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.

A field trial with an experimental vaccine against Staphylococcus aureus mastitis in cattle. 1. Clinical parameters.

A total of 108 heifers were included in a placebo-controlled multicenter study on the use of an experimental Staphylococcus aureus mastitis vaccine containing whole, inactivated bacteria with pseudocapsule, alpha and beta toxoids, and a mineral oil as adjuvant. The heifers were injected in the area of the supramammary lymph nodes twice before calving and were observed and sampled throughout the first lactation. None of the vaccinated cows suffered from clinical Staph. aureus mastitis, and only 8.6% suffered from subclinical Staph. aureus mastitis, but a total of 16.0% of the control cows suffered from clinical or subclinical Staph. aureus mastitis. Mean SCC in vaccinated and control cows were the same throughout the lactation. Local swellings at the injection site were palpable in a substantial proportion of the vaccinated cows. In the statistical analyses, when cow was used as the unit of concern, no significant differences occurred between groups. However, when all parameters on udder health were considered together, the results indicated a potential protective effect of this vaccine during the entire lactation.

A field trial with an experimental vaccine against Staphylococcus aureus mastitis in cattle. 2. Antibody response.

A Staphylococcus aureus vaccine containing whole, inactivated bacteria with pseudocapsule and alpha and beta toxoids with a mineral oil as adjuvant, was used in a field trial. Heifers were injected in the area of the supramammary lymph nodes with vaccine or placebo twice before calving and observed and sampled throughout their first lactation. Antibody response toward the pseudocapsule and the alpha toxin was significant in serum from the vaccinated cows. These antibody concentrations were significantly higher in serum and milk during the entire lactation compared with that of the controls. The antibody response to the beta toxin was moderate in serum from vaccinated cows; no differences in antibody concentrations in milk were significant between groups. The antibody response to the pseudocapsule consisted of the IgG1 and IgG2 isotypes, but, in milk, only the concentration of IgG1 was significantly increased in the vaccinated cows during the lactation compared with the control cows.

Production of enterotoxins and toxic shock syndrome toxin by bovine mammary isolates of Staphylococcus aureus.

The production of staphylococcal enterotoxin A (SEA), SEB, SEC, SED, and SEE and toxic shock syndrome toxin 1 by bovine mammary isolates of Staphylococcus aureus was evaluated. Enterotoxin secretion was detected by immunodiffusion using specific polyclonal antisera. Of 262 isolates examined, 75 (28.6%) produced one or more toxins. The most common pattern was secretion of both SEC and SED and toxic shock syndrome toxin 1. No isolates secreted SEE, one produced SEA, and seven secreted SEB.

Secretion of alpha-hemolysin by bovine mammary isolates of Staphylococcus aureus.

A total of 262 strains of Staphylococcus aureus isolated from the mammary gland of dairy cows were examined for the production of alpha-hemolysin. Strains were cultured in a liquid medium of casein hydrolysate and yeast extract in an atmosphere of 7% (v/v) CO2 in air. The assay consisted of a dot immunoblotting technique employing bacterial culture supernatants and a mouse monoclonal antibody specific for alpha-hemolysin. Ninety-four percent (247) of 262 strains were positive for alpha-hemolysin by this method, when cultured in the laboratory. This figure is compared with those obtained in previous studies which typically based their results on the hemolytic patterns of isolates on blood agar plates.

Effect of specific antibody on adherence of Staphylococcus aureus to bovine mammary epithelial cells.

Staphylococcus aureus is a major pathogen in the bovine mammary gland. The ability of S. aureus to adhere to epithelial cells in the ductules and alveoli of the bovine mammary gland is believed to add greatly to its virulence and may be necessary for colonization. Two in vitro methods were developed for the purposes of quantifying adherence and of determining the effect which specific antibody may have in inhibiting the adherence of this organism. Both methods utilize bovine mammary epithelial primary cells as targets for labeled bacteria. In one assay, the bacteria are labeled with [methyl-3H]thymidine and incubated on the primary epithelial monolayers. The second assay involves labeling the bacteria with biotin. An enzyme-linked immunosorbent assay is then performed with streptavidin conjugated to horseradish peroxidase. Both methods have proven to be reliable and allow for the testing of many criteria in one assay. Cows were immunized with a whole-cell vaccine, and immune serum and milk were collected. The bacteria were then incubated in the presence of serum or milk as a test for antiadherent capability. By using the methods described, distinct antiadherent activity in both serum and milk was demonstrated.

