Comparison of the Clinical Manifestation of HPAI H5Nx in Different Poultry Types in the Netherlands, 2014-2022.

This study describes clinical manifestations of highly pathogenic avian influenza (HPAI) H5N1, H5N8 and H5N6 outbreaks between 2014 and 2018 and 2020 and 2022 in the Netherlands for different poultry types and age groups. Adult duck (breeder) farms and juvenile chicken (broiler and laying pullet) farms were not diagnosed before 2020. Outbreaks in ducks decreased in 2020-2022 vs. 2014-2018, but increased for meat-type poultry. Neurological, locomotor and reproductive tract signs were often observed in ducks, whereas laying- and meat-type poultry more often showed mucosal membrane and skin signs, including cyanosis and hemorrhagic conjunctiva. Juveniles (chickens and ducks) showed neurological and locomotor signs more often than adults. Diarrhea occurred more often in adult chickens and juvenile ducks. Mortality increased exponentially within four days before notification in chickens and ducks, with a more fluctuating trend in ducks and meat-type poultry than in layers. For ducks, a mortality ratio (MR) > 3, compared to the average mortality of the previous week, was reached less often than in chickens. A lower percentage of laying flocks with MR > 3 was found for 2020-2022 vs. 2014-2018, but without significant differences in clinical signs. This study provides a basis for improvements in mortality- and clinical-sign-based early warning criteria, especially for juvenile chickens and ducks.

Comparing the transmission of carbapenemase-producing and extended-spectrum beta-lactamase-producing Escherichia coli between broiler chickens.

The emergence of carbapenemase-producing Enterobacteriaceae (CPE) is a threat to public health, because of their resistance to clinically important carbapenem antibiotics. The emergence of CPE in meat-producing animals is particularly worrying because consumption of meat contaminated with resistant bacteria comparable to CPE, such as extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, contributed to colonization in humans worldwide. Currently, no data on the transmission of CPE in livestock is available. We performed a transmission experiment to quantify the transmission of CPE between broilers to fill this knowledge gap and to compare the transmission rates of CPE and other antibiotic-resistant E. coli. A total of 180 Ross 308 broiler chickens were distributed over 12 pens on the day of hatch (day 0). On day 5, half of the 10 remaining chickens in each pen were orally inoculated with 5·102 colony-forming units of CPE, ESBL, or chloramphenicol-resistant E. coli (catA1). To evaluate the effect of antibiotic treatment, amoxicillin was given twice daily in drinking water in 6 of the 12 pens from days 2-6. Cloacal swabs of all animals were taken to determine the number of infectious broilers. We used a Bayesian hierarchical model to quantify the transmission of the E. coli strains. E. coli can survive in the environment and serve as a reservoir. Therefore, the susceptible-infectious transmission model was adapted to account for the transmission of resistant bacteria from the environment. In addition, the caecal microbiome was analyzed on day 5 and at the end of the experiment on day 14 to assess the relationship between the caecal microbiome and the transmission rates. The transmission rates of CPE were 52 - 68 per cent lower compared to ESBL and catA1, but it is not clear if these differences were caused by differences between the resistance genes or by other differences between the E. coli strains. Differences between the groups in transmission rates and microbiome diversity did not correspond to each other, indicating that differences in transmission rates were probably not caused by major differences in the community structure in the caecal microbiome. Amoxicillin treatment from day 2-6 increased the transmission rate more than three-fold in all inoculums. It also increased alpha-diversity compared to untreated animals on day 5, but not on day 14, suggesting only a temporary effect. Future research could incorporate more complex transmission models with different species of resistant bacteria into the Bayesian hierarchical model.

Monitoring Wind-Borne Particle Matter Entering Poultry Farms via the Air-Inlet: Highly Pathogenic Avian Influenza Virus and Other Pathogens Risk.

