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Abstract Background An uneven spatial distribution of leprosy can be caused by the influence of geography on the distribution of risk factors over the area, or by population characteristics that are heterogeneously distributed over the area. We studied the distribution of leprosy cases detected by a control program to identify spatial and spatio-temporal patterns of occurrence and to search for environmental risk factors for leprosy. Methods The houses of 11,060 leprosy cases registered in the control area during a 15-year period (1989–2003) were traced back, added to a geographic database (GIS), and plotted on digital maps. We looked for clusters of cases in space and time. Furthermore, relationships with the proximity to geographic features, such as town center, roads, rivers, and clinics, were studied. Results Several spatio-temporal clusters were observed for voluntarily reported cases. The cases within and outside clusters did not differ in age at detection, percentage with multibacillary leprosy, or sex ratio. There was no indication of the spread from one point to other parts of the district, indicating a spatially stable endemic situation during the study period. The overall risk of leprosy in the district was not associated with roads, rivers, and leprosy clinics. The risk was highest within 1 kilometer of town centers and decreased with distance from town centers. Conclusion The association of a risk of leprosy with the proximity to towns indicates that rural towns may play an important role in the epidemiology of leprosy in this district. Further research on the role of towns, particularly in rural areas, is warranted.

Abstract Background There is a higher case-detection rate for leprosy among spatially proximate contacts such as household members and neighbors. Spatial information regarding the clustering of leprosy can be used to improve intervention strategies. Identifying high-risk areas within villages around known cases can be helpful in finding new cases. Methods Using geographic information systems, we created digital maps of four villages in a highly endemic area in northwest Bangladesh. The villages were surveyed three times over four years. The spatial pattern of the compounds – a small group of houses – was analyzed, and we looked for spatial clusters of leprosy cases. Results The four villages had a total population of 4,123. There were 14 previously treated patients and we identified 19 new leprosy patients during the observation period. However, we found no spatial clusters with a probability significantly different from the null hypothesis of random occurrence. Conclusion Spatial analysis at the microlevel of villages in highly endemic areas does not appear to be useful for identifying clusters of patients. The search for clustering should be extended to a higher aggregation level, such as the subdistrict or regional level. Additionally, in highly endemic areas, it appears to be more effective to target complete villages for contact tracing, rather than narrowly defined contact groups such as households.

Effective control of infectious bronchitis is a challenge in commercial poultry operations due to the high transmissibility of the virus. Although multiple IBV lineages are circulating in the United States, the DMV1639-type IBV strain (GI-17) is currently the major circulating variant, creating production losses in the poultry industry. This study aimed to test whether the combination of a GA08 (GI-27) and a Mass-type (GI-1) IB vaccines could significantly reduce the transmission of a DMV1639-type (GI-17) field IBV strain in 4-week-old commercial broilers. Half of the birds were directly challenged, whereas the other half of the groupmates were put in contact 24 hours later. Two replicates of the same study setup, including 10 directly challenged and 10 contact birds per group, were run. Transmission of the challenge virus was significantly reduced in vaccinates ( R  = 0.0), whereas all unvaccinated birds became infected ( R  = 9.6). Reduced transmission of the DMV1639 IB challenge virus by the combined vaccination programme in broiler chickens was also accompanied by clinical protection. These data are important because prevention of IBV transmission by vaccination will result in overall reduced viral replication and consequently in reduced likelihood of genetic changes that can lead to new variants. This is the first published evidence of the successful transmission control of a DMV1639 IBV strain in chickens.

Information on the epidemiological and economic consequences of control measures is fundamental to design effective foot and mouth disease (FMD) control measures. One approach to obtaining this information is through bioeconomic modelling. In this study, a bioeconomic model was used to evaluate FMD control in two different study areas in Thailand: a high farm density area predominantly consisting of dairy farms and a low farm density area with mixed farm types. The bioeconomic model consists of an epidemiological part and an economic part. For the epidemiological part, a stochastic between-farm transmission model was constructed with transmission parameters estimated from FMD outbreaks in Thailand. The outputs from the epidemiological model, i.e. the number of infected farms, the number of affected farms and the outbreak duration, are used as inputs for economic model to calculate the economic consequences. We applied the simulation model with four FMD control measures: culling the animals of infected farms, ring vaccination, animal movement restrictions and isolation of infected farms. Furthermore, we included effect of farmers' compliance to asses its effect on control measures. The simulated FMD outbreaks in the low farm density area were small, thus control measures did not greatly affect the size of outbreaks and, therefore, did not have a positive economic return. In contrast, in the high farm density area, FMD outbreaks were large without control measures. All measures reduced the size of the outbreaks but resulted in different total costs. In terms of outbreak control, culling infectious farms was the best option, but its total cost was higher than ring vaccination or isolation of infected farms. In terms of cost-effectiveness, ring vaccination was the best measure. If farmers' compliance were low, all control measures would be ineffective, resulting in high total cost of the outbreak. The cost distribution between compliant and non-compliant farms showed that non-compliant farms paid more than compliant farms, except for the ring vaccination scenario. The results emphasize the economic significance to customize control measures specific to the area's conditions and highlight the importance of farmers' compliance when designing control measures., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Simulating resistant bacteria transmission in livestock informs surveillance strategies for emerging threats like Carbapenem-resistant Enterobacteriaceae (CPE), aiding targeted surveillance and detecting CPE through active methods. We employed a simulation model to assess three potential scenarios for introducing CPE: 1) a single import of live animals, 2) the use of contaminated feed, and 3) multiple imports of live animals. Employing the SimInf package, we constructed a population model for broiler production, encompassing rearing farms, multiplier farms, hatcheries, and broiler farms. Subsequently, we introduced CPE and allowed it to spread throughout the population using the Susceptible-Colonized (Infectious)-Susceptible model. The model ran for 10 years with 100 runs. In the single import scenario, 1-2 rearing and multiplier farms saw major outbreaks in all 100 runs, while the broiler farm experienced major outbreaks in only 10 out of 100 runs; in the feed scenario, major outbreaks occurred in rearing farms in 32 runs and in multiplier farms in 26 runs, with major outbreaks in broiler farms observed in all 100 runs; in the multiple import scenario, outbreaks in rearing and multiplier farms happened in all 100 runs, with these major outbreaks reaching the broiler farm in 91 out of 100 runs. CPE transmission from imported or colonized broilers is rapid but short-lived within the broiler population, contrasting with the sporadic and prolonged emergence of CPE from contaminated feed, resulting in lower cumulative probabilities of detection from imported or colonized animals (0-0.50) compared to contaminated feed (0.9-0.97) over a 10-year period. Sensitivity analysis indicated that key outcomes such as farm outbreaks, chicken colonization, and outbreak duration are highly correlated with age-associated reductions in transmission (ψ)., Competing Interests: None., (© 2024 The Authors. Published by Elsevier B.V.)

