Cambios en analitos de leche en vacas con mamitis clínica y subclínica: un estudio piloto
PEDRO JAVIER VALLEJO MATEO1, Anelise Salina2, Camila Peres Rubio3, Lorena Franco Martínez4, Jose Joaquín Cerón Madrigal5, María Dolores Contreras Aguilar5
(1)-Department of Animal Medicine and Surgery, Veterinary School, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, Campus de Espinardo, Espinardo, Murcia 30100, Spain (2)-Departamento de produção animal e medicina veterinária preventive, FMVZ-UNESP Botucatu, Brasil (3)-Interdisciplinary Laboratory of Clinical Analysis of the University of Murcia (Interlab-UMU), Department of Animal Medicine & Surgery, Veterinary School, Campus Mare Nostrum, University of Murcia, Murcia 30100, Spain
(4)-Interdisciplinary Laboratory of Clinical Analysis of the University of Murcia (Interlab-UMU), Department of Animal Medicine & Surgery, Veterinary School, Campus Mae Nostrum, University of Murcia,Murcia 30100, Spain. Laboratory of Proteomics, Clinic for Internal Diseases, Faculty of Veterinary Medici (5)-Department of Animal Medicine and Surgery, Veterinary School, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, Campus de Espinardo, Espinardo, Murcia 30100, Spain. Interdisciplinary Laboratory of Clinical Analysis of the University of Murcia (Interlab-UMU), Depa
Early and reliable mastitis detection in dairy herds requires diagnostic approaches that are simple, low-cost, and analytically robust. From a welfare perspective, there is additional value in techniques based on minimally invasive sampling and low-stress handling, ideally using routinely obtainable fluids. Building on the concept of adapting routine biochemical panels to alternative matrices (e.g., “sialochemistry”), milk may provide a practical source of biomarkers for both clinical and subclinical mastitis.
We hypothesised that biochemical changes in bovine milk could serve as diagnostic biomarkers for bovine mastitis. Therefore, this study aims (1) to analytically validate, using spectrophotometric techniques, a 17-analyte panel integrating enzymes, metabolites, and proteins in bovine milk whey; (2) to evaluate potential changes in these analytes in dairy cows with clinical or subclinical mastitis.
For this purpose, 18 dairy cows were enrolled as healthy (H group, n=6), clinical mastitis (C-M group, n=6) and subclinical mastitis (SC-M group, n=6), defined as clinical vs. subclinical mastitis when a somatic cell count >200,000 cells/mL and positive PCR and microbiological culture for Mollicutes spp. and/or Mycoplasma bovis results in milk were observed with or without sick signs and/or milk changes compatible with mastitis, respectively; and as healthy when no sick signs and milk changes compatible with mastitis were observed. The milk was collected and stored at −20 °C. Then, milk serum was obtained by collecting the supernatant after centrifugation at 10,000g and 4 °C for 30 min, and stored at -80 °C until analysis. The analytes measured were creatinine, albumin, calcium, amylase, aspartate aminotransferase (AST), lipase, triglycerides, γ-glutamyl transferase (GGT), lactate, uric acid, creatine kinase (CK), lactate dehydrogenase (LDH), total proteins, adenosine deaminase (ADA), total esterase activity (TEA), ferritin and unsaturated iron-binding capacity (UIBC), using spectrophotometric colorimetric assays adapted to an automated clinical chemistry analyser.
The proposed analytes measured in serum milk showed adequate intra-assay precision and a high correlation coefficient (mean R2 = 0.994 ± 0.0108) during the dilution study. In addition, in this pilot cohort study, 10 analytes showed significant differences between groups. Compared with healthy cows, the C-M group showed increases in amylase (median [interquartile ranges (IQR)] = 1.77 [1.61-8.39] vs. 0.00 [0.00-0.00] UI/L; P = 0.002), AST (mean ± Standard deviation [SD] = 280.6 ± 201.70 vs. 7.4 ± 2.65 UI/L; P = 0.007), triglycerides (median [IQR] = 163.6 [44.72-334.63] vs. 36.0 [30.55-41.01] mg/dL; P = 0.039), lactate (median [IQR] = 3.36 [1.19-5.88] vs. 0.04 [0.03-0.04] mmol/L; P < 0.001), CK (mean ± SD = 186.8 ± 148.19 vs. 0.7 ± 1.67 UI/L; P = 0.014), LDH (median [IQR] = 3,942.0 [2,705.60-4,662.90] vs. 115.8 [93.5-105.10] UI/L; P < 0.001), ADA (median [IQR] = 34.20 [7.68-66.28] vs. 0.00 [0.00-0.00] UI/L; P < 0.001) and ferritin (median [IQR] = 1,155.0 [260.25-2,880.00] vs. 65.0 [2.00-263.00] µg/L; P = 0.009). Decreases between the C-M vs. H groups were observed in creatinine (mean ± SD = 1.39 ± 0.31 vs. 1.79 ± 0.16 mg/dL; P = 0.036) and lipase (mean ± SD = 49.6 ± 42.18 vs. 173.8 ± 76.65 mg/dL; P = 0.005). Finally, similar changes to those observed in the H group were also observed between the C-M vs. SC-M groups in AST (mean ± Standard deviation [SD] = 280.6 ± 201.70 vs. 28.2 ± 24.42 UI/L; P = 0.012), lactate (median [IQR] = 3.36 [1.19-5.88] vs. 0.06 [0.04-0.17] mmol/L; P = 0.003), CK (mean ± SD = 186.8 ± 148.19 vs. 5.6 ± 5.47 UI/L; P = 0.015), LDH (median [IQR] = 3,942.0 [2,705.60-4,662.90] vs. 182.3 [103.03-210.73] UI/L; P < 0.001), ADA (median [IQR] = 34.20 [7.68-66.28] vs. 0.00 [0.00-0.00] UI/L; P < 0.001) and ferritin (median [IQR] = 1,155.0 [260.25-2,880.00] vs. 00.0 [00.0-00.0] µg/L; P < 0.001).
In conclusion, in this pilot study, all 17 evaluated analytes demonstrated to be able to be measured in bovine milk serum using economical spectrophotometric methods on an automated analyser. In addition, at least 10 analytes in serum milk showed differences between healthy cows and cows with clinical mastitis, with potential diagnostic value for mastitis of AST, lactate, CK, LDH, ADA, and ferritin. Nevertheless, these findings warrant confirmation in a larger population.
