Search

The emergence and spread of β-lactamase-producing Enterobacteriaceae poses a significant threat to public health, necessitating the rapid detection and investigation of the molecular epidemiology of these pathogens. We modified a multiplex real-time (RT)-PCR to concurrently detect β-lactamase genes (blaCTX-M, blaTEM, and blaSHV) and Enterobacteriaceae 16S ribosomal RNA. qPCR probes and primers were validated using control isolates, and the sensitivity and specificity assessed. The optimised multiplex qPCR was used to screen 220 non-clinical Enterobacteriaceae from food animals and in-contact humans in Southeast Nigeria selected on cefotaxime-supplemented agar plates. Binary logistic regression was used to explore factors associated with the presence of the blaTEM and blaSHV genes in these isolates, and a subset of isolates from matched sampling sites and host species were whole genome sequenced, and their antimicrobial resistance (AMR) and plasmid profiles determined. The sensitivity and specificity of the qPCR assay was 100%. All isolates (220/220) were positive for Enterobacteriaceae ribosomal 16S rRNA and blaCTX-M, while 66.4% (146/220) and 9% (20/220) were positive for blaTEM and blaSHV, respectively. The prevalence of blaTEM and blaSHV varied across different sampling sites (farm, animal market and abattoirs). Isolates from Abia state were more likely to harbour blaTEM (OR = 2.3, p = 0.04) and blaSHV (OR = 5.12,p = 0.01) than isolates from Ebonyi state; blaTEM was more likely to be detected in isolates from food animals than humans (OR = 2.34, p = 0.03), whereas the reverse was seen for blaSHV (OR = 7.23, p = 0.02). Furthermore, Klebsiella and Enterobacter isolates harboured more AMR genes than Escherichia coli, even though they were isolated from the same sample. We also identified pan resistant Klebsiella harbouring resistance to ten classes of antimicrobials and disinfectant. Therefore, we recommend ESKAPE pathogens are included in AMR surveillance in future and suggest qPCRs be utilised for rapid screening of Enterobacteriaceae from human and animal sources., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Olorunleke 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.)

Bovine tuberculosis (bTB) continues to be the costliest, most complex animal health problem in England. The effectiveness of the test-and-slaughter policy is hampered by the imperfect sensitivity of the surveillance tests. Up to half of recurrent incidents within 24 months of a previous one could have been due to undetected infected cattle not being removed. Improving diagnostic testing with more sensitive tests, like the interferon (IFN)-gamma test, is one of the government's top priorities. However, blanket deployment of such tests could result in more false positive results (due to imperfect specificity), together with logistical and cost-efficiency challenges. A targeted application of such tests in higher prevalence scenarios, such as a subpopulation of high-risk herds, could mitigate against these challenges. We developed classification machine learning algorithms (using 80% of 2012-2019 bTB surveillance data as the training set) to evaluate the deployment of IFN-gamma testing in high-risk herds (i.e. those at risk of an incident in England) in two testing data sets: i) the remaining 20% of 2012-19 data, and ii) 2020 bTB surveillance data. The resulting model, classification tree analysis, with an area under a receiver operating characteristic (ROC) curve (AUC) > 95, showed a 73% sensitivity and a 97% specificity in the 2012-2019 test dataset. Used on 2020 data, it predicted eight percent (3 510 of 41 493) of eligible active herds as at-risk of a bTB incident, the majority of them (66% or 2 328 herds) experiencing at least one. Whilst all predicted at-risk herds could have preventive measures applied, the additional application of IFN-gamma test in parallel interpretation to the statutory skin test, if the risk materialises, would have resulted in 8 585 additional IFN-gamma reactors detected (a 217% increase over the 2 710 IFN-gamma reactors already detected by tests carried out). Only 18% (330 of 1 819) of incidents in predicted high-risk herds had the IFN-gamma test applied in 2020. We therefore conclude that this methodology provides a better way of directing the application of the IFN-gamma test towards the high-risk subgroup of herds. Classification tree analysis ensured the systematic identification of high-risk herds to consistently apply additional measures in a targeted way. This could increase the detection of infected cattle more efficiently, preventing recurrence and accelerating efforts to achieve eradication by 2038. This methodology has wider application, like targeting improved biosecurity measures in avian influenza at-risk farms to limit damage to the industry in future outbreaks., Competing Interests: Declaration of Competing Interest This research was funded by the Animal and Plant Health Agency and undertaken with the Royal Veterinary College, manuscript approval number 1557012. The Animal and Plant Health Agency employs the following authors: Dr M. Pilar Romero, Dr Jessica Parry, Alison Prosser, and Paul Upton. None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

