Vaccines against S. aureus bovinemastitis are scarce and show limited protection only. All currently available vaccines are
applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to
specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study,
immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous
(suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and
milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the
recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit
LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and
LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations.
Furthermore, the harmful effects of Efb and leukotoxin LukMF’ on host-defense were neutralized by serum antibodies in a
route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum
antibodies towards Efb and LukMF’, shown by increased phagocytosis of S. aureus and increased viability of bovine
leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity
against S. aureus mastitis in cattle.