Fluoroquinolone resistance in E. coli isolates from livestock in Europe remains high despite EMA restrictions on fluoroquinolone use in animals. However, flumequine, a quinolone not classified as a fluoroquinolone by various regulatory bodies, is still used in livestock in the Netherlands, Belgium, Greece and France. We investigated whether flumequine selects for the same resistance mechanisms in E. coli. Resistant and non-resistant E. coli isolates were obtained from caecal fermentation assays and broilers exposed to concentrations of flumequine and enrofloxacin. Flumequine usage leads to an approximately 3-fold increase in resistant E. coli in the caecal fermentation, similar to enrofloxacin. In vitro exposure to both flumequine and enrofloxacin revealed the same amino acid substitutions (S83L, D87G) in GyrA. Additionally, the same resistance-causing substitutions were found in phenotypically resistant E. coli isolates from broilers treated with either enrofloxacin or flumequine. Flumequine induces similar resistance mechanisms as enrofloxacin, warranting equivalent restrictions on its use., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Antimicrobial resistance (AMR) is an emerging worldwide problem and a health threat for humans and animals. Antimicrobial usage in human and animal medicine or in agriculture results in selection for AMR. The selective concentration of antimicrobial compounds can be lower than the minimum inhibitory concentration and differs between environments, which can be a reason for bacterial resistance. Therefore, knowledge of the minimal selective concentration (MSC), under natural conditions, is essential to understand the selective window of bacteria when exposed to residual antimicrobials. In this study, we estimated the MSCs of three antimicrobials, amoxicillin, doxycycline, and enrofloxacin in a complex microbial community by conducting fermentation assays with cecal material derived from broilers. We examined the phenotypic resistance of Escherichia coli , resistome, and microbiome after 6 and 30 hours of fermenting in the presence of the antimicrobials of interest. The concentrations were estimated to be 10-100 times lower than the epidemiological cut-off values in E. coli for the respective antimicrobials as determined by EUCAST, resulting in an MSC between 0.08 and 0.8 mg/L for amoxicillin, 0.4 and 4 mg/L for doxycycline, and 0.0125 and 0.125 mg/L for enrofloxacin. Additionally, resistome analysis provided an MSC for doxycycline between 0.4 and 4 mg/L, but amoxicillin and enrofloxacin exposure did not induce a significant difference. Our findings indicate at which concentrations there is still selection for antimicrobial-resistant bacteria. This knowledge can be used to manage the risk of the emergence of antimicrobial-resistant bacteria.IMPORTANCEAntimicrobial resistance possibly affects human and animal health, as well as economic prosperity in the future. The rise of antimicrobial-resistant bacteria is a consequence of using antimicrobial compounds in humans and animals selecting for antimicrobial-resistant bacteria. Concentrations reached during treatment are known to be selective for resistant bacteria. However, at which concentrations residues are still selective is important, especially for antimicrobial compounds that remain in the environment at low concentrations. The data in this paper might inform decisions regarding guidelines and regulations for the use of specific antimicrobials. In this study, we are providing these minimal selective concentrations for amoxicillin, doxycycline, and enrofloxacin in complex environments., Competing Interests: The authors declare no conflict of interest.