The rise in antimicrobial resistance (AMR) in bacteria is reducing therapeutic options for livestock and human health, with a paucity of information globally. To fill this gap, a One-Health approach was taken by sampling livestock on farms ( n = 52), abattoir ( n = 8), and animal markets ( n = 10), and in-contact humans in Southeast Nigeria. Extended spectrum cephalosporin (ESC)-resistant (ESC-R) Escherichia coli was selectively cultured from 975 healthy livestock faecal swabs, and hand swabs from in-contact humans. Antimicrobial susceptibility testing (AST) was performed on all ESC-R E. coli . For isolates showing a multi-drug resistance (MDR) phenotype ( n  = 196), quantitative real-time PCR (qPCR) was performed for confirmation of extended-spectrum β-lactamase (ESBL) and carbapenemase genes. Whole-genome sequencing (WGS) was performed on a subset ( n  = 157) for detailed molecular characterisation. The results showed ESC-R E. coli was present in 41.2% of samples, with AST results indicating 48.8% of isolates were phenotypically MDR. qPCR confirmed presence of ESBL genes, with bla CTX-M present in all but others in a subset [ bla TEM (62.8%) and bla SHV (0.5%)] of isolates; none harboured transferable carbapenemase genes. Multi-locus sequence typing identified 34 Sequence Types (ST) distributed among different sampling levels; ST196 carrying bla CTX-M-55 was predominant in chickens. Large numbers of single nucleotide polymorphisms (SNPs) in the core genome of isolates, even within the same clade by phylogenetic analysis, indicated high genetic diversity. AMR genotyping indicated the predominant bla CTX-M variant was bla CTX-M-15 (87.9%), although bla CTX-M-55 , bla CTX-M-64, and bla CTX-M-65 were present; it was notable that bla CTX-M-1 , common in livestock, was absent. Other predominant AMR genes included: sul 2, qnr S1, str B, bla TEM-1b , tet A-v2, and dfr A14, with prevalence varying according to host livestock species. A bla CTX-M-15 harbouring plasmid from livestock isolates in Ebonyi showed high sequence identity to one from river/sewage water in India, indicating this ESBL plasmid to be globally disseminated, being present beyond the river environment. In conclusion, ESC-R E. coli was widespread in livestock and in-contact humans from Southeast Nigeria. WGS data indicated the isolates were genetically highly diverse, probably representing true diversity of wild type E. coli ; they were likely to be MDR with several harbouring bla CTX-M-15. Surprisingly, human isolates had highest numbers of AMR genes and pigs the least., 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 © 2022 Olorunleke, Kirchner, Duggett, AbuOun, Okorie-Kanu, Stevens, Card, Chah, Nwanta, Brunton and Anjum.)

Bovine tuberculosis (bTB) remains one of the most complex, challenging, and costly animal health problems in England. Identifying and promptly removing all infected cattle from affected herds is key to its eradication strategy; the imperfect sensitivity of the diagnostic testing regime remaining a serious obstacle. The main diagnostic test for bTB in cattle in England, the Single Intradermal Comparative Cervical Tuberculin Test (SICCT: also known as the skin test), can produce inconclusive results below the reactor threshold. The immediate isolation of inconclusive reactor (IR) animals followed by a 60-day retest may not prevent lateral spread within the herd (if it is substandard, allowing transmission) or transmission to wildlife. Over half of IR-only herds that went on to have a positive skin test result (a bTB herd 'incident') in 2020, had it triggered by at least one IR not clearing their 60-day retest, instead of by another test within the previous 15 months. Machine learning classification algorithms (classification tree analysis and random forest), applied to England's 2012-2020 IR-only surveillance herd tests, identified at-risk tests for an incident at the IRs' 60-day retest. In this period, 4 739 out of 22 946 (21 %) IR-only surveillance tests disclosing 6 296 out of 42 685 total IRs, had an incident at retest (2 716 IRs became reactors and 3 580 IRs became two-time IRs). Both models showed an AUC above 80 % in the 2012-2019 dataset. Classification tree analysis was preferred due to its easy-to-interpret outputs, 70 % sensitivity, and 93 % specificity in the 20 % of 2019-2020 testing dataset. The paper aimed to identify IR-only surveillance tests at-risk of an incident at the 60-day retest to target them with appropriate measures to mitigate the IRs' risk. Sixteen percent (341 out of 2 177) of IR-only herd tests were identified as high-risk in the 2020 dataset, with 265 (78 %) of these having at least one reactor or IR at retest. Severe-level reinterpretation of the high-risk IR-only disclosing tests identified in this dataset would turn 68 out of the 590 (12 %) IRs into reactors, generating 23 incidents, the majority (19 or 83 %) part of the 265 incidents that would have been declared at the retest. Classification tree analysis used to identify IR-only high-risk tests in herds eligible for severe interpretation would enhance the sensitivity of the test-and-slaughter regime, cornerstone of the bTB eradication programme in England, further mitigating the risk of disease spread posed by IRs., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