Antibody and lymphoblastogenic responses of dogs experimentally infected with Trypanosoma cruzi isolates from North American mammals.

The humoral and cellular immune responses of dogs infected with either a non-pathogenic Trypanosoma cruzi isolated from a North American dog (Tc-D) or a pathogenic T. cruzi isolate from an opossum (Tc-O) were studied over a 240 day period. Antibody to T. cruzi epimastigote antigens prepared from Tc-O or Tc-D isolates were first detected by ELISA by Day 26 post infection (PI), peaked by day 175 PI and remained elevated throughout the experimental period in both Tc-O and Tc-D infected dogs. Differences in antibody levels between infected groups were not detected. Western blot analyses were performed using Tc-O and Tc-D epimastigote antigens probed with pooled sera and sera from individual Tc-O and Tc-D infected dogs prior to infection (Day 0), and during the acute (Day 16-35 PI), indeterminate (Day 50-135 PI) and chronic (Day 235 PI) stages of infection. Generally, the patterns, number of protein bands, and temporal appearance of the protein bands identified by pooled sera and sera from individual dogs within each antigen preparation were similar. However, similarities and differences were present in antibody responses between sera from Tc-O and Tc-D infected dogs. Blastogenic responses of peripheral blood mononuclear cells (PBMC) from Tc-O and Tc-D infected dogs to mitogens (concanavalin A, phytohemagglutinin and pokeweed) were not significantly different from controls at any time during the experimental period. The PBMC from both groups of dogs were unresponsive to epimastigote antigens during the acute stage of infection. Statistically significant differences (P less than 0.05) in PBMC responsiveness from controls were observed on Days 70 and 175 PI. Responses decreased to pre-infection levels by Day 240 PI. These studies demonstrate that although two North American T. cruzi isolates have markedly different virulence for dogs, some aspects of their cellular and humoral immune responses are similar while other responses, such as antibody recognition of specific T. cruzi antigens, vary.

Effects of preventing periparturient hypocalcemia in cows by parathyroid hormone administration on hematology, conglutinin, immunoglobulin, and shedding of Staphylococcus aureus in milk.

The effects of hypocalcemia at parturition on concentrations of serum immunoglobulin and conglutinin, number of bacteria shed into milk, and leukograms of dairy cows were investigated from -4 wk prepartum to 4 wk postpartum. Ten healthy multiparous Holstein cows were fed a high calcium diet to induce hypocalcemia at parturition. Five cows received intramuscular parathyroid hormone to prevent hypocalcemia at parturition. All cows experienced a leukopenia (attributable to an absolute and relative neutropenia) during the 1st wk after calving, decreased serum conglutinin activity during the first 3 wk postpartum, and decreased concentration of serum IgG1 during the 3 wk before calving. At parturition, a large increase in organisms was found in foremilk (1000 to 10,000 times more than prepartum values). Neither the hematological changes nor the decreased immunoglobulin concentration was influenced by hypocalcemia or the development of milk fever. This implies that the degree of hypocalcemia observed did not have a large or irreversible influence on bacterial infection, hematological, or humoral immunity changes in periparturient cows.

Determination by enzyme-linked immunosorbent assay of the optimal dose of staphylococcal leukocidin for systemic immunization of dairy cows.

A dose-response study was conducted to determine the optimal dose of staphylococcal leukocidin toxin to use for systemic vaccination of lactating dairy cows. Each of 5 groups of cows (8 cows/group) were given 2 injections of crude leukocidin (dose range, 9 to 2,700 mg). Antileukocidin antibody concentration in milk samples collected before vaccination and at 4 and 10 weeks after vaccination was determined by use of an ELISA. The highest antibody concentration at postvaccination sample collection dates was observed in cows of the group immunized with 900 mg of leukocidin. This appeared to be the optimal vaccination dose for production of antileukocidin antibodies in the mammary gland of lactating cows.

