Immunization with Staphylococcus aureus lysate incorporated into microspheres.

Antibiotics are of limited value against Staphylococcus aureus due to development of resistant strains, scar tissue formation, and blockage of ducts due to inflammation. Though macrophages are the predominant cell type in the mammary gland, they are primarily scavenger cells and are not effective against bacteria entering the gland. Neutrophil phagocytosis is the bovine's primary defense against S. aureus mastitis. Attempts to develop vaccines that enhance neutrophil phagocytosis by stimulating production of opsonizing antibodies to S. aureus have met with limited success because of the low immunogenicity of the exopolysaccharide capsule surrounding S. aureus. Staphylococcus aureus can also adhere to and penetrate epithelial tissue. This study was conducted to determine whether lysates of S. aureus encapsulated in biodegradable microspheres would increase the production of opsonizing antibodies to capsule and block adherence. Four groups of four cows each were injected with 1 ml of the respective treatment in the area of the supramammary lymph node and 1 ml in the hip muscle. The treatments were: lysate in NaCl, lysate in Freund's incomplete adjuvant (FICA), lysate in microspheres in NaCl, and lysate in microspheres in FICA. Antigen in microspheres produced a similar antibody response to antigen emulsified in FICA, but to a lesser magnitude. Antigen in microspheres produced antibodies that were more opsonic for neutrophils at 20 and 52 wk postimmunization and inhibited S. aureus adherence to mammary epithelium. Ability to control antigen release and presentation, and the benefit of a single injection for long-term immunity using microspheres warrants additional studies.

Production of antibodies to Staphylococcus aureus serotypes 5, 8, and 336 using poly(DL-lactide-co-glycolide) microspheres.

Staphylococcus aureus is responsible for a major portion of the economic losses due to mastitis. Attempts to produce a vaccine to prevent S. aureus mastitis have been hampered by the low immunogenicity of the polysaccharide, which forms on the surface of the organism when it enters the mammary gland. The polysaccharide inhibits phagocytosis and destruction of the organism by neutrophils. This study was conducted to determine if S. aureus polysaccharide serotypes 5, 8, and 336 conjugated to a protein and incorporated in poly(DL-lactide-co-glycolide) microspheres would enhance the production of opsonizing antibodies to the polysaccharide. Cows were immunized with either polysaccharide conjugates emulsified in Freund's incomplete adjuvant or polysaccharide conjugates encapsulated in poly (DL-lactide-co-glycolide) microspheres emulsified in Freund's incomplete adjuvant. All cows produced sustained antibody titers to the three polysaccharide serotypes. Cows immunized with microspheres had higher antibody titers. Cows in both groups produced increased concentrations of IgG1 and IgG2 antibodies; neither group produced an increase in IgM. Immune sera from cows immunized with conjugates alone increased phagocytosis, which decreased at the end of the study. Sera from cows immunized with conjugates in microspheres increased phagocytosis, which was sustained at the end of the study. Immune sera from both groups decreased bacterial adherence to bovine mammary epithelial cells. These data showed that a single injection of antigen in microspheres produced higher titers and more sustained enhancement of phagocytosis, which could aid in the defense of the cow against S. aureus infections.

Adhesion receptor CD11b/CD18 contributes to neutrophil diapedesis across the bovine blood-milk barrier.

epithelium. Neutrophil migration across mammary arterial endothelial cells was almost completely dependent on CD18, the beta-chain of the beta(2) integrins, and to a lesser extent on CD11b, one of the alpha-chains of the beta(2) integrins. Neutrophil migration across collagen was partially blocked by monoclonal antibodies to CD18. No inhibition was observed by monoclonal antibodies to CD11b. Conversely, neutrophil diapedesis across mammary epithelial cells was dependent to a greater extent on CD11b. These results provide evidence for different CD11b/CD18-dependent mechanisms for neutrophil diapedesis across the various cell layers of the blood-milk barrier.

Diapedesis across mammary epithelium reduces phagocytic and oxidative burst of bovine neutrophils.