Antibiotics are used in aquaculture to maintain the health and welfare of stocks; however, the emergence and selection of antibiotic resistance in bacteria poses threats to humans, animals and the environment. Mitigation of antibiotic resistance relies on understanding the flow of antibiotics, residues, resistant bacteria and resistance genes through interconnecting systems, so that potential solutions can be identified and issues around their implementation evaluated. Participatory systems-thinking can capture the deep complexity of a system while integrating stakeholder perspectives. In this present study, such an approach was applied to Nile tilapia ( Oreochromis niloticus ) production in the Nile Delta of Egypt, where disease events caused by antibiotic-resistant pathogens have been reported. A system map was co-produced with aquaculture stakeholders at a workshop in May 2018 and used to identify hotspots of antibiotic use, exposure and fate and to describe approaches that would promote fish health and thus reduce antibiotic use. Antibiotics are introduced into the aquaculture system via direct application for example in medicated feed, but residues may also be introduced into the system through agricultural drainage water, which is the primary source of water for most fish farms in Egypt. A follow-up survey of stakeholders assessed the perceived feasibility, advantages and disadvantages of potential interventions. Interventions that respondents felt could be implemented in the short-term to reduce antibiotic usage effectively included: more frequent water exchanges , regular monitoring of culture water quality parameters , improved storage conditions for feed , use of probiotics and greater access to farmer and service providers training programmes . Other potential interventions included greater access to suitable and rapid diagnostics , high quality feeds , improved biosecurity measures and genetically-improved fish , but these solutions were expected to be achieved as long-term goals, with cost being of one of the noted barriers to implementation. Identifying feasible and sustainable interventions that can be taken to reduce antibiotic use, and understanding implementation barriers, are important for addressing antibiotic resistance and ensuring the continued efficacy of antibiotics. This is vital to ensuring the productivity of the tilapia sector in Egypt. The approach taken in the present study provides a means to identify points in the system where the effectiveness of interventions can be evaluated and thus it may be applied to other food production systems to combat the problem of antibiotic resistance., Competing Interests: 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., (© 2021 The Authors.)

Nearly a decade into Defra's current eradication strategy, bovine tuberculosis (bTB) remains a serious animal health problem in England, with c.30,000 cattle slaughtered annually in the fight against this insidious disease. There is an urgent need to improve our understanding of bTB risk in order to enhance the current disease control policy. Machine learning approaches applied to big datasets offer a potential way to do this. Regularized regression and random forest machine learning methodologies were implemented using 2016 herd-level data to generate the best possible predictive models for a bTB incident in England and its three surveillance risk areas (High-risk area [HRA], Edge area [EA] and Low-risk area [LRA]). Their predictive performance was compared and the best models in each area were used to characterize herds according to risk. While all models provided excellent discrimination, random forest models achieved the highest balanced accuracy (i.e. average of sensitivity and specificity) in England, HRA and LRA, whereas the regularized regression LASSO model did so in the EA. The time since the last confirmed incident was resolved was the only variable in the top-ten ranking in all areas according to both types of models, which highlights the importance of bTB history as a predictor of a new incident. Risk categorisation based on Receiver Operating Characteristic (ROC) analysis was carried out using the best predictive models in each area setting a 99 % threshold value for sensitivity and specificity (97 % in the LRA). Thirteen percent of herds in the whole of England as well as in its HRA, 14 % in its EA and 31 % in its LRA were classified as high-risk. These could be selected for the deployment of additional disease control measures at national or area level. In this way, low-risk herds within the area considered would not be penalised unnecessarily by blanket control measures and limited resources be used more efficiently. The methodology presented in this paper demonstrates a way to accurately identify high-risk farms to inform a targeted disease control and prevention strategy in England that supplements existing population strategies., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

