Differences in the coinfective process of Staphylococcus aureus and Streptococcus agalactiae in bovine mammary epithelial cells infected by Mycobacterium avium subsp. paratuberculosis

Abstract

The interactions between Mycobacterium avium subsp. paratuberculosis (MAP) and the causative agents of bovine mastitis are still relatively unknown. Still, it is suspected that they may contribute to the worsening and persistence of mastitis within the mammary epithelial cells. Considering the growing economic implications of paratuberculosis and subclinical mastitis in dairy herds, this study aimed to determine the coinfection interaction between MAP and S. aureus or S. agalactiae in bovine mammary epithelial cells (MAC-T) in an ex-vivo model. For this purpose, internalisation tests of MAP + S. aureus or MAP + S. agalactiae were performed in MAC-T cells for 10, 30 and 120 min. The qPCR was performed to quantify internalised MAP at the time of exposure. Colony-forming units were counted on BHI agar medium for internalised subclinical mastitis bacteria at each time of infection. Viability tests of MAC-T cells, using the lactate dehydrogenase assay, were performed. The results showed that in the MAC-T cells previously infected by MAP and subsequently by S. aureus, there was a rapid internalisation in the first 10 min, maintaining a higher number of internalised bacteria during all exposure times. Regarding MAP + S. agalactiae, there were no changes in the internalisation patterns. The amount of MAP remained constant at all times evaluated, and there was no compromise in the viability of MAC-T cells during the tests. Thus, the results demonstrate the existence of an interaction between MAP + S. aureus, favouring internalisation and being able to contribute to the persistence of subclinical mastitis in dairy herds.