RESUMEN
Whole blood stimulation assay (WBA) with killed gram-positive and gram-negative udder pathogens were used to investigate the interference of the endotoxin-binding antibiotic polymyxin B (PMB) on the ex vivo TNF-α response. Blood samples were collected from first to third lactating dairy cows in their early lactation (<50 days in milk, n = 32) period. The WBA was stimulated with both inactivated bacteria (e.g., dead Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis), at a concentration of 2.5 × 106/mL; and pathogen-associated molecular pattern molecules, namely E. coli LPS (10 µg/mL), and S. aureus peptidoglycan (PG, 10 µg/mL). The PMB was added at a concentration of 0, 12.5, 25, 50, 100, and 200 µg/mL to each stimulant, respectively. All bacteria stimulants resulted in an increased TNF-α response compared to the negative control. The PMB affected the TNF-α responses of gram-positive (except S. dysgalactaie), gram-negative bacteria; and bacterial cell wall components at a PMB concentration of 25-50 µg/mL. The LPS and E. coli had similar TNF-α response but PG had a lower TNF-α response than gram-positive bacteria. The doses of PMB (≥ 25 µg/mL) should be used with caution when using different types of pathogens or should be avoided in ex vivo TNF-α studies.
Asunto(s)
Bovinos , Leucocitos/efectos de los fármacos , Polimixina B/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proteínas Bacterianas/inmunología , Citocinas/genética , Citocinas/metabolismo , Femenino , Lipopolisacáridos/toxicidadRESUMEN
A whole blood stimulation assay was used to investigate the effects of parity, number of weeks after calving and Gram-positive and Gram-negative bacteria on the ex vivo TNF-α responsiveness of Danish Holstein-Friesian cows of first to third lactation (n = 28). Blood samples were collected in weeks 2, 3, 5 and 8 after parturition and stimulated with Escherichia coli LPS (10 µg/mL), Staphylococcus aureus peptidoglycan (PGN, 10 µg/mL) and dead Escherichia coli, Streptococcus uberis, Staphylococcus aureus, and Streptococcus dysgalactiae at a concentration of 2.5 × 106/mL. The antibiotic polymyxin-B (100 µg/mL) was added to the Gram-positive bacteria to avoid the influence of environmental endotoxin by ELISA test. Overall, parity had no effect, whereas number of weeks after calving altered the TNF-α responsiveness of the majority of the stimulants. Ex vivo, Gram-positive bacteria always resulted in a higher TNF-α response than Gram-negative bacteria with large differences within the individual cows. High correlations were found within the Gram-negative stimulants panel (r = 0.83) and within the Gram-positive (r = 0.81 to 0.86) stimulants panel except PGN. The higher TNF-α responsiveness by Gram-positive bacteria is in agreement with in vitro studies in human but in contrast to the in vivo TNF-α responsiveness in bovine udder.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Bacterias Gramnegativas/inmunología , Bacterias Grampositivas/inmunología , Leucocitos/inmunología , Leucocitos/microbiología , Mastitis Bovina/microbiología , Leche/microbiología , Factor de Necrosis Tumoral alfa/análisis , Animales , Bovinos , Dinamarca , Femenino , Bacterias Gramnegativas/clasificación , Bacterias Grampositivas/clasificación , Lactancia , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The objective of this study was to determine the lipolytic activity on milk fat of 2 bovine mastitis pathogens, that is, Staphylococcus aureus and Streptococcus agalactiae. The lipolytic activity was determined by 2 different techniques, that is, thin-layer chromatography and an extraction-titration method, in an experimental model using the most commonly occurring field strains of the 2 mastitic bacteria isolated from Swedish dairy farms. The microorganisms were inoculated into bacteria-free control milk and incubated at 37°C to reflect physiological temperatures in the mammary gland. Levels of free fatty acids (FFA) were analyzed at time of inoculation (t=0) and after 2 and 6h of incubation, showing significant increase in FFA levels. After 2h the FFA content had increased by approximately 40% in milk samples inoculated with Staph. aureus and Strep. agalactiae, and at 6h the pathogens had increased FFA levels by 47% compared with the bacteria-free control milk. Changes in lipid composition compared with the bacteria-free control were investigated at 2 and 6h of incubation. Diacylglycerols, triacylglycerols, and phospholipids increased significantly after 6h incubation with the mastitis bacteria, whereas cholesterol and sterol esters decreased. Our results suggest that during mammary infections with Staph. aureus and Strep. agalactiae, the action of lipases originating from the mastitis pathogens will contribute significantly to milk fat lipolysis and thus to raw milk deterioration.