Development of an on-farm test method to detect betalactam antibiotic residues in raw milk

Abstract

There exist different methods of determining antibiotic residues in milk. However, the ideal testing methods such as HPLC and rapid kits have been found to be expensive, takes long to give results and needs definite expertise posing a challenge to their utility among farmers and the cooperatives societies in the Kenyan milk industry. The Hardy Diagnostics Beta-Lactamase Test (HDBT) is recommended for testing beta-lactamase production by Neisseria gonorrhoeae, Haemophilus, and Staphylococcus species. HDBT is an acidometric method that utilizes a reagent consisting of penicillin, sodium chloride, trisodium citric acid, trisodium phosphate, and phenol red dissolved in distilled water. The objective of this study was to modify the ingredients of the HDBT reagent in order to develop a test method for detecting beta-lactam antibiotic residues in raw milk. The first part of the study involved modifying the composition of the ingredients to achieve the best colour differentiation between positive and negative raw milk samples, determining the appropriate mixing ratios for the raw milk and reagent, and investigating the impact of different breeds on the test results. The second part of the study focused on determining the shelf life of the test reagent for its applicability in the dairy sector. In the third part of the study, the sensitivity and specificity of four rapid antibiotic test methods were compared to the developed test method. Pooled raw milk samples were collected from 3 Friesian and 3 Ayrshire lactating cows that had previously been treated with beta-lactam antibiotics after being diagnosed to have subclinical mastitis. Trained panelists conducted evaluations to assess the colour differences between positive and negative raw milk samples in all experiments. The results demonstrated that the gradual addition of trisodium phosphate and phenol red in the reagent resulted in a significant difference (P ≤ 0.05) between positive and negative raw milk samples in terms of colour assessment. Additionally, mixing equal portions of milk and reagent showed a significant difference (P ≤ 0.05) compared to other proportions, and there was no significant difference (P ≤ 0.05) in the test method results between Friesians and Ayrshires raw milk samples. The second part of the study revealed that the reagent remained effective for 5 hours, but when used in powder form and packaged under semi-vacuum conditions, it had a shelf life of over 4 months. There was no significant difference (P ≤ 0.05) between the results obtained using the powder and reagent. In the third part of the study, the developed test method exhibited a sensitivity of 66.7% and a specificity of 100%, while the other test methods achieved a sensitivity and specificity of 100% each. The Kappa coefficient, according to the Landis- Koch scale, indicated a moderate agreement of 0.5882 between the developed test method and the other test methods. A fuchsia purple colour indicated a beta-lactam negative sample, while peach or pink colour indicated a positive sample. This test method can be employed along raw milk collection routes to accept, set aside, or reject raw milk suspected of containing antibiotic residues. Further research on the developed test method can be carried out to investigate the possibility of obtaining quantitative results.