Identifying and understanding the risk factors for endemic bovine tuberculosis (TB) in cattle herds is critical for the control of this disease. Exploratory machine learning techniques can uncover complex non-linear relationships and interactions within disease causation webs, and enhance our knowledge of TB risk factors and how they are interrelated. Classification tree analysis was used to reveal associations between predictors of TB in England and each of the three surveillance risk areas (High Risk, Edge, and Low Risk) in 2016, identifying the highest risk herds. The main classifying predictor for farms in England overall related to the TB prevalence in the 100 nearest cattle herds. In the High Risk and Edge areas it was the number of slaughterhouse destinations and in the Low Risk area it was the number of cattle tested in surveillance tests. How long ago the last confirmed incident was resolved was the most frequent classifier in trees; if within two years, leading to the highest risk group of herds in the High Risk and Low Risk areas. At least two different slaughterhouse destinations led to the highest risk group of herds in England, whereas in the Edge area it was a combination of no contiguous low-risk neighbours (i.e. in a 1 km radius) and a minimum proportion of 6-23 month-old cattle in November. A threshold value of prevalence in 100 nearest neighbours increased the risk in all areas, although the value was specific to each area. Having low-risk contiguous neighbours reduced the risk in the Edge and High Risk areas, whereas high-risk ones increased the risk in England overall and in the Edge area specifically. The best classification tree models informed multivariable binomial logistic regression models in each area, adding statistical inference outputs. These two approaches showed similar predictive performance although there were some disparities regarding what constituted high-risk predictors. Decision tree machine learning approaches can identify risk factors from webs of causation: information which may then be used to inform decision making for disease control purposes., Competing Interests: Declaration of Competing Interest None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

The single intradermal comparative cervical tuberculin (SICCT) test is the primary test for ante-mortem diagnosis of bovine tuberculosis (TB) in England and Wales. When an animal is first classified as an inconclusive reactor (IR) using this test, it is not subject to compulsory slaughter, but it must be isolated from the rest of the herd. To understand the risk posed by these animals, a case-control study was conducted to measure the association between IR status of animals and the odds of them becoming a reactor to the SICCT at a subsequent test. The study included all animals from herds in which only IR animals were found at the first whole herd test in 2012 and used data from subsequent tests up until the end of 2016. Separate mixed-effects logistic regression models were developed to examine the relationship between IR status and subsequent reactor status for each risk area of England and for Wales, adjusting for other explanatory variables. The odds of an animal becoming a subsequent reactor during the study period were greater for IR animals than for negative animals in the high-risk area (odds ratio (OR): 6.85 (5.98-7.86)) and edge area (OR: 8.79 (5.92-13.04)) of England and in Wales (OR: 6.87 (5.75-8.22)). In the low-risk area of England, the odds were 23 times greater, although the confidence interval around this estimate was larger due to the smaller sample size (11-48, p < 0.001). These findings support the need to explore differential controls for IR animals to reduce the spread of TB, and they highlight the importance of area-specific policies.

