Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells.

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis. In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.

Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells.

Surface-associated protein expression by Streptococcus uberis was influenced by the presence of collagen, laminin and bovine mammary epithelial cells in the culture medium. After electrophoresis and silver staining, four proteins stained more intensively in samples from S. uberis cultivated with epithelial cells and extracellular matrix components than in samples from S. uberis cultivated alone. Induction of these proteins was more obvious after multiple bacterial passages. The correlation between the phenotype of S. uberis and its potential virulence status as illustrated by an immunoblotting study with sera obtained from infected cows revealed that these proteins are probably expressed in vivo during infection.

Incubation of Streptococcus uberis with extracellular matrix proteins enhances adherence to and internalization into bovine mammary epithelial cells.

Two strains of Streptococcus uberis (UT 888 and UT 366) isolated from cows with clinical mastitis were co-cultured with bovine mammary epithelial cells (MAC-T) with and without laminin, fibrinogen, fibronectin or collagen. Incubation of S. uberis with extracellular matrix proteins (ECMPs) increased adherence to and internalization into MAC-T cells. Both strains of S. uberis exhibited greater adherence when co-cultured in the presence of collagen than with any other ECMP. However, adherence was always higher when strains were co-cultured with ECMP than in medium alone. S. uberis UT 888 adhered better to MAC-T cells than S. uberis UT 366. The influence of ECMPs on bacterial internalization into MAC-T cells was similar to adherence, however, differences among ECMPs were less noticeable. S. uberis UT 888 had a higher internalization index than S. uberis UT 366. It is possible that ECMPs induce or up-regulate proteins that selectively adhere to ECMPs which could serve as a bridge between the eukaryotic cell and the bacterial pathogen that leads to internalization of the ECMP-bound pathogen into the mammary epithelial cell.

Binding of bovine lactoferrin to Streptococcus dysgalactiae subsp. dysgalactiae isolated from cows with mastitis.

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (UT516, UT519, ATCC 27957) were used to determine if bovine lactoferrin (Lf) binds to bacterial cells by biotin avidin binding assay (BABA), enzyme-linked immunosorbent assay (ELISA), and binding inhibition assay. Binding assays revealed that all strains of S. dysgalactiae subsp. dysgalactiae (S. dysgalactiae) evaluated in this study bound to Lf. However, differences in Lf binding capability among strains and between methods used were detected. Binding of Lf was not inhibited by transferrin (Tf) and Lf moiety molecules (mannose, galactose, and lactose) but by Lf. This study demonstrates that S. dysgalactiae bound to bovine Lf in a specific manner.

Predicted antigenic regions of Streptococcus uberis adhesion molecule (SUAM) are involved in adherence to and internalization into mammary epithelial cells.

Streptococcus uberis is a significant cause of bovine mastitis throughout the world. Previous work from our laboratory demonstrated that S. uberis adhesion molecule (SUAM) is an important factor in adherence to and internalization of S. uberis into bovine mammary epithelial cells. Antibodies directed against SUAM significantly reduced bacterial adherence to and internalization into bovine mammary epithelial cells implying that SUAM is surface exposed. Objectives of this research were to: (1) predict surface exposed peptides, and (2) select peptide sequences for production of synthetic peptides with the final aim of evaluating their role in adherence and internalization and immunogenic potential. The Kyte/Doolittle hydropathicity prediction method; Chou/Fasman ß-turn prediction method; and output from Coils, Paircoil and MultiCoil scores for prediction of secondary and tertiary structures were used. Prediction algorithms resulted in identification of five overlapping regions of the SUAM sequence with the most hydrophilic valleys and the highest peaks for ß-turns. The five 15-mer SUAM epitopes selected by bioinformatic analysis were produced to evaluate the immunogenic value and pathogenic role of these putative domains. Peptides were bound to fluorescent latex beads, incubated with MAC-T bovine mammary epithelial cells, and internalization into MAC-T cells was evaluated using confocal laser and transmission electron microscopy. All peptides evaluated induced some degree of internalization of fluorescent beads into MAC-T cells; however, 2 peptides induced significantly more internalization of fluorescent beads than the other peptides evaluated. These peptides, designated III and IV, were located in the central region of SUAM, between two coiled-coil regions. Convalescent sera were tested against these biotinylated peptides for SUAM specific immune response using an indirect ELISA format. Among the 5 peptides evaluated, peptides I, II and V elicited significant serological response suggesting that the N-terminal region (peptide I), central region (peptide II) and C-terminal region (peptide V) are immunodominant epitopes of SUAM. Results will be useful to design immunotherapeutic tools based on immunodominant epitopes.

Vaccination of dairy cows with recombinant Streptococcus uberis adhesion molecule induces antibodies that reduce adherence to and internalization of S. uberis into bovine mammary epithelial cells.