Objectives: Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens., Methods: The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism., Results: Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations., Conclusions: Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The transmission rate per hour between hosts is a key parameter for simulating transmission dynamics of antibiotic-resistant bacteria, and might differ for antibiotic resistance genes, animal species, and antibiotic usage. We conducted a Bayesian meta-analysis of resistant Escherichia coli (E. coli) transmission in broilers and piglets to obtain insight in factors determining the transmission rate, infectious period, and reproduction ratio. We included bla CTX-M-1, bla CTX-M-2, bla OXA-162, catA1, mcr-1, and fluoroquinolone resistant E. coli. The Maximum a Posteriori (MAP) transmission rate in broilers without antibiotic treatment ranged from 0.4∙10 -3 to 2.5∙10 -3 depending on type of broiler (SPF vs conventional) and inoculation strains. For piglets, the MAP in groups without antibiotic treatment were between 0.7∙10 -3 and 0.8∙10 -3 , increasing to 0.9∙10 -3 in the group with antibiotic treatment. In groups without antibiotic treatment, the transmission rate of resistant E. coli in broilers was almost twice the transmission rate in piglets. Amoxicillin increased the transmission rate of E. coli carrying bla CTX-M-2 by three-fold. The MAP infectious period of resistant E. coli in piglets with and without antibiotics is between 971 and 1065 hours (40 - 43 days). The MAP infectious period of resistant E. coli in broiler without antibiotics is between 475 and 2306 hours (20 - 96 days). The MAP infectious period of resistant E. coli in broiler with antibiotics is between 2702 and 3462 hours (113 - 144 days) which means a lifelong colonization. The MAP basic reproduction ratio in piglets of infection with resistant E. coli when using antibiotics is 27.70, which is higher than MAP in piglets without antibiotics between 15.65 and 18.19. The MAP basic reproduction ratio in broilers ranges between 3.46 and 92.38. We consider three possible explanations for our finding that in the absence of antibiotics the transmission rate is higher among broilers than among piglets: i) due to the gut microbiome of animals, ii) fitness costs of bacteria, and iii) differences in experimental set-up between the studies. Regarding infectious period and reproduction ratio, the effect of the resistance gene, antibiotic treatment, and animal species are inconclusive due to limited data., Competing Interests: Declaration of Competing Interest None, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) in animals are considered a human health threat, because this type of bacteria can serve as a reservoir of antibiotic resistant genes and act as a continuous threat of the emergence of new resistant bacteria, in addition to the direct effect of making infection untreatable. Although the prevalence of ESBL producing bacteria in broilers was drastically reduced in the Netherlands, chicken meat still has the highest prevalence among meat products. Therefore, further control of the ESBL-producing E. coli in the broiler production chain is important to reduce public health risks. The main objectives of this study were to evaluate the effectiveness of intervention scenarios to reduce the transmission of ESBL-producing E. coli in the broiler production chain and to quantitatively estimate the risk to public health. In this study, we developed two different types of transmission models that described the observed time-related decline in prevalence during a production round: one with time-dependent decline in susceptibility and one with partial immunity to phylogenetic groups. Both models incorporated the environmental contamination effect between production rounds and within flocks. The parameter values, including transmission rate and recovery rate, were estimated by Approximate Bayesian computation (ABC) method using data from a longitudinal study in a Dutch organic broiler farm. We applied the models to the three production stages in the broiler production chain, beginning from the Parent Stock (PS) farms, the hatcheries, and to the broiler farms. In our models, eggs were collected from different parent stock farms and transported to the hatchery and from there to a broiler farm.The size of a flock and the number of farms were adjusted to the Dutch situation. Both models were able to describe the observed dynamics within and between the production stages equally well, with estimated ESBL-producing E. coli prevalence of 8.98% and 11.47% in broilers at slaughter and 0.12% and 0.15% in humans due to chicken consumption. Both models indicated that improving farm management to eliminate the bacteria from the environment was the most effective intervention, making this outcome robust. Although chicken meat consumption is not a major risk factor for human carriage of the bacteria according to our models, reducing the bacteria in the PS and broiler farm environment to at least one percent can further decrease the prevalence in humans., Competing Interests: Declaration of Competing Interest none., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Knowledge of who infected whom during an outbreak of an infectious disease is important to determine risk factors for transmission and to design effective control measures. Both whole-genome sequencing of pathogens and epidemiological data provide useful information about the transmission events and underlying processes. Existing models to infer transmission trees usually assume that the pathogen is introduced only once from outside into the population of interest. However, this is not always true. For instance, SARS-CoV-2 is suggested to be introduced multiple times in mink farms in the Netherlands from the SARS-CoV-2 pandemic among humans. Here, we developed a Bayesian inference method combining whole-genome sequencing data and epidemiological data, allowing for multiple introductions of the pathogen in the population. Our method does not a priori split the outbreak into multiple phylogenetic clusters, nor does it break the dependency between the processes of mutation, within-host dynamics, transmission, and observation. We implemented our method as an additional feature in the R-package phybreak. On simulated data, our method correctly identifies the number of introductions, with an accuracy depending on the proportion of all observed cases that are introductions. Moreover, when a single introduction was simulated, our method produced similar estimates of parameters and transmission trees as the existing package. When applied to data from a SARS-CoV-2 outbreak in Dutch mink farms, the method provides strong evidence for independent introductions of the pathogen at 13 farms, infecting a total of 63 farms. Using the new feature of the phybreak package, transmission routes of a more complex class of infectious disease outbreaks can be inferred which will aid infection control in future outbreaks., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Van der Roest et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Foot and mouth disease (FMD) is an important livestock disease in Thailand, with outbreaks occurring every year. However, the effects of FMD control measures in Thailand have received little research attention. Epidemiological models have been widely used to evaluate FMD outbreak control, but such a model has never been developed for Thailand. We constructed a stochastic between-farm transmission model to evaluate FMD control measures. The epidemiological unit of the model was the farm, which could be in different states: susceptible, latent, undetected infectious, detected infectious and recovered. The between-farm transmission was calculated by the sum of distance-dependent transmission and trade network transmission using parameters derived from FMD outbreaks in 2016-2017. We used this model to simulate the outbreaks with and without the implementation of the following control measures: culling all animals on infected farms, ring vaccination, animal movement restrictions and isolation of infected farms. The control measures were evaluated by estimating the number of secondarily infected farms and the outbreak duration for each scenario. The model was simulated in two study areas located in the Lamphaya Klang subdistrict (high farm density) and the Bo Phloi district (low farm density). The effects of control measures differed between the two study areas. When farm density was high, rigid control measures were required to prevent a major outbreak. Among all options, culling the animals on infected farms resulted in the lowest number of infected farms and the shortest outbreak duration. In contrast, for an area with a low farm density, less stringent control measures were sufficient to control the usually minor outbreaks. The results indicate that different areas require a different approach to control an outbreak of FMD., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: SARS-CoV-2 is known to jump across species. The occurrence of transmission in households between humans and companion animals has been shown, but the contribution of companion animals to the overall transmission within a household is unknown. The basic reproduction number ( R 0 ) is an important indicator to quantify transmission. For a pathogen with multiple host species, such as SARS-CoV-2, the basic reproduction number needs to be calculated from the partial reproduction numbers for each combination of host species., Method: In this study, the basic and partial reproduction numbers for SARS-CoV-2 were estimated by reanalyzing a survey of Dutch households with dogs and cats and minimally one SARS-CoV-2-infected human., Results: For households with cats, a clear correlation between the number of cats and the basic reproduction number (Spearman's correlation: p 0.40, p -value: 1.4 × 10 -5 ) was identified, while for dogs, the correlation was smaller and not significant (Spearman's correlation: p 0.12, p -value: 0.21). Partial reproduction numbers from cats or dogs to humans were 0.3 (0.0-2.0) and 0.3 (0.0-3.5) and from humans to cats or dogs were 0.6 (0.4-0.8) and 0.6 (0.4-0.9)., Discussion: Thus, the estimations of within-household transmission indicated the likelihood of transmission from these companion animals to humans and vice versa, but the observational nature of this study limited the ability to establish conclusive evidence. This study's findings support the advice provided during the pandemic to COVID-19 patients to maintain distance from companion animals as a precautionary measure and given the possibility of transmission, although there is an overall relatively limited impact on the pandemic when compared to human-to-human transmission., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fischer, Broens, Kooistra, De Rooij, Stegeman and De Jong.)