The effect of diapedesis on the phagocytic and oxidative burst activity of polymorphonuclear neutrophil (PMN) was examined, using an in vitro cell culture model consisting of a monolayer of primary mammary epithelial cells. Isolated blood PMN from 10 cows were added to the basal side of the epithelial cell monolayer. Diapedesis was induced by the addition of complement factor C5a to the apical side of the monolayer. PMN phagocytosis of Staphylococcus aureus and oxidative burst were measured before diapedesis on PMN that were non-activated and activated by incubation with C5a and on PMN after diapedesis, using flow cytometry. The percentages of PMN fluorescing due to phagocytosis of S. aureus and oxidative burst were reduced by 21.2 and 14.4%, respectively, after diapedesis. Pre-incubation in the presence of C5a had no effect on percentage PMN fluorescing due to phagocytosis or oxidative burst. The capacity for individual migrated PMN to phagocytose S. aureus and to produce an oxidative burst, as measured by the intensity of fluorescence, decreased by 34.2 and 30.3%. Activation of PMN with C5a increased intensity due to the oxidative burst, but had no effect on intensity due to phagocytosis. These data show that PMN diapedesis across mammary epithelium results in decreased phagocytosis and oxidative burst of the PMN.

In vitro expression of adhesion receptors and diapedesis by polymorphonuclear neutrophils during experimentally induced Streptococcus uberis mastitis.

The expression of adhesion receptors and diapedesis by polymorphonuclear neutrophils (PMN) were studied before and during experimentally induced Streptococcus uberis mastitis. Both quarters of the left half of the udders of five midlactation cows were inoculated with a suspension containing approximately 500 CFU of S. uberis 0140J. Clinical signs of an inflammatory reaction and leukocyte influx were observed 24 h after challenge. The expression of CD11b/CD18 adhesion receptors, determined by flow cytometry, was upregulated 24 h after challenge. A confluent monolayer of bovine secretory mammary epithelial cells on collagen-coated inserts was used to study PMN diapedesis. Bovine C5a was used as the chemoattractant. An 80% decrease in PMN diapedesis was observed 24 h after challenge. The decrease in diapedesis continued for 3 weeks after challenge.

A bovine mammary endothelial/epithelial cell culture model of the blood/milk barrier.

The complex nature of the mammary gland has hampered in-depth studies of the relationship of the circulatory system to cells lining the teat ducts and alveoli of the gland. This study reports an in vitro model of endothelial and epithelial cells separated by a subcellular matrix that simulates the blood milk barrier of the bovine mammary gland. Dual chamber culture dishes with a porous membrane separating the upper and lower chamber were used. Endothelial and epithelial cells were cultured on opposite sides of the porous membrane. A collagen and fibroblast subcellular matrix, separating the 2 cell layers, simulated the in vivo interstitial tissue. Changes in surface binding of anti-bodies to polymorphonuclear neutrophils (PMN) following their migration from the upper to the lower chamber simulated the passage of PMN from blood to milk. Changes in the binding of antibodies to PMN agreed with results observed following the migration of PMN from blood to milk in vivo. This gives credence to the model's potential value for studies where more direct observation of the blood/milk barrier is required. The model will be further tested for its usefulness as an assay for determining: 1) antibiotic diffusion from milk to blood and from blood to milk, 2) cytotoxicity of prophylactic and therapeutic mammary infusion products, 3) factors affecting bacterial adhesion and penetration of mammary epithelial tissue, 4) effectiveness of antibodies present in lacteal secretions in preventing bacterial adhesion, and 5) the feasibility of gene constructs to induce synthesis and secretion of mastitis-preventing compounds and prophylactic and therapeutic compounds for treatment of human disorders.

The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells.

A flow cytometric technique was developed to measure the relative concentration of whey protein and beta-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (alpha-lactalbumin + beta-lactoglobulin) or beta-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more beta-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.

Bovine mammary explant versus primary cell cultures: effect of bovine somatotropin and insulin-like growth factor-I on DNA content and protein synthesis.

Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 +/- 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulin-like growth factor-I, and somatotropin + insulin-like growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G0G1 phase was increased by: (a) insulin-like growth factor-I in explant cells; (b) somatotropin, insulin-like growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulin-like growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G0G1 or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more beta-casein than primary cells but secretion trends for beta-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.

Formulation of poly(DL-lactide-co-glycolide) microspheres and their ingestion by bovine leukocytes.

This study determined optimal parameters for producing controlled-release microspheres and examined their suitability for vaccines via ingestion by bovine leukocytes. Microspheres elicit an immune response when ingested by antigen-presenting cells and provide sustained exposure of antigen to sensitized cells. Ingestion of microspheres is determined by their size (< 10 microns), and antigen release is governed by composition. Poly(DL-lactide-co-glycolide) microspheres were prepared using different polymer concentrations, stir rates, emulsifier concentrations, and emulsifier molecular masses. Microspheres that were < 10 microns were prepared using a 6% 50:50 lactide to glycolide polymer solution emulsified at 12,000 rpm in a low molecular mass, 5% polyvinyl alcohol solution. Microspheres that were > 20 microns were prepared using a 10% 85:15 lactide to glycolide polymer solution emulsified at 1200 rpm in a low molecular mass, 3% polyvinyl alcohol solution. Small microspheres released 90% of the antigen after 7 d, and large microspheres released only 24% of the antigen after 56 d. Both monocytes and neutrophils selectively ingested small microspheres that were opsonized with normal bovine serum (heated and unheated). Ingestion of microspheres that had been opsonized with fetal bovine serum (heated and unheated) was minimal. Lymphocytes did not ingest microspheres. Ingestion of small microspheres by bovine monocytes and sustained release of antigen by large microspheres suggested that microspheres have the ability to produce a sustained immune response with a single injection.

