N.L. Norcross

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.

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

The effect of staphylococcal β 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 α and β toxins and with culture supernatants from S. aureus M60 and two mutant strains that are negative for either the production of α (DU5789α−) or β (DU5846β−) toxin. Lysis of bovine erythrocytes was due primarily to β toxin. Alpha toxin increased the lysis of bovine erythrocytes by purified β toxin, but the presence of α toxin in culture supernatants from S. aureus did not increase the lysis of bovine erythrocytes. Purified β toxin was cytotoxic to mammary secretory epithelial cells, but to a lesser extent than α toxin. Together they exhibited an additive effect on mammary epithelial cells. Inactivation of the α toxin-gene of S. aureus M60 decreased the cytotoxic effect on mammary epithelial cells to a greater extent than the inactivation of the β toxin-gene. Also, the relative percentages of DU5789α− and DU5846β− 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 β toxin damages bovine mammary secretory epithelial cells, increases the damaging effects of α toxin, increases the adherence of S. aureus to mammary epithelial cells and increases the proliferation of S. aureus.

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.

BOVINE MYCOPLASMA MASTITIS

One of the more difficult problems encountered by investigators interested in the pathogenicity of the genus Mycoplasma is distinguishing between the mere association of these organisms with disease and a true cause-effect relationship. In this context, diseases that have been unequivocally shown to be due to mycoplasma infection alone assume particular significance, especially if such diseases occur naturally. An infectious mastitis of cattle is in this category. Mycoplasma infection of the bovine mammary gland was probably first observed by Alstrom (1955) in Sweden. He isolated pleomorphic organisms from bovine milk on serum-enriched media and considered them variants of bacteria present in some of the same milk samples. Retrospectively, it seems likely that these serum-dependent organisms were mycoplasma. The English investigators Davidson and Stuart (1960), in a brief report, described the isolation of mycoplasma from milk samples taken from cows with mastitis, but did not clearly establish an etiological relationship. Hale et al. ( 1962) at the University of Connecticut cultured mycoplasma from the milk of cattle suffering from severe mastitis. Similar results were reported by Carmichael et al. (1963), who isolated mycoplasma from severe mastitis cases in IS New York dairy herds. Both groups showed that the disease could be reproduced readily with pure cultures and described clinical and pathological changes that occurred in infected udders. In 1961, Langer isolated a hemadsorbing agent in tissue culture from the milk of a cow with bacteria-free mastitis. Langer initially considered this agent a virus, and designated it strain 56-R. Further study (Langer and Carmichael, 1963) showed that 56-R could be cultivated on artificial serum-enriched media, and that this isolate possessed characteristics of mycoplasma. Other milk samples drawn in 1961 from cows with mastitis had given negative results on conventional bacteriological media, but produced definite, though slight, changes in inoculated bovine kidney cell cultures. Although no microorganisms were identified, the milk samples were considered suspicious and were frozen for study at a later time. After the identification of 56-R as a mycoplasma, the stored samples were recultured on serum-enriched artificial media, and a portion of them yielded mycoplasma. These isolates, however, were different from strain 56-R. Severe, purulent bovine mastitis was produced experimentally with pure cultures of each strain. In 1963, Stuart et al. published the results of further studies on isolates from bovine mastitis in England, and presented proof that the mycoplasma which they had isolated were responsible for the outbreaks of severe mastitis that had been observed earlier (Davidson and Stuart, 1960). Subsequent outbreaks have been diagnosed in Massachusetts, California, Israel ( Bar-Moshe, 1964), as well as in additional herds in Connecticut and New York.

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.

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, Freunds 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 Freunds incomplete adjuvant.

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.