Diagnosis of bovine mastitis: performance of chromogenic medium compared to convencional methods and MALDI-TOF as gold standard technique

Abstract

Bovine mastitis is a highly prevalent, multifactorial inflammatory disease affecting the mammary gland of dairy cattle. Brazil has one of the most competitive dairy industries worldwide, and the economic impact of mastitis represents a major challenge for the dairy production chain. The disease negatively affects producers through reduced milk quality and animal welfare, milk discard, and increased expenditures on antimicrobial treatments. The microorganisms most commonly associated with bovine mastitis include Staphylococcus spp. (S. aureus and non-aureus Staphylococcus [NAS]), Streptococcus spp. (S. agalactiae, S. dysgalactiae, S. uberis, among others), coliforms (Escherichia coli, Klebsiella spp., and Enterobacter spp.), and Pseudomonas spp. Accurate identification of these pathogens is essential for guiding antimicrobial use on dairy farms. In the first chapter of this dissertation, we evaluated the diagnostic performance [diagnostic sensitivity (DSe), diagnostic specificity (DSp), and accuracy (Ac)] of a commercial chromogenic culture system (Mamitest, inLab, Brazil) compared with the conventional microbiological culture method recommended by the National Mastitis Council (NMC). Additionally, agreement (Cohen’s kappa coefficient) between two evaluators, one highly experienced in microbiology and one novice was assessed when interpreting the rapid test results. A total of 632 milk samples were analyzed using both diagnostic methods. The evaluation of Ac, DSe, DSp, and inter-observer agreement demonstrated a high level of agreement for most outcomes (Kappa > 0.80). Furthermore, DSe values were satisfactory for the main pathogen groups, particularly Gram-positive bacteria [0.86 (95% CI: 0.82–0.89)], staphylococci [0.88 (95% CI: 0.82–0.93)], and streptococci [0.93 (95% CI: 0.88–0.96)]. In the second chapter, we expanded upon the previous study by comparing both diagnostic methods with a reference standard consisting of bacterial culture and isolation followed by identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Diagnostic performance was assessed through the calculation of DSe, DSp, and Ac using milk samples cryopreserved at −80°C. In this phase, 526 bacterial isolates were recovered, of which 490 were successfully identified and classified into 61 different species. The chromogenic culture system demonstrated good performance as a screening tool for the major bacterial groups, particularly Gram-positive and Gram-negative organisms. Conventional microbiological culture showed superior performance in species-level differentiation; however, it exhibited only moderate DSe values for some critical pathogens, including S. aureus, non-aureus Staphylococcus, and S. agalactiae. Overall, the results obtained with the evaluated chromogenic culture system support its use as an effective on-farm screening tool for milk samples. This approach can assist dairy producers and veterinarians in making timely treatment decisions, particularly by supporting more judicious antimicrobial use and contributing to antimicrobial stewardship in dairy herds.