Expression of influenza A virus glycan receptor candidates in mallard, chicken, and tufted duck.

Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Receptor incompatibility due to differently expressed glycan structures between species has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative sialic acid-containing glycan receptors for IAV in mallard, chicken and tufted duck; three bird species with different roles in the zoonotic ecology of IAV. The methodology used pinpoints specific glycan structures to specific glycosylation sites of identified glycoproteins and was also used to successfully discriminate α2-3- from α2-6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides in tandem MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex N-glycans including α2-3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both α2-3- and α2-6-linked Neu5Ac. We also found the recently identified putative IAV receptor structures, Man-6P N-glycopeptides, in all tissues of the three bird species. Furthermore, we found many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures, previously anticipated to be mammalian specific, in all three bird species may have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV.

An oseltamivir-resistant avian H1N1 influenza A virus can transmit from mallards to chickens similarly to a wild-type strain: implications for the risk of resistance transmission to humans.

The neuraminidase inhibitor (NAI) oseltamivir is stockpiled globally as part of influenza pandemic preparedness. However, oseltamivir carboxylate (OC) resistance develops in avian influenza virus (AIV) infecting mallards exposed to environmental-like OC concentrations, suggesting that environmental resistance is a real concern. Herein we used an in vivo model to investigate if avian influenza H1N1 with the OC-resistant mutation NA-H274Y (51833/H274Y) as compared to the wild-type (wt) strain (51833 /wt) could transmit from mallards, which would potentially be exposed to environmentally contaminated environments, to and between chickens, thus posing a potential zoonotic risk of antiviral-resistant AIV. Regardless of whether the virus had the OC-resistant mutation or not, chickens became infected both through experimental infection, and following exposure to infected mallards. We found similar infection patterns between 51833/wt and 51833/H274Y such that, one chicken inoculated with 51833/wt and three chickens inoculated with 51833/H274Y were AIV positive in oropharyngeal samples more than 2 days consecutively, indicating true infection, and one contact chicken exposed to infected mallards was AIV positive in faecal samples for 3 consecutive days (51833/wt) and another contact chicken for 4 consecutive days (51833/H274Y). Importantly, all positive samples from chickens infected with 51833/H274Y retained the NA-H274Y mutation. However, none of the virus strains established sustained transmission in chickens, likely due to insufficient adaptation to the chicken host. Our results demonstrate that an OC-resistant avian influenza virus can transmit from mallards and replicate in chickens. NA-H274Y does not constitute a barrier to interspecies transmission per se, as the resistant virus did not show reduced replicative capacity compared to the wild-type counterpart. Thus, responsible use of oseltamivir and surveillance for resistance development is warranted to limit the risk of an OC-resistant pandemic strain.

Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

OBJECTIVES: In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. METHODS: WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. RESULTS: mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. CONCLUSIONS: The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

Genome-associations of extended-spectrum ß-lactamase producing (ESBL) or AmpC producing E. coli in small and medium pig farms from Khon Kaen province, Thailand.

Thailand is undergoing rapid intensification of livestock production where small subsistence farms and medium sized commercial farms coexist. In medium farms, antimicrobials are prescribed by a veterinarian, whereas in small farms antimicrobial use remains largely unsupervised. The impact of these differences as well as other farming practices on the emergence and composition of antimicrobial resistance genes (ARGs) remains largely unknown. We analyzed 363 genomes of extended-spectrum ß-lactamase producing (ESBL) and/or AmpC producing Escherichia coli recovered from humans and pigs at small and medium farms from the Khon Kaen province, Thailand. We tested for genome-wide associations to identify links between ARGs, host, and farm size. Pig isolates from small farms were associated with mcr and qnr genes conferring resistance to colistin and fluoroquinolones, respectively. In contrast, pig isolates from medium farms were associated with ARGs conferring resistance to drugs commonly used on medium farms (i.e., streptomycin). ESBL plasmids from small farms co-carried ARGs conferring resistance to critically important antimicrobials more frequently compared to plasmid from medium farms. Frequent ARG combinations included blaCTX-M-55 + qnrS1 (29.8% vs 17.5% in small and medium farms, respectively), blaCTX-M-55 + qnrS1 + mcr-3.19 (5% vs 0%), blaCTX-M-14 + qnrS1 (9.3% vs 6.2%), and blaCTX-M-14 + qnrS1 + mcr-1.1 (3.1% vs 0%). The co-location on plasmids of ARGs conferring resistance to critically important antimicrobials as defined by the World Health Organization is concerning, and actions to curb their spread are urgently needed. Legislation on limiting antimicrobial sales and initiatives to better inform farmers and veterinarians on appropriate antimicrobial usage and farm biosecurity could help reduce antimicrobial use on farms.

