Concentrations and remediation of cephalosporin residues in waste milk from dairy farms in England and Wales

Robert A. Horton*1, Victoria Bailey-Horne2, Meenaxi Sharma1, Luke P. Randall1, Lucy Brunton1, Jeffrey R. Jones1, Roberto La Ragione1, Katharina Heinrich2 and Matthew Sharman2 1Animal Health and Veterinary Laboratories Agency (AHVLA), Addlestone, Surrey, KT15 3NB, UK 2The Food and Environment Research Agency, Sand Hutton, York, YO41 1LZ, UK

Introduction and Purpose Waste milk is milk that is not suitable for human consumption, often because cows have been treated with antimicrobial drugs, and antimicrobial residues are therefore present in milk. The feeding of waste milk to calves is a common practice on dairy farms. It has been suggested that cephalosporin residues in waste milk could select for cephalosporin resistant E. coli in the calves' gut. Therefore the objectives of this study were: • To determine the concentrations of cephalosporin residues in waste milk from dairy farms in England and Wales. • To investigate the degradation of cefquinome in spiked-unpasteurised milk by heat treatment.

Methods • Samples of waste milk were collected from 103 dairy farms in England and Wales, together with information on the antibiotics administered to the cows. • The concentrations of cephalosporin residues in samples of waste milk were measured. • The degradation of cefquinome in fresh, unpasteurised milk was determined over 10 days using milk that had been spiked with cefquinome sulphate (final cefquinome concentration ~2 µg/ml). • 2x10 ml aliquots of spiked milk were maintained at approximately 4°C, 18°C, 37°C or 50°C (± 2°C) throughout the study. • Aliquots were sampled at the following times 0, 2, 4, 6, 8, 24, 48, 72, 120, and 240 hours, and were stored frozen at -80°C prior to analysis for cefquinome residues. • All residue analysis was performed quantitatively by liquid chromatography–tandem mass spectrometry.

Results Waste milk cephalosporin residues survey • Cefquinome (a 4th generation cephalosporin) was the most prevalent cephalosporin detected in waste milk, being detected in ~21% of samples tested (Table 1). • For cefquinome positive samples, the mean cefquinome concentration was ~1.5 µg/ml (range from 0.006 – 4.7 µg/ml). • Cefalonium, cefalexin and cefapirin (all first generation cephalosporins) were detected in 7.8%, 5.8%, and 2.9% of samples respectively. • There was good agreement with the reported use of cephalosporins and the detection of cephalosporin residues (agreement for 95% of samples tested). Temperature mediated degradation of cefquinome in spiked milk • Cefquinome concentrations were unaffected by incubation at 4°C. • Incubation at higher temperatures caused a decrease in cefquinome concentrations over time. • At the 24 hour time point, 37° C was the most effective temperature for cefquinome degradation.

Table 1 Summary of cephalosporin concentrations in waste 3.0 milk from 103 dairy farms in England and Wales 2.5

Cephalosporin concentration in waste milk (µg/ml)a Cephalosporin reportedly 2.0 Farm used in cows contributing ID to sample cefalexin cefalonium Cefapirin cefquinome 9 cefquinome * * * 3.914 4°C 13 cefalexin 0.041 * * * 1.5 16 cefapirin, cefqinome * * 0.005 0.618 18°C 20 cefalexin 0.005 * * * 37°C 23 cefquinome * * * 0.028 1.0 26 none reported 0.010 * * * 50°C 27 cefquinome * * * 3.605

30 cefquinome * * * 1.339 cefquinome mean 31 cefquinome * * * 2.575 0.5 concentration (µ g/ml) 38 cefquinome * * * 1.442 42 cefquinome * * * 0.896 43 cefquinome * * * 4.738 0.0 47 cefquinome * * * 0.024 48 cefalonium * 0.036 * * 49 cefquinome * * * 0.330 0 24 48 72 96 120 144 168 192 216 240 264 288 50 cefalonium * 0.072 * * 51 none reported * * * 0.006 Time (hours) cefalexin, cefquinome and 54 cefalonium * 0.013 * 0.855 55 cefquinome * 0.597 * * 57 cefquinome, cefalonium * 0.018 * 1.133 58 cefquinome * * * 0.608 Figure 1 Temperature mediated degradation of cefquinome 61 cefalexin 0.070 * * * in spiked-unpasteurised milk. All data points are means of 62 cefalexin 0.680 * * * 63 cefquinome * * * 4.223 n=2 replicates 64 cefquinome * * * 0.134 69 cefquinome * * * 0.065 74 cefquinome * * * 2.060 78 cefalonium, cefquinome * 0.042 * * 84 cefquinome * * * 2.266 86 cefquinome * * * 1.339 90 cefalexin 0.008 * * * 92 cefquinome * * * 0.134 Conclusions 97 none reported * 0.019 * * • The survey of a limited number of dairy farms in England and 100 cefapirin * * 0.144 * Wales confirms that cephalosporin residues are present in waste milk from a proportion of those farms studied. 101 cefapirin * * 2.575 * • The results from the temperature mediated degradation of 102 none reported * 0.006 * * Mean concentration (µg/ml) 0.136 0.100 0.908 1.470 cefquinome experiment indicate that incubating milk at ~18°C Proportion of positive results from 5.8 7.8 2.9 21.4 for 72 hours or at ~37°C for 24 hours can effectively decrease 103 farms (%) the concentration of cefquinome in spiked-unpasteurised milk. Proportion of unexpected positive 1.0 2.9 0.0 1.0 samples (%)b • This study has identified a potential method for decreasing the concentration of cefquinome residues in waste milk and further * indicates below detection limit (~ 0.004 µg/ml). work is required to assess whether this can be used practically a Data obtained originally as µg/kg milk and converted to µg/ml by a factor of 1.03/1000. i.e. assuming density of milk to be 1.03 g/ml. on farm. b waste milk samples found to be positive for cephalosporin residues from farms without reported use of cephalosporins *Correspondence: [email protected] Acknowledgments: UK Government, Department for Environment, Food and Rural Affairs and the Veterinary Medicines Directorate funded project OD2031.