Wubshet Mamo

Vaccination against Staphylococcus aureus mastitis : immunological response of mice vaccinated with fibronectin-binding protein (FnBP-A) to challenge with S. aureus

Mice were immunized with fusion proteins encompassing the fibronectin-binding domain of a staphylococcal fibronectin-binding protein (FnBP-A). A specific antibody response against the fibronectin-binding part of the fusion proteins was detected in the serum of all vaccinated animals. The protective potential of these vaccinations was evaluated in a mouse mastitis model, using Staphylococcus aureus, strain SA113, for challenge. The mice vaccinated with FnBP fusion proteins showed a decreased number of bacteria recovered from the mammary glands and significantly reduced cases of severe mastitis. Histopathological examination of tissue from challenged glands of vaccinated mice revealed either no pathological reactions or disseminated inflammatory reactions with focal necrosis whereas four of six examined tissues from challenged glands of non-vaccinated animals showed total necrosis. A combination of FnBP fusion protein with staphylococcal alpha-toxoid did not increase the efficacy of the vaccination and animals vaccinated with alpha-toxoid alone were as sensitive to challenge as those from the non-vaccinated control group. Thus vaccination of mice with recombinant FnBP resulted in significant protection against challenge with S. aureus.

Interaction of sub-epithelial connective tissue components with Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from bovine mastitis were examined for their ability to interact with 125I-labelled fibronectin, fibrinogen and type II collagen. Their relative surface hydrophobicity and production of extracellular capsule were also investigated. Almost all S. aureus strains bound fibronectin (mean value 23%), fibrinogen (mean value 12%) and type II collagen (mean value 16%). CNS bound fibronectin (mean value 6%) and type II collagen (mean value 7%), but not fibrinogen (mean value 2%). The specificity of binding of these proteins to S. aureus strain F1440 and to coagulase-negative Staphylococcus chromogenes strain BO52 was studied by adding an excess of unlabelled proteins. Fibronectin and collagen binding were observed to be specific, varying between 50 and 75%, whereas the specificity of fibrinogen binding to S. aureus strain F1440 was lower (26%). Most of the S. aureus strains (63%) showed very high surface hydrophobicity (autoaggregation) or lower hydrophobicity (29% of the strains) and the rest were hydrophilic. None of the CNS strains autoaggregated, 44% were classified as hydrophilic strains. Hydrophilic strains (except the reference strains) did not show extracellular capsule production. However, the encapsulated (reference) strains showed low binding to these proteins as compared to their unencapsulated variants. Pre-treatment of S. aureus strain F1440 and S. chromogenes strain BO52 with trypsin decreased their fibronectin binding capacity and surface hydrophobicity, whereas pre-treatment with bovine milk (except on collagen binding to strain F1440) did not significantly affect binding to these proteins. These data indicate that S. aureus and CNS isolated from bovine udder infection have the ability to bind to tissue matrix and plasma proteins which may be exposed in the traumatized or toxin-damaged udder epithelial lesions.

Binding of fibronectin, fibrinogen and type II collagen to streptococci isolated from bovine mastitis

Binding of 125I-labelled fibronectin, fibrinogen and type II collagen to group B (S. agalactiae), group C (S. dysgalactiae and S zooepidemicus), group E (S. uberis) and nontypable streptococci isolated from bovine mastitis was studied. S. agalactiae and S. uberis were found to bind low levels of all three proteins, while S. zooepidemicus bound high levels. Binding of the proteins to S. dysgalactiae varied, i.e. fibronectin was high, fibrinogen moderate and collagen low. Nontypable strains showed moderate or low binding of all proteins. Both hydrophobic and hydrophilic strains were found to bind fibronectin. For S. dysgalactiae the specific fibronectin binding ranged from 70% to 10% and for S. zooepidemicus it was more than 80% and this binding was sensitive to papain treatment. The binding of 29K-fibronectin fragment to one S. dysgalactiae strain showed an affinity of KD = 2.6 x 10(-8) M and the number of binding sites per colony forming unit (CFU) was calculated at 11,000.

Enhanced virulence of Staphylococcus aureus from bovine mastitis induced by growth in milk whey

The virulence towards mice of Staphylococcus aureus strains from bovine mastitis was enhanced upon growth in milk whey compared to homologous organisms grown in tryptic soy broth (TSB). In the mouse mastitis model, S. aureus grown in milk whey caused more severe lesions than homologous strains grown in TSB. Staphylococcus aureus strain F1440 grown in milk whey induced 75% mortality and local necrotic reaction in subcutaneously inoculated mice, whereas the homologous strain grown in TSB caused only 5% mortality and slight skin reaction. Extracellular capsule on milk whey-grown, S. aureus could not be demonstrated. However, diffuse type colony morphology could be correlated with an increased virulence of S. aureus towards mice.

