Persistency of adenoviral-mediated lysostaphin expression in goat mammary glands.

Gene therapy has great potential to enable synthesis of protein molecules in targeted cells of an animal. One application may be the production of antibacterial enzymes by the mammary gland as a means of preventing or treating mastitis. We have previously demonstrated that goat mammary cells are capable of producing lysostaphin, an antistaphylococcal enzyme, after being transduced in vivo with a recombinant adenoviral vector containing a modified lysostaphin gene (Ad-lys). The current study examined duration of expression, and antibody response to lysostaphin and the adenoviral vector. Following intramammary infusion into nonlactating goats (n = 4), recovery of transducible adenoviral vector in mammary secretions persisted for 11 d. Transducible vector was not detected in serum, saliva, urine, or feces. Peak lysostaphin concentrations (< 20 microg/mL) in mammary secretions of infused udders were detected approximately 1 wk postinfusion, and generally returned to undetectable levels after an additional 1 to 2 wk. The poor persistency of expression was likely due to the very potent immune response to both the adenovirus and the expressed lysostaphin. Serum IgG antibodies recognizing the adenoviral vector developed within 7 d of the infusion, and titers rose dramatically to greater than 1:1 x 10(5). Similar titers of serum IgG antibodies to lysostaphin developed in 3 goats, with more moderate titers in the fourth goat. The antibody response to lysostaphin was delayed by approximately 4 d in comparison to the response to the adenovirus. Serum IgG antibody profiles were reflected in mammary secretions. No IgA antibodies to adenovirus or lysostaphin were detected in sera or mammary secretion. We demonstrate that while the lysostaphin gene can be introduced to the mammary gland using an adenoviral-mediated gene transfer technique, the strong immune response that it provokes makes the approach unsuitable for combating mastitis.

Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland.

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.

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.

Staphylococcus aureus stimulates urokinase-type plasminogen activator expression by bovine mammary cells.

Staphylococcus aureus commonly causes bovine mastitis, but bovine strains, unlike human isolates of S. aureus, do not produce the bacterial plasminogen activator, staphylokinase. By use of bovine mammary epithelial and myoepithelial cell lines, it was found that bovine S. aureus M60 and its culture filtrates induce a 3- to 10-fold increase in urokinase-type plasminogen activator activity in mammary cell-conditioned media and cellular lysates. Furthermore, transcytosis of S. aureus M60 across a mammary epithelial cell monolayer was significantly enhanced by the addition of bovine plasminogen and inhibited by aprotinin. These findings provide evidence that S. aureus M60 can trigger superactivation of host plasminogen activator production and may then utilize the plasminogen activator-plasmin(ogen) system to facilitate tissue invasion without producing staphylokinase.

Invasion of bovine epithelial cells by verocytotoxin-producing Escherichia coli O157:H7.

The ability of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 to enter selected human (RPMI-4788 and HeLa) and bovine (MAC-T, mammary secretory; MDBK, kidney) epithelial cell lines was evaluated. All VTEC evaluated efficiently entered RPMI-4788 and MAC-T cell lines. VTEC entered MDBK cells at approximately 4% of MAC-T cells. VTEC were not able to invade HeLa cells. Presence of plasmid had no influence on efficiency of entry, nor did production of shiga-like toxin (SLT I or SLT II). Internalization required microfilaments, but not microtubules. Two types of adherence, localized and diffuse, were exhibited depending on isolate and cell line evaluated. Ability of VTEC to invade bovine mammary epithelial cells may be important in pathogenesis in the bovine, may indicate a route by which raw milk may potentially become contaminated, and may provide a reservoir of bacteria for the contamination of workers, equipment and carcass at time of slaughter.

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.

Cell cycle regulation of mammary epithelial cell detachment by Staphylococcus aureus.

The effect of Staphylococcus aureus on detachment of bovine mammary epithelial cells in culture was examined. Mammary epithelial cells became detached from fresh monolayers following a 3 h incubation in the presence of Staph. aureus M60. Two different procedures indicated that cell detachment coincided with the S-phase of the cell cycle. The roles of proteinases, toxins and Ca availability in inducing cell detachment were examined. Addition of the proteinase inhibitor phenylmethylsulphonyl fluoride (1 mM) to the culture medium prevented cell detachment. Addition of a combination of purified staphylococcal proteinases XVI and XVII-B to the culture medium of mammary epithelial cells induced cell detachment in the absence of Staph. aureus. Cell detachment may be caused by a staphylococcal proteinase. However, addition of Ca (10 mM) to the culture medium abolished Staph. aureus-induced cell detachment, despite the fact that proteinase activity was still apparently present. Isogenic mutants of Staph. aureus M60, expressing either alpha or beta toxins but not both, induced cell detachment, but to a lesser extent than the wild type. Thus, Ca and toxins play some role during cell detachment. Clones established from detached cells that were washed and replated showed the same susceptibility to Staph. aureus-induced cell detachment as the parental cells. This indicated that there is no subclone of mammary epithelial cells more sensitive to this effect.

