Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

Cell cycle-dependent fluctuation of urokinase-type plasminogen activator, its receptor, and inhibitors in cultured bovine mammary epithelial and myoepithelial cells.

Bovine mammary epithelial (BME-UV1, clone E-T and BME-UV, clone E-T2) and myoepithelial (BMM-UV, clone m-T2) cell lines were used to study the modulation of cell-associated activity of urokinase-type plasminogen activator (u-PA), as well as mRNA transcripts of u-PA, its receptor (u-PAR), and inhibitors (PAI-1 and PAI-2) during the cell cycle. After release from a growth arrest accomplished by growth factor deprivation, the length of the cell cycle was determined as 19-21 h, with G1, S, and G2+M phases of 6-7, 7-9, and 5-6 h respectively. As the cell cycle progressed, accumulated cell-associated u-PA activity increased. Maximal activity occurred at the S/G2 boundary and decreased during the G2/M phases. All cell lines tested produced plasmin-specific inhibitor(s). Accumulation of u-PA mRNA peaked 3 h after stimulation into the growth cycle for m-T2 and E-T and during 3-6 h for E-T2 cells. Maximum levels of u-PAR mRNA were observed at 3 h for the E-T cell line, 6-9 h for E-T2 cells, and 3-9 h for m-T2 cells. The cell cycle distribution of the PAI-1 mRNA was similar to that of u-PA for both epithelial cell lines, while for m-T2 cells maximal accumulation of PAI-1 mRNA was detected at 3-9 h after growth initiation. The increase of PAI-2 mRNA transcription for m-T2 and E-T cells was detected at 3-6 h. The PAI-2 mRNA in E-T2 cells was under detectable levels. The data indicate that the expression of the constituents of the PA system in bovine mammary epithelial and myoepithelial cells is not cell type-dependent but is tightly connected to the phase of the cell cycle.

An in vitro approach to ruminant mammary gland biology.

This review discusses both fundamental and applied in vitro studies on ruminant mammary gland biology and summarizes progress made over the last decade in development of in vitro techniques to study growth, function and pathology of the mammary gland. The advantages and limitations of different in vitro systems are considered including explant cultures, primary cell cultures and immortalized lines of mammary-derived cells from cow, sheep and goat. The cell growth, differentiation and response to lactogenic hormones and growth factors are discussed as well as the relevance of the cell behavior in different culture conditions.

Adhesion of fimbriate Escherichia coli to bovine mammary-gland epithelial cells in vitro.

The adhesion of bovine and a human isolate of Escherichia coli to epithelial cells from the teat and lactiferous sinuses of the udder was examined. Adhesion was detected with bacterial suspensions that produced mannose-sensitive agglutination of guinea-pig red cells. Adhesion to epithelial cells could be inhibited by mannose and the degree of adhesion occurring with a suspension correlated with its haemagglutinating activity. This demonstrated that fimbriae were responsible for the adhesion. The observation that whole milk inhibited attachment of E. coli to cells in vitro indicates that such attachment may not occur in vivo in the lactating cow.

Differences in intramammary pathogenicity of four strains of Streptococcus dysgalactiae.

The intramammary pathogenicity of four strains of Streptococcus dysgalactiae was measured by infusion of small numbers of bacteria (8-16 colony-forming units) into the teat sinus after milking. Significant differences in the infectivity of strains were detected.

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.

Carriage of Corynebacterium pyogenes by the cattle nuisance flies Hydrotaea irritans (Fallén) and Musca autumnalis (De Geer).

Two species of cattle-visiting Muscidae were experimentally contaminated with C. pyogenes, a pathogen involved in the aetiology of summer mastitis. Surface contamination persisted for at least 4 days. Since M. autumnalis would not feed on media containing C. pyogenes the bacterium did not persist internally. All C. pyogenes were eliminated from the gut of H. irritans in 4 days. H. irritans is thus more likely to transmit C. pyogenes than is M. autumnalis but only by mechanical transfer, and is not a true vector.

The pathogenesis of a high-virulence and a low-virulence strain of Corynebacterium bovis in the mammary gland of the mouse.

A comparison was made of the pathogenesis of experimental mastitis in mice caused by a strain of Corynebacterium bovis (P3) isolated from clinical mastitis in the cow and a strain (NCDO 1930) isolated from the teat of a symptomless carrier cow. Strain NCDO 1930 elicited a neutrophil response which controlled the infection so that, after 6 to 8 days, 9 of 10 glands were sterile and one abscess was found. In contrast, the neutrophil response to strain P3 failed to control the infection and, by 6 to 8 days, 8 of 10 glands were infected and there were abscesses in 11 of 20 glands. The virulence of strain P3 was associated with its ability to colonize on and in milk fat globules, from which the organisms multiplied in the alveolar lumen irrespective of the neutrophil infiltration.