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., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Plasmids are important vectors for the spread of genes among diverse populations of bacteria. However, there is no standard method to determine the rate at which they spread horizontally via conjugation. Here, we compare commonly used methods on simulated and experimental data, and show that the resulting conjugation rate estimates often depend strongly on the time of measurement, the initial population densities, or the initial ratio of donor to recipient populations. Differences in growth rate, e.g. induced by sub-lethal antibiotic concentrations or temperature, can also significantly bias conjugation rate estimates. We derive a new 'end-point' measure to estimate conjugation rates, which extends the well-known Simonsen method to include the effects of differences in population growth and conjugation rates from donors and transconjugants. We further derive analytical expressions for the parameter range in which these approximations remain valid. We present an easy to use R package and web interface which implement both new and previously existing methods to estimate conjugation rates. The result is a set of tools and guidelines for accurate and comparable measurement of plasmid conjugation rates., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Early detection of emerging carbapenem-resistant Enterobacteriaceae (CPE) in food-producing animals is essential to control the spread of CPE. We assessed the risk of CPE introduction from imported livestock, livestock feed, companion animals, hospital patients, and returning travelers into livestock farms in The Netherlands, including (1) broiler, (2) broiler breeder, (3) fattening pig, (4) breeding pig, (5) farrow-to-finish pig, and (6) veal calf farms. The expected annual number of introductions was calculated from the number of farms exposed to each CPE source and the probability that at least one animal in an exposed farm is colonized. The total number of farms with CPE colonization was estimated to be the highest for fattening pig farms, whereas the probability of introduction for an individual farm was the highest for broiler farms. Livestock feed and imported livestock are the most likely sources of CPE introduction into Dutch livestock farms. Sensitivity analysis indicated that the number of fattening pig farms determined the number of high introductions in fattening pigs from feed, and that uncertainty on CPE prevalence impacted the absolute risk estimate for all farm types. The results of this study can be used to inform risk-based surveillance for CPE in livestock farms.

Understanding under which conditions conjugative plasmids encoding antibiotic resistance can invade bacterial communities in the gut is of particular interest to combat the spread of antibiotic resistance within and between animals and humans. We extended a one-compartment model of conjugation to a two-compartment model, to analyse how differences in plasmid dynamics in the gut lumen and at the gut wall affect the invasion of plasmids. We compared scenarios with one and two compartments, different migration rates between the lumen and wall compartments, and different population dynamics. We focused on the effect of attachment and detachment rates on plasmid dynamics, explicitly describing pair formation followed by plasmid transfer in the pairs. The parameter space allowing plasmid invasion in the one-compartment model is affected by plasmid costs and intrinsic conjugation rates of the transconjugant, but not by these characteristics of the donor. The parameter space allowing plasmid invasion in the two-compartment model is affected by attachment and detachment rates in the lumen and wall compartment, and by the bacterial density at the wall. The one- and two-compartment models predict the same parameter space for plasmid invasion if the conditions in both compartments are equal to the conditions in the one-compartment model. In contrast, the addition of the wall compartment widens the parameter space allowing invasion compared with the one-compartment model, if the density at the wall is higher than in the lumen, or if the attachment rate at the wall is high and the detachment rate at the wall is low. We also compared the pair-formation models with bulk-conjugation models that describe conjugation by instantaneous transfer of the plasmid at contact between cells, without explicitly describing pair formation. Our results show that pair-formation and bulk-conjugation models predict the same parameter space for plasmid invasion. From our simulations, we conclude that conditions at the gut wall should be taken into account to describe plasmid dynamics in the gut and that transconjugant characteristics rather than donor characteristics should be used to parameterize the models., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2021. The Author(s).)

Plasmid-mediated antimicrobial resistance is a major contributor to the spread of resistance genes within bacterial communities. Successful plasmid spread depends upon a balance between plasmid fitness effects on the host and rates of horizontal transmission. While these key parameters are readily quantified in vitro , the influence of interactions with other microbiome members is largely unknown. Here, we investigated the influence of three genera of lactic acid bacteria (LAB) derived from the chicken gastrointestinal microbiome on the spread of an epidemic narrow-range ESBL resistance plasmid, IncI1 carrying bla CTX-M-1 , in mixed cultures of isogenic Escherichia coli strains. Secreted products of LAB decreased E. coli growth rates in a genus-specific manner but did not affect plasmid transfer rates. Importantly, we quantified plasmid transfer rates by controlling for density-dependent mating opportunities. Parametrization of a mathematical model with our in vitro estimates illustrated that small fitness costs of plasmid carriage may tip the balance towards plasmid loss under growth conditions in the gastrointestinal tract. This work shows that microbial interactions can influence plasmid success and provides an experimental-theoretical framework for further study of plasmid transfer in a microbiome context.