Aquaculture systems are highly complex, dynamic and interconnected systems influenced by environmental, biological, cultural, socio-economic and human behavioural factors. Intensification of aquaculture production is likely to drive indiscriminate use of antibiotics to treat or prevent disease and increase productivity, often to compensate for management and husbandry deficiencies. Surveillance or monitoring of antibiotic usage (ABU) and antibiotic resistance (ABR) is often lacking or absent. Consequently, there are knowledge gaps for the risk of ABR emergence and human exposure to ABR in these systems and the wider environment. The aim of this study was to use a systems-thinking approach to map two aquaculture systems in Vietnam - striped catfish and white-leg shrimp - to identify hotspots for emergence and selection of resistance, and human exposure to antibiotics and antibiotic-resistant bacteria. System mapping was conducted by stakeholders at an interdisciplinary workshop in Hanoi, Vietnam during January 2018, and the maps generated were refined until consensus. Thereafter, literature was reviewed to complement and cross-reference information and to validate the final maps. The maps and component interactions with the environment revealed the grow-out phase, where juveniles are cultured to harvest size, to be a key hotspot for emergence of ABR in both systems due to direct and indirect ABU, exposure to water contaminated with antibiotics and antibiotic-resistant bacteria, and duration of this stage. The pathways for human exposure to antibiotics and ABR were characterised as: occupational (on-farm and at different handling points along the value chain), through consumption (bacterial contamination and residues) and by environmental routes. By using systems thinking and mapping by stakeholders to identify hotspots we demonstrate the applicability of an integrated, interdisciplinary approach to characterising ABU in aquaculture. This work provides a foundation to quantify risks at different points, understand interactions between components, and identify stakeholders who can lead and implement change., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The objective was to measure the association between badger culling and bovine tuberculosis (TB) incidents in cattle herds in three areas of England between 2013-2017 (Gloucestershire and Somerset) and 2015-2017 (Dorset). Farming industry-selected licensed culling areas were matched to comparison areas. A TB incident was detection of new Mycobacterium bovis infection (post-mortem confirmed) in at least one animal in a herd. Intervention and comparison area incidence rates were compared in central zones where culling was conducted and surrounding buffer zones, through multivariable Poisson regression analyses. Central zone incidence rates in Gloucestershire (Incidence rate ratio (IRR) 0.34 (95% CI 0.29 to 0.39, p < 0.001) and Somerset (IRR 0.63 (95% CI 0.58 to 0.69, p < 0.001) were lower and no different in Dorset (IRR 1.10, 95% CI 0.96 to 1.27, p = 0.168) than comparison central zone rates. The buffer zone incidence rate was lower for Gloucestershire (IRR 0.64, 95% CI 0.58 to 0.70, p < 0.001), no different for Somerset (IRR 0.97, 95% CI 0.80 to 1.16, p = 0.767) and lower for Dorset (IRR 0.45, 95% CI 0.37 to 0.54, p < 0.001) than comparison buffer zone rates. Industry-led culling was associated with reductions in cattle TB incidence rates after four years but there were variations in effects between areas.

Bovine tuberculosis (TB) is an important animal health issue in many parts of the world. In England and Wales, the primary test to detect infected animals is the single intradermal comparative cervical tuberculin test, which compares immunological responses to bovine and avian tuberculins. Inconclusive test reactors (IRs) are animals that demonstrate a positive reaction to the bovine tuberculin only marginally greater than the avian reaction, so are not classified as reactors and immediately removed. In the absence of reactors in the herd, IRs are isolated, placed under movement restrictions and re-tested after 60 days. Other animals in these herds at the time of the IR result are not usually subject to movement restrictions. This could affect efforts to control TB if undetected infected cattle move out of those herds before the next TB test. To improve our understanding of the importance of IRs, this study aimed to assess whether median survival time and the hazard of a subsequent TB incident differs in herds with only IRs detected compared with negative-testing herds. Survival analysis and extended Cox regression were used, with herds entering the study on the date of the first whole herd test in 2012. An additional analysis was performed using an alternative entry date to try to remove the impact of IR retesting and is presented in the Supplementary Material. Survival analysis showed that the median survival time among IR only herds was half that observed for clear herds (2.1 years and 4.2 years respectively; p < 0.001). Extended Cox regression analysis showed that IR-only herds had 2.7 times the hazard of a subsequent incident compared with negative-testing herds in year one (hazard ratio: 2.69; 95% CI: 2.54, 2.84; p < 0.001), and that this difference in the hazard reduced by 63% per year. After 2.7 years the difference had disappeared. The supplementary analysis supported these findings showing that IR only herds still had a greater hazard of a subsequent incident after the IR re-test, but that the effect was reduced. This emphasizes the importance of careful decision making around the management of IR animals and indicates that re-testing alone may not be sufficient to reduce the risk posed by IR only herds in England and Wales.