Effects of lysostaphin on Staphylococcus aureus infections of the mouse mammary gland.

A 5 to 6 log10 reduction in the viable count of Staphylococcus aureus was produced in vitro with 10 micrograms lysostaphin ml-1 milk. Infusion of the lactating murine mammary gland with 10 micrograms lysostaphin, immediately following inoculation with 10(8) colony forming units of S aureus, resulted in a significant 2 to 3 log10 reduction in viable S aureus (P less than 0.02) within 30 minutes. Pre-infusion with 10 micrograms lysostaphin either immediately before or one hour before staphylococcal challenge reduced the recovery of S aureus by more than 6 log10 and greatly reduced pathological changes typical of S aureus mastitis. This clearly demonstrates that lysostaphin has considerable potential for the therapeutic or prophylactic control of staphylococcal mastitis.

Use of predictive modeling to evaluate the manipulation of milking frequency, temperature, and oxygen tension on growth of Escherichia coli in an artificial intramammary environment.

A method was developed to evaluate frequent milking as a means of controlling intramammary infection. An artificial intramammary environment was used to determine growth responses of Escherichia coli (P4) to natural changes in the mammary gland resulting from bacterial invasion. Physical conditions manipulated in this model were growth medium, temperature, and oxygen tension. Mathematical modeling was then incorporated to generate predictions concerning growth dynamics of the organism when milking frequency was changed. To test accuracy of the model, initial predictions were derived from bacterial growth data in which E coli was incubated in tryptose soy broth for 12 hours at 37 C and PO2 equal to 23.3 mm of Hg. These predictions matched closely with experimental data in which 12-, 4-, and 2-hour milking intervals were simulated in the artificial intramammary environment. The mathematical model was then used to characterize growth rate data from in vitro experiments in ultra-high temperature-treated milk and in vivo experimental infection data generated with E coli (P4). Predictions generated from this model suggested that increasing milking frequency to 4 or 6 times daily controls growth of E coli for a prolonged period and that 12 times daily milking may lead to elimination of the bacterium.

The bactericidal activity of a teat dip containing chlorhexidine and cetrimide.

Using an in vivo test on teat skin the disinfectant activity of a teat dip containing chlorhexidine and cetrimide was compared with two iodophor solutions, one containing the recommended concentration of 0.5 per cent available iodine and the other a 10-fold dilution of this (0.05 per cent iodine). The test organisms used were Staphylococcus aureus and Escherichia coli and for both species the 0.5 per cent iodophor was significantly more bactericidal than either the diluted iodophor or the chlorhexidine/cetrimide teat dip (P less than 0.01). In the test against S aureus, chlorhexidine/cetrimide and the 0.05 per cent iodophor showed similar bactericidal activity, but the iodophor was significantly more bactericidal against E coli (P less than 0.01). It is argued that due to its low bactericidal activity this formulation of chlorhexidine/cetrimide is likely to be inferior to 0.5 per cent iodophor solution as a disinfectant teat dip.

Mastitis in a large, zero-grazed dairy herd.

A zero-grazed herd of approximately 400 cows had a significant mastitis problem associated with Escherichia coli and Streptococcus uberis during a study over three and a half years. Dry cow therapy and post-milk teat dipping effectively controlled staphylococci and the bulk milk cell count averaged less than 400 X 10(3) cells/ml, but over 1800 clinical cases of mastitis occurred over this period, 32 per cent of which were associated with E coli and 25 per cent with Str uberis. Only 8 per cent of the cases associated with E coli showed obvious systemic disturbance and 75 per cent were cured following penicillin and streptomycin treatment. The incidence was highest during spring and summer when the housed cows were dirtiest. Gross teat-end contamination came mainly from sources other than cubicle bedding, and changing the bedding from sawdust to sand did not alter the incidence of clinical mastitis. It was not possible to maintain adequate cleanliness either inside or outside the parlour, nor maintain a trouble-free milking apparatus. The costs of mastitis in this herd during one year are calculated.

Corynebacterium pyogenes mastitis among heifer calves.

In August 1975 an outbreak of C pyogenes mastitis occurred involving 18 heifer calves at the National Institute for Research in Dairying. This paper describes the outbreak which affected animals varying in age from five to 22 months. Fourteen of the cases (77%) were among calves less than 10 months old. The advisability of using fly control measures among young stock in the summer months is discussed.