Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages

Streptococcus uberis is frequently isolated from the mammary gland of dairy cattle. Infection with some strains can induce mild subclinical inflammation whilst others induce severe inflammation and clinical mastitis. We compared here the inflammatory response of primary cultures of bovine mammary epithelial cells (pbMEC) towards S. uberis strains collected from clinical or subclinical cases (seven strains each) of mastitis with the strong response elicited by Escherichia coli. Neither heat inactivated nor live S. uberis induced the expression of 10 key immune genes (including TNF, IL1B, IL6). The widely used virulent strain 0140J and the avirulent strain, EF20 elicited similar responses; as did mutants defective in capsule (hasA) or biofilm formation (sub0538 and sub0539). Streptococcus uberis failed to activate NF-κB in pbMEC or TLR2 in HEK293 cells, indicating that S. uberis particles did not induce any TLR-signaling in MEC. However, preparations of lipoteichoic acid (LTA) from two strains strongly induced immune gene expression and activated NF-κB in pbMEC, without the involvement of TLR2. The immune-stimulatory LTA must be arranged in the intact S. uberis such that it is unrecognizable by the relevant pathogen receptors of the MEC. The absence of immune recognition is specific for MEC, since the same S. uberis preparations strongly induced immune gene expression and NF-κB activity in the murine macrophage model cell RAW264.7. Hence, the sluggish immune response of MEC and not of professional immune cells to this pathogen may aid establishment of the often encountered belated and subclinical phenotype of S. uberis mastitis

A novel subtraction diversity array distinguishes between clinical and non-clinical Streptococcus uberis and identifies potential virulence determinants

Streptococcus uberis is an important bovine mastitis pathogen, but not all isolates have equal capacity to cause disease. The aims of this study were to identify possible virulence-associated genes that could be used to identify isolates with enhanced virulence. DNA from a pool of putative commensals was subtracted from a clinical pool resulting in a set of DNA sequences (probes) that were enriched in the clinical mastitis group. The probes were hybridised with DNA from a collection 29 isolates from cases of clinical mastitis and isolates not associated with disease. Hybridization revealed five major clusters. The first cluster (7 isolates) consisted almost entirely of commensals, while the second (7 isolates) was mixed. The remaining three clusters contained 15 S. uberis isolates from cows with clinical mastitis. Twenty-six probes were selected for sequencing based on principal component analysis (PCA) or their presence mainly in clinical isolates. PCA identified five probes with clear differences in intensity between signals from clinical isolates and commensals; these probes could represent novel virulence determinants. Manual inspection of arrays identified genes prominent among clinical isolates that specify carbohydrate and lipid metabolism (possible role in the growth or survival of S. uberis in milk) and genes specifying hypothetical proteins, possibly novel virulence factors. The common occurrence, among clinical isolates, of probes having homology with transposases and insertion sequences suggests recent acquisition of factors that could be associated with virulence. These results suggest the existence of a subset of S. uberis with enhanced virulence, due possession of virulence-associated gene sequences

Molecular epidemiology of recurrent clinical mastitis due to Streptococcus uberis: Evidence of both an environmental source and recurring infection with the same strain

This study was undertaken because clinicians and farmers have observed that a considerable number of cows diagnosed with Streptococcus uberis mastitis have recurrences of mastitis in the same or a different quarter. The study was an attempt to answer whether these recurring cases were due to treatment failure (in which case a search would have begun for a better treatment for Strep. uberis mastitis) or due to reinfection with a different strain of Strep. uberis. Using pulsed-field gel electrophoresis (PFGE), we determined that the majority of recurrences (20 of 27) were caused by a new strain of Strep. uberis, indicating that treatment of the initial infection had been successful. A small number of recurrences (5 of 27) were caused by the initial strain, indicating persistence. The remaining 2 recurrences occurred in a new quarter but with the initial strain of Strep. uberis, indicating either spread between quarters or reactivation of a previous subclinical infection. Analysis of the PFGE profiles failed to reveal any strain-specific propensity to persist, because strains causing recurrences occurred in most of the major clusters