Culling badgers to control the transmission of bovine tuberculosis (TB) between this wildlife reservoir and cattle has been widely debated. Industry-led culling began in Somerset and Gloucestershire between August and November 2013 to reduce local badger populations. Industry-led culling is not designed to be a randomized and controlled trial of the impact of culling on cattle incidence. Nevertheless, it is important to monitor the effects of the culling and, taking the study limitations into account, perform a cautious evaluation of the impacts. A standardized method for selecting areas matched to culling areas in factors found to affect cattle TB risk has been developed to evaluate the impact of badger culling on cattle TB incidence. The association between cattle TB incidence and badger culling in the first 2 years has been assessed. Descriptive analyses without controlling for confounding showed no association between culling and TB incidence for Somerset, or for either of the buffer areas for the first 2 years since culling began. A weak association was observed in Gloucestershire for Year 1 only. Multivariable analysis adjusting for confounding factors showed that reductions in TB incidence were associated with culling in the first 2 years in both the Somerset and Gloucestershire intervention areas when compared to areas with no culling (incidence rate ratio (IRR): 0.79, 95% CI: 0.72-0.87, p  < .001 and IRR: 0.42, 95% CI: 0.34-0.51, p  < .001, respectively). An increase in incidence was associated with culling in the 2-km buffer surrounding the Somerset intervention area (IRR: 1.38, 95% CI: 1.09-1.75, p  = .008), but not in Gloucestershire (IRR: 0.91, 95% CI: 0.77-1.07, p  = .243). As only 2 intervention areas with 2 years of data are available for analysis, and the biological cause-effect relationship behind the statistical associations is difficult to determine, it would be unwise to use these findings to develop generalizable inferences about the effectiveness of the policy at present.

The aim of this study was to investigate the bacterial strains and farm environment that may contribute to the persistence of ESBL-producing E. coli on a single UK dairy farm. A longitudinal study was conducted comprising 6 visits, between August and October 2010, followed by a further visit at approximately 69weeks after the initial visit. Faecal and environmental samples were collected from different parts of the farm. The persistence and extent of faecal shedding of ESBL E. coli by individual calves was also determined. Twenty two different PFGE types were identified. Four of these were persistent during the study period and were associated with serotypes: O98, O55, O141 and O33. The counts suggest that shedding in calf faeces was an important factor for the persistence of strains, and the data will be useful for parameterising mathematical models of the spread and persistence of ESBL strains within a dairy farm., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

While much is known about the risk factors for bovine tuberculosis (bTB) in herds located in high incidence areas, the drivers of bTB spread in areas of emerging endemicity are less well established. Epidemiological analysis and intensive social research identified natural and social risk factors that may prevent or encourage the spread of disease. These were investigated using a case-control study design to survey farmers in areas defined as recently having become endemic for bTB (from or after 2006). Telephone surveys were conducted for 113 farms with a recent history of a bTB incident where their officially tuberculosis free status had been withdrawn (OTFW) (cases) and 224 controls with no history of a bTB incident, matched on location, production type and the rate of endemic bTB spread. Farmers were questioned about a range of farm management strategies, farm characteristics, herd health, wildlife and biosecurity measures with a focus on farmer attitudes and behaviours such as farmers' perception of endemicity and feelings of control, openness and social cohesion. Data generated in the telephone surveys was supplemented with existing herd-level data and analysed using conditional logistic regression. Overall, herd size (OR 1.07), purchasing an animal at a cattle market compared to purchasing outside of markets (OR 2.6), the number of contiguous bTB incidents (2.30) and the number of inconclusive reactors detected in the 2 years prior to the case incident (OR 1.95) significantly increased the odds of a bTB incident. Beef herds using a field parcel more than 3.2km away from the main farm and dairy herds reporting Johne's disease in the previous 12 months were 3.0 and 4.7 times more likely to have a recent history of a bTB incident, respectively. Beef herds reporting maize growing near, but not on, their farm were less likely to be case herds. Operating a closed farm in the two years prior to the case breakdown did not reduce the odds of a bTB incident. Farmers that had recently experienced a bTB incident were more likely to have implemented badger biosecurity in the previous year, but no more likely than control farms to have implemented cattle biosecurity. Case farmers felt significantly less likely to be influenced by government, vets or other farmers compared to those with no history of bTB. This suggests that alternative methods of engaging with farmers who have recently had a breakdown may need to be developed., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Between 2005 and 2007, E. coli obtained from clinical diagnostic submissions from cattle, goats, pigs and sheep to government laboratories in England and Wales were tested for sensitivity to 16 antimicrobials. Resistance was most commonly observed against ampicillin, streptomycin, sulphonamides and tetracyclines. Resistance levels varied significantly between species, with isolates from cattle frequently showing the highest levels. Verocytotoxigenic E. coli (VTEC) expressed less resistance than non-VTEC. Only 19·3% of non-VTEC and 43·5% of VTEC were susceptible to all antimicrobials, while 47·1% and 30·4%, respectively, were resistant to ⩾5 antimicrobials. The resistance phenotype SSuT was commonly observed, and isolates resistant to third-generation cephalosporins were also identified. We recommend judicious antimicrobial usage in the livestock industry in order to preserve efficacy.