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.

Effects of Staphylococcus aureus products on growth and function of bovine mammary myoepithelial cells in vitro.

The effects of culture supernatants conditioned by the growth of Staphylococcus aureus M60 on in vitro growth and functional properties of bovine mammary myoepithelial cells were examined. Myoepithelial cell proliferation was reduced by Staph. aureus M60 culture supernatants. Exposure of myoepithelial cells to culture supernatants of isogeneic mutants of Staph. aureus M60 that produced either alpha or beta toxins reduced proliferation, but to a lesser extent than supernatants from the wild type strain. Thus, alpha and beta toxins may play some role in affecting myoepithelial cell proliferation. Of the cells tested, 42% contracted following addition of oxytocin (10(-7) M) in the culture medium. Treatment of myoepithelial cells for 15 min with Staph. aureus M60 supernatants, prior to addition of oxytocin in the culture medium, increased the number of cells that contracted to 92%. Exposure of cells for 3 h to the same supernatant, prior to addition of oxytocin in the culture medium, abolished oxytocin responsiveness, had no effect on immunolocalization of actin and vimentin, but affected the localization of alpha-actinin within myoepithelial cells. Treatment of myoepithelial cells for 3 h with a combination of purified staphylococcal proteinases XVI and XVII-B abolished oxytocin responsiveness and mimicked the effect of the Staph. aureus culture supernatant. We conclude that Staph. aureus M60 culture supernatant affected proliferation and functional properties of myoepithelial cells.

Effect of oxygen tension on killing of Escherichia coli by bovine polymorphonuclear neutrophil leucocytes in vitro.

A batch fermenter modified to simulate the physical conditions of an inflamed v. uninflamed mammary gland was utilized to evaluate the effect of oxygen tension on killing of Escherichia coli P4 by bovine polymorphonuclear neutrophil leucocytes. Leucocytosis was simulated in vitro 4 h after inoculation with Escherichia coli by adding bovine neutrophils isolated from peripheral blood to the culture medium. Experiments were conducted at 39 degrees C in UHT treated milk. At micro-aerophilic oxygen tension (oxygen partial pressure, 3.11 kPa), bacterial numbers declined during the first hour following addition of the neutrophils. Oxygen tension declined rapidly following PMN addition. Once oxygen was depleted, neutrophil activity was presumably diminished and Esch. coli numbers began to increase. Under anaerobic conditions (oxygen partial pressure, 0.17 kPa), no reduction in population was observed. Photomicrographs taken at the time of neutrophil addition and at subsequent time intervals demonstrated a specific association between neutrophils and the pathogen. Subsequent lysis of neutrophils associated with Esch. coli growth was seen coincident with oxygen depletion.

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.

Patterns of intramammary infection and clinical mastitis over a 5 year period in a closely monitored herd applying mastitis control measures.

The udder health of a research herd of between 160 and 220 Friesian cows run on a commercial basis has been monitored closely, including detailed bacteriological study, over 5 years. The five point mastitis control plan had been in use for several years prior to this study and was continued with minor alterations to the management of the plan, more detailed bacteriological monitoring and increased encouragement to apply it. It has proved possible to make a substantial improvement in the udder health of the herd. The percentage of infected cows fell from 21.9 to 12.0 and the percentage of infected quarters from 7.3 to 3.3. The main benefit has been a drastic reduction in the rate of clinical and subclinical mastitis caused by coagulase-positive staphylococci. However the total incidence of clinical mastitis did not change substantially, averaging around 30 cases/100 cows per year. This was largely because environmental mastitis organisms were responsible for 65% of all clinical cases. The results showed marked differences in the patterns of infection due to the environmental mastitis pathogens, Gram-negative bacteria and aesculin-hydrolysing streptococci, suggesting different mechanisms of invasion of the gland.

Effects of Staphylococcus aureus toxins on the growth of bovine mammary epithelial cells (MAC-T) in culture.