To evaluate and compare the risk of emerging vector-borne diseases (VBDs), a Model for INTegrated RISK assessment, MINTRISK, was developed to assess the introduction risk of VBDs for new regions in an objective, transparent and repeatable manner. MINTRISK is a web-based calculation tool, that provides semi-quantitative risk scores that can be used for prioritization purposes. Input into MINTRISK is entered by answering questions regarding entry, transmission, establishment, spread, persistence and impact of a selected VBD. Answers can be chosen from qualitative answer categories with accompanying quantitative explanation to ensure consistent answering. The quantitative information is subsequently used as input for the model calculations to estimate the risk for each individual step in the model and for the summarizing output values (rate of introduction; epidemic size; overall risk). The risk assessor can indicate his uncertainty on each answer, and this is accounted for by Monte Carlo simulation. MINTRISK was used to assess the risk of four VBDs (African horse sickness, epizootic haemorrhagic disease, Rift Valley fever, and West Nile fever) for the Netherlands with the aim to prioritise these diseases for preparedness. Results indicated that the overall risk estimate was very high for all evaluated diseases but epizootic haemorrhagic disease. Uncertainty intervals were, however, wide limiting the options for ranking of the diseases. Risk profiles of the VBDs differed. Whereas all diseases were estimated to have a very high economic impact once introduced, the estimated introduction rates differed from low for Rift Valley fever and epizootic haemorrhagic disease to moderate for African horse sickness and very high for West Nile fever. Entry of infected mosquitoes on board of aircraft was deemed the most likely route of introduction for West Nile fever into the Netherlands, followed by entry of infected migratory birds., Competing Interests: The authors have declared that no competing interests exist.

Foot-and-mouth disease (FDM) is a disease of cloven-hoofed animals with high costs in animal welfare and animal production. Up to now, transmission between farms in FMD-endemic areas has been given little attention. Between farm transmission can be quantified by distance independent transmission parameters and a spatial transmission kernel indicating the rate of transmission of an infected farm to susceptible farms depending on the distance. The spatial transmission kernel and distance-independent transmission parameters were estimated from data of an FMD outbreak in Lamphaya Klang subdistrict in Thailand between 2016 and 2017. The spatial between-farm transmission rate in Lamphaya Klang subdistrict was higher compared with the spatial between-farm transmission rate from FMDV in epidemic areas. The result can be explained by the larger size of the within-farm outbreak in the endemic area due to no culling. The inclusion of distance-independent transmission parameters improved the model fit, which suggests the presence of transmission sources from outside the area and spread within the area independent of the distance between farms. The remaining distance-dependent transmission was mainly local and could be due to over-the-fence transmission or other forms of contact between nearby farms. Farm size on the kernel positively affects the transmission rate, by increasing both infectivity and susceptibility with increasing farm size. The results showed that both distance-dependent transmission and distance-independent transmission were contributed to FMDV transmission in Lamphaya Klang outbreak. These transmission parameters help to gain knowledge about FMD transmission dynamic in the endemic area., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Staphylococcus aureus and Streptococcus agalactiae are important contagious mastitis pathogens and are considered to mainly transmit between cows through the milking machine. Controlling contagious mastitis on dairy farms requires a reduction of the transmission rate or the duration of intramammary infections (IMI), or both. These parameters may differ in dairy herds milked with an automatic milking system (AMS) as compared to those milked with a conventional milking system (CMS). The aims of this prospective longitudinal study were to estimate the transmission rate, the median duration of IMI and the basic reproduction number (R 0 ) of Staph. aureus and Strep. agalactiae in a Dutch AMS herd. Bacteriological cultures of quarter milk samples were collected every 2 wks. Using 3 different definitions of IMI, we estimated the transmission rate for Staph. aureus to be within the range of 0.002 (95 % CI: 0-0.005) quarter-day -1 to 0.019 (95 % CI: 0.010-0.032) quarter-day -1 , and for Strep. agalactiae of 0.007 (95 % CI: 0.005-0.010) quarter-day -1 to 0.019 (95 % CI: 0.011-0.032) quarter-day -1 , the median duration of chronic IMI at 95 (95 % CI: 72-125) days for Staph. aureus and at 86 (95 % CI: 67-111) days for Strep. agalactiae, and the R 0 between 0.16 (95 % CI: 0.05-0.27) and 0.34 (95 % CI: 0.20-0.48) for Staph. aureus, and between 0.64 (95 % CI: 0.41-0.87) and 0.68 (95 % CI: 0.48-0.88) for Strep. agalactiae. Transmission of these two contagious pathogens in this herd was limited and theoretically the IMI would not sustain, given that R 0 of both pathogens was lower than 1. The estimated transmission rate of Staph. aureus in this AMS herd was found to be comparable to those described for CMS herds, while for Strep. agalactiae, it was slightly higher than in CMS herds. The duration of Staph. aureus IMI was in line with results from CMS farms, while the duration of Strep. agalactiae was lower than what has been described in CMS herds. The R 0 of these contagious pathogens was found to be lower than the estimates in CMS herds. Our study suggests that the transmission rate of these two contagious pathogens in this AMS herd were comparable to what has been reported about well-performing CMS herds that have a low rate of transmission., (Copyright © 2021. Published by Elsevier B.V.)

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 (log 10 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., (Copyright © 2020 Dame-Korevaar, Kers, van der Goot, Velkers, Ceccarelli, Mevius, Stegeman and Fischer.)