Wind-supported transport of particle matter (PM) contaminated with excreta from highly pathogenic avian influenza virus (HPAIv)-infected wild birds may be a HPAIv-introduction pathway, which may explain infections in indoor-housed poultry. The primary objective of our study was therefore to measure the nature and quantity of PM entering poultry houses via air-inlets. The air-inlets of two recently HPAIv-infected poultry farms (a broiler farm and a layer farm) were equipped with mosquito-net collection bags. PM was harvested every 5 days for 25 days. Video-camera monitoring registered wild bird visits. PM was tested for avian influenza viruses (AIV), Campylobacter and Salmonella with PCR. Insects, predominantly mosquitoes, were tested for AIV, West Nile, Usutu and Schmallenberg virus. A considerable number of mosquitoes and small PM amounts entered the air-inlets, mostly cobweb and plant material, but no wild bird feathers. Substantial variation in PM entering between air-inlets existed. In stormy periods, significantly larger PM amounts may enter wind-directed air-inlets. PM samples were AIV and Salmonella negative and insect samples were negative for all viruses and bacteria, but several broiler and layer farm PM samples tested Campylobacter positive. Regular wild (water) bird visits were observed near to the poultry houses. Air-borne PM and insects-potentially contaminated with HPAIv or other pathogens-can enter poultry air-inlets. Implementation of measures limiting this potential introduction route are recommended.

Manifestation of SARS-CoV-2 Infections in Mink Related to Host-, Virus- and Farm-Associated Factors, The Netherlands 2020.

SARS-CoV-2 outbreaks on 69 Dutch mink farms in 2020 were studied to identify risk factors for virus introduction and transmission and to improve surveillance and containment measures. Clinical signs, laboratory test results, and epidemiological aspects were investigated, such as the date and reason of suspicion, housing, farm size and distances, human contact structure, biosecurity measures, and presence of wildlife, pets, pests, and manure management. On seven farms, extensive random sampling was performed, and age, coat color, sex, and clinical signs were recorded. Mild to severe respiratory signs and general diseases such as apathy, reduced feed intake, and increased mortality were detected on 62/69 farms. Throat swabs were more likely to result in virus detection than rectal swabs. Clinical signs differed between virus clusters and were more severe for dark-colored mink, males, and animals infected later during the year. Geographical clustering was found for one virus cluster. Shared personnel could explain some cases, but other transmission routes explaining farm-to-farm spread were not elucidated. An early warning surveillance system, strict biosecurity measures, and a (temporary) ban on mink farming and vaccinating animals and humans can contribute to reducing the risks of the virus spreading and acquisition of potential mutations relevant to human and animal health.

Conserved developmental trajectories of the cecal microbiota of broiler chickens in a field study.

There is great interest in identifying gut microbiota development patterns and underlying assembly rules that can inform strategies to improve broiler health and performance. Microbiota stratification using community types helps to simplify complex and dynamic ecosystem principles of the intestinal microbiota. This study aimed to identify community types to increase insight in intestinal microbiota variation between broilers and to identify factors that explain this variation. A total of 10 well-performing poultry flocks on four farms were followed. From each flock, the cecal content of nine broilers was collected at 7, 14, and 35 days posthatch. A total of two robust community types were observed using different clustering methods, one of which was dominated by 7-day-old broilers, and one by 35-day-old broilers. Broilers, 14-day-old, were divided across both community types. This is the first study that showed conserved cecal microbiota development trajectories in commercial broiler flocks. In addition to the temporal development with age, the cecal microbiota variation between broilers was explained by the flock, body weight, and the different feed components. Our data support a conserved development of cecal microbiota, despite strong influence of environmental factors. Further investigation of mechanisms underlying microbiota development and function is required to facilitate intestinal health promoting management, diagnostics, and nutritional interventions.

Chicken-derived RSPO1 and WNT3 contribute to maintaining longevity of chicken intestinal organoid cultures.

