Bovine mastitis caused by coagulase-negative staphylococci: host response and bacterial factors

Coagulase-negative staphylococci (CNS) are the most common bacteria isolated from subclinical mastitis in several countries and are also a frequent cause of clinical mastitis. The prevalence of CNS mastitis is higher in primiparous cows and the most common species isolated are S. chromogenes, S. epidermidis, S. xylosus and S. simulans. The distribution of species differs among countries and herds. Half of CNS intramammary infections (IMI) persist in the udder for long periods and although cure rates after antimicrobial therapy are usually high, antimicrobial resistance among CNS can be a problem. The inflammation in the infected quarter is usually mild, and treatment at dryingoff is recommended rather than during lactation. Some risk factors for CNS mastitis have been recognised in epidemiological studies that should be taken into account in herd health plans. The pathogenesis of CNS intramammary infection is poorly understood. This dissertation focuses on host response to S. chromogenes, S. epidermidis and S. simulans in bovine experimental intramammary infection. A model for mild experimentally induced mastitis was developed with S. chromogenes (study I). This infection model revealed a diurnal rhythm in milk amyloid A (MAA) concentrations, the content of which was higher in the mornings despite dilution in a greater volume of milk. The dose of bacteria used for inoculation in study II was higher in order to induce clinical mastitis. The innate immune response of S. epidermidis and S. simulans was compared in eight cows using a crossover design. All cows became infected and showed mild to moderate clinical signs of mastitis. S. simulans causes a slightly stronger innate immune response than S. epidermidis, with significantly higher concentrations of IL-1 and IL-8. The spontaneous elimination rate of the 16 infections was 31%, with no difference between species. Infections triggered a local cytokine response in the experimental udder quarters, but cytokines were not detected in the uninfected control quarters or in systemic circulation. The IL-8 response could be divided into early, delayed, or combined types of response. Neither the CNS species nor persistence of infection were associated with the type of IL-8 response. No significant differences were recorded between spontaneously eliminated and persistent infections, although the response was stronger with spontaneously eliminated infections, except with tumor necrosis factor (TNF), which remained elevated in persistent infections. Lactoferrin (Lf) is a component of the humoral defence of the host and is present at low concentrations in the milk. It is an iron-binding glycoprotein, which has bacteriostatic and bacteriosidic activity, as well as immunoregulatory functions. The concentration of Lf in milk is high during the dry period, and in colostrum and mastitic milk. The effect of an inherent, high concentration of lactoferrin in the milk in experimental CNS intramammary infection was studied in transgenic cows that expressed recombinant human Lf in their milk. Human Lf did not prevent S. chromogenes IMI, but the host response was milder in transgenic animals compared with normal cows and the infection was eliminated sooner. Biofilm and slime formation assist bacteria in avoiding host immune defences and antimicrobial therapy. Biofilm has been suspected of affecting the severity or persistence of CNS mastitis. The phenotypic biofilm formation and slime producing ability of CNS isolates from bovine mastitis was investigated in vitro. Genes encoding the adhesion proteins MSCRAMM (microbial surface components recognizing adhesive matrix molecules) and biofilm-associated proteins (bap) were detected. One-third of mastitis isolates produced biofilm. The Kappa test value, measuring the agreement between two phenotypic biofilm tests, differed among CNS species. Slime production was less frequent for isolates of the common mastitis causing species S. chromogenes (0.2% of isolates produced slime) and S. simulans (3.5%) compared with S. epidermidis (40%). No association was found between the phenotypic ability to form biofilm and the persistence of IMI or severity of mastitis. Slime production was associated with persistent infections, but as a rare event its clinical relevance remains uncertain. The eno gene, encoding laminin-binding protein, was most frequently detected among the mastitis isolates, 75% of which have this gene. Only a few other MSCRAMM genes were detected. The results of this dissertation indicate that experimental infection using three CNS species, S. chromogenes, S. epidermidis and S. simulans, triggers a systemic and local immune response of the cow. Differences among species were found in host response, and in biofilm and slime production of CNS isolated in mastitis. A high lactoferrin concentration in milk contributed to milder inflammation and more rapid elimination of infection from the quarters, but did not protect the mammary gland from infection. The common CNS species isolated in mastitis produce less biofilm and slime than isolates from other sources, except S. epidermidis. Biofilm or slime producing ability has no effect on the clinical severity of mastitis or on the persistence of CNS infection. This dissertation provides knowledge on the pathogenesis of the most important CNS species that can be useful in developing control strategies for CNS mastitis.