Streptococcus uberis is an important environmental mastitis pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows and heifers throughout the world. Previous work from our laboratory suggests that S. uberis adhesion molecule (SUAM) is involved in S. uberis pathogenesis and may be an excellent target for vaccine development. The objective of this study was to evaluate the antibody response of cattle vaccinated with recombinant SUAM (rSUAM). Uninfected primiparous dairy cows (n=30) in late lactation were divided randomly into three groups of 10 cows each: control, 200 µg rSUAM, and 400 µg rSUAM. Cows in groups vaccinated with 200 µg and 400 µg rSUAM received an emulsion containing adjuvant, phosphate-buffered saline (PBS) and affinity purified rSUAM. Cows in the control group received an emulsion containing adjuvant and PBS. Cows were vaccinated subcutaneously in the neck region at drying off (D-0), 28 d after drying off (D+28) and within 7 d after calving. Serum was collected at D-0, D+28, at calving (C-0), calving vaccination (CV), and during early lactation (CV+14). Serum antibody responses were measured by an ELISA against rSUAM. Following the first vaccination a significant increase in anti-rSUAM antibodies was detected at D+28 in cows from groups vaccinated with 200 µg and 400 µg rSUAM when compared to the control group. This increase in anti-rSUAM antibodies continued following the second immunization at D+28; reaching the highest levels in the post-parturient sampling period (C0), after which antibodies appeared to plateau. S. uberis UT888 pretreated with several dilutions of heat-inactivated serum from cows vaccinated with rSUAM, affinity purified antibodies against rSUAM, and to a 17 amino acid long peptide from the N terminus of SUAM (pep-SUAM) were co-cultured with bovine mammary epithelial cells and adherence to and internalization of S. uberis into epithelial cells was measured. Compared to untreated controls, opsonization of two strains of S. uberis with sera from cows vaccinated with rSUAM, with affinity purified rSUAM antibodies, or with affinity purified pep-SUAM antibodies significantly reduced adherence to and internalization of this pathogen into bovine mammary epithelial cells. In conclusion, subcutaneous vaccination of dairy cows with rSUAM during physiological transitions of the mammary gland either from or to a state of active milk synthesis induced antibodies in serum and milk and these antibodies reduced adherence to and internalization of S. uberis into mammary epithelial cells under in vitro conditions. SUAM appears to be an excellent candidate for vaccine development.

pGh9:ISS1 transpositional mutations in Streptococcus uberis UT888 causes reduced bacterial adherence to and internalization into bovine mammary epithelial cells.

Streptococcus uberis is an important mastitis pathogen that affects dairy cows worldwide. In spite of the economic impact caused by the high prevalence of S. uberis intramammary infections (IMI) in many well-managed dairy herds, pathogenic strategies and associated virulence factors of S. uberis are not well understood. It has been shown that S. uberis attaches to and internalizes into mammary epithelial cells and can survive inside cells for extended periods of time. We hypothesize that early attachment to and internalization into mammary epithelial cells is a critical step for the establishment of intramammary infection. The aim of this study is to identify and characterize chromosomally encoded virulence factors of S. uberis that allow early bacterial attachment to and internalization into mammary epithelial cells. A common approach used to identify virulence factors is by generating random insertion mutants that are defective in adherence to and internalization into mammary epithelial cells using pGh9:ISS1 mutagenesis system. A random insertion mutant library of S. uberis strain UT888 was created using a thermo-sensitive plasmid pGh9:ISS1 carrying ISS1 insertion sequence. Integration of the insertion sequence into the chromosome of these mutant clones was confirmed by PCR and Southern blot. Southern blot analysis of mutant clones also showed that insertional integration was random. Of 1000 random chromosomal insertion mutants of S. uberis strain UT888 screened, 32 had significantly reduced ability to adhere to and internalize into mammary epithelial cells. Chromosomal mapping of insertion sequence integration sites in some of these defective mutants showed integration into penicillin binding protein 2A (pbp2A), sensor histidine kinase, tetR family regulatory protein, phosphoribosylaminoimidazole carboxylase catalytic subunit (purE), lactose phosphotransferase, phosphoribosylamine glycine ligase (purD), and other genes involved in metabolic activities. These proteins may have a significant role in early bacterial colonization of the mammary gland during infection.

Identification and Differentiation of Coagulase-Negative Staphylococcus aureus by Polymerase Chain Reaction.

Staphylococcus aureus is an etiological agent of a wide variety of human and animal infections. The majority of S. aureus are coagulase-positive; however, some may be atypical in that they do not produce coagulase. Incorrect identification of an isolate can impact implementation of effective treatment and/or control measures. In this study, polymerase chain reaction-based DNA fingerprinting was used to differentiate coagulase-positive Staphylococcus aureus (CPSA) from coagulase-negative Staphylococcus aureus (CNSA). A total of 29 CNSA and 50 CPSA were evaluated. PCR-based DNA fingerprinting differentiated CNSA from CPSA on the basis of visible observation and densitometric evaluation. The method is rapid and accurate, eliminating variability associated with conventional techniques.

Decreased growth of Streptococcus uberis in milk from mammary glands of cows challenged with the same mastitis pathogen.