Development and evaluation of an enzyme-linked immunosorbent assay for endotoxin in milk.

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of endotoxin in milk samples. Bovine and rabbit antisera raised in response to vaccination with the J5 mutant of Escherichia coli 0111:B4 were used. Antiserum to this mutant has been shown to be cross-reactive with endotoxin from other gram-negative organisms. Known quantities of endotoxin were added to milk samples to generate a standard curve. Acid treatment of whole milk enhanced the detection of endotoxin as compared to untreated whole milk, skim milk and chloroform-treated milk. Milk samples from experimentally induced mastitic cows were then assayed for endotoxin content. Recovery of endotoxin, as measured by ELISA, positively correlated with the amount of endotoxin infused and the time post-infusion of sampling. However, when endotoxin from these samples was quantitated using the Limulus Amebocyte Lysate (LAL) assay, readings tended to increase, suggesting false-positive reactions with the LAL assay. Milk samples from cases of clinical mastitis were assayed by ELISA with 64% of these showing measurable levels of endotoxin. While further studies of this assay are needed, refinements may produce an assay important for clinical applications.

Enzyme-linked immunosorbent assay for detection of leukocidin toxin from Staphylococcus aureus in bovine milk samples.

An enzyme-linked immunosorbent assay was developed for the detection of leukocidin toxin from Staphylococcus aureus. The minimum concentration of leukocidin detectable with the assay was 30 ng/ml. The enzyme-linked immunosorbent assay was found to be a more sensitive method, by a mean of 45-fold, for leukocidin detection than was observation of cytolytic effects of the toxin on bovine neutrophils. A mean toxin concentration of 974 ng/ml was required to produce observable cytolytic effects on neutrophils. Although the enzyme-linked immunosorbent assay was able to detect leukocidin in milk samples from toxin-infused mammary glands, the toxin was detectable in only 2 of 27 S. aureus-infected milk samples (7%) from cows with chronic staphylococcal mastitis. To determine whether leukocidin antibodies in the mastitic milk samples were preventing toxin detection, leukocidin was mixed with milk with a high antileukocidin antibody titer (from a vaccinated cow) and evaluated with the immunoassay. Leukocidin was readily detected in this sample, indicating that milk antileukocidin antibodies were not sufficient to prevent detection of any leukocidin present in the mastitic milk samples. Failure to detect leukocidin in most mastitic milk samples with this assay indicated that, if leukocidin is produced in the bovine mammary gland during chronic staphylococcal mastitis, the concentration of the toxin may be too low to produce cytolytic effects on neutrophils.

Evaluation by enzyme-linked immunosorbent assay of local and systemic production of milk immunoglobulins to surface exopolysaccharide antigen in cows with staphylococcal mastitis.

An enzyme-linked immunosorbent assay (ELISA) was developed to evaluate milk immunoglobulin levels to surface exopolysaccharide antigen of Staphylococcus aureus in cows with staphylococcal mastitis. Quarter milk samples were obtained from 24 lactating dairy cows on two occasions, one month apart. Cows were classified as S. aureus-positive (S. aureus cultured from at least one quarter on both sample dates) or S. aureus-negative. Individual quarter samples were tested for IgA (representing local synthesis) and IgG1 (primarily of systemic origin) specific for staphylococcal surface exopolysaccharide antigen. No significant differences were found for specific IgA or IgG1 between S. aureus-positive and S. aureus-negative cows, nor between infected and non-infected quarters of S. aureus-positive cows. The data indicate that, in cows with staphylococcal mastitis, milk immunoglobulins specific for exopolysaccharide antigen are not significantly increased by either the systemic or the local immune response.

Antibody specific for Escherichia coli J5 cross-reacts to various degrees with an Escherichia coli clinical isolate grown for different lengths of time.