Intestinal organoids are advanced cellular models, which are widely used in mammalian studies to mimic and study in vivo intestinal function and host-pathogen interactions. Growth factors WNT3 and RSPO1 are crucial for the growth of intestinal organoids. Chicken intestinal organoids are currently cultured with mammalian Wnt3a and Rspo1, however, maintaining their longevity has shown to be challenging. Based on the limited homology between mammalian and avian RSPO1, we expect that chicken-derived factors are required for the organoid cultures. Isolated crypts from embryonic tissue of laying hens were growing in the presence of chicken WNT3 and RSPO1, whereas growth in the presence of mammalian Wnt3a and Rspo1 was limited. Moreover, the growth was increased by using Prostaglandin E2 (PGE2) and a Forkhead box O1-inhibitor (FOXO1-inhibitor), allowing to culture these organoids for 15 passages. Furthermore, stem cells maintained their ability to differentiate into goblets, enterocytes and enteroendocrine cells in 2D structures. Overall, we show that chicken intestinal organoids can be cultured for multiple passages using chicken-derived WNT3 and RSPO1, PGE2, and FOXO1-inhibitor.

Wild Bird Densities and Landscape Variables Predict Spatial Patterns in HPAI Outbreak Risk across The Netherlands.

Highly pathogenic avian influenza viruses' (HPAIVs) transmission from wild birds to poultry occurs globally, threatening animal and public health. To predict the HPAI outbreak risk in relation to wild bird densities and land cover variables, we performed a case-control study of 26 HPAI outbreaks (cases) on Dutch poultry farms, each matched with four comparable controls. We trained machine learning classifiers to predict outbreak risk with predictors analyzed at different spatial scales. Of the 20 best explaining predictors, 17 consisted of densities of water-associated bird species, 2 of birds of prey, and 1 represented the surrounding landscape, i.e., agricultural cover. The spatial distribution of mallard (Anas platyrhynchos) contributed most to risk prediction, followed by mute swan (Cygnus olor), common kestrel (Falco tinnunculus) and brant goose (Branta bernicla). The model successfully distinguished cases from controls, with an area under the receiver operating characteristic curve of 0.92, indicating accurate prediction of HPAI outbreak risk despite the limited numbers of cases. Different classification algorithms led to similar predictions, demonstrating robustness of the risk maps. These analyses and risk maps facilitate insights into the role of wild bird species and support prioritization of areas for surveillance, biosecurity measures and establishments of new poultry farms to reduce HPAI outbreak risks.

Long-chain glucomannan supplementation modulates immune responsiveness, as well as intestinal microbiota, and impacts infection of broiler chickens with Salmonella enterica serotype Enteritidis.

The zoonotic pathogen Salmonella enterica serotype Enteritidis (SE) causes severe disease in young chickens. Restriction on antibiotic use requires alternative SE control strategies such as nutritional solutions to improve the resistance of chickens. In this study, chickens were fed long-chain glucomannan (GM) or standard diet and challenged with SE at seven days of age. During 21 days post-infection (dpi), we determined numbers and responsiveness of natural killer (NK) and T cells in ileum and spleen, and SE-specific antibody titers in serum. Microbiota compositions in ileum and caeca were determined, as well as correlations of these with numbers and function of immune cells. Some of the samples in the control group had numerically higher CFUs than the GM-treated group. In addition, the relative abundance of SE based on DNA assessment was significantly lower at 21 dpi upon GM supplementation. At 3 dpi, numbers of intraepithelial NK cells were significantly higher, while activation of intraepithelial NK cells (7 dpi), numbers of intraepithelial cytotoxic CD8+ T cells (14 dpi) and SE-specific antibodies (14 dpi) were numerically higher. Furthermore, relative abundance of the commensal lactic acid bacteria (LAB) significantly increased with GM supplementation post-infection. Higher relative abundance of streptococci was associated with reduced SE in ileal and caecal contents at 21 dpi. Relative abundance of streptococci negatively correlated with SE counts and positively correlated with NK cell activation and SE-specific antibodies, which suggests involvement of the commensal LAB in NK cell responsiveness. These results indicate that GM supplementation modulates the immune system, intestinal microbiota and impacts SE infection of young chickens.