Milk samples from mammary glands challenged with Streptococcus uberis and from unchallenged mammary glands were selected for analyses of bacterial growth, antibody response, and lactoperoxidase activity. All challenged mammary glands became infected with isolation of S. uberis and elevated somatic cell counts in milk during the first week after challenge. In vitro growth of the homologous challenge strain and a heterologous strain of S. uberis was significantly lower in milk from challenged mammary glands than in milk from control mammary glands at 3, 5, and 7 days after challenge. Removal of casein significantly reduced bacterial growth. In general, antibodies specific to S. uberis started to increase at day 3 post-challenge and were higher in milk from challenged mammary glands than in milk from control mammary glands. There was also a marked increase in total IgG in milk from challenged mammary glands. Growth of S. uberis increased following heat treatment at 56 degrees C of pooled milk or whey samples from challenged mammary glands. Growth of S. uberis correlated negatively with the specific antibody response to the bacteria (P < 0.001). Lactoperoxidase activity varied among cows and among different samples over time and did not appear to contribute to decreased growth of S. uberis. These results suggest that decreased growth of S. uberis in milk from challenged mammary glands in comparison to milk from control mammary glands could result from the interaction of antibodies with complement components.

Changes in bovine mammary-secretion composition during early involution following intramammary infusion of recombinant bovine cytokines.

This study was designed to determine whether intramammary infusion of recombinant bovine interleukin-1 beta or interleukin-2 had any adverse influence on bovine mammary function during the early non-lactating period. Mammary glands of eight Jersey cows were infused with either 10 micrograms of interleukin-1 beta or 1.0 mg of interleukin-2 following abrupt cessation of milking. Mammary secretions from each gland were collected from cows frequently during early involution and evaluated for changes in secretion composition. Percentage of milk fat and solids-not-fat during the experimental period was variable for all treatments. Percentage of protein increased throughout the study for all treatments, but mammary glands treated with interleukin-1 beta had higher protein percentages. All interleukin-treated mammary glands had lower concentrations of lactose than controls. Mammary secretions from interleukin-1 beta treated glands had higher concentrations of lactoferrin at 7 and 14 days of involution. Similarly, mammary secretions from interleukin-2 treated glands had higher concentrations of serum albumin on day 3 of involution and throughout the remainder of the study. Concentration of citrate and the citrate: lactoferrin molar ratio generally decreased throughout the study for all treatment groups. Concentration of alpha-lactalbumin in mammary secretions decreased throughout the study and was not different between treatment groups. Though significant changes in mammary secretion composition due to the presence of cytokines were observed in this study, resulting in an apparent acceleration of involution, there was no indication that these changes adversely affected normal bovine mammary function during the early non-lactating period.

Adherence of Streptococcus uberis to bovine mammary epithelial cells and to extracellular matrix proteins.

Adherence of an encapsulated (UT 101) and a non-encapsulated (UT 102) strain of Streptococcus uberis to a bovine mammary epithelial cell line (MAC-T) and to extracellular matrix proteins (ECMP) including fibronectin, collagen and laminin was investigated. S. uberis was co-cultured at 4 degrees C with MAC-T cell monolayers. Both strains of S. uberis adhered to MAC-T cells. However, the non-encapsulated strain of S. uberis adhered better to MAC-T cells than the encapsulated strain. Preincubation of MAC-T cells with lipoteichoic acid (LTA) and/or treatment of S. uberis with antibodies directed against the carboxyl-terminal half of type 24 M protein reduced adherence of both strains of S. uberis to MAC-T cells. Adherence to ECMP was measured by incubating bis-carboxyethyl-carboxyfluorescein acetomethyl ester (BCECF-AM) labelled S. uberis in 96-well plates coated with fibronectin, collagen or laminin. Both strains adhered to ECMP, however, the encapsulated strain adhered better to ECMP than the non-encapsulated strain. Results of this investigation demonstrated that both strains of S. uberis evaluated were capable of adhering to bovine mammary epithelial cells and to ECMP. Adherence of S. uberis to mammary epithelium may be an extremely important mechanism in the establishment and progression of bovine intramammary infections.

Efficacy of a new premilking teat disinfectant containing a phenolic combination for the prevention of mastitis.

A teat disinfectant containing a phenolic combination was evaluated in a natural exposure study in two dairy research herds. Premilking teat disinfection was compared with a negative control using a split-udder experimental design. In both herds, premilking and postmilking teat disinfections with the phenolic combination were significantly more effective in preventing new intramammary infection (IMI) than was postmilking teat disinfection only. Clinical mastitis and new IMI by Streptococcus uberis, Streptococcus dysgalactiae, Gram-negative pathogens, and coagulase-negative Staphylococcus species were significantly lower in quarters of cows with teats predipped and postdipped than in quarters with teats postdipped only. No chapping or teat skin irritation was observed. Premilking teat disinfection with the phenolic combination in association with good udder preparation and postmilking teat disinfection can further reduce the occurrence of new IMI by numerous mastitis pathogens during lactation.