Encapsulation of Staphylococcus aureus isolated from bovine milk.

Evidence is presented that nearly all Staphylococcus aureus infections of the bovine mammary gland are by encapsulated organisms (94% of isolates examined). This observation is based on the demonstration of diffuse growth in serum-soft agar, of cultures taken directly from mastitic milk without subculture on artificial media. Only milks containing pure cultures of S. aureus were examined. It was previously reported that only 6% had capsules. Evidence is presented that as few as 3 or 4 subcultures and/or a short storage on artificial media results in loss of encapsulation of most S. aureus of bovine origin. Antisera raised against encapsulate strains inhibit the expression of capsule formation on bacteria that are encapsulated in the presence of normal serum.

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.

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.

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.

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.

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.

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.

Immunoelectrophoresis of duck sera and immunoglobulins.

Duck whole sera (DWS) and immunoglobulins (DIg) were studied in immunoelectrophoresis (IE) and immunodiffusion (ID) tests by rabbit (R) anti-DWS and R anti-DIg sera, as well as by R antisera monospecific for DIgM and DIgG classes. Immunoelectrophoresis of adult DWS against R anti-DWS illustrated the presence of DIgM and two subclasses of DIgG. These observations were verified by the use of monospecific R anti-DIgM and R anti-DIgG sera. It appeared that DIgM is an electrophoretically heterogeneous protein with components that migrate more slowly than IgM of other species. The cathodal tip of the DIgM extended into the gamma2 migration zone almost as far as the DigG arc. Changes in the IE pattern of DWS from 1-to-14-day-old ducklings were observed that reflected a reduction in DIgG content from the age of 1 to 7 days followed by a gradual change toward the IE appearance of adult sera. Immunoelectrophoresis and ID failed to detect DIgM in sera of newly hatched ducklings. This class of DIg appeared in sera of 7-day-old duckling and seemed to be more concentrated in sera of 14-day-old ducklings.

Precipitating and agglutinating activity in duck anti-soluble protein immune sera.

Ducks were inoculated parenterally, up to 4 times, with bovine serum albumin (BSA), B gamma globulin (BGG), or horse gamma globulin (HGG). Their sera were tested in immunodiffusion tests for precipitins. Only BSA and BGG induced precipitins. Only 7 of 443 sera tested, obtained from 3 of 62 inoculated ducks, developed a precipitin line with homologous antigen. All 7 sera were obtained from the earliest bleeding (7 days) after inoculation. Sera were also tested for agglutinins in direct passive hemagglutination (DPHA) and direct red-cell-linked antigen tests (DRCLAT) and for nonagglutinating antibodies in indirect red-cell-linked antigen tests (IRCLAT). No duck had a passive-hemagglutination-demonstrable primary immune response. Demonstrable DPHA titers after subsequent inoculations were very low compared with responses to these antigens of other species noted by other workers. Duck immune response was greatly diversified: all inoculation regimens that induced agglutinins in some ducks left others completely unstimulated. Precipitins and agglutinins of the same ducks correlated well: those 7 sera that had precipitins also had the highest DPHA titers for the 3 donor ducks. However, hemagglutinating titer and presence of precipitins in sera of different ducks correlated poorly. Increasing the age of the ducks at the first inoculation from 6 to 10 weeks increased the number of responders and DPHA titers of their sera. Non-agglutinating antibodies were demonstrated in anti-BSA sera: in IRCLAT, the sera had titers 2-to-256-fold higher than the sera in DPHA or DRCLAT had. Duck immunoglobulins were deficient in those immunological reactions (precipitation and agglutination) that require functional bivalency.

Humoral immune response of the duck to duck hepatitis virus: virus-neutralizing vs. virus-precipitating antibodies.

Ducks were induced to develop high-level duck hepatitis virus (DHV)-neutralizing antibodies by inculation with a chicken-embryo-adapted DHV via subcutaneous, intramuscular, and intratracheal routes. Administration of the DHV orally in a gelatin capsule failed to stimulate immune response in the ducks. Contact controls of these ducks also remained negative for anti-DHV antibodies. These observations indicated that the DHV administered orally, in gelatin capsule, failed to infect the ducks. None of numerous duck anti-DHV immune sera, with virus-neutralizing activity in the range of 1.8 to 5.57 log10 median- embryo-infective-dose (EID50) neutralization index, developed precipitin lines against a variety of DHV preparations tested in low- and high-ionic-strength agar. The results suggest that the agar-gel immunodiffusion test is unsuitable for serologic testing of duck sera for anti-DHV antibody activity. Virus-neutralizing activity was revealed in both immunoglobulin M (IgM) and IgG classes of sera of actively immunized ducks. Immunodiffusion tests of Sephadex G-200 fractions of 1-day-old duckling sera with monospecific rabbit anti-duck IgM (DIgM) serum failed to detect DIgM. These results demonstrated that the IgM is not being transferred from the dam to the newly hatched ducklings. Seven- and 14-day-old ducks had DIgM in their sera. However, this IgM had no DHV-neutralizing activity, indicating that it was newly developed by the ducklings, which had no active DHV immune response, not having been exposed to DHV.

Enzyme immunoassay for detection of antibodies specific for staphylococcal alpha haemolysin in bovine milk.