SARS-CoV-2 infection in cats and dogs in infected mink farms.

Animals like mink, cats and dogs are susceptible to SARS-CoV-2 infection. In the Netherlands, 69 out of 127 mink farms were infected with SARS-CoV-2 between April and November 2020 and all mink on infected farms were culled after SARS-CoV-2 infection to prevent further spread of the virus. On some farms, (feral) cats and dogs were present. This study provides insight into the prevalence of SARS-CoV-2-positive cats and dogs in 10 infected mink farms and their possible role in transmission of the virus. Throat and rectal swabs of 101 cats (12 domestic and 89 feral cats) and 13 dogs of 10 farms were tested for SARS-CoV-2 using PCR. Serological assays were performed on serum samples from 62 adult cats and all 13 dogs. Whole Genome Sequencing was performed on one cat sample. Cat-to-mink transmission parameters were estimated using data from all 10 farms. This study shows evidence of SARS-CoV-2 infection in 12 feral cats and 2 dogs. Eleven cats (18%) and two dogs (15%) tested serologically positive. Three feral cats (3%) and one dog (8%) tested PCR-positive. The sequence generated from the cat throat swab clustered with mink sequences from the same farm. The calculated rate of mink-to-cat transmission showed that cats on average had a chance of 12% (95%CI 10%-18%) of becoming infected by mink, assuming no cat-to-cat transmission. As only feral cats were infected it is most likely that infections in cats were initiated by mink, not by humans. Whether both dogs were infected by mink or humans remains inconclusive. This study presents one of the first reports of interspecies transmission of SARS-CoV-2 that does not involve humans, namely mink-to-cat transmission, which should also be considered as a potential risk for spread of SARS-CoV-2.

Adaptation, spread and transmission of SARS-CoV-2 in farmed minks and associated humans in the Netherlands.

In the first wave of the COVID-19 pandemic (April 2020), SARS-CoV-2 was detected in farmed minks and genomic sequencing was performed on mink farms and farm personnel. Here, we describe the outbreak and use sequence data with Bayesian phylodynamic methods to explore SARS-CoV-2 transmission in minks and humans on farms. High number of farm infections (68/126) in minks and farm workers (>50% of farms) were detected, with limited community spread. Three of five initial introductions of SARS-CoV-2 led to subsequent spread between mink farms until November 2020. Viruses belonging to the largest cluster acquired an amino acid substitution in the receptor binding domain of the Spike protein (position 486), evolved faster and spread longer and more widely. Movement of people and distance between farms were statistically significant predictors of virus dispersal between farms. Our study provides novel insights into SARS-CoV-2 transmission between mink farms and highlights the importance of combining genetic information with epidemiological information when investigating outbreaks at the animal-human interface.

Development of the in vitro Cecal Chicken ALIMEntary tRact mOdel-2 to Study Microbiota Composition and Function.

The digestive system of the chicken plays an important role in metabolism, immunity, and chicken health and production performance. The chicken ceca harbor a diverse microbial community and play a crucial role in the microbial fermentation and production of energy-rich short-chain fatty acids (SCFA). For humans, dogs, and piglets in vitro digestive system models have been developed and are used to study the microbiota composition and metabolism after intervention studies. For chickens, most research on the cecal microbiota has been performed in in vivo experiments or in static in vitro models that may not accurately resemble the in vivo situations. This paper introduces an optimized digestive system model that simulates the conditions in the ceca of the chicken, i.e., the Chicken ALIMEntary tRact mOdel-2 (CALIMERO-2). The system is based on the well-validated TNO in vitro model of the colon-2 (TIM-2) and is the first dynamic in vitro digestion model for chickens species. To validate this model, the pH, temperature, and different types of microbial feeding were compared and analyzed, to best mimic the conditions in the chicken ceca. The bacterial composition, as well as the metabolite production at 72 h, showed no significant difference between the different microbial feedings. Moreover, we compared the CALIMERO-2 digestive samples to the original inoculum and found some significant shifts in bacterial composition after the fermentation started. Over time the bacterial diversity increased and became more similar to the original inoculum. We can conclude that CALIMERO-2 is reproducible and can be used as a digestive system model for the chicken ceca, in which the microbial composition and activity can be maintained and shows similar results to the in vivo cecum. CALIMERO-2 can be used to study effects on composition and activity of the chicken cecum microbiota in response to in-feed interventions.

