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Lactoferrin (Lf) is a molecule naturally present in bovine milk that affects the availability and transport systems of iron. Lf also binds endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria and modulates the immunological response. In the present study, concentrations of bovine Lf (bLf) and citrate in milk were determined in early (EL) and late (LL) lactating dairy cows, using an experimentally induced endotoxin mastitis model and a crossover design. Nine clinically healthy Finnish Ayrshire cows were challenged twice with 100 μg endotoxin infused into one udder quarter. Milk samples were collected from the challenged and control quarters of each cow before and after endotoxin infusion during 3 d, and bLf and citrate concentrations were measured. In all cows, clinical signs of mastitis were seen at both times of challenge, but the response was more severe in EL than in LL. Concentration of bLf in the milk started to rise approximately 8 h after endotoxin infusion and was still higher than normal on the third day, especially in the late-lactating cows. In milk of the LL group, concentrations of bLf were significantly higher than in the EL group. In contrast, concentrations of citrate were higher in milk of the EL cows compared with the LL cows. Concentration of bLf and citrate varied substantially among cows. The molar ratio of citrate to bLf before and after challenge was significantly higher during the EL period. The results of this study partly explain why cows in early lactation are more susceptible to intramammary infections and why mastitis is more severe in them.

Coagulase-negative staphylococci (CNS) have emerged as bovine mastitis pathogens in many countries. CNS mastitis is generally mild but can persist in the udder for long periods. Pathogenesis of CNS intramammary infection is not well understood. In the present study, adhesion, invasion and intracellular replication of twenty-two CNS strains isolated from bovine mastitis and the effect of bovine lactoferrin (bLf) on the internalization were studied in vitro in a bovine mammary epithelial (BME) cell model. The CNS strains were ofStaphylococcus chromogenes, Staph. simulans, Staph. epidermidis, Staph. haemolyticusandStaph. cohnii urealyticus; two strains ofStaph. aureuswere used as controls. Seven of the CNS strains originated from persistent and five from transient mastitis infections. The in-vitro susceptibility of the strains to bLf was also investigated. All CNS species examined had an adhesive ability equal to that ofStaph. aureus, but internalization varied among staphylococcal strains. The antagonistic effect of bLf on the adhesion and invasion of CNS strains was weak, but bLf significantly decreased intracellular replication and replication rates of CNS. No correlation between the in-vitro susceptibility of the strain to bLf or internalization among clinical signs of mastitis was established. No difference between the persistent and transient CNS strains in adhesion, invasion or replication rate was recorded. This in-vitro BME cell model can be used to study the virulence potential of mastitis pathogens, although the severity and persistence of eventual infections shall be further investigated in vivo. The role of bLf in intramammary infection caused by CNS may be limited.

Isolates of various species of coagulase-negative staphylococci (CNS) from extramammary swab samples were compared with isolates of bovine mastitis CNS species. Swab samples were taken from perineum skin and udder skin, teat apices and teat canals of lactating dairy cows of the research dairy herd of the University of Helsinki in 1999 and 2002. In addition, hands of herd staff and liners of teat cups were sampled for CNS. CNS isolates from milk samples of subclinical or clinical mastitis in the same herd were collected during 1998–2002. Species identification was performed using phenotyping (API Staph ID 32 test) and by constructing a 16 and 23S rRNA RFLP library (ribotyping). Based on phenotype, 84% of mastitis isolates and 57% of extramammary isolates were identified at species level with >90% probability. Ribotype patterns formed 24 clusters, and 15 of them included a CNS type strain. If the ribotype clusters contained isolates of both extramammary and mastitis origin, they were further typed using pulsed-field gel electrophoresis (PFGE). The predominant CNS species in mastitis, based both on phenotyping and genotyping, wereStaph. chromogenesandStaph. simulans. Phenotyping failed to identify half of the extramammary isolates. Based on phenotyping,Staph. equorumandStaph. sciuri, and based on ribotyping,Staph. succinusandStaph. xylosus, were the predominant CNS species in extramammary samples. The most common species in milk samples,Staph. chromogenes, was also isolated from several extramammary samples, and five out of ten pulsotypes were shared between mastitis and extramammary isolates, indicating that strains from udder skin are highly similar. The second commonest mastitis species,Staph. simulans, was isolated only from three extramammary samples, indicating thatStaph. simulansmay be more specifically associated with mastitis. Consequently, the origin of CNS mastitis may vary depending on the causing CNS species.