Effect of antibodies to staphylococcal alpha and beta toxins and Staphylococcus aureus on the cytotoxicity for and adherence of the organism to bovine mammary epithelial cells.

OBJECTIVE: To determine the effect of antibodies to staphylococcal alpha and beta toxins and Staphylococcus aureus on the toxicity for and adherence of S aureus to bovine mammary epithelial cells. SAMPLE POPULATION: Cultured bovine mammary epithelial cells and Staphylococcus aureus obtained from a cow with mastitis. PROCEDURE: Cultured bovine epithelial cells were incubated with antisera to alpha and beta toxins of S aureus and culture supernatant; cell damage and S aureus adherence to cells were measured. RESULTS: Antisera to alpha, beta, and alpha + beta toxins inhibited cytotoxicity of S aureus culture supernatant. Antiserum to alpha + beta toxin was the most effective inhibitor of cytotoxicity and antiserum to beta toxin was the least effective. All 3 antisera decreased the percentage of S aureus adhered to the mammary epithelial cell monolayers and numbers of organisms per cluster of adhered bacteria. In this study, antisera to alpha and alpha + beta toxins decreased the number of S aureus clusters per dish, but antiserum to beta toxin had no significant effect. Antiserum to alpha + beta toxin decreased the percentage of epithelial cells with adhered S aureus, but neither antiserum to alpha nor beta toxin had significant effect. Antiserum to S aureus decreased the percentage of S aureus adhered, number of clusters perdish, number of organisms per cluster, and percentage of epithelial cells with S aureus adhered. CONCLUSIONS: Antibodies to staphylococcal alpha and beta toxins inhibit adherence to and cytotoxicity of S aureus for bovine mammary epithelial cells, and antibodies to S aureus inhibit adherence of S aureus to bovine mammary epithelial cells.

Cell culture system for studying bovine neutrophil diapedesis.

Neutrophils are the major defense against bacterial infection in the bovine mammary gland. Neutrophils migrate from blood into the lumen of the gland in response to inflammatory stimuli. This study describes the development of a system of cell culture that can be used to study neutrophil diapedesis through secretory and ductal mammary epithelial barriers. The culture system consists of successive layers of collagen, fibroblasts, collagen, and a confluent monolayer of secretory or ductal epithelial cells layered on a porous membrane. Confluence was determined by electrical resistance and trypan blue diffusion. Neutrophil diapedesis occurred from the basal to the apical surface of the monolayers. Purified complement C5a, fetal bovine serum that had been activated by zymosan, and fetal bovine serum that had been activated by Escherichia coli induced neutrophil diapedesis. Neutrophil diapedesis was greater across ductal cell monolayers. Blood neutrophils from five cows differed in their ability to migrate through the multilayered culture system in response to C5a. Monoclonal antibodies to C5a blocked diapedesis induced by purified C5a but had no effect on diapedesis induced by fetal bovine serum that had been activated by zymosan or by fetal bovine serum that had been activated by E. coli endotoxin, indicating that factors other than C5a were chemotactic for neutrophils. Monomeric IgG2, immune complexes, and E. coli endotoxin did not induce neutrophil diapedesis.

Staphylococcus aureus invasion of bovine mammary epithelial cells.

Staphylococcus aureus is a frequent cause of mastitis in dairy cows. However, pathogenesis of the infection has not been completely defined. We report the invasion of two strains of S. aureus into a bovine mammary epithelial cell line and a bovine mammary epithelial cell primary culture. Invasion of S. aureus into bovine mammary cells was time-dependent. Transmission electron microscopy of bovine mammary cells invaded by S. aureus showed intracellular replication of the bacterium within membrane-bound vacuoles. Invasion was reduced significantly when bovine mammary epithelial cells were treated with inhibitors of F-actin microfilament polymerization but not when these cells were treated with inhibitors of microtubule formation. Results indicated that S. aureus is capable of invading and replicating inside bovine mammary epithelial cells. Data also suggested that S. aureus invasion of bovine mammary epithelial cells requires active participation of specific components of the cytoskeleton of the epithelial cell.

