Research Phd Theses

Intramammary immunity against Staphylococcus aureus in cattle

Mastitis in cattle is a worldwide problem in dairy farming with a major impact on animal welfare, life span and milk production. Interestingly, quarters of the same cow can differ in their susceptibility to mastitis. This indicates that susceptibility is defined on the quarter level rather than the cow level.

To investigate whether milk characteristics play a role in quarter susceptibility to mastitis, we collected quarter milk samples of morning and evening milk, analysed the milk composition, and determined in vitro growth of Staphylococcus aureus (S. aureus) a major mastitis causing pathogen, in these fresh milk samples. We found that almost all measured milk components (protein, fat, lactose, urea, lactoferrin, lactoperoxidase, and β-lactoglobulin concentrations, and S. aureus-specific antibodies) differed significantly between

quarters and that they showed a diurnal pattern. Furthermore, we observed that quantitative growth of S. aureus differed significantly between morning milk and evening milk. Mixed model analyses revealed that replication of S. aureus was negatively associated with S. aureus-specific IgG1 antibodies, and morning milk.

Next, we studied whether the milk composition also influenced in vivo growth of S. aureus. Our results revealed that the numbers of S. aureus re-isolated from milk post-experimental intramammary inoculation was positively associated with the milk fat percentage and negatively associated with the S. aureus-specific IgG1 titer pre-inoculation.

Although the exact mechanism leading to the negative association between the S. aureus specific IgG1 titer and decreased S. aureus replication, both in vitro and in vivo, could not be identified, our finding supports the on-going effort to develop a prophylactic vaccine against bovine S. aureus-induced mastitis and indicates that stimulation of the humoral immune response in milk against this bacterium might contribute to protection.

Driven by these observations, we explored immunization strategies to increase the presence of specific antibodies in the bovine mammary gland. First, we determined the impact of vaccine administration via different routes and showed that vaccination via the subcutaneous route results in higher levels of specific, neutralizing antibodies compared to vaccination via the intramuscular, intranasal, and intramammary route. We also showed that subcutaneous immunizations resulted in higher antibody titers when administered near the udder compared to administration in the neck. Furthermore, we observed that an Alum-Saponin-Oil based adjuvant stimulates the induction of specific antibody titers, favouring both IgG1 and IgG2 responses, and neutralization titers in milk and serum more efficiently compared to adjuvants comprising Alum-Saponin or Alum-Oil as adjuvant.

In conclusion, the subcutaneous administration near the udder of a vaccine comprising Alum-Saponin-Oil as adjuvant efficiently increases intramammary antibody responses. Increased levels of functional antibodies allow the investigation of the role of antibodies in bovine intramammary immunity during S. aureus infections.


Eveline Boerhout studied Medical Biology at the Radboud University Nijmegen, The Netherlands. She fulfilled her final internship at the department of Microbiological Research and Development (R&D) of MSD Animal Health (MSD-AH), where she analysed the immune responses induced by Neospora caninum-based DNA vaccines in a murine model. In 2011, she graduated with honours. After her Master studies, Eveline continued to work at MSD-AH. In 2012 she started her PhD studies on Staphylococcus aureus mastitis in cattle, a research collaboration between the department of Ruminant R&D of MSD-AH and the department of Infectious Diseases and Immunology of the Faculty of Veterinary Medicine of the Utrecht University. Eveline recently obtained her PhD degree with her dissertation, Intramammary immunity against Staphylococcus aureus in cattle.