Genetic and phenotypic analysis of the pathogenic potential of two novel Chlamydia gallinacea strains compared to Chlamydia psittaci.

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and have at least 99.5% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight into the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is a ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs, C. gallinacea induced mortality was observed, potentially strain dependent, but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of potential virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as an opportunistic pathogen than an innocuous commensal.

Coaching Belgian and Dutch Broiler Farmers Aimed at Antimicrobial Stewardship and Disease Prevention.

A reduction in antimicrobial use (AMU) is needed to curb the increase in antimicrobial resistance in broiler production. Improvements in biosecurity can contribute to a lower incidence of disease and thereby lower the need for AMU. However, veterinary advice related to AMU reduction or biosecurity is often not complied with, and this has been linked to the attitudes of farmers. Behavior change promoted by coaching may facilitate uptake and compliance regarding veterinary advice. Thirty broiler farms in Belgium and the Netherlands with high AMU were included in this study for 13 months. For each farmer, the attitude towards AMU reduction was quantified using an adjusted Awareness, Desire, Knowledge, Ability, and Reinforcement (ADKAR®) change management model, and farm biosecurity was assessed with the Biocheck.UGent™ tool. Subsequently, farmers were coached to improve disease prevention and antimicrobial stewardship. After the individual coaching of farmers, there was a change in their attitudes regarding AMU, reflected by an increase in ADKAR® scores. Biosecurity levels improved by around 6% on average, and AMU was reduced by 7% on average without negative effects on performance parameters. Despite these improvements, no significant association could be found between higher ADKAR® scores and lower AMU. Further investigation into sociological models is needed as a tool to reduce AMU in livestock production.

Analysis of chicken intestinal natural killer cells, a major IEL subset during embryonic and early life.

Restrictions on antimicrobials demand alternative strategies to improve broiler health, such as supplying feed additives which stimulate innate immune cells like natural killer (NK) cells. The main objective of this study was to characterize intestinal NK cells in broiler chickens during embryonic and early life and compare these to NK cells in spleen, blood and bone marrow. Also T-cell subsets were determined. The majority of intestinal NK cells expressed IL-2Rα rather than 20E5 and 5C7, and showed low level of activation. Within intestinal NK cells the activation marker CD107 was mostly expressed on IL-2Rα+ cells while in spleen and blood 20E5+ NK cells primarily expressed CD107. High percentages of intestinal CD8αα+, CD8αß+ and from 2 weeks onward also gamma delta T cells were found. Taken together, we observed several intestinal NK subsets in broiler chickens. Differences in NK subsets were mostly observed between organs, rather than differences over time. Targeting these intestinal NK subsets may be a strategy to improve immune-mediated resistance in broiler chickens.

Association of wild bird densities around poultry farms with the risk of highly pathogenic avian influenza virus subtype H5N8 outbreaks in the Netherlands, 2016.