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., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Newcastle Disease is one of the most important infectious poultry diseases worldwide and is associated with high morbidity, mortality, and economic loss. In several countries, vaccination is applied to prevent and control outbreaks; however, information on the ability of vaccines to reduce transmission of ND virus (NDV) is sparse. Here we quantified the transmission of velogenic NDV among 42-day-old broilers. Chickens were either vaccinated with a single dose of a vector vaccine expressing the F protein (rHVT-ND) at day-old in the presence of maternally derived antibodies or kept unvaccinated. Seeders were challenged 8 h before the co-mingling with the corresponding contacts from the same group. Infection was monitored by daily testing of cloacal and oro-nasal swabs with reverse transcription-real-time PCR and by serology. Vaccinated birds were completely protected against clinical disease and virus excretion was significantly reduced compared to the unvaccinated controls that all died during the experiment. The reproduction ratio, which is the average number of secondary infections caused by an infectious bird, was significantly lower in the vaccinated group (0.82 (95% CI 0.38-1.75)) than in the unvaccinated group (3.2 (95% CI 2.06-4.96)). Results of this study demonstrate the potential of rHVT-ND vaccine in prevention and control of ND outbreaks.

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 10 1 or 10 2  cfu/mL E. coli encoding the beta-lactamase gene bla CTX-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 (log 10  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 log 10  cfu/g), and cecal content (up to -2.80 log 10  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., (Copyright © 2020. Published by Elsevier Inc.)

Background: Conjugation plays a major role in the transmission of plasmids encoding antibiotic resistance genes in both clinical and general settings. The conjugation efficiency is influenced by many biotic and abiotic factors, one of which is the taxonomic relatedness between donor and recipient bacteria. A comprehensive overview of the influence of donor-recipient relatedness on conjugation is still lacking, but such an overview is important to quantitatively assess the risk of plasmid transfer and the effect of interventions which limit the spread of antibiotic resistance, and to obtain parameter values for conjugation in mathematical models. Therefore, we performed a meta-analysis on reported conjugation frequencies from Escherichia coli donors to various recipient species., Results: Thirty-two studies reporting 313 conjugation frequencies for liquid broth matings and 270 conjugation frequencies for filter matings were included in our meta-analysis. The reported conjugation frequencies varied over 11 orders of magnitude. Decreasing taxonomic relatedness between donor and recipient bacteria, when adjusted for confounding factors, was associated with a lower conjugation frequency in liquid matings. The mean conjugation frequency for bacteria of the same order, the same class, and other classes was 10, 20, and 789 times lower than the mean conjugation frequency within the same species, respectively. This association between relatedness and conjugation frequency was not found for filter matings. The conjugation frequency was furthermore found to be influenced by temperature in both types of mating experiments, and in addition by plasmid incompatibility group in liquid matings, and by recipient origin and mating time in filter matings., Conclusions: In our meta-analysis, taxonomic relatedness is limiting conjugation in liquid matings, but not in filter matings, suggesting that taxonomic relatedness is not a limiting factor for conjugation in environments where bacteria are fixed in space.

Plasmid-mediated extended-spectrum β-lactamase and AmpC β-lactamase (ESBL/pAmpC) producing bacteria are present at all levels of the broiler production pyramid. Young birds can be found positive for ESBL/pAmpC-producing Escherichia coli shortly after arrival at farm. The aim of this study was to determine the effect of different challenge doses of ESBL/pAmpC-producing E. coli on time-until-colonization and the level of excretion in young broilers. One-day-old broilers (specific-pathogen free (SPF) and conventional Ross 308) were housed in isolators and challenged with 0.5 ml ESBL/pAmpC-producing E. coli strains of varying doses (10 1 -10 5  CFU/ml). Presence and concentration (CFU/gram feces) of ESBL/pAmpC-producing E. coli and total E. coli were determined longitudinally from cloacal swabs, and in cecal content 72 h after challenge. Higher challenge doses resulted in shorter time-until-colonization. However, even the lowest dose (10 1  CFU/ml) resulted in colonization of the broilers which excreted >10 6  CFU/gram feces 72 h after inoculation. Conventional broilers were colonized later than SPF broilers, although within 72 h after challenge all broilers were excreting ESBL/pAmpC-producing E. coli. A probabilistic model was used to estimate the probability of colonization by initial inoculation or transmission. The higher the dose the higher the probability of excreting ESBL/pAmpC-producing E. coli as a result of inoculation. In conclusion, low initial doses of ESBL/pAmpC-producing E. coli can result in rapid colonization of a flock. Interventions should thus be aimed to eliminate ESBL/pAmpC-producing bacteria in the environment of the hatchlings and measures focusing at reducing colonization and transmission of ESBL/pAmpC-producing E. coli should be applied shortly after hatching., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the short life of broiler chickens, their intestinal microbiota undergoes many changes. To study underlying biological mechanisms and factors that influence the intestinal microbiota development, longitudinal data from flocks and individual birds is needed. However, post-mortem collection of samples hampers longitudinal data collection. In this study, invasively collected cecal and ileal content, cloacal swabs collected from the same bird, and boot sock samples and cecal droppings from the litter of the broilers' poultry house, were collected on days 0, 2, 7, 14 and 35 post-hatch. The different sample types were evaluated on their applicability and reliability to characterize the broiler intestinal microbiota. The microbiota of 247 samples was assessed by 16S ribosomal RNA gene amplicon sequencing. Analyses of α and β measures showed a similar development of microbiota composition of cecal droppings compared to cecal content. Furthermore, the composition of cecal content samples was comparable to that of the boot socks until day 14 post-hatch. This study shows that the value of non-invasive sample types varies at different ages and depends on the goal of the microbiota characterization. Specifically, cecal droppings and boot socks may be useful alternatives for cecal samples to determine intestinal microbiota composition longitudinally.