Aims: The aims of this work were to develop a model of dairy farm waste milk and to investigate methods for the bioremediation of milk containing cefquinome residues., Methods and Results: Unpasteurized milk and UHT milk that had both been spiked with cefquinome at a concentration of 2 μg ml(-1) were used as a model for waste milk containing cephalosporin residues. Adjustment of the spiked UHT milk to pH 10 or treatment with conditioned medium from bacterial growth producing cefotaximase, were the most effective methods for decreasing the cefquinome concentrations within 24 h. A large-scale experiment (10 l of cefquinome-spiked unpasteurized milk) suggested that fermentation for 22 h at 37°C followed by heating at 60°C for 2 h was sufficient to decrease cefquinome concentrations to below the limit of quantification (<125 μg kg(-1) ) and to kill the majority of the enriched bacterial population., Conclusions: One or a combination of the bioremediation methods described may have potential as a practical treatment for dairy farm waste milk., Significance and Impact of the Study: Treatment of waste milk to decrease cephalosporin residue concentrations and also to kill bacteria prior to feeding to dairy calves could decrease the risk of selection for ESBL bacteria on dairy farms., (© 2015 Crown copyright. Journal of Applied Microbiology © 2015 The Society for Applied Microbiology. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.)

A mathematical method for estimating the endemic status of bovine tuberculosis (bTB) in cattle in England and Wales has been developed. 6.25km(2) hexagonal cells were used as the base resolution. Maps were produced for overlapping two-year periods spanning 2001/03 to 2009/11. Distance from a farm to the ten nearest 'Officially Tuberculosis Free status - Withdrawn' incidents within the same time period was measured. Endemic areas were defined as those hexagons containing farms where the 3rd nearest incident occurred within 7km. Temporal spread of endemic bTB was estimated by creating a contour map displaying the spread of endemic bTB over the two-year periods, and using boundary displacement to calculate the rate of spread across each hexagon. A rate was obtained for ∼2300 cells and varied between 0.04km and 15.9km per year (median=3.3km per year). This work will enable further analysis of the factors associated with this expansion., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

A longitudinal field trial was carried out on a farm known to harbour cefotaximase (CTX-M)-positive Escherichia coli, in order to assess the impact of feeding waste milk containing antibiotic residues (WM+AR) on the prevalence of these bacteria in the faeces of calves. Fifty calves were alternately assigned to one of two groups at birth and fed either milk replacer (control group) or WM+AR (treatment group). Faecal samples were collected from all calves daily for the first week after enrolment, twice weekly until weaning, then weekly for a further six weeks. Environmental samples from the calf housing were collected weekly. WM+AR and powdered milk samples were examined for antibiotic residues and CTX-M-positive E. coli. Total E. coli and CTX-M-positive E. coli in faecal samples were enumerated using selective media. Regression analyses were performed on the bacterial count data using a population-averaged approach based on generalised estimating equations (GEE) to account for repeated measurements on individual calves over time. Cefquinome, a fourth generation cephalosporin, was detected in 87% of WM+AR samples at a mean concentration of 0.746 mg/l. All environmental sampling locations yielded CTX-M-positive E. coli. Significantly more pen floor samples were positive in the treatment group. Calves in the treatment group shed greater numbers of CTX-M-positive E. coli than calves in the control group throughout the study, and shedding decreased at a slower rate in the treatment group. CTX-M-positive E. coli persisted in a larger number of calves fed WM+AR compared with calves fed milk replacer where the prevalence in the treatment group declined significantly slower over time. There was no difference between calves fed WM+AR or calves fed milk replacer in the proportion of E. coli isolates that were CTX-M-positive. These findings indicate that feeding WM+AR increased the amount of resistant bacteria shed in the faeces. Shedding of CTX-M-positive E. coli persisted for longer in calves fed WM+AR, and persisted after weaning., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

