Demonstration of intracellular Staphylococcus aureus in bovine mastitis alveolar cells and macrophages isolated from naturally infected cow milk

Numerous in vitro studies have demonstrated that Staphylococcus aureus may be internalized and survive in a bovine mammary epithelial cell line. We re port here the presence of internalized and living S. aureus in alveolar cel ls and macrophages in milk samples of bovine mastitis. We used fluorochrome labeled monoclonal antibodies, specifically recognizing surface cell marke rs of bovine alveolar cells and macrophages, to isolate these two types of cells using fluorescence activated cell sorting. Extracellular bacteria and DNA were previously eliminated to exclude possible contamination, In order to detect intracellular bacterial DNA inside the isolated cells, we used P CR amplification of bacterial DNA and the PCR products were analyzed by Sou thern blot with a specific probe for Staphylococcus. The results showed the presence of Staphylococcus DNA inside the two isolated populations of cell s, confirming that S. aureus could penetrate alveolar cells and macrophages . The demonstration of the presence of intracellular living S. aureus was d etermined by bacteriological culture of positive samples plated onto blood agar plates and by its further identification. Our results showed for the f irst time that living S. aureus and its DNA are present in both alveolar ce lls and macrophages in chronically infected cow milk. (C) 2000 Federation o f European Microbiological Societies. Published by Elsevier Science B.V. Al l rights reserved.