Six lactating cows were inoculated in the external inguinal lymph node region with a staphylococcal cell-toxoid preparation. An additional three cows were inoculated with saline as control animals. Enzyme immunoassay (EIA) was used for detection of IgG antibodies in the milk, specific for staphylococcal alpha haemolysin. The positive EIA response in the inoculated cows was followed over an 84 day period. Saline controls showed no response.

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.

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.

Frequency and immunologic cross-reactivity of encapsulated Staphylococcus aureus in bovine milk in New York.

A total of 213 milk samples, which were positive by bacteriologic cultural examination for Staphylococcus aureus, were obtained from lactating cows on 55 farms throughout the dairying areas of New York. The S aureus isolates were evaluated for encapsulation directly from milk by the serum-soft agar technique. Results indicated that 93% of S aureus field isolates showed evidence of encapsulation on direct evaluation from milk. Suppression of capsule formation in vitro was seen in 72.7% of isolates when grown in the presence of 1% of anticapsular sera. One of the 3 anticapsular sera used exhibited a significantly higher level of cross-reactivity with the field isolates than did the 2 other sera. Increasing the concentrations of the antisera to 2% and 4% in the serum-soft agar had no effect on the expression of capsule in those isolates which had shown no evidence of capsule suppression with the specific antisera at the 1% level. Limited subculture of randomly selected encapsulated isolates on artificial media resulted in a decrease in the percentage of encapsulation from 100 to 33.3.

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.

Bovine antibody against Streptococcus agalactiae, type Ia, produced by preparturient intramammary and systemic vaccination.

Four pregnant heifers were immunized by the intramammary route with killed or live Streptococcus agalactiae vaccine, and a 5th heifer was vaccinated by the intramuscular route with killed vaccine. Antibody in the colostrum from vaccinated and non-vaccinated glands was compared. Antibacterial glands was compared. Antibacterial antibody titers of the 4 immunoglobulin classes were determined by indirect fluorescent antibody assay. Although the content of immunoglobulin G1 (IgG1), IgG2, and IgM in the colostrum from the vaccinated glands was not substantially different from the nonvaccinated glands, IgA content was considerably greater in the former. Antibody specific to S agalactiae was isolated from all colostrum samples. The mouse passive protection test and Ouchterlony analysis were used to demonstrate the presence of type-specific antibody to Ia strain used for vaccination. The passive mouse protection test also was useful to compare the protective capacity of specific S agalactiae, type Ia, antibodies of immunoglobulin classes IgG, IgM, and IgA. Increased protective capacity of IgM and IgA over IgG1, on a weight basis, was demonstrated. The present study indicates that S agalactiae preparations, when introduced into the mammary gland, can give rise to local antibody synthesis in the vaccinated glands.

Antibody response in lacteal secretions of cows after immunization with various concentrations of staphylococcal and streptococcal antigens.

A total of 55 lactating Holstein cows were randomly allocated to 6 groups. Five of these groups (No. 2 through 6) were inoculated on 2 occasions in the region of the external inguinal lymph node with various concentrations of 3 bacterial antigens. Saline solution was administered to group 6 as a control. The antigen preparations consisted of a Staphylococcus aureus bacterin, a Streptococcus agalactiae bacterin, and staphylococcal alpha-toxoid. These antigens were administered as a composite preparation suspended in saline solution. The concentration of antibody in the lacteal secretions, represented by immunoglobulin G specific for each of the 3 vaccine antigens, was monitored during the 18-week experimental period by enzyme-linked immunosorbent assay. The concentration of each of the 3 vaccine components which was required to stimulate a maximal immune response in the lactating gland appears to have been established.

Antibody-producing cells in bovine lacteal secretions after local immunization.

The indirect Jerne plaque assay was used to determine the presence of antibody-forming cells in lacteal secretions of 2 cows inoculated in the mammary gland with T4 phage. Two adjacent mammary glands of 2 nonlactating 7-months pregnant cows were inoculated by intramammary injection 4 times at 3- to 5-day intervals. The presence of plaque-forming cells (PFC) was assessed in each quarter beginning on postinoculation day 8 and at 4- to 14-day intervals thereafter. Amounts of antibodies in serum and secretions were measured by an indirect hemagglutination test. The PFC were detected in secretions from all quarters of both cows between postinoculation days 8 and 32. Concentrations of PFC fluctuated within quarters during the course of the experiment but no relationship was evident between numbers of PFC in a quarter and its inoculation status. The use of monospecific antiglobulin sera at 1 sample-collection period revealed that cells synthesizing immunoglobulin (Ig)G and IgA antibodies were predominant in lacteal secretions. Antibody amounts in serum and secretions rose after inoculation, and titers in secretions were markedly higher in most instances. Antibody-forming cells were thus demonstrated to accumulate in the mammary gland after intramammary inoculation. The presence of antibody-forming cells in non-inoculated glands may have been the result of antigen transfer among quarters, but was considered more likely to have resulted from systemic migration of antigen-stimulated cells with migration into all quarters, regardless of inoculation status. Antibodies in lacteal secretions may have accumulated through a combination of local synthesis and selective transport from the bloodstream.

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.

Secretion and composition of colostrum and milk.

Lacteal fluids contain antibodies of immunoglobulin (Ig) A, IgG, and IgM classes, and these proteins are complete and biologically active. Lymphocytic cells have also been demonstrated that are capable of an array of biological activity characteristic of similar cells in the blood. Lacteal fluids are rich in phagocytic cells that function less efficiently than do their corresponding blood cells.