Highly pathogenic (HP) avian influenza viruses (AIV) can spread globally through migratory birds and cause massive outbreaks in commercial poultry. AIV outbreaks have been associated with proximity to waterbodies, presence of waterfowl or wild bird cases near poultry farms. In this study, we compared densities of selected HPAI high-risk wild bird species around 7 locations (H farms) infected with HPAIV H5N8 in the Netherlands in 2016-2017 to densities around 21 non-infected reference farms. Nine reference farms were in low-lying water-rich areas (R-W) and 12 in higher non-water-rich areas (R-NW). Average monthly numbers/km2 of Eurasian wigeons, tufted ducks, Anatidae (ducks, geese and swans) and Laridae (gulls) were calculated between September and April in rings of 0-1, 1-3, 3-6 and 6-10 km around the farms. Linear mixed model analyses showed generally higher bird densities for H and R-W compared to R-NW farms between October and March. This was most striking for Eurasian wigeons, with in peak month December 105 (95% CI:17-642) and 40 (7-214) times higher densities around H and R-W farms, respectively, compared to R-NW farms. Increased densities around H farms for Eurasian wigeons and Anatidae were more pronounced for distances up to 10 km compared to 0-1 km that mostly consists of the farm yard, which is an unattractive habitat for waterfowl. This distance effect was not observed in gulls, nor in tufted ducks that live on large open waterbodies which are unlikely to be within 0-1 km of farms. This study provides insights into spatio-temporal density dynamics of HPAI high-risk birds around farms and their associations with poultry outbreaks. The outcomes indicate that knowledge of environmental and ecological drivers for wild bird presence and abundance may facilitate identification of priority areas for surveillance and biosecurity measures and decisions on establishments of poultry farms to reduce risk of HPAI outbreaks.

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.

Comparing Farm Biosecurity and Antimicrobial Use in High-Antimicrobial-Consuming Broiler and Pig Farms in the Belgian-Dutch Border Region.

As antimicrobial resistance is a worldwide problem, threatening both livestock and public health, understanding the drivers for resistance in different settings and countries is essential. Therefore, 30 pig and 30 poultry farms with country-specific high antimicrobial use (AMU) were recruited in the Belgian-Dutch border region. Information regarding production parameters, farm characteristics, biosecurity, and AMU was collected. On average, more biosecurity measures were implemented on Dutch farms, compared to Belgian farms in both animal species. In addition, more opportunities were found to increase the level of internal biosecurity compared to external biosecurity in both countries. AMU, quantified as treatment incidence (TI), differed marginally significant between broiler farms in Belgium and the Netherlands (median BE: 8; NL: 3), whereas in weaned piglets (median BE: 45 and NL: 14) and finishing pigs (median BE: 5 and NL: 1), there was a substantial difference in AMU between farms from both countries. Overall, Dutch farms showed less between-farm variation in TI than did Belgian farms. In both poultry and pig production, the majority of antimicrobials used were extended-spectrum penicillins (BE: 32 and 40%; NL: 40 and 24% for poultry and pigs, respectively). Compared to Belgian farms, Dutch poultry farms used high amounts of (fluoro)quinolones (1 and 15% of total AMU, respectively). None of the production parameters between broiler farms differed significantly, but in pig production, weaning age in Belgian farms (median: 23) was lower than in Dutch farms (median: 27). These results indicate considerable room for improvement in both countries and animal species. Farm-specific preventive strategies can contribute to lowering the risk for animal disease and hence the need for AMU.

Early Life Inoculation With Adult-Derived Microbiota Accelerates Maturation of Intestinal Microbiota and Enhances NK Cell Activation in Broiler Chickens.

