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

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.

Advancements in Dairy Research: Exploring Nutritional Strategies, Enhancing Raw Milk Quality and Innovations—Unveiling the Topic “New Insights into Milk 2.0”

“…This editorial highlights the study by Flórez et al [5] on the importance of rapidly quantifying endotoxins in raw milk samples for differentiating between Gram-positive and Gram-negative mastitis, aiding in their clinical management. Mastitis, a common disease in dairy cattle, results in significant losses in the dairy industry and affects animal welfare.…”

“…While the assay shows promise for diagnosing coliform mastitis, further research is required to assess its efficacy in other Gram-negative-associated mastitis cases and throughout the clinical course of these infections, including postantibiotic treatment. Overall, the developed assay by Flórez et al [5] offers a standard-ized method for detecting endotoxins in raw and commercial cow milk, potentially improving mastitis management in dairy cattle.…”

Visual Detection of LPS at the Femtomolar Level Based on Click Chemistry-Induced Gold Nanoparticles Electrokinetic Accumulation

“…The limulus amebocyte lysate (LAL) 6 test is the standard method for detecting LPS. Although the method is simple and rapid, it relies mainly on enzymatic reaction, and the results are greatly affected by factors such as pH and temperature.…”