Background: The intestinal microbiota is shaped by many interactions between microorganisms, host, diet, and the environment. Exposure to microorganisms present in the environment, and exchange of microorganisms between hosts sharing the same environment, can influence intestinal microbiota of individuals, but how this affects microbiota studies is poorly understood. We investigated the effects of experimental housing circumstances on intestinal microbiota composition in broiler chickens, and how these effects may influence the capacity to determine diet related effects in a nutrition experiment. A cross-sectional experiment was conducted simultaneously in a feed research facility with mesh panels between pens (Housing condition 1, H1), in an extensively cleaned stable with floor pens with solid wooden panels (H2), and in isolators (H3). In H1 and H2 different distances between pens were created to assess gut microbiota exchange between pens. Feed with and without a blend of medium-chain fatty acids (MCFA) was used to create differences in cecal microbiota between pens or isolators within the same housing condition. Male one-day-old Ross broiler chickens (n = 370) were randomly distributed across H1, H2, and H3. After 35 days cecal microbiota composition was assessed by 16S ribosomal RNA gene amplicon sequencing. Metabolic functioning of cecal content was assessed based on high-performance liquid chromatography., Results: Microbial alpha diversity was not affected in broilers fed +MCFA in H1 but was increased in H2 and H3. Based on weighted UniFrac distances, the nutritional intervention explained 10%, whereas housing condition explained 28% of cecal microbiota variation between all broilers. The effect size of the nutritional intervention varied within housing conditions between 11, 27, and 13% for H1, H2, and H3. Furthermore, performance and metabolic output were significantly different between housing conditions. The distance between pens within H1 and H2 did not influence the percentage of shared genera or operational taxonomic units (OTUs)., Conclusions: The cecal microbiota of broilers was modifiable by a nutritional intervention, but the housing condition affected microbiota composition and functionality stronger than the diet intervention. Consequently, for interpretation of intestinal microbiota studies in poultry it is essential to be aware of the potentially large impact of housing conditions on the obtained results.

Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the bla CTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 10 6 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene bla CTX-M-1 At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying bla CTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli , but no major increase occurred as observed in vitro Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation. IMPORTANCE Bacteria that produce extended-spectrum beta-lactamases are resistant to an important class of antimicrobials in human and veterinary medicine. Reduction in antibiotic use is expected to decrease the prevalence of resistance. However, resistance genes often lie on plasmids which can be copied and transferred to other bacteria by conjugation, so in vitro resistance was observed to increase in the absence of antimicrobials. We sought to determine whether this also occurs in the chicken gut and if competitive exclusion by similar E. coli variants without the resistance occurred. We studied the excretion of E. coli carrying IncI1 plasmids with the bla CTX-M-1 resistance gene in small groups of broiler chickens, after inoculating the chickens with E. coli suspensions containing different fractions of plasmid-carrying cells. Our results showed little variation between chickens within groups but large differences between groups that were independent of the ratio of variants with and without the plasmid and with persistence or extinction of the plasmid. However, there was no major plasmid increase as observed in vitro We conclude that in vivo studies with sufficient independent replications are important for intervention studies on plasmid-mediated antimicrobial resistance., (Copyright © 2019 American Society for Microbiology.)

Objectives: To explore the dynamics of faecal ESBL/AmpC shedding in dairy cattle and farmers, a study was conducted to examine changes in shedding by individual animals, as well as environmental exposure, and to study the association between antimicrobial use (AMU) and ESBL/AmpC shedding., Methods: The study comprised a cross-sectional survey of 20 farms and a 1 year follow-up of 10 farms. Faecal samples were cultured by both direct inoculation on MacConkey agar + 1 mg/L cefotaxime (MC+) and enrichment in LB-broth + 1 mg/L cefotaxime with subsequent inoculation on MC+. Dust samples were collected using electrostatic dustfall collectors (EDCs). Human faecal samples were collected by the farmers. Presence of ESBL/AmpC genes was screened for by PCR and sequencing. Using mixed effects logistic regression, ORs were determined and population-attributable fractions (PAFs) calculated subsequently., Results: In Phase 1, 8/20 farms were positive for ESBL/AmpC and, with 2 negative farms, were selected for Phase 2. Transient shedding of dominant allele variants was observed in the animals. EDCs and human faecal samples did not reflect what was observed in the animals. AMU was related to shedding of ESBLs in the next sampling moment [OR 14.6 (95% CI 3.0-80.0)] and the PAF of AMU was 0.36 (95% CI 0.08-0.77). Calves fed with colostrum from cows on dry-off therapy was not a risk factor [OR 1.7 (95% CI 0.7-4.9, P = 0.28)]., Conclusions: The presence of ESBL/AmpC could only be partly explained by AMU. No link was shown between shedding in cattle and humans or the environment. Interventions should focus on prevention of introduction., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Plasmid mediated Extended Spectrum Beta-Lactamase and AmpC Beta-Lactamase (ESBL/pAmpC) producing bacteria are resistant to beta-lactam antimicrobials and are widespread in humans, the environment and animals. Animals, especially broilers, are an important reservoir of ESBL/pAmpC producing bacteria. To control ESBL/pAmpC prevalence in broilers, transmission within the entire broiler production pyramid should be considered. This study, including 103 articles originating from two electronic databases, searched for evidence for possible routes of transmission of ESBL/pAmpC producing bacteria in the broiler production pyramid. Possible routes of transmission were categorised as 1) vertical between generations, 2) at hatcheries, 3) horizontal on farm, and 4) horizontal between farms and via the environment of farms. This review presents indications for transmission of ESBL/pAmpC producing bacteria for each of these routes. However, the lack of quantitative results in the literature did not allow an estimation of the relative contribution or magnitude of the different routes. Future research should be specifically targeted towards such information as it is crucial to guide reduction strategies for the spread of ESBL/pAmpC producing bacteria in the broiler production chain., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The initial development of intestinal microbiota in poultry plays an important role in production performance, overall health and resistance against microbial infections. Multiplexed sequencing of 16S ribosomal RNA gene amplicons is often used in studies, such as feed intervention or antimicrobial drug trials, to determine corresponding effects on the composition of intestinal microbiota. However, considerable variation of intestinal microbiota composition has been observed both within and across studies. Such variation may in part be attributed to technical factors, such as sampling procedures, sample storage, DNA extraction, the choice of PCR primers and corresponding region to be sequenced, and the sequencing platforms used. Furthermore, part of this variation in microbiota composition may also be explained by different host characteristics and environmental factors. To facilitate the improvement of design, reproducibility and interpretation of poultry microbiota studies, we have reviewed the literature on confounding factors influencing the observed intestinal microbiota in chickens. First, it has been identified that host-related factors, such as age, sex, and breed, have a large effect on intestinal microbiota. The diversity of chicken intestinal microbiota tends to increase most during the first weeks of life, and corresponding colonization patterns seem to differ between layer- and meat-type chickens. Second, it has been found that environmental factors, such as biosecurity level, housing, litter, feed access and climate also have an effect on the composition of the intestinal microbiota. As microbiota studies have to deal with many of these unknown or hidden host and environmental variables, the choice of study designs can have a great impact on study outcomes and interpretation of the data. Providing details on a broad range of host and environmental factors in articles and sequence data repositories is highly recommended. This creates opportunities to combine data from different studies for meta-analysis, which will facilitate scientific breakthroughs toward nutritional and husbandry associated strategies to improve animal health and performance.

