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.</p

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

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+, CD8αβ+ 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.SUPPLEMENTARY MATERIAL : Table S1. Characterization of immune cells generated in broiler chickens in the present study compared to data known in layer chickens.The Dutch Research Council (NWO) and by Cargill Animal Nutrition and Health.http://www.elsevier.com/locate/desalhj2022Veterinary Tropical Disease

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

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

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

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 5·10 2 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

Comparing the transmission of carbapenemase-producing and extended-spectrum beta-lactamase-producingEscherichia colibetween broiler chickens

Abstract 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 similar 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 on the day of hatch (day 0) over 12 pens. On day 5, half of the chickens in each pen were orally inoculated with 5·102 colony-forming units of CPE, ESBL, or chloramphenicol-resistant E. coli (catA1). Amoxicillin drinking water treatment was given twice daily in 6 of the 12 pens from days 2 to 6 to evaluate the effect of antibiotic treatment on the transmission rates. 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 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 to 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

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

Effectiveness of Passive and Active Surveillance for Early Detection of SARS-CoV-2 in Mink during the 2020 Outbreak in the Netherlands

Starting December 2019, a novel coronavirus (SARS-CoV-2) spread among humans across the world. From 2020 onward, farmed mink were found susceptible to the virus. In this paper, we describe the Dutch surveillance system and the added surveillance components for early detection of SARS-CoV-2 outbreaks and their results in Dutch mink farms. In the Netherlands, a surveillance system was in place in which mink farmers could submit carcasses for postmortem evaluation and could contact a telephone helpdesk for veterinary advise. Through this system, the first SARS-CoV-2 outbreak in two mink farms was detected in April 2020. Immediately, the Dutch Ministry of Agriculture commissioned a consortium of statutory and research institutes to intensify the surveillance system. The program consisted of both passive surveillance, i.e., mandatory notifications and active surveillance components, i.e., serological screenings and weekly risk-based sampling of dead mink for early detection of new SARS-CoV-2 infections. When one of the surveillance components indicated a suspicion of a possible SARS-CoV-2 infection, follow-up samplings were conducted and at confirmation, all mink were culled. During 2020, 67 out of 124 mink farms that were under surveillance became infected with SARS-CoV-2 (54%). Of these, 31 were detected based on clinical signs (passive surveillance of clinical signs) and 36 were detected through active surveillance. From the mink farms with a new SARS-CoV-2 outbreak that was detected through the surveillance, in 19% of the farms (n = 7), the mink never showed any clinical signs of SARS-CoV-2 and might have been missed by the passive notification system. This study underlines the added value of a surveillance system that can quickly be intensified. The subsequent combination of both passive and active surveillance has shown to be effective in the early detection of emerging pathogens, which is important to minimize the risk of zoonotic spill-over