The effects of Staphylococcus aureus M60 culture supernatant on growth of mammary epithelial cells were tested in vitro. Exposure of MAC-T cells to S. aureus culture supernatant reduced cell proliferation and colony-forming ability because of reduced ability to adhere to plastic. Growth-inhibiting effects of S. aureus culture supernatant were abolished by pretreatment with trypsin. Lysis of S. aureus cells with lysostaphin demonstrated that the inhibition was also present in cell lysates. Treatment of MAC-T cells with culture supernatant from isogeneic mutants of S. aureus M60 that produced either alpha or beta toxins implicated alpha toxin as the main factor inhibiting cell proliferation.

Effects of temperature and oxygen tension on growth of Escherichia coli in milk.

An in vitro system was developed to mimic conditions within normal and mastitic mammary glands. The system consisted of a modified batch fermenter that allowed for manipulation of temperature, oxygen tension, and pH. Experiments in tryptose-soy broth and UHT-treated milk were conducted to evaluate growth characteristics of Escherichia coli P4 as physical conditions were manipulated. The effect of bacterial growth on oxygen tension and pH were also evaluated. Growth of E. coli was inhibited as temperature was increased from 37 to 41 degrees C and as oxygen tension was decreased from microaerophilic to anaerobic levels. At bacterial populations > 6 log10 cfu/ml, microaerophilic cultures became anaerobic. The pH followed a similar trend; however, after a significant decrease in pH, mean bacterial populations were 7.1 log10 cfu/ml in tryptosesoy broth and 8.2 log10 cfu/ml in UHT-treated milk. This dynamic model demonstrated potential use in evaluation of growth characteristics of mammary gland pathogens in the lactating mammary gland.

Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system.

The lysostaphin gene of Staphylococcus simulans was cloned into Escherichia coli. The 5' end of the gene was modified to include a eukaryotic start codon, the Kozak expression start site consensus sequence, and an enzyme site to facilitate manipulation of the gene. Transcription of the modified gene in vitro yielded an RNA transcript which, when added to a rabbit reticulocyte cell-free translation system, directed the synthesis of several products. The largest product, migrating at approximately 93 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was probably preprolysostaphin, since it was cleaved in the presence of an S. simulans culture supernatant to yield a polypeptide of a size similar to that of mature lysostaphin. When canine pancreatic microsomal vesicles were added to the translation system, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. The gene was also expressed transiently from the human cytomegalovirus promoter in COS-7 cells. Active enzyme could be detected in the cell lysate, and the prokaryotic signal appeared to target secretion of active enzyme to the culture medium. The successful expression of the lysostaphin gene and processing of the precursor to produce active secreted enzyme open up the possibility of controlling staphylococcal mastitis by targeting expression of this gene to the mammary glands of transgenic animals.

Effect of milking without pulsation on teat duct colonization with Streptococcus agalactiae and penetrability to endotoxin.

Forty quarters of 10 cows were milked for a 9-d period with one of four treatments: 1) no liner pulsation, 2) conventional milking, 3) no pulsation for 4 d followed by conventional milking for 5 d, and 4) conventional milking for 4 d followed by milking without pulsation for 5 d. All teat orifices were inoculated with approximately .5 million cfu of Streptococcus agalactiae and Streptococcus dysgalactiae on d 1 and 5. Recoveries of Strep. agalactiae from the teat end were increased for teats milked without pulsation. Recoveries of Strep. dysgalactiae were lower than those of Strep. agalactiae and not increased by milking without pulsation. In a second experiment, teats of 20 cows were milked for a 15-d period with or without liner pulsation. For 10 successive milkings, all teats were inoculated with 1.0 micrograms of Escherichia coli endotoxin either immediately or 2.5 h after each milking. The frequency of endotoxin penetration, measured by the Wisconsin Mastitis Test, in pulsated quarters and in nonpulsated quarters was similar. For quarters milked without pulsation but not for pulsated quarters, inoculation of endotoxin immediately after milking led to greater incidence of teat duct penetration than for inoculation 2.5 h after milking.

The effect of udder preparation before milking and contamination from the milking plant on bacterial numbers in bulk milk of eight dairy herds.

The effect of teat washing and drying on bacterial numbers in bulk milk was compared with that of no teat preparation in eight commercial herds over one year. Using in-line milk samplers, milk was collected at various points during its passage through the milking plant and the samples were used to establish the relative significance of the sources of contamination of raw milk. Teat washing and drying of cows housed during winter reduced the total counts by 40