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The emergence of carbapenemase-producingEnterobacteriaceae(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-resistantE. 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 5·102colony-forming units of CPE, ESBL, or chloramphenicol-resistantE. 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 theE. colistrains.E. colican 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 theE. colistrains. 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.

Broilers are an important reservoir of extended spectrum beta-lactamase and AmpC beta-lactamase (ESBL/pAmpC)-producing bacteria. In previous studies, a single supply of a competitive exclusion (CE) product before challenge with a high dose of ESBL/pAmpC-producing Escherichia coli led to reduced colonization, excretion, and transmission, but could not prevent colonization. The hypothesized mechanism is competition; therefore, in this study the effect of a prolonged supply of CE products on colonization, excretion, and transmission of ESBL-producing E. coli after challenge with a low dose at day 0 or day 5 was investigated. Day-old broilers (Ross 308) (n = 220) were housed in isolators. Two CE products, containing unselected fermented intestinal bacteria (CEP) or a selection of pre- and probiotics (SYN), were supplied in drinking water from day 0 to 14. At day 0 or 5, broilers were challenged with 0.5 mL with 101 or 102 cfu/mL E. coli encoding the beta-lactamase gene blaCTX-M-1 on an IncI plasmid (CTX-M-1-E. coli). Presence and concentration of CTX-M-1-E. coli were determined using cloacal swabs (days 0–14, 16, 19, and 21) and cecal content (day 21). Cox proportional hazard model and a mixed linear regression model were used to determine the effect of the intervention on colonization and excretion (log10 cfu/g). When challenged on the day of hatch, no effect of CEP was observed. When challenged at day 5, both CEP and SYN led to a prevention of colonization with CTX-M-1-E. coli in some isolators. In the remaining isolators, we observed reduced time until colonization (hazard ratio between 3.71 × 10−3 and 3.11), excretion (up to −1.60 log10 cfu/g), and cecal content (up to −2.80 log10 cfu/g), and a 1.5 to 3-fold reduction in transmission rate. Colonization after a low-dose challenge with ESBL-producing E. coli can be prevented by CE products. However, if at least 1 bird is colonized it spreads through the whole flock. Prolonged supply of CE products, provided shortly after hatch, may be applicable as an intervention to reduce the prevalence of ESBL/pAmpC-producing bacteria in the broiler production chain.

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 related humans on farms. High number of farm infections (68/126) in minks and farm related personnel (>50% of farms) were detected, with limited spread to the general human population. Three of five initial introductions of SARS-CoV-2 lead to subsequent spread between mink farms until November 2020. The largest cluster acquired a mutation in the receptor binding domain of the Spike protein (position 486), evolved faster and spread more widely and longer. 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 combing genetic information with epidemiological information at the animal-human interface.

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.

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 showed 99.1% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight in the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is an 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 a (conditional) pathogen than an innocuous commensal.ImportanceChlamydiaceae are a family of bacteria comprising human and animal pathogens including the recently recognized Chlamydia gallinacea. C. gallinacea is widespread in poultry without causing clinical signs, which raises questions about its pathogenic potential. To assess this potential, two novel C. gallinacea strains were isolated, tested in infection experiments in embryonated chicken eggs and compared to C. psittaci. C. psittaci infection in poultry can result in severe systemic illness, depending on the conditions, and infections can be transmitted to humans. In the experiments C. gallinacea infection induced mortality of the embryo, but to a lower extent than infection with C. psittaci. Subsequent genome comparisons confirmed both C. gallinacea strains possess potential virulence genes typical for chlamydia, but fewer than C. psittaci. These results indicate C. gallinacea does have a pathogenic potential which warrants further research to elucidate its role as a poultry pathogen.

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.

The zoonotic origin of the SARS-CoV-2 pandemic is still unknown. Animal experiments have shown that non-human primates, cats, ferrets, hamsters, rabbits and bats can be infected by 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 of outbreaks on 16 mink farms and humans living or working on these farms, using whole genome sequencing. We conclude that the virus was initially introduced from humans and has evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period several weeks prior to detection. At the moment, despite enhanced biosecurity, early warning surveillance and immediate culling of infected farms, there is ongoing transmission between mink farms with three big transmission clusters with unknown modes of transmission. We also describe the first animal to human transmissions of SARS-CoV-2 in mink farms.One sentence summarySARS-CoV-2 transmission on mink farms.

Disturbances in intestinal health are a common problem affecting commercial broiler chickens worldwide. Several studies have revealed associations between health, production performance, and intestinal microbiota. This study aimed to describe the development of the intestinal microbiota of broilers within a production cycle to evaluate to what extent clinical parameters and phenotypic characteristics can explain the intestinal microbiota variation. Of four well‐performing flocks within two farms, the cecal content was collected of nine broilers at 0, 2, 4, or 5, 7, 11, or 12, 14, 21, 28, 35, and 40 days of the production cycle. In total, 342 samples were analyzed using 16S ribosomal RNA gene amplicon sequencing. Variables as macroscopic gut abnormalities, gut lesions, age, individual body weight, sex, footpad integrity, the color of ceca, and foam in cecal content were determined. Ileum tissue was collected for histological quantification of villus length and crypt depth. Flock infection levels of the intestinal disease coccidiosis were measured in pooled feces from the poultry house. Increases in phylogenetic diversity were observed from hatch until day 21 of age. Constrained multivariate analysis indicated that age, farm, body weight, ileum crypt depth, cecal color, and the coccidiosis lesion score were important variables to describe the variation in cecal microbiota. These results contribute to determining relevant variables in flocks that may be indicative of the intestinal microbiota composition. Moreover, this knowledge increases the awareness of interactions between the intestinal microbiota and broiler health as well as their relative importance., The microbiota composition in the ceca of broiler chickens developed strongly in the first week of life, but the phylogenetic diversity stabilized around 21 days of age. The variation in cecal microbiota composition in well‐performing broiler flocks under field conditions was mostly explained by age, farm, broiler body weight, ileum crypt depth, the color of the cecal content, and coccidiosis lesion scores. This study contributes to the understanding of the development and the interaction between intestinal microbiota and broiler health.

