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AimsTo investigate whether on-farm antibacterial usage (ABU), environmental antibacterial resistant (ABR) Escherichia coli prevalence, and sampling and sample handling methodologies are associated with ABR E. coli positivity in individual faecal samples from dairy heifers.Methods and ResultsThree hundred and sixty-four heifers from 37 farms were sampled via rectal or faecal pat sampling. Samples were stored at -80°C for variable periods before microbiological analysis. Data analysis was through a multilevel, multivariable logistic regression approach.Individual rectal samples had increased odds of positivity for amoxicillin, cefalexin and tetracycline-resistant E. coli. Sample storage for 6-12 month was associated with decreased odds of finding amoxicillin and tetracycline-resistant E. coli. On-farm ABU had little influence, and environmental ABR E. coli prevalence had no significant influence on on the odds of sample-level positivity for ABR E. coli.ConclusionsSampling methodology and sample handling have a greater association than on-farm factors with the detection of ABR E. coli in individual faecal samples from dairy heifers.Significance and Impact of the StudySampling and storage methodologies should be considered carefully at the point of designing ABR surveillance studies in livestock and their environments and, where possible, standardised between and within future studies.

Third-generation cephalosporin resistance (3GC-R) in Escherichia coli is a rising problem in human and farmed animal populations. We conducted whole genome sequencing analysis of 138 representative 3GC-R isolates previously collected from dairy farms in South West England and confirmed by PCR to carry acquired 3GC-R genes. This analysis identified blaCTX-M (131 isolates: encoding CTX-M-1, −14, −15, −32 and the novel variant, CTX-M-214), blaCMY-2 (6 isolates) and blaDHA-1 (one isolate). A highly conserved plasmid was identified in 73 isolates, representing 27 E. coli sequence types. This novel ~220 kb IncHI2 plasmid carrying blaCTX-M-32 was sequenced to closure and designated pMOO-32. It was found experimentally to be stable in cattle and human transconjugant E. coli even in the absence of selective pressure and was found by multiplex PCR to be present on 26 study farms representing a remarkable range of transmission over 1500 square kilometres. However, the plasmid was not found amongst human urinary E. coli we have recently characterised from people living in the same geographical location, collected in parallel with farm sampling. There were close relatives of two blaCTX-M plasmids circulating amongst eight human and two cattle isolates, and a closely related blaCMY-2 plasmid found in one cattle and one human isolate. However, phylogenetic evidence of recent sharing of 3GC-R strains between farms and humans in the same region was not found.ImportanceThird-generation cephalosporins (3GCs) are critically important antibacterials and 3GC-resistance (3GC-R) threatens human health, particularly in the context of opportunistic pathogens such as Escherichia coli. There is some evidence for zoonotic transmission of 3GC-R E. coli through food, but little work has been done examining possible transmission (e.g. via interaction of people with the local near-farm environment). We characterised acquired 3GC-R E. coli found on dairy farms in a geographically restricted region of the United Kingdom and compared these with E. coli from people living in the same region, collected in parallel. Whilst there is strong evidence for recent farm-to-farm transmission of 3GC-R strains and plasmids – including one epidemic plasmid that has a remarkable capacity to transmit – there was no evidence that 3GC-R found on study farms had a significant impact on circulating 3GC-R E. coli strains or plasmids in the local human population.

ObjectivesTo characterise putative AmpC hyper-producing 3rd generation cephalosporin-resistant E. coli from dairy farms and their phylogenetic relationships as well as to identify risk factors for their presence; to assess evidence for their zoonotic transmission into the local human populationMethodsProteomics was used to explain differences in antimicrobial susceptibility. Whole genome sequencing allowed phylogenetic analysis. Multilevel, multivariable logistic regression modelling was used to identify risk factors.ResultsIncreased use of amoxicillin-clavulanate was associated with an increased risk of finding AmpC hyper-producers on farms. Expansion of cephalosporin resistance in AmpC hyper-producers was seen in farm isolates with marR mutations (conferring cefoperazone resistance) or when AmpC was mutated (conferring 4th generation cephalosporin and cefoperazone resistance). Phylogenetic analysis confirmed the dominance of ST88 amongst farm AmpC hyper-producers but there was no evidence for acquisition of farm isolates by members of the local human population.ConclusionsIn this two-year surveillance study of 53 dairy farms, AmpC hyper-production was the cause of cefotaxime resistance in 46.2% of E. coli. There was evidence of recent farm-to-farm transmission and of adaptive mutations to expand resistance. Whilst there was no evidence of isolates entering the local human population, efforts to reduce 3rd generation cephalosporin resistance on dairy farms must address the high prevalence of AmpC hyper-producers. The finding that amoxicillin-clavulanate use was associated with increased risk of finding AmpC hyper-producers is important because this is not currently categorised as a highest-priority critically important antimicrobial and so is not currently targeted for specific usage restrictions in the UK.

