Multi-drug residues and antimicrobial resistance patterns in waste milk from dairy farms in Central California PN Tempini 1, SS Aly1,2, BM Karle3, RV Pereira1 1 Department of Population Health and Reproduction, School of Veterinary Medicine, UC Davis, Davis, CA. 2 Veterinary Medicine Teaching and Research Center, School of Veterinary Medicine, UC Davis, Tulare, CA. 3 Cooperative Extension, Division of Agriculture and Natural Resources, University of California, Orland,CA

Introduction Waste milk in dairy farms Drug residues in Waste Milk Tolerance of Drug Residues in Milk Source : cdc.gov; en.wikipedia.org; enasco.com

Introduction Waste milk in dairy farms Drug residues in Waste Milk 1 out of 3 Dairy Farms (USDA, 2008) $0.69 savings per calf per day (Godden et al., 2005) Health Hazards? Source : allontario.ca ;

Introduction Feeding trial: Impacts of feeding milk with drug residues No drug residues Added drugs at low concentrations Impacts on Antimicrobial Resistance β-lactams & Tetracycline Cephalosporins Multidrug Resistant Source: Pereira RV et al. PloS one. 2014;9(12):e115223;

Objective Identify and measure the concentration of antimicrobial residues in Waste milk (WM) samples on dairy farms in the Central Valley of California; Survey farm management practices for factors that could suggest occurrence of specific antimicrobial residues in WM Characterize the antimicrobial resistance patterns of E. coli cultured from WM samples. http://photosinbox.com/sign-and-symbol/question-symbol Source : shutterstock.com; photosinbox.com

Drug Screening of Waste Milk Material & Methods Drug Screening of Waste Milk Waste Milk Samples: 25 farms Multi-Residues Screening & Quantification LC-MS/MS On-Farm Questionnaire Microbiological Testing Source :; johnlund.com; farmbuildingguide.org; agilent.com

Results & Discussion Description Distribution (%) Table. Distribution of drug residues in raw waste milk samples detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS) Description Distribution (%) LC-MS/MS test (n = 25) Positive Negative   15 10 Distribution by drug class Only β-lactams Only Sulfamethazine Only Tetracyclines β-Lactams and Tetracycline 1 3

Results & Discussion Table. Drug residues in waste milk measured by LC-MS/MS and distributed by individual drug class. Drug Residue Number Mean SE Tolerance   (%) (ng/mL) β-Lactams: 11 (44) Ceftiofur 7 (28) 251.14 177.0 100 Cephapirin 3 (12) 39 24.5 20 Penicillin G 1 (4) 10 - Sulfonamides: Sulfadimethoxine 7 Tetracyclines: 4 (16) Tetracycline 590 300 Oxytetracycline 378.5 308.7

Results & Discussion Ceftiofur 13 22 19 10 17 Penicillin G 6 1 9 8 3 Table. Answers to “what are the two most common drugs used for/to treat”: dry cow treatment, mastitis, reproductive diseases, lameness and pneumonia (BRD). Antimicrobial Agent Dry cow TX Mastitis Reproduct. Diseases Lameness BRD Ceftiofur 13 22 19 10 17 Penicillin G 6 1 9 8 3 Tetracycline Cephapirin Amoxicillin 7 2 4 Oxytetracycline 5 Sulfadimethoxine Other Drugs   No Treatment

Non-fermentative gram Staphylococcus aureus Results & Discussion Table. Milk culture results for waste milk samples collected at dairy farms. Bacteria Positives (%) (n = 25) Streptococcus spp. 21 (84) Staphylococcus spp. 20 (80) Escherichia coli 10 (40) Non-fermentative gram negatives 8 (32) Staphylococcus aureus 5 (20) Bacillus spp. Klebsiella spp. 4 (16) Trueperella pyogenes 3 (12) Mycoplasma spp. 2 (8) Enterobacter spp. 1 (4) Prothoteca spp. Yeasts Antimicrobial % Resistant β-Lactams Ampicillin 30 Cephalothin 20 Ceftiofur 10 Tetracycline Sulfonamides Sulfisoxazole

Conclusion LC-MS/MS revealed that ceftiofur was the most prevalent detectable drug residues, followed by cephapirin and tetracycline. Multiple bacteria cultured were from waste milk, supporting the importance of pasteurization before feeding. Furthermore, the most common antimicrobials drug for which E. coli was resistant was ampicillin. Most common drug used to treat sick cows on the farm was ceftiofur, followed by penicillin G and tetracycline. Ceftiofur was most the common drug used to treatment mastitis on farms (88%), followed by cephapirin (40%).

Questions / Comments Funding Sources: U.S. Department of Agriculture Project number CA-V-PHR-4708-AH408