Intralaboratory Validation of a Kinetic Turbidimetric Assay Based on Limulus Amebocyte Lysate (LAL) for Assessing Endotoxin Activity in Cow Milk

SIMPLE SUMMARY: Rapid quantification of endotoxins in raw milk samples would be of interest for differentiating Gram-positive and Gram-negative mastitis and would contribute to their clinical management. In this research, we have validated a kinetic turbidimetric assay based on Limulus amebocyte lysate for endotoxin quantification in milk samples. This assay was demonstrated to be robust and useful in avoiding the effect of colour and interfering substances in milk samples and allowing for the identification of coliform mastitis in milk samples from affected cows. ABSTRACT: Mastitis, one of the most common diseases in dairy cattle, causes severe losses in the dairy sector worldwide and affects animal welfare. The disease is characterized by an inflammatory reaction of the mammary gland and is mainly caused by bacterial infections, including both Gram-negative and Gram-positive bacteria. The release of endotoxins associated to bacterial lysis is a weighty factor in the clinical course of Gram-negative associated mastitis and should be taken into consideration when using antibiotics in the treatment of these infections. Therefore, endotoxin detection in milk samples would be of help in the management of bovine mastitis. With this aim, we have validated a kinetic turbidimetric assay based on Limulus amebocyte lysate (LAL) for the quantification of endotoxins in milk samples. The assay was adapted to this particular matrix by incorporating filtration and dilution of the milk samples in the procedure. Our results demonstrate the robustness and usefulness of the assay, which allows the identification of coliform mastitis in milk samples from affected cows and the quantification of endotoxin activity in bulk and commercial milk samples. Further studies are required to evaluate the performance of the assay in mastitis milk samples associated to Gram-negative bacteria other than Escherichia coli as well as during the clinical course of these Gram-negative mastitis or after their treatment with antibiotics.