Evaluación de la liberación de endotoxinas bacterianas inducida por antibióticos utilizados en el tratamiento de mamitis en ganado bovino

Résumé

Mastitis is one of the most common diseases in dairy cattle and causes significant economic losses in the dairy industry worldwide, as well as negative effects on animal welfare. It is characterized by an inflammatory reaction of the mammary gland that produces significant changes in the biochemical composition of milk, being associated with infections by both Gram-negative and Gram-positive bacteria. The release of endotoxins associated with bacterial lysis is a key factor in the clinical course of mastitis caused by Gram-negative bacteria and should be taken into consideration when using antibiotics in the management of these infections. Therefore, the detection of endotoxins in milk samples would be of great interest in the management of bovine mastitis. Similarly, the study of the inhibition kinetics of different pathogenic bacteria as well as endotoxin release kinetics after exposure to different antibiotics would provide relevant information for the clinical approach of this important disease. In this PhD thesis we have optimized a kinetic turbidimetric assay based on Limulus amebocyte lysate (LAL) for the quantification of endotoxins in milk samples, introducing into the procedure a pre-treatment by filtration through a polyvinyl filter and dilution in order to minimize the interferences associated with this complex matrix. In a second stage, we have investigated, using this technique, the effect of some antibiotics commonly used in the treatment of bovine mastitis on bacterial growth and endotoxins release, using a milk culture system or mini-udder cultures which reproduce the conditions of the infection of the mammary gland. The optimized endotoxin detection and quantification assay was validated following the indications established by the standards of the International Organization for Standardization and was used for the determination in milk samples from animals with clinical mastitis associated with both Gram-negative and Gram-positive bacteria, in bulk milk and in commercial UHT (ultra high temperature) treated milk. Our results demonstrate the robustness and usefulness of this assay for the quantification of endotoxins in a wide range of concentrations, allowing the identification of coliform mastitis in milk samples from animals with clinical mammary gland disease, as well as detecting endotoxins in bulk milk or even in some commercial milks. The experimental model or mini-udder cultures allow the reenactment of the conditions of an intramammary infection with two species of Gram-negative bacteria, previously isolated from the milk of cows with mastitis, Escherichia coli and Enterobacter aerogenes, and to assess the effect of the exposition to four antibiotics commonly used in the management of clinical mastitis in cattle: benzylpenicillin, dihydrostreptomycin, cephalosporin and cephapirin. The kinetics of growth inhibition and endotoxin release were investigated in these cultures and compared with the kinetics determined in controls with identical conditions, but without antibiotic treatment. As expected, all antibiotics tested caused a significant reduction in growth, more pronounced after 3-4 hours of exposure, with differences in these inhibition kinetics depending on each antibiotic and its mode of action. Furthermore, all antibiotics produced a release of endotoxins compared to the controls which also varied depending on the antibiotic. Thus, exposure to cephalosporins was associated with a slower bacterial death and, at the same time, a greater release of endotoxins, probably as a result of bacterial filamentation phenomena due to changes in the morphology of the bacterial cell membrane. In contrast, dihydrostreptomycin combined a quick bactericidal activity with a reduced release of endotoxin, probably as a result of rapid cell lysis.