ARS | Publication request: Evaluation of endotoxin (LPS) activity in bovine blood using neutrophil dependent chemiluminescence

Submitted to: Journal of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: March 21, 2011
Publication Date: July 10, 2011
Citation: Kahl, S., Elsasser, T.H., Obiezu-Forster, C.V. 2011. Evaluation of endotoxin (LPS) activity in bovine blood using neutrophil dependent chemiluminescence [abstract]. Journal of Animal Science. 89(E-Suppl. 1):515.

Technical Abstract: The purpose of this study was to evaluate the applicability of a neutrophil chemiluminescence-based assay for the measurement of LPS stimulatory activity in bovine whole blood. The assay is based on the capacity for LPS to trigger the respiratory oxidative burst activity (RBA) of autologous neutrophils. This RBA is then detected as photons released from oxidized added luminol in a chemiluminometer. In the protocol, chemiluminescence (CL) of blood samples without (CLBL) and with an added reference quantity of LPS (100 ng/ml, CLBL+LPS) was measured in luminol/zymosan solution and the relative LPS blood activity (EAR) was expressed as a CLBL/CLBL+LPS ratio. EDTA-stabilized whole blood was collected from 16 healthy steers, 9 cows diagnosed with advanced mastitis of Gram(-) bacterial etiology, and 4 steers given an i.v. bolus of low (0.25 µg/kg BW) or high (2.0 µg/kg BW) LPS. In the in vitro recovery studies a linear relationship was observed between added LPS (up to 100 ng/ml) and EAR (R2 = 0.99, P < 0.01). Estimated mean (± SE) blood EAR in healthy cows was 0.058 ± 0.010 and increased to 0.353 ± 0.062 (P < 0.01) in cows with mastitis. Individual EAR values exceeded a calculated clinical cut-off value of 0.137 (upper limit of 95% confidence interval of observed EAR in healthy group) in 78% of infected cows. After the in vivo injection of high LPS dose, estimated blood EAR values exceeded clinical cut-off value at 2 min (0.317 ± 0.057; n = 2) and 5 min (0.163 ± 0.018), but returned to baseline at 10 min (0.084 ± 0.027). Within 10 min after low LPS dose injection, estimated EAR values remained below cut-off value, although animals showed clinical symptoms of initial proinflammatory response. Results indicate that bovine neutrophil chemiluminescence assay has the potential to detect LPS stimulation of neutrophil respiratory burst activity in bovine blood and to discriminate between healthy cows and cows infected with Gram(-) bacteria displaying clinical mastitis. Additional studies are needed to evaluate the specificity of the assay while further modifications to this protocol may result in increased sensitivity.