdoor flocks with the same rearing flock of origin, age, and breed were selected. In each flock, cloacal swabs were taken from ten layers, and microbiota were analysed with 16S rRNA gene amplicon sequencing. RESULTS: Housing type (indoor vs outdoor), rearing farm, farm and poultry house within the farm all significantly contributed to bacterial community composition. Poultry house explained most of the variation (20.9%), while housing type only explained 0.2% of the variation in community composition. Bacterial diversity was higher in indoor-layers than in outdoor-layers, and indoor-layers also had more variation in their bacterial community composition. No phyla or genera were found to be differentially abundant between indoor and outdoor poultry houses. One amplicon sequence variant was exclusively present in outdoor-layers across all outdoor poultry houses, and was identified as Dietzia maris. CONCLUSIONS: This study shows that exposure to an outdoor environment is responsible for a relatively small proportion of the community variation in the microbiota of layers. The poultry house, farm, and rearing flock play a much greater role in determining the cloacal microbiota composition of adult laying hens. Overall, measuring differences in cloacal microbiota of layers as an indicator for the level of exposure to potential pathogens and biosecurity seems of limited practical use. To gain more insight into environmental drivers of the gut microbiota, future research should aim at investigating community composition of commercial layer flocks over time.

Temporal Dynamics of Cloacal Microbiota in Adult Laying Chickens With and Without Access to an Outdoor Range.

Associations between animal health and performance, and the host's microbiota have been recently established. In poultry, changes in the intestinal microbiota have been linked to housing conditions and host development, but how the intestinal microbiota respond to environmental changes under farm conditions is less well understood. To gain insight into the microbial responses following a change in the host's immediate environment, we monitored four indoor flocks of adult laying chickens three times over 16 weeks, during which two flocks were given access to an outdoor range, and two were kept indoors. To assess changes in the chickens' microbiota over time, we collected cloacal swabs of 10 hens per flock and performed 16S rRNA gene amplicon sequencing. The poultry house (i.e., the stable in which flocks were housed) and sampling time explained 9.2 and 4.4% of the variation in the microbial community composition of the flocks, respectively. Remarkably, access to an outdoor range had no detectable effect on microbial community composition, the variability of microbiota among chickens of the same flock, or microbiota richness, but the microbiota of outdoor flocks became more even over time. Fluctuations in the composition of the microbiota over time within each poultry house were mainly driven by turnover in rare, rather than dominant, taxa and were unique for each flock. We identified 16 amplicon sequence variants that were differentially abundant over time between indoor and outdoor housed chickens, however none were consistently higher or lower across all chickens of one housing type over time. Our study shows that cloacal microbiota community composition in adult layers is stable following a sudden change in environment, and that temporal fluctuations are unique to each flock. By exploring microbiota of adult poultry flocks within commercial settings, our study sheds light on how the chickens' immediate environment affects the microbiota composition.

Limited changes in the fecal microbiome composition of laying hens after oral inoculation with wild duck feces.

Interspecies transmission of fecal microbiota can serve as an indicator for (indirect) contact between domestic and wild animals to assess risks of pathogen transmission, e.g., avian influenza. Here, we investigated whether oral inoculation of laying hens with feces of wild ducks (mallards, Anas platyrhynchos) resulted in a hen fecal microbiome that was detectably altered on community parameters or relative abundances of individual genera. To distinguish between effects of the duck inoculum and effects of the inoculation procedure, we compared the fecal microbiomes of adult laying hens resulting from 3 treatments: inoculation with wild duck feces (duck), inoculation with chicken feces (auto), and a negative control group with no treatment. We collected cloacal swabs from 7 hens per treatment before (day 0), and 2 and 7 D after inoculation, and performed 16S rRNA amplicon sequencing. No distinguishable effect of inoculation with duck feces on microbiome community (alpha and beta diversity) was found compared to auto or control treatments. At the individual taxonomic level, the relative abundance of the genus Alistipes (phylum Bacteroidetes) was significantly higher in the inoculated treatments (auto and duck) compared to the control 2 D after inoculation. Seven days after inoculation, the relative abundance of Alistipes had increased in the control and no effect was found anymore across treatments. These effects might be explained by the perturbation of the hen's microbiome caused by the inoculation procedure itself, or by intrinsic temporal variation in the hen's microbiome. This experiment shows that a single inoculation of fecal microbiota from duck feces to laying hens did not cause a measurable alteration of the gut microbiome community. Furthermore, the temporary change in relative abundance for Alistipes could not be attributed to the duck feces inoculation. These outcomes suggest that the fecal microbiome of adult laying hens may not be a useful indicator for detection of single oral exposure to wild duck feces.

Subpopulations of bovine WC1(+) gammadelta T cells rather than CD4(+)CD25(high) Foxp3(+) T cells act as immune regulatory cells ex vivo.

Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis. Especially CD4(+)CD25(high) T cells are considered to be important regulators of immune reactivity. In humans and rodents these natural Treg are characterized by their anergic nature, defined as a non-proliferative state, suppressive function and expression of Foxp3. In this study the potential functional role of flowcytometry-sorted bovine white blood cell populations, including CD4(+)CD25(high) T cells and gammadelta T cell subpopulations, as distinct ex vivo regulatory cells was assessed in co-culture suppression assays. Our findings revealed that despite the existence of a distinct bovine CD4(+)CD25(high) T cell population, which showed Foxp3 transcription/expression, natural regulatory activity did not reside in this cell population. In bovine co-culture suppression assays these cells were neither anergic nor suppressive. Subsequently, the following cell populations were tested functionally for regulatory activity: CD4(+)CD25(low) T cells, WC1(+), WC1.1(+) and WC1.2(+) gammadelta T cells, NK cells, CD8(+) T cells and CD14(+) monocytes. Only the WC1.1(+) and WC1.2(+) gammadelta T cells and CD14(+) monocytes proved to act as regulatory cells in cattle, which was supported by the fact that these regulatory cells showed IL-10 transcription/expression. In conclusion, our data provide first evidence that cattle CD4(+)CD25(high)Foxp3(+) and CD4(+)CD25(low) T cells do not function as Treg ex vivo. The bovine Treg function appears to reside in the gammadelta T cell population, more precisely in the WC1.1(+) and the WC1.2(+) subpopulation, major populations present in blood of cattle in contrast to non-ruminant species.