The cause for the high prevalence of cefotaximase-producing Escherichia coli reported in dairy calves is unknown but may be partly due to the selective pressure of antimicrobial residues in waste milk (milk unfit for human consumption) fed to the calves. Antimicrobial use and waste milk feeding practices were investigated in 557 dairy farms in 2010/2011 that responded to a randomised stratified postal survey. The mean number of cases of mastitis per herd in the previous year was 47, and 93 per cent of respondents used antibiotic intra-mammary tubes to treat mastitis. The most frequently used lactating cow antibiotic tubes contained dihydrostreptomycin, neomycin, novobiocin, and procaine penicillin (37 per cent), and cefquinome (29 per cent). Ninety-six per cent of respondents used antibiotic tubes at the cessation of lactation ('drying off'). The most frequently used dry cow antibiotic tube (43 per cent) contained cefalonium. Frequently used injectable antibiotics included tylosin (27 per cent), dihydrostreptomycin and procaine penicillin (20 per cent) and ceftiofur (13 per cent). Eighty-three per cent of respondents (413) fed waste milk to calves. Of these 413, 87 per cent fed waste milk from cows with mastitis, and only one-third discarded the first milk after antibiotic treatment. This survey has shown that on more than 90 per cent of the farms that feed waste milk to calves, waste milk can contain milk from cows undergoing antibiotic treatment. On some farms, this includes treatment with third- and fourth-generation cephalosporins. Further work is underway to investigate the presence of these antimicrobials in waste milk.

Many tumors overexpress the NQO1 gene, which encodes DT-diaphorase (NADPH:quinone oxidoreductase; EC 1.6.99.2). This obligate two-electron reductase deactivates toxins and activates bioreductive anticancer drugs. We describe the establishment of an isogenic human tumor cell model for DT-diaphorase expression. An expression vector was used in which the human elongation factor 1alpha promoter produces a bicistronic message containing the genes for human NQO1 and puromycin resistance. This was transfected into the human colon BE tumor line, which has a disabling point mutation in NQO1. Two clones, BE2 and BE5, were selected that were shown by immunoblotting and enzyme activity to stably express high levels of DT-diaphorase. Drug response was determined using 96-h exposures compared with the BE vector control. Functional validation of the isogenic model was provided by the much greater sensitivity of the NQO1-transfected cells to the known DT-diaphorase substrates and bioreductive agents streptonigrin (113- to 132-fold) and indoloquinone EO9 (17- to 25-fold) and the inhibition of this potentiation by the DT-diaphorase inhibitor dicoumarol. A lower degree of potentiation was seen with the clinically used agent mitomycin C (6- to 7-fold) and the EO9 analogs, EO7 and EO2, that are poorer substrates for DT-diaphorase (5- to 8-fold and 2- to 3-fold potentiation, respectively), and there was no potentiation or protection with menadione and tirapazamine. Exposure time-dependent potentiation was seen with the diaziquone analogs methyl-diaziquone and RH1 [2, 5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], the latter being an agent in preclinical development. In contrast to the in vitro potentiation, there was no difference in the response to mitomycin C when BE2 and BE vector control were treated as tumor xenografts in vivo. This isogenic model should be valuable for mechanistic studies and bioreductive drug development.

Three lipophilic quinazoline-based aminomethyl pyridine compounds, which differ only in the position of the nitrogen in their pyridine ring, are described. CB300179 (2-pyridine), CB300189 (4-pyridine) and CB30865 (3-pyridine) all inhibited isolated mammalian TS with IC50 values of 508, 250 and 156 nM respectively. CB30865 was the most potent growth inhibitory agent (IC50 values in the range 1-100 nM for several mouse and human cell types). CB300179 and CB300189 were active in the micromolar range. Against W1L2 cells, CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. Furthermore, combinations of precursors and end products of folate metabolism, e.g. dThd/hypoxanthine (HX) or leucovorin (LV), did not prevent activity. CB30865 did not interfere with the incorporation of tritiated dThd, uridine or leucine after 4 h. A cell line was raised with acquired resistance to CB30865 (W1L2:R865; > 200-fold), which was not cross-resistant to CB300179 or CB300189. In addition, W1L2:R865 cells were as sensitive as parental cells to agents from all the major chemotherapeutic drug classes. CB300179 and CB300189 induced an S phase accumulation (preventable by co-administration of dThd). No cell cycle redistribution was observed following exposure (4-48 h) to an equitoxic concentration of CB30865. In the NCI anticancer drug-discovery screen, CB30865 displayed a pattern of activity which was not consistent with known anti-tumour agents. These data suggest that CB30865 represents a class of potent potential anti-tumour agents with a novel mechanism of action.