Live vaccines for coccidiosis control are infrequently used in broilers, mainly due to variability in efficacy and relatively high costs. More insight in transmission of vaccine and wild-type strains can facilitate optimization of vaccination strategies and might increase its use as an alternative for anticoccidial drugs. The aim of this study was to quantify transmission of a live Eimeria acervulina vaccine strain and to determine the degree of protection against a subsequent infection with a wild-type E. acervulina strain. An experiment was carried out with 4 groups of 22 SPF broilers. At 2 days of age, 11 birds of groups 2 to 4 were vaccinated directly by oral application of E. acervulina oocysts of the Paracox (TM) vaccine and 11 birds were placed in contact with these birds (contact-vaccinated). Birds in group 1 remained unvaccinated (controls) and were not exposed to vaccinated birds. At day 28 of age, 6 groups of 10 birds were formed, with 2 groups (duplo) for each treatment group, i.e. vaccinated, contact-vaccinated or unvaccinated control birds. Five birds of each group were orally inoculated with wild-type E. acervulina oocysts and five were contact-exposed. Single droppings were examined daily from days 5 to 49 of age for oocyst output and to determine the time of infection. The transmission rate of the vaccine strain was estimated to be 1.6 per day and of the wild-type strain 2.3, 8.7 and 20.8 per day for vaccinated, contact-vaccinated and unvaccinated birds, respectively. Although transmission of wild-type coccidia was not significantly reduced in vaccinated or contact-vaccinated groups, both groups were equally protected against high oocyst output after infection compared to unvaccinated groups. These results suggest that factors influencing transmission of live vaccine strains in flocks may be important targets for improvement of vaccine efficacy and warrant further research.

The infection dynamics of Eimeria species determine the clinical manifestation of the disease coccidiosis in poultry flocks, and a better understanding of the dynamics may contribute to improvement of control measures. Our aim was to study the course of infection and the transmission of Eimeria acervulina in groups of broilers by quantifying the transmission rate parameter and oocyst output. Three transmission experiments were carried out with groups of 20 male SPF broilers. At 2 days of age, one bird in each trial was orally inoculated with five sporulated E. acervulina oocysts (DO post-inoculation, pi). One day after inoculation (D1 pi), the inoculated bird was housed with 19 non-inoculated contact birds. Individual faecal droppings were examined daily from D3-D32 pi to quantify the number of oocysts per gram faeces. The inoculated bird started shedding oocysts at D5 pi and contact birds between D10 and D17 pi. Contact birds that became infected due to oocyst excretion by the inoculated bird were characterized as first generation contact birds (C1). Contact birds excreting from D15 pi onwards (C2) became infected after the first Cl birds had started shedding and were considered to belong to a successive generation of the flock infection. Oocyst output was significantly lower for C1 compared to C2 birds, but the transmission rate parameter remained constant for both infection generations. These results suggest that although oocyst load increases, the transmission rate of E. acervulina remains constant between successive generations of infection in a flock.

This case report describes the course of an outbreak of avian influenza on a Dutch turkey farm. When clinical signs were observed their cause remained unclear. However, serum samples taken for the monitoring campaign launched during the epidemic of highly pathogenic avian influenza in 2003, showed that all the remaining turkeys were seropositive against an h7 strain of avian influenza virus, and the virus was subsequently isolated from stored carcases. The results of a reverse-transcriptase pcr showed that a h7n3 strain was involved, and it was characterised as of low pathogenicity. However, its intravenous pathogenicity index was 2·4, characterising it as of high pathogenicity, suggesting that a mixture of strains of low and high pathogenicity may have been present in the isolate. The outbreak remained limited to three farms.

This study sought to determine the efficacy of isopathic and pluralist homeopathic treatment of colibacillosis in broiler chickens and thereby contribute to the evaluation of homeopathy in general. In each of two experiments three groups of broilers, infected intratracheally at 8 days of age with E. coli (O78:K80), were treated with different combinations of homeopathic remedies. Control groups and an infected, doxycyline-treated group were included. Experiments differed only in the dose of E. coli. Efficacy of treatment was evaluated based on the parameters mortality, body weight gain and colibacillosis lesions. In both experiments doxycyline prevented mortality and reduced E. coli lesions and stunting. None of the homeopathically treated groups differed significantly with respect to any of the parameters from the non-medicated, infected control group. It is concluded that the results of this study do not justify use of these homeopathic remedies for treatment of colibacillosis in broilers. Furthermore, no significant effects of this homeopathic treatment were established.