Little is known about the drivers of critically important antibacterial resistance in species with zoonotic potential present on farms (e.g. CTX-M □-lactamase-positive Escherichia coli). Here, we collected samples, monthly over a two-year period, on 53 dairy farms in the South West of England, and data for 610 variables concerning antimicrobial usage, management practices and meteorological factors. We detected E. coli resistant to amoxicillin, ciprofloxacin streptomycin and tetracycline, respectively, in 2754/4145 (66%), 263/4145 (6%), 1475/4145 (36%) and 2874/4145 (69%) of all samples from faecally contaminated sites. E. coli positive for blaCTX-M were detected in 224/4145 (5.4%) of samples. Multilevel, multivariable logistic regression showed antibiotic dry cow therapeutic choice (including use of cefquinome or framycetin) to be associated with increased odds of blaCTX-M positivity. Low temperature was associated with reduced odds of blaCTX-ME. coli positivity in samples and to reduced odds of finding E. coli resistant to each of the four test antibacterials. This was additional to the effect of temperature on total E. coli density. Furthermore, samples collected close to calves had increased odds of having E. coli resistance to each antibacterial or positive for blaCTX-M. Samples collected on pastureland had reduced odds of having E. coli resistant to amoxicillin or tetracycline, and being positive for blaCTX-M.ImportanceAntibacterial resistance poses a significant threat to human and animal health and global food security. Surveillance for resistance on farms is important for many reasons, including to track the impacts of interventions aimed at reducing the prevalence of resistance. In this epidemiological survey of dairy farm antibacterial resistance, we show that local temperature, as it changes over the course of a year, is associated with the prevalence of antibacterial resistant E. coli. Also, that prevalence of resistant E. coli is higher in indoor environments and in environments inhabited by young animals. These findings have profound implications for routine surveillance and for surveys carried out for research. They provide important evidence that sampling at a single time-point and/or single location on a farm is unlikely to be adequate to accurately determine the status of the farm with regard to the presence or number of resistant E. coli.

IntroductionKnowledge of accurate weights of cattle is crucial for effective dosing of individual animals with medicine and for reporting antimicrobial usage metrics, amongst other uses. The most common weight for dairy cattle presented in current literature is 600 kg, but this is not evidenced by data. For the first time, we provide an evidence-based estimate of the average weight of UK dairy cattle to better inform decisions by farmers, veterinarians and the scientific community.MethodsWe collected data for 2,747 dairy cattle from 20 farms in the UK, 19 using Lely Automatic Milking Systems with weigh floors and 1 using a crush with weigh scales. These data covered farms with different breed types, including Holstein, Friesian, Holstein-Friesian and Jersey, as well as farms with dual purpose breeds and cross-breeds. Data were used to calculate a mean weight for dairy cattle by breed, and a UK-specific mean weight was generated by scaling to UK-specific breed proportions. Trends in weight by lactation number, DIM and production level were also explored using individual cattle-level data.ResultsMean weight for adult dairy cattle included in this study was 617 kg (standard deviation (sd) 85.6 kg). Mean weight varied across breeds, with a range of 466 kg (sd=56.0 kg, Jersey) to 636 kg (sd=84.1, Holsteins). When scaled to UK breed proportions, the estimated mean UK dairy cattle weight was 620 kg. Overall, first-lactation heifers weighed 9% less than cows. Mean weight declined for the first 30 days post-calving, before steadily increasing. For cattle at peak production, mean weight increased with production level.ConclusionsThis study is the first to calculate a mean weight of adult dairy cattle in the UK based on on-farm data. Overall mean weight was higher than that most often proposed in the literature (600 kg). Evidence-informed weights are crucial as the UK works to better monitor and report metrics to monitor antimicrobial use and are useful to farmers and veterinarians to inform dosing decisions.

The issue of antimicrobial resistance is of global concern across human and animal health. In 2016 the UK government committed to new targets for reducing antimicrobial use (AMU) in livestock. However, though a number of metrics for quantifying AMU are defined in the literature, all give slightly different interpretations.This paper reviews a selection of metrics for AMU in the dairy industry: total mg, total mg/kg, daily dose and daily course metrics. Although the focus is on their application to the dairy industry, the metrics and issues discussed are relevant across livestock sectors.In order to be used widely, a metric should be understandable and relevant to the veterinarians and farmers who are prescribing and using antimicrobials. This means that clear methods, assumptions (and possible biases), standardised values and exceptions should be published for all metrics. Particularly relevant are assumptions around the number and weight of cattle at risk of treatment and definitions of dose rates and course lengths; incorrect assumptions can mean metrics over- or under-represent AMU.The authors recommend that the UK dairy industry work towards UK-specific metrics using UK-specific medicine dose and course regimens as well as cattle weights in order to monitor trends nationally.