Rabbit antiserum raised against the rough mutant of Escherichia coli O111:B4, designated J5, was examined for cross-reactivity to an E. coli clinical isolate (A2385). In whole-cell enzyme-linked immunosorbent assays, J5 antiserum reacted to a greater extent with A2385 grown for 5 h than with the same bacteria grown for 19 h, while the homologous antiserum reacted similarly with bacteria grown for different lengths of time. J5 antiserum reacted to the greatest extent with lipopolysaccharide (LPS) from A2385 grown for up to 10 h, and reactivity greatly diminished thereafter; homologous antiserum showed no difference in reaction over time. LPS from smooth bacteria grown for 19 h showed no reaction with J5 antiserum in immunoblots, while LPS from A2385 grown for 5 or 10 h showed a positive reaction. Little or no difference among the three LPS samples could be seen when homologous antiserum was used. Mice vaccinated with J5 LPS before lethal challenge with live A2385 were protected from this challenge, whereas most nonimmunized mice died. Toxicity tests in mice showed LPS from A2385 grown for 19 h to be twice as lethal as LPS from A2385 grown for 3 h. Mice vaccinated with J5 LPS were protected to a greater extent when challenged with a lethal dose of LPS from A2385 grown for 3 h than when challenged with LPS from A2385 grown for 19 h. The results reported here may explain the means by which J5 vaccination (active or passive) sometimes protects against heterologous challenge.

The expression of capsule in serum-soft agar by Staphylococcus aureus isolated from bovine mastitis.

Strains of Staphylococcus aureus were isolated from bovine mastitis, subcultured and maintained in the laboratory for up to 3 years. Encapsulation was assessed by production of a diffuse colony in serum-soft agar. Eight (4%) of 200 strains were encapsulated. Three rapid passages of the remaining 192 strains through either brain-heart infusion broth containing 30% serum or modified 110 medium retrieved the capsule in 75%, but this was rapidly lost after subculture on blood agar. The stimulation of capsule production was studied in 18 of these strains by addition of various components to the passaging medium. Heat-labile factors in serum, milk and mastitic milk enhanced capsule production while bovine serum albumin, an extract of polymorphonuclear leucocytes, NaCl and immunoglobulins had minimal effect. The results indicate that encapsulation is common in bovine staphylococci and while it is lost on subculture, may be retrieved under appropriate conditions.

Use of enzyme-linked immunosorbent assay to measure bovine milk and serum antibodies to alpha toxin, beta toxin, and capsular antigens of Staphylococcus aureus.

An enzyme-linked immunosorbent assay (ELISA) procedure was used to quantitate milk and serum antibodies (IgG) to Staphylococcus aureus alpha and beta toxins, and S. aureus 2-8 and Smith diffuse strain capsular antigens. Milk samples were collected on two occasions. A comparison was made between levels of milk antibodies specific for the two toxins and capsular antigens for 41 cows that were infected with S. aureus on both sampling dates, and 18 cows not S. aureus-infected on either date. Staphylococcus aureus-infected cows were grouped according to somatic cell counts. All groups of infected cows, regardless of somatic cell counts, had significantly higher milk antibody levels to alpha and beta toxins than did the non-infected cows (P less than .002). Serum samples taken for 13 infected and 4 non-infected cows also indicated that significant elevations in anti-alpha toxin and anti-beta toxin IgG were present in S. aureus-infected cows, compared to non-infected cows. A similar immune response was not seen to capsular antigens, however. No significant differences were present between the two groups of cows for either milk or serum antibodies to Smith diffuse strain capsular antigens. Milk antibodies to 2-8 capsule were significantly elevated only in infected cows with somatic cell counts greater than 10(6)/ml, compared to non-infected cows; no differences were present for serum antibodies to 2-8 capsule between infected and non-infected cows. These results indicate that significant increases in milk (and possibly serum) antibodies to alpha and beta toxins are present in cows with chronic staphylococcal mastitis, apparently resulting from a systemic immune response to these toxins. There does not appear to be a similar immune response to capsular antigens.

Use of 51Cr release to measure the cytotoxic effects of staphylococcal leukocidin and toxin neutralization on bovine leukocytes.