Protection Induced in Mice Vaccinated with Recombinant Collagen‐Binding Protein (CnBP) and Alpha‐Toxoid against Intramammary Infection with Staphylococcus aureus

Mice vaccinated with a combination of two Staphylococcus aureus antigens consisting of a recombinant collagen‐binding protein (CnBP) and alpha‐toxoid (α‐toxoid) were significantly protected from intramammary challenge infection with S. aureus. The average number of bacteria recovered from the glands of mice vaccinated with the combination of CnBP/α‐toxoid was significantly lower compared to the average number of bacteria recovered from the glands of mice vaccinated with only CnBP or α‐toxoid or controls (P ≤ 0.01). Histopathological examination of mammary glands of mice vaccinated with CnBP together with α‐toxoid showed no pathological changes, whereas glands of mice vaccinated with CnBP or α‐toxoid alone developed severe mastitis and showed both focal and disseminated necrosis.

Adhesion of Staphylococcus aureus to Bovine Mammary Epithelial Cells Induced by Growth in Milk Whey

Staphylococcus aureus strains isolated from bovine intramammary infection (mastitis) were tested for adhesion to bovine mammary epithelial cells after growth in milk whey or TSB. Bacteria grown in milk whey adhered more efficiently to mammary gland epithelial cells in vitro than the corresponding homologous bacteria grown in TSB. Trypsin treatment of milk whey‐grown S. aureus had no effect on their adherence. Whereas, pretreatment with periodate significantly decreased bacterial adherence capacity. Periodate treatment of TSB‐grown bacteria had no effect on adhesion to the mammary gland epithelial cells.

Bacterial Cell-Surface Hydrophobicity

The methodology to determine bacterial cell-surface hydrophobicity is discussed as well as the possible contribution of various surface-bound bacterial components to the hydrophobic nature of a cell surface. A number of agents and conditions affect cell-surface hydrophobicity, such as growth media, growth-phases and bacterial species. Studies are presented on hydrophobicities of staphylococcal cells of different species in connection with slime production and growth in serum-soft agar. Experimental data support a correlation between hydrophobicity and phagocytosis. The genetic origin of hydrophobicity, and the therapeutical implication of hydrophobic interactions are also discussed.

Binding of fibronectin and type II collagen to Staphylococcus aureus from bovine mastitis: reduction of binding after growth in milk whey☆

The binding of fibronectin and type II collagen to Staphylococcus aureus strains isolated from bovine mastitis was found to be 20-80% lower for organisms grown in milk whey compared to those grown in tryptic soy broth (TSB). The reduced binding was accompanied by reduced surface hydrophobicity. The observed changes, after growth in milk whey, were not due to a mere adsorption of milk whey components. The binding of fibronectin and the degree of surface hydrophobicity of milk whey-grown bacteria became similar to that of TSB-grown bacteria after periodate treatment, whereas trypsin or papain treatments had no effect.

A periodate-sensitive anti-phagocytic surface structure, induced by growth in milk whey, on Staphylococcus aureus isolated from bovine mastitis

The phagocytic and chemiluminescent activity of purified bovine neutrophils in response to two Staphylococcus aureus strains isolated from mastitic bovine milk and grown in milk whey was studied. The activity was significantly reduced compared with the response elicited by the same strains grown in tryptic soy broth (TSB). A mild periodate treatment of the milk whey-grown strains resulted in a significant increase of both chemiluminescence and phagocytosis, whereas trypsin, subtilisin or papain treatment had no effect. The decreased binding of complement factor C3 to milk-whey-grown bacteria was restored to the level of TSB-grown homologous organisms by periodate treatment. Moreover, this treatment, but not treatment with trypsin, increased the surface hydrophobicity of milk-whey-grown bacteria. The chemiluminescent activity was as high towards heat-killed as towards live bacteria. Also, incubation of heat-killed TSB-grown bacteria in milk whey did not alter the chemiluminescent response, indicating that the reduced neutrophil activity towards milk-whey-grown bacteria was not due to binding of milk components to the microorganisms. These results strongly suggest that bovine mastitis S. aureus strains grown in milk whey produce an anti-phagocytic surface structure. This structure is heat- and protease-resistant and renders the bacterial surface hydrophilic. The anti-phagocytic material is altered or, more likely, released from the bacterial surface on periodate treatment and is probably of carbohydrate nature.

In Vivo-Like Antigenic Surface Properties of Staphylococcus aureus from Bovine Mastitis Induced upon Growth in Milk Whey

Antigenic surface properties of Staphylococcus aureus strains grown in milk whey were compared with TSB‐grown bacteria using immuno‐gold electron microscopy. It is shown that colloidal gold (CG) particles coated with polyclonal antibody raised against Staphylococcus aureus surface antigen expressed in vivo bound to the surface of S. aureus strain F1440 grown in milk whey, but not to homologous bacteria grown in TSB. S. aureus strains grown in milk whey agglutinated in the presence of the polyclonal antibody, whereas the corresponding bacteria grown in TSB did not agglutinate. Immuno‐gold particles did not bind to milk whey‐grown bacteria treated with periodate. Periodate‐treated milk whey‐grown bacteria did not agglutinate in the presence of the polyclonal antibody, whereas periodate treatment had no effect on TSB‐grown bacteria.