A method for measuring specific antibodies in bovine lacteal secretions during the nonlactating period.

A large portion of new IMI in dairy cattle occurs during the nonlactating period. Because antibiotic infusions at the beginning of the nonlactating period are only partially effective, attempts have been made to stimulate the production of protective antibodies in lacteal secretions during this period. However, measurement of antibodies in mammary secretions during the nonlactating period has been hampered by the complex, viscous nature of these secretions. This report describes the use of caprylic acid to clarify secretions from the bovine mammary gland during the nonlactating period to provide a more accurate measurement of specific antibody. Six healthy Jersey cows were injected in the area of the supramammary lymph node with an encapsulated strain of Staphylococcus aureus in dextran sulfate at the beginning of the nonlactating period and 15 and 30 d later. Seven healthy unimmunized Jersey cows served as controls. Lacteal secretions taken at the beginning of the nonlactating period; at 15, 30, and 45 d into the nonlactating period; and at calving were treated with caprylic acid prior to assay for specific antibodies using ELISA. Purified S. aureus capsule was used as the antigen in the ELISA. Caprylic acid lowered non-specific binding of IgG1 and IgM in secretions during the dry period from unimmunized control cows and lowered IgM from immunized cows. The most pronounced effect of caprylic acid was an increase in IgG2 binding in secretions from immunized cows. Treatment with caprylic acid more accurately measured specific activity of Ig in mammary secretions during the nonlactating period.

Effect of staphylococcal beta toxin on the cytotoxicity, proliferation and adherence of Staphylococcus aureus to bovine mammary epithelial cells.

The effect of staphylococcal beta toxin on the cytotoxicity, proliferation and adherence of S. aureus. to bovine mammary epithelial cells was studied. Bovine erythrocytes and mammary epithelial cells were incubated with purified staphylococcal alpha and beta toxins and with culture supernatants from S. aureus M60 and two mutant strain that are negative for either the production of alpha (DU5789 alpha-) or beta (DU5846 beta-) toxin. Lysis of bovine erythrocytes was due primarily to beta toxin. Alpha toxin increased the lysis of bovine erythrocytes by purified beta toxin, but the presence of alpha toxin in culture supernatants from S. aureus did not increase the lysis of bovine erythrocytes. Purified beta toxin was cytotoxic to mammary secretory epithelial cells, but to a lesser extent than alpha toxin. Together they exhibited an additive effect on mammary epithelial cells. Inactivation of the alpha toxin-gene of S. aureus M60 decreased the cytotoxic effect on mammary epithelial cells to a greater extent than the inactivation of the beta toxin-gene. Also, the relative percentages of DU5789 alpha- and DU5846 beta- adhering to mammary cell monolayers, the number and size of colonies and the number of infected epithelial cells decreased. This in vitro study showed that beta toxin damages bovine mammary secretory epithelial cells, increased the damaging effects of alpha toxin, increases the adherence of S. aureus to mammary epithelial cells and increases the proliferation of S. aureus.

Effect of alpha-toxin and capsular exopolysaccharide on the adherence of Staphylococcus aureus to cultured teat, ductal and secretory mammary epithelial cells.

Cultures of teat, ductal and secretory epithelial cells were used to study the role of alpha-toxin and the capsular exopolysaccharide on the adherence of Staphylococcus aureus to mammary epithelium. The adherence of S aureus to the cells and their susceptibility to damage by alpha-toxin increased from teat to ductal to secretory cells. Alpha-toxin increased the susceptibility of epithelial cell monolayers to adherence by S aureus, and the extent of the adherence increased with the time of exposure to alpha-toxin. The exopolysaccharide capsule deterred the adherence of S aureus to mammary epithelial cells and to collagen. Organisms with a rigid capsule adhered to a smaller extent than those with a flaccid capsule. Both encapsulated and unencapsulated S aureus adhered more readily to collagen than to either healthy monolayers of epithelial cells or monolayers of cells damaged by alpha-toxin.

Effect of whole Staphylococcus aureus and mode of immunization on bovine opsonizing antibodies to capsule.

