Streptococcus uberis is a prevalent causative organism of mastitis and resides naturally in the environment of the dairy cow making prevention of the disease difficult. A bovine cDNA microarray comprising approximately 22,000 expressed sequence tags was used to evaluate the transcriptional changes that occur in the mammary gland after the onset of clinical Strep. uberis mastitis. Five lactating Friesian heifers were intramammary infused in an uninfected quarter with approximately 1,000 to 1,500 cfu of a wild-type strain of Strep. uberis. Microarray results showed that Strep. uberis mastitis led to the differential expression of more than 2,200 genes by greater than 1.5-fold compared with noninfected control quarters. The most highly upregulated genes were associated with the immune response, programmed cell death, and oxidative stress. Quantitative real-time reverse transcription PCR analysis confirmed the increase in mRNA expression of immune-related genes complement component 3, clusterin, IL-8, calgranulin C, IFN-gamma , IL-10, IL-1beta, IL-6, toll-like receptor-2, tumor necrosis factor-alpha, serum amyloid A3, lactoferrin, LPS-bonding protein, and oxidative stress-related genes metallothionein 1A and superoxide dimutase 2. In contrast, a decrease of mRNA levels was observed for the major milk protein genes. Bovine mammary epithelial cells in culture challenged with the same Strep. uberis strain used to induce clinical mastitis in the in vivo animal experiment did not cause a change in the mRNA levels of the immune-related genes. This suggests that the expression of immune-related genes by mammary epithelial cells may be initiated by host factors and not Strep. uberis. However, challenging epithelial cells with different Strep. uberis strains and Staphylococcus aureus resulted in an increase in the mRNA expression of a subset of the immune-related genes measured. In comparison, an Escherichia coli challenge caused an increase in the majority of immune-related genes measured. Results demonstrate the complexity of the bovine mammary gland immune response to an infecting pathogen and indicate that a coordinated response exists between the resident, recruited, and inducible immune factors.

Streptococcus uberis is commonly found in the environment and in association with various bovine body sites and is a major cause of bovine mastitis. Moreover, S. uberis is known to produce a variety of bacteriocin-like inhibitory substances, antimicrobial agents that generally inhibit closely related bacterial species. In this respect, S. uberis strain 42 has previously been shown to produce a novel nisin variant named nisin U. This paper reports that, in addition to nisin U, S. uberis strain 42 produces a second bacteriocin that induces the lysis of metabolically active, susceptible target bacteria and which has therefore been named uberolysin. Isolation of the native active antimicrobial agent revealed that uberolysin is a 7048 Da peptide that is refractory to sequence analysis by Edman degradation. Transposon mutagenesis was used to generate a uberolysin-negative mutant of S. uberis 42 and sequencing of DNA flanking the insertion site revealed, in addition to the structural gene (ublA), several open reading frames likely to be involved in post-translational modification, transport and producer self-protection (immunity), and possibly in regulation of the biosynthetic gene cluster. In addition, a pair of direct repeats that may be involved in bacteriocin acquisition were identified; indeed, ublA could be identified in 18 % of tested S. uberis strains. Enzymic hydrolysis of uberolysin was used to confirm that ublA does indeed encode the precursor of uberolysin, that an unusually short leader sequence of only six amino acids is cleaved during processing of the mature peptide and that uberolysin is post-translationally covalently modified to form a head-to-tail monocycle. Thus, uberolysin is a unique cyclic bacteriocin, belonging to the same family of bacteriocins as enterocin AS-48 and circularin A.

The expression of a ␤-defensin, the lingual antimicrobial peptide (LAP), in response to mastitis was investigated by real-time PCR of RNA from mastitic and control udder quarters. There was a positive relationship between somatic cell count in milk and LAP expression. In situ hybridization showed that LAP mRNA was expressed in epithelial cells of mastitic tissue. These results suggest that LAP plays a role in the innate immune response to mastitis.

We have used cDNA microarray analysis to identify genes that play a role in bovine mammary involution. Involution was induced by termination of milking, and alveolar tissue was collected from 48 nonpregnant Friesian cows in mid lactation sacrificed at 0, 6, 12, 18, 24, 36, 72, and 192 h (n = 6/group) postmilking. The most highly upregulated genes were those associated with oxidative stress. Quantitative real-time reverse-transcription PCR analysis confirmed that mRNA expression of spermidine/spermine N(1)-acetyltransferase was increased by 24 h, superoxide dismutase 2 and metallothionein 1A by 36 h, and glutathione peroxidase by 72 h postmilking. The mRNA expression of the host defense proteins lactoferrin and lingual antimicrobial peptide were increased by 192 h postmilking. A dramatic increase in the protein expression of lactoferrin by 192 h postmilking was also detected by Western analysis. Decreased mRNA expression of the milk protein genes alpha(S1)-, beta-, and kappa-casein, and alpha-lactalbumin were early events in the process of involution occurring within 24 to 36 h postmilking, whereas beta-lactoglobulin mRNA was decreased by 192 h postmilking. Decreases in alpha-lactalbumin and beta-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192 h postmilking, respectively, and the cell survival factors beta1-integrin and focal adhesion kinase were decreased by 72 and 192 h postmilking, respectively. The results demonstrate that in the bovine mammary gland, decreased milk protein gene expression and cell survival signaling are associated with multiple protective responses to oxidative stress that occur before the induction of immune responses and mammary epithelial cell apoptosis during involution.