African horse sickness (AHS) and equine encephalosis (EE) are Culicoides-borne viral diseases that could have the potential to spread across Europe if introduced, thus being potential threats for the European equine industry. Both share similar epidemiology, transmission patterns and geographical distribution. Using stochastic spatiotemporal models of virus entry, we assessed and compared the probabilities of both viruses entering France via two pathways: importation of live-infected animals or importation of infected vectors. Analyses were performed for three consecutive years (2010-2012). Seasonal and regional differences in virus entry probabilities were the same for both diseases. However, the probability of EE entry was much higher than the probability of AHS entry. Interestingly, the most likely entry route differed between AHS and EE: AHS has a higher probability to enter through an infected vector and EE has a higher probability to enter through an infectious host. Consequently, different effective protective measures were identified by 'what-if' scenarios for the two diseases. The implementation of vector protection on all animals (equine and bovine) coming from low-risk regions before their importation was the most effective in reducing the probability of AHS entry. On the other hand, the most significant reduction in the probability of EE entry was obtained by the implementation of quarantine before import for horses coming from both EU and non-EU countries. The developed models can be useful to implement risk-based surveillance., (© 2016 Blackwell Verlag GmbH.)

Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (pAmpC) are enzymes able to hydrolyze a large variety of β-lactam antibiotics, including third-generation cephalosporins and monobactams. Broilers and broiler meat products can be highly contaminated with ESBL- and pAmpC-producing Escherichia coli strains, also known as extended-spectrum cephalosporin (ESC)-resistant E. coli strains, and can be a source for human infections. As few data on interventions to reduce the presence of ESC-resistant E. coli in broilers are available, we used transmission experiments to examine the role of competitive exclusion (CE) on reducing transmission and excretion in broilers. A broiler model to study the transmission of ESC-resistant E. coli was set up. Day-old chickens were challenged with an ESBL-producing E. coli strain isolated from healthy broilers in the Netherlands. Challenged and not challenged chicks were housed together in pairs or in groups, and ESBL-producing E. coli transmission was monitored via selective culturing of cloacal swab specimens. We observed a statistically significant reduction in both the transmission and excretion of ESBL-producing E. coli in chicks treated with the probiotic flora before E. coli challenge compared to the transmission and excretion in untreated controls. In conclusion, our results support the use of competitive exclusion as an intervention strategy to control ESC-resistant E. coli in the field. IMPORTANCE Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases are a primary cause of resistance to β-lactam antibiotics among members of the family Enterobacteriaceae in humans, animals, and the environment. Food-producing animals are not exempt from this, with a high prevalence being seen in broilers, and there is evidence pointing to a possible foodborne source for human contamination. We investigated the effect of administration of a commercial probiotic product as an intervention to reduce the amount of ESBL-producing Escherichia coli in broilers. Our results showed a substantial reduction in the level of colonization of broiler intestines by ESBL-producing E. coli after administration of commercial probiotic product. The protective effect provided by these probiotics could be implemented on a larger scale in poultry production. Reductions in the levels of ESBL-producing Enterobacteriaceae in the food chain would considerably benefit public health., (Copyright © 2017 American Society for Microbiology.)

Extended-spectrum β-lactamase and plasmid mediated AmpC β-lactamase (ESBL/pAmpC) producing bacteria are resistant to Extended Spectrum Cephalosporins (ESC), and are present in all levels of the broiler production chain. We determined the prevalence, concentration, and persistence of ESBL/pAmpC-Escherichia coli in a broiler parent flock during the rearing and laying period. One-day old chickens were housed in four separate pens. Until week 33 no antibiotics or coccidiostatics were used. During rearing 57 chickens in each pen (n=228), and in the laying period two groups of 33 chickens were individually sampled (n=66). Environmental samples were taken from week 16 onwards. ESBL/pAmpC-E. coli presence was determined by selective culturing. In the samples of week 16-19 the concentration of ESBL/pAmpC-E. coli was determined. All ESC-resistant isolates found were positive for pAmpC gene bla CMY-2 located on IncA/C plasmids, in several E. coli MLST types. CMY-2-E. coli prevalence decreased from 91% (95%CI 86-94%) at day 7 (week 1) to 0% (95%CI 0-5%) in week 21. However, CMY-2-E. coli remained present in the environmental samples during the whole study. CMY-2-E. coli concentration varied between detection limit (<10^3) and 2·10^4 cfu/g faeces. The sharp reduction of CMY-2-E. coli in this broiler parent flock in absence of antibiotics suggests a selective disadvantage of bla CMY-2 on IncA/C plasmids on animal level. The underlying mechanism should be studied further as this may provide new insights on how to reduce ESBL/pAmpC prevalence and transmission in the broiler production chain., (Copyright © 2017 Elsevier B.V. All rights reserved.)