Exopolysaccharide capsule is a major virulence factor of Staphylococcus aureus because it inhibits neutrophil recognition of antibodies to highly antigenic S. aureus cell wall. To circumvent this inhibition, two modes of immunization were tested for ability to induce anticapsular opsonins. Cows were immunized at drying off and boosted on d 14 and 28 by injection of Smith diffuse S. aureus plus dextran sulfate in the area of the supramammary lymph node or intramammarily. In cows immunized in the area of the supramammary lymph node, IgG1 and IgG2 sera antibody titers to capsule increased and remained elevated to the end of the study, 120 d postcalving. The IgM titers increased during the dry period but declined to preimmunization levels at calving. Response of serum IgG1 and IgM to intramammary immunization was similar to that with supramammary lymph node immunization, but more delayed and lower in magnitude. Antibodies of all four isotypes, IgG1, IgG2, IgA, and IgM, increased in dry secretions following immunization via lymph node. In cows immunized in the lymph node, IgG1 antibodies remained elevated throughout the study, but IgG2 antibodies dropped to baseline 15 d postcalving. In cows immunized intramammarily, only IgA antibodies increased significantly in lacteal secretions and remained elevated throughout the study. Immunization of cows in the lymph node during the dry period enhanced the ability of dry secretions and colostrum to support phagocytosis.

Encapsulation of Streptococcus uberis: influence of storage and cultural conditions.

Streptococcus uberis (n = 100) isolated from bovine mammary secretions were assessed by India ink for expression of capsule. Organisms were evaluated under four conditions; (1) after primary culture on blood agar, (2) following 5 passages on blood agar, (3) after 5 passages in Trypticase Soy Broth (TSB), and (4) after storage in 10% skim milk. Strains from primary culture (44 of 100) were positive for an unstained halo (capsule) by the India ink method. Number of strains expressing capsule decreased greatly after passage and following storage. Freeze-etching followed by electron microscopy confirmed results of India ink preparations. Strains were also cultured in various media to determine influence of medium components on capsule expression. Todd-Hewitt medium supplemented with either serum or egg yolk enhanced the size of capsule expressed. Results of this study may aid researchers investigating the pathogenicity of S. uberis.

Adherence of Staphylococcus aureus to cultured bovine mammary epithelial cells.

Bovine mammary secretory cells, isolated at necropsy, were cultured in vitro and used as a model to study the mode of adherence of Staphylococcus aureus to mammary epithelium. Cultured cells were characterized by their morphology and physiology as secretory epithelial cells. Cells showed characteristic growth patterns when grown on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma. Cells cultured on collagen formed confluent monolayers and were the most suitable for bacterial adherence studies. Cultured cells stained intensely for cytokeratin and for specific milk proteins, i.e., alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin. The effect of frozen storage for 10 mo on cell viability or presence of milk proteins was minimal. Staphylococcus aureus showed large affinity for extracellular matrix components, i.e., fibronectin, laminin, and collagen. Adherence to confluent cell monolayers was minimal. In preconfluent cell monolayers, most S. aureus adhered more readily to the exposed matrix than to the epithelial cells. Overnight exposure to staphylococcal alpha-toxin greatly increased adherence of S. aureus to confluent monolayers. However, whether bacteria adhered to alpha-toxin damaged cells or to exposed matrix is not clear. Unencapsulated S. aureus adhered in larger numbers than did encapsulated S. aureus.

Role of the neutrophil leucocyte in the local and systemic reactions during experimentally induced E. coli mastitis in cows immediately after calving.

Mammary leucocytes are the major contributors to natural defence against mastitis after a microorganism has entered the gland. This paper reviews the role of the neutrophil granulocyte during acute coliform mastitis in cows in the periparturient period. Qualitative and quantitative aspects of several neutrophil cell functions before and during experimentally induced infections are briefly discussed.

Bovine mammary teat and ductal epithelial cell cultures.

Bovine mammary epithelial cells from teat and ductal tissue were isolated at necropsy and were grown in culture. Cells were characterized by the presence of cytokeratin filaments, cell morphologic features, synthesis of milk proteins, esterase activity, DNA content, and growth patterns on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma. Cultured teat and ductal cells stained intensely for cytokeratin and had similar morphologic features. Both cell types synthesized alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin to variable degrees. Cell type and culture conditions did not affect the DNA content of the cells, as indicated by similar amounts of DNA in G0G1 and G2M phases of the mitotic cycle in cultured cells and in cells from freshly isolated mammary explants. Cells cultured on polystyrene, fibronectin, laminin, and collagen formed pavement-like cell monolayers suitable for cytotoxicity and bacterial adherence studies. Cells cultured on the reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma formed three-dimensional structures closely resembling lactiferous ducts and alveoli, which could be used for studying lactogenesis and galactopoiesis. Freshly isolated cells and cultured cells were stored at -70 C or in liquid nitrogen. The latter storage method affected the cells less than did freezing at -70 C.