Evidence of Mycobacterium avium subsp. paratuberculosis binding to albumin: technical and biological implications.

Albumin binding ability is a well-characterized feature of many bacteria. To the best of our knowledge, there are no previous reports about this ability among mycobacteria, even when bovine serum albumin (BSA) is a common component of supplements used for the enrichment of synthetic media for mycobacterial growth in vitro and also of buffers used in laboratory techniques. In this work we explored the albumin binding ability of Mycobacterium avium subsp. paratuberculosis (MAP), a pathogenic bacterium causing a known and relevant ruminant disease worldwide, by immunizing rabbits with MAP (grown in media containing or not BSA) or BSA and conducting ELISA and immunoblot experiments with the obtained sera. As a result, we found that MAP can bind BSA when cultured in a conventional BSA-containing medium and when incubated for a short time in the presence of the protein. We also evaluated the host specificity of MAP interaction with albumin and found a preference for the protein of bovine origin when compared with its horse and rabbit homologs. Considerations about its technical and biological implications are discussed.

Effect of different glycerol concentrations on phosphatidylserine translocation and mitochondrial membrane potential in chilled boar spermatozoa.

Boar spermatozoa are extremely sensitive to low temperatures and the cryopreservation causes dramatic changes in sperm survivability, but it is not clear which part of the cryopreservation process affects the most. The aim of this work was to assess early events of apoptotic changes as damage indicators in boar sperm cooled to 5 °C and exposed to different glycerol (GLY) concentrations. For this purpose, progressive sperm motility (CASA), plasmatic and acrosome membranes integrity (CFDA/PI; phase contrast), plasma membrane functionality (HOS), phosphatidylserine translocation (Annexin-V/FITC) and reduction of mitochondrial membrane potential (Ψm) (JC-10) were carried out at 37 °C, 17 °C and 5 °C in eight boar sperm pools. Afterwards, three aliquots were diluted in different freezing extenders (control: 0% GLY; A: 2% GLY and B: 3% GLY); sperm quality and early apoptotic changes were assessed. Motility was negatively affected during cooling to 5 °C. Furthermore, plasma membrane functionality was the most affected by cooling. The number of necrotic cells was higher at 5 °C. However, no differences were observed in phosphatidylserine translocation. The extender with 3% GLY at 5 °C presented better Ψm than 0 and 2% GLY. Based on this analysis, boar sperm cooling to 5 °C does not modify the rate of early apoptotic changes, although alterations in the Ñ°m were evident.