Leukocidin toxin from Staphylococcus aureus produces specific cytolytic effects on neutrophils and macrophages. The most commonly used method for determination of leukocidin activity is microscopic examination for characteristic morphological changes in toxin-treated cells. The 51Cr release assay was modified to allow quantitation of the cytolytic effects of leukocidin on bovine peripheral blood neutrophils and lymphocytes. Toxin neutralization by serum and milk samples was quantitated by this method. The neutralizing abilities of the various samples were found to correlate with the levels of immunoglobulin G (IgG1) specific for leukocidin. Undiluted normal serum samples, however, were capable of partially preventing the cytotoxic effects of leukocidin. The assay was shown to be an effective means of quantitating the cytotoxic activity of leukocidin on neutrophils as well as demonstrating neutralization of cytotoxicity by milk and serum samples.

Enzyme-linked immunosorbent assay for detection of milk immunoglobulins to leukocidin toxin of Staphylococcus aureus.

Leukocidin toxin from a bovine strain of Staphylococcus aureus was partially purified by ion exchange chromatography. An enzyme-linked immunosorbent assay was developed to quantitate antibodies specific for leukocidin in bovine milk. This was used to assay quarter samples from 88 cows in a S aureus-infected herd for antibody levels to the toxin. Milk samples from 65 cows with S aureus infections in at least one quarter produced a mean optical density of 1.054, whereas milk samples from 23 cows that were free of bacteria on cultural examination had a mean optical density of 0.584. There was a significant difference (P less than 0.001) in milk anti-leukocidin levels between these 2 groups. Evaluation of serum samples from 40 of these cows indicated that the milk anti-leukocidin concentrations were reflective of systemic anti-leukocidin values. The capability of 57 milk samples to neutralize the cytolytic effect of minimal amounts of leukocidin on bovine peripheral blood neutrophils was examined. Good correlation existed between the enzyme-linked immunosorbent assay antibody concentration and toxin-neutralizing capability of individual milk samples.

Antibodies in bovine serum and lacteal secretions to capsular antigens of Staphylococcus aureus.

Pregnant cows were immunized systemically with an encapsulated strain of Staphylococcus aureus (Smith diffuse strain). Antibodies in serum and colostrum were detectable by enzyme-linked immunosorbent assay and prevented capsule production by the Smith diffuse strain in a soft agar medium. Antibody in milk, although detectable by enzyme-linked immunosorbent assay, did not affect the production of capsule in vitro. Antibodies were absorbed from milk and serum, using staphylococcal surface antigen. In a 2nd experiment, lactating cows were immunized, using Smith diffuse strain antigens in the form of a bacterin or as a surface extract; the bacterin or extract was emulsified in Freund's incomplete adjuvant. Antibody titers in the milk of cows given bacterin were significantly (P less than 0.001) greater than titers in the milk of animals immunized with surface extract. The soft agar technique was insufficiently sensitive to detect antibody in the milk of any of the cattle.

Immunogenic properties of Staphylococcus aureus and Streptococcus agalactiae administered separately and in combination to lactating cows.

Composite bacterins of Staphylococcus aureus and Streptococcus agalactiae, or either bacterin alone, were administered systemically to groups of lactating cows. The response to each bacterin was unaffected by the simultaneous administration of the antigens when assessed by comparing the antibody levels in milk by enzyme-linked immunosorbent assay. There was no evidence of cross reactivity of the antigens studied, nor immunopotentiation by either bacteria.

Comparative effect of selected adjuvants on the response in the bovine mammary gland to staphylococcal and streptococcal antigens.

The effect of 4 adjuvants on the response in the lactating bovine mammary gland to an antigenic stimulus was examined. Fifty four lactating Holstein Friesian cows were randomly allocated to 6 groups. Four of these groups received a staphylococcal and streptococcal bacterin-toxoid vaccine administered systemically in association with an adjuvant preparation. The adjuvants used were: aluminum hydroxide gel, Freund's incomplete adjuvant, a metabolizable lipid emulsion and Bordetella pertussis. Two further groups serving as controls received saline, or the vaccine suspended in saline only. The immunoglobulin G response specific for each of 3 vaccine antigens, was monitored in the milk by means of enzyme-linked immunosorbent assay for a period of 23 weeks. The results indicated that high levels of antibody may be maintained in the milk, throughout the average lactation, if cows are vaccinated in the region of the supramammary lymph node with an optimum dose of antigen emulsified in Freund's incomplete adjuvant.