Studies in mammals, including chickens, have shown that the development of the immune system is affected by interactions with intestinal microbiota. Early life microbial colonization may affect the development of innate and adaptive immunity and may contribute to lasting effects on health and resilience of broiler chickens. We inoculated broiler chickens with adult-derived-microbiota (AM) to investigate their effects on intestinal microbiota composition and natural killer (NK) cells, amongst other immune cells. We hypothesized that AM inoculation directly upon hatch (day 0) would induce an alteration in microbiota composition shortly after hatch, and subsequently affect (subsets of) intestinal NK cells and their activation. Microbiota composition of caecal and ileal content of chickens of 1, 3, 7, 14, 21, and 35 days of age was assessed by sequencing of 16S ribosomal RNA gene amplicons. In parallel, subsets and activation of intestinal NK cells were analyzed by flow cytometry. In caecal content of 1- and 3-day-old AM chickens, a higher alpha-diversity (Faith's phylogenetic diversity) was observed compared to control chickens, whereas ileal microbiota were unaffected. Regarding beta-diversity, caecal microbiota profiles could be clustered into three distinct community types. Cluster A represented caecal microbiota of 1-day-old AM chickens and 1- and 3-day-old control chickens. Cluster B included microbiota of seven of eight 3- and 7-day-old AM and 7-day-old control chickens, and cluster C comprised microbiota of all chickens of 14-days and older, independent of inoculation. In 3-day-old AM chickens an increase in the percentages of intestinal IL-2Rα+NK cells and activated NK cells was observed compared to control chickens of the same age. In addition, an increase in relative numbers of intestinal cytotoxic CD8αα+T cells was observed in 14- and 21-day-old AM chickens. Taken together, these results indicate that early exposure to AM shapes and accelerates the maturation of caecal microbiota, which is paralleled by an increase in IL-2Rα+NK cells and enhanced NK cell activation. The observed association between early life development of intestinal microbiota and immune system indicates possibilities to apply microbiota-targeted strategies that can accelerate maturation of intestinal microbiota and strengthen the immune system, thereby improving the health and resilience of broiler chickens.

Competitive Exclusion Prevents Colonization and Compartmentalization Reduces Transmission of ESBL-Producing Escherichia coli in Broilers.

Extended spectrum beta-lactamase (ESBL)-producing bacteria are resistant to extended-spectrum cephalosporins and are common in broilers. Interventions are needed to reduce the prevalence of ESBL-producing bacteria in the broiler production pyramid. This study investigated two different interventions. The effect of a prolonged supply of competitive exclusion (CE) product and compartmentalization on colonization and transmission, after challenge with a low dose of ESBL-producing Escherichia coli, in broilers kept under semi-field conditions, were examined. One-day-old broilers (Ross 308) (n = 400) were housed in four experimental rooms, subdivided in one seeder (S/C1)-pen and eight contact (C2)-pens. In two rooms, CE product was supplied from day 0 to 7. At day 5, seeder-broilers were inoculated with E. coli strain carrying bla CTX-M- 1 on plasmid IncI1 (CTX-M-1-E. coli). Presence of CTX-M-1-E. coli was determined using cloacal swabs (day 5-21 daily) and cecal samples (day 21). Time until colonization and cecal excretion (log10 CFU/g) were analyzed using survival analysis and linear regression. Transmission coefficients within and between pens were estimated using maximum likelihood. The microbiota composition was assessed by 16S ribosomal RNA gene amplicon sequencing in cecal content of broilers on days 5 and 21. None of the CE broilers was CTX-M-1-E. coli positive. In contrast, in the untreated rooms 187/200 of the broilers were CTX-M-1-E. coli positive at day 21. Broilers in C2-pens were colonized later than seeder-broilers (Time to event Ratio 3.53, 95% CI 3.14 to 3.93). The transmission coefficient between pens was lower than within pens (3.28 × 10-4 day-2, 95% CI 2.41 × 10-4 to 4.32 × 10-4 vs. 6.12 × 10-2 day-2, 95% CI 4.78 × 10-2 to 7.64 × 10-2). The alpha diversity of the cecal microbiota content was higher in CE broilers than in control broilers at days 5 and 21. The supply of a CE product from day 0 to 7 prevented colonization of CTX-M-1-E. coli after challenge at day 5, likely as a result of CE induced effects on the microbiota composition. Furthermore, compartmentalization reduced transmission rate between broilers. Therefore, a combination of compartmentalization and supply of a CE product may be a useful intervention to reduce transmission and prevent colonization of ESBL/pAmpC-producing bacteria in the broiler production pyramid.