Selection of Resistant Bacteria in Mallards Exposed to Subinhibitory Concentrations of Ciprofloxacin in Their Water Environment.

Emergence and selection of antibiotic resistance following exposure to high antibiotic concentrations have been repeatedly shown in clinical and agricultural settings, whereas the role of the weak selective pressures exerted by antibiotic levels below the MIC (sub-MIC) in aquatic environments due to anthropogenic contamination remains unclear. Here, we studied how exposure to sub-MIC levels of ciprofloxacin enriches for Escherichia coli with reduced susceptibility to ciprofloxacin using a mallard colonization model. Mallards were inoculated with two isogenic extended-spectrum-ß-lactamase (ESBL)-encoding E. coli strains, differing only by a gyrA mutation that results in increased MICs of ciprofloxacin, and exposed to different levels of ciprofloxacin in their swimming water. Changes in the ratios of mutant to parental strains excreted in feces over time and ESBL plasmid spread within the gut microbiota from individual birds were investigated. Results show that in vivo selection of gyrA mutants occurred in mallards during exposure to ciprofloxacin at concentrations previously found in aquatic environments. During colonization, resistance plasmids were readily transferred between strains in the intestines of the mallards, but conjugation frequencies were not affected by ciprofloxacin exposure. Our results highlight the potential for enrichment of resistant bacteria in wildlife and underline the importance of reducing antibiotic pollution in the environment.

Characterization of Klebsiella pneumoniae complex isolates from pigs and humans in farms in Thailand: population genomic structure, antibiotic resistance and virulence genes.

OBJECTIVES: To define characteristics of Klebsiella pneumoniae complex (hereafter KP) isolates from healthy pigs, farm workers and their household members in Thailand. METHODS: A total of 839 individual rectal swabs from pigs on 164 farms and 271 faecal samples of humans working on pig farms and persons living in the same household in Khon Kaen, Thailand were screened for gut colonization by KP. Genomic sequences were investigated for antibiotic resistance and virulence genes. Phylogenetic analyses were performed in addition to comparison with isolates from previous studies from Thailand. RESULTS: KP was detected in approximately 50% of pig and human samples. In total, 253 KP isolates were obtained: 39% from pigs, 34% from farmers and 26% from individuals living on the same farm but without animal contact. MLST revealed high genetic diversity with 196 different STs distributed over four phylogroups (Kp1 to Kp4). Low prevalence of ESBL-KP (7.5%) and colistin-resistant KP (3.2%) was observed among pigs and humans. Remarkably, four convergent MDR and hypervirulent strains were observed: one from pigs (ST290) and three from humans [ST35, ST3415 (strain 90CP1), ST17 (strain 90CM2)]. Sharing of KP clones among pigs and humans was identified for some STs including ST4788, ST661, ST3541 and ST29. CONCLUSIONS: The study indicated a low prevalence of ESBL and mcr genes among KP isolated from pigs and healthy humans in Thailand and suggested the possibility of zoonotic transmission for a subset of circulating KP clones.

Residual antimicrobial agents in food originating from animals.

BACKGROUND: The agricultural food products industry in Bangladesh depends on utilizing antimicrobials indiscriminately as growth promoters and for controlling infectious diseases. Thus, there is always a risk of antimicrobial agent accumulation in food sources that originate from agricultural production. METHODS: In the present study, we collected data from published articles between January, 2013 and December, 2019 on antimicrobial residues in human food sources such as meat, milk, eggs, and fishes. RESULTS: Liver contained the highest percentage of antimicrobial residues (74%; 95% CI: 59.66-85.37) against the in vitro enteric pathogen Escherichia coli in layer chickens. Similar results were demonstrated in liver (68%; 95% CI: 53.30-80.48) and kidney (66%, 95% CI: 51.23-78.79) of layer chickens against Bacillus cereus and Bacillus subtilis. Amongst all antibiotics, the highest concentrations of ciprofloxacin were detected in kidney (48.57%; 95% CI: 31.38-66.01), followed by liver (47.56; 95% CI: 40.88-54.30) of broiler chickens. Ciprofloxacin was also present in liver (46.15%; 95% CI: 33.70-58.96) of layer chickens. The percentage of ciprofloxacin in thigh and breast meat in broiler bird were 41.54% (95% CI: 34.54-48.79) and 37.95% (95% CI: 31.11-45.15) respectively. Enrofloxacin was the second most dominant antimicrobial agent and was present in the liver of both types of poultry (Broiler and Layer chickens: 41.54%; 95% CI: 29.44-54.4 and 437.33%; 95% CI: 30.99-44.01). The prevalence rates of enrofloxacin in thigh and breast meat of broiler chickens were 24.10% (95% CI: 18.28-30.73) and 20.51% (95% CI: 15.08-26.87), respectively. Tetracycline, a commonly used antibiotic in livestock, was present in the liver (49.23%; 95% CI: 36.60-61.93) of layer chickens. In case of aquaculture food products, the highest amount of amoxicillin (683.2 mg/kg) was detected in Tilapia fish (Oreochromis niloticus), followed by 584.4 mg/kg in climbing perch (Anabas testudineus) and 555.6 mg/kg in Rui fish (Labeo rohita). Among the five types of fishes, Rui fish (0.000515 mg/kg) contained the highest concentrations of chloramphenicol antibiotic residues. CONCLUSIONS: The presence of antimicrobial residues in meat, milk, egg, and fish is a serious public health threat due to the potential induction of antimicrobial resistance. It can negatively impact the food supply chain, especially with the current strain that it is already facing with the current COVID-19 pandemic. The findings of the present study highlight the ongoing risk of residual antimicrobial agents in food of animal origin in Bangladesh and countries with similar practices. This can draw the attention of public health officials to propose plans to mitigate or stop this practice.

Influenza A/H4N2 mallard infection experiments further indicate zanamivir as less prone to induce environmental resistance development than oseltamivir.

Neuraminidase inhibitors (NAIs) are the gold standard treatment for influenza A virus (IAV). Oseltamivir is mostly used, followed by zanamivir (ZA). NAIs are not readily degraded in conventional wastewater treatment plants and can be detected in aquatic environments. Waterfowl are natural IAV hosts and replicating IAVs could thus be exposed to NAIs in the environment and develop resistance. Avian IAVs form the genetic basis for new human IAVs, and a resistant IAV with pandemic potential poses a serious public health threat, as NAIs constitute a pandemic preparedness cornerstone. Resistance development in waterfowl IAVs exposed to NAIs in the water environment has previously been investigated in an in vivo mallard model and resistance development was demonstrated in several avian IAVs after the exposure of infected ducks to oseltamivir, and in an H1N1 IAV after exposure to ZA. The N1 and N2 types of IAVs have different characteristics and resistance mutations, and so the present study investigated the exposure of an N2-type IAV (H4N2) in infected mallards to 1, 10 and 100 µg l-1 of ZA in the water environment. Two neuraminidase substitutions emerged, H274N (ZA IC50 increased 5.5-fold) and E119G (ZA IC50 increased 110-fold) at 10 and 100 µg l-1 of ZA, respectively. Reversion towards wild-type was observed for both substitutions in experiments with removed drug pressure, indicating reduced fitness of both resistant viruses. These results corroborate previous findings that the development of resistance to ZA in the environment seems less likely to occur than the development of resistance to oseltamivir, adding information that is useful in planning for prudent drug use and pandemic preparedness.

Multi-laboratory evaluation of ReaScan TBE IgM rapid test, 2016 to 2017.

BackgroundTick-borne encephalitis (TBE) is a potentially severe neurological disease caused by TBE virus (TBEV). In Europe and Asia, TBEV infection has become a growing public health concern and requires fast and specific detection.AimIn this observational study, we evaluated a rapid TBE IgM test, ReaScan TBE, for usage in a clinical laboratory setting.MethodsPatient sera found negative or positive for TBEV by serological and/or molecular methods in diagnostic laboratories of five European countries endemic for TBEV (Estonia, Finland, Slovenia, the Netherlands and Sweden) were used to assess the sensitivity and specificity of the test. The patients' diagnoses were based on other commercial or quality assured in-house assays, i.e. each laboratory's conventional routine methods. For specificity analysis, serum samples from patients with infections known to cause problems in serology were employed. These samples tested positive for e.g. Epstein-Barr virus, cytomegalovirus and Anaplasma phagocytophilum, or for flaviviruses other than TBEV, i.e. dengue, Japanese encephalitis, West Nile and Zika viruses. Samples from individuals vaccinated against flaviviruses other than TBEV were also included. Altogether, 172 serum samples from patients with acute TBE and 306 TBE IgM negative samples were analysed.ResultsCompared with each laboratory's conventional methods, the tested assay had similar sensitivity and specificity (99.4% and 97.7%, respectively). Samples containing potentially interfering antibodies did not cause specificity problems.ConclusionRegarding diagnosis of acute TBEV infections, ReaScan TBE offers rapid and convenient complementary IgM detection. If used as a stand-alone, it can provide preliminary results in a laboratory or point of care setting.

Genetic analyses of Seoul hantavirus genome recovered from rats (Rattus norvegicus) in the Netherlands unveils diverse routes of spread into Europe.

Seoul virus (SEOV) is the etiologic agent of hemorrhagic fever with renal syndrome. It is carried by brown rats (Rattus norvegicus), a commensal rodent that closely cohabitates with humans in urban environments. SEOV has a worldwide distribution, and in Europe, it has been found in rats in UK, France, Sweden, and Belgium, and human cases of SEOV infection have been reported in Germany, UK, France, and Belgium. In the search of hantaviruses in brown rats from the Netherlands, we found both serological and genetic evidence for the presence of SEOV in the local wild rat population. To further decipher the relationship with other SEOV variants globally, the complete genome of SEOV in the Netherlands was recovered. SEOV sequences obtained from three positive rats (captured at close trapping locations at the same time) were found highly similar. Phylogenetic analyses demonstrated that two lineages of SEOV circulate in Europe. Strains from the Netherlands and UK, together with the Baxter strain from US, constitute one of these two, while the second includes strains from Europe and Asia. Our results support a hypothesis of diverse routes of SEOV spread into Europe. These findings, combined with other indications on the expansion of the spatial European range of SEOV, suggest an increased risk of this virus for the public health, highlighting the need for increased surveillance.

Alternate routes of influenza A virus infection in Mallard (Anas platyrhynchos).

The natural reservoir for all influenza A viruses (IAVs) is wild birds, particularly dabbling ducks. During the autumn, viral prevalence can be very high in dabbling ducks (> 30%) in the Northern Hemisphere, and individuals may be repeatedly infected. Transmission and infection is through the fecal-oral route, whereby birds shed viruses in feces and conspecifics are infected though feeding in virus-contaminated water. In this study we wanted to assess two alternative infection routes: cloacal drinking and preening. Using experimental infections, we assessed patterns of infection using a combination of virus shedding, as assessed by real-time PCR from cloacal swabs, and patterns of viral replication using virus-immunohistochemistry of gastrointestinal tissues. The cloacal drinking experiment consisted of two trials using cloacal inoculation at two different time points to account for age differences, as well as a trial whereby ducks were allowed to take up virus-laden water through the cloaca. All ducks became infected, and rather than the bursa of Fabricius being the main site of replication, the colon had the highest intensity of replication, as inferred through immunohistochemistry. In experiments assessing preening, feathers were contaminated with virus-laden water and all ducks became infected, regardless of whether they were kept individually or together. Further, naive contacts were infected by the individuals whose feathers were virus-contaminated. Overall, we reinforce that IAV transmission in dabbling ducks is multifactorial-if exposed to virus-contaminated water ducks may be infected through dabbling, preening of infected feathers, and cloacal drinking.

In vivo mallard experiments indicate that zanamivir has less potential for environmental influenza A virus resistance development than oseltamivir.

Neuraminidase inhibitors are a cornerstone of influenza pandemic preparedness before vaccines can be mass-produced and thus a neuraminidase inhibitor-resistant pandemic is a serious threat to public health. Earlier work has demonstrated the potential for development and persistence of oseltamivir resistance in influenza A viruses exposed to environmentally relevant water concentrations of the drug when infecting mallards, the natural influenza reservoir that serves as the genetic base for human pandemics. As zanamivir is the major second-line neuraminidase inhibitor treatment, this study aimed to assess the potential for development and persistence of zanamivir resistance in an in vivo mallard model; especially important as zanamivir will probably be increasingly used. Our results indicate less potential for development and persistence of resistance due to zanamivir than oseltamivir in an environmental setting. This conclusion is based on: (1) the lower increase in zanamivir IC50 conferred by the mutations caused by zanamivir exposure (2-17-fold); (2) the higher zanamivir water concentration needed to induce resistance (at least 10 µg l-1); (3) the lack of zanamivir resistance persistence without drug pressure; and (4) the multiple resistance-related substitutions seen during zanamivir exposure (V116A, A138V, R152K, T157I and D199G) suggesting lack of one straight-forward evolutionary path to resistance. Our study also adds further evidence regarding the stability of the oseltamivir-induced substitution H275Y without drug pressure, and demonstrates the ability of a H275Y-carrying virus to acquire secondary mutations, further boosting oseltamivir resistance when exposed to zanamivir. Similar studies using influenza A viruses of the N2-phylogenetic group of neuraminidases are recommended.

Influenza A(H7N9) virus acquires resistance-related neuraminidase I222T substitution when infected mallards are exposed to low levels of oseltamivir in water.

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 µg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

Oseltamivir-resistant influenza A (H1N1) virus strain with an H274Y mutation in neuraminidase persists without drug pressure in infected mallards.

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.

Understanding Vietnamese chicken farmers' knowledge and practices related to antimicrobial resistance using an item response theory approach.

Introduction: Antimicrobial resistance (AMR) poses a threat to animal and human health, as well as food security and nutrition. Development of AMR is accelerated by over- and misuse of antimicrobials as seen in many livestock systems, including poultry production. In Vietnam, high AMR levels have been reported previously within poultry production, a sector which is dominated by small-scale farming, even though it is intensifying. This study focuses on understanding small- and medium-scale chicken farmers' knowledge and practices related to AMR by applying an item response theory (IRT) approach, which has several advantages over simpler statistical methods. Methods: Farmers representing 305 farms in Thai Nguyen province were interviewed from November 2021 to January 2022, using a structured questionnaire. Results generated with IRT were used in regression models to find associations between farm characteristics, and knowledge and practice levels. Results: Descriptive results showed that almost all farmers could buy veterinary drugs without prescription in the local community, that only one third of the farmers received veterinary professional advice or services, and that the majority of farmers gave antibiotics as a disease preventive measure. Regression analysis showed that multiple farm characteristics were significantly associated to farmers' knowledge and practice scores. Conclusion: The study highlights the complexity when tailoring interventions to move towards more medically rational antibiotic use at farms in a setting with high access to over-the-counter veterinary drugs and low access to veterinary services, since many on-farm factors relevant for the specific context need to be considered.

Understanding veterinary drug shop workers' knowledge and practices to identify drivers of antibiotic use in Vietnamese livestock farms.

BACKGROUND: Antimicrobial resistance (AMR) is a One Health issue and a major threat to animal and human health. Antibiotic use (ABU) drives AMR development, and several hotspots for ABU, and AMR, in livestock have been identified in Southeast Asia, including Vietnam. There are often multiple drivers of ABU at farms, and to identify all of them there is a need to look beyond farm level. OBJECTIVES: The overall aim of this study was to identify routines and/or competencies, related to antibiotic sales, among veterinary drug shop workers that may be improved in order to decrease the medically non-rational use of antibiotics in livestock production. METHODS: A questionnaire-based survey was conducted at 50 veterinary drug shops in northern Vietnam. RESULTS: Results showed high education and knowledge levels. According to the respondents, antibiotic treatment advice was almost always provided to the farmers, and the recommended treatment was most commonly based on recommendations for the specific disease. However, farmers had almost never had their animals properly diagnosed. Antibiotics were the most sold drug category, penicillins being the most common. Several broad-spectrum antibiotics were also quite frequently sold. Further, >50% of respondents recommended antibiotics for disease prevention. CONCLUSIONS: Even though education and knowledge levels might be high, several challenges can prevent drug shop workers from contributing to more prudent ABU at farms, for example, lack of proper diagnosis, commercial interests and individual farmer motives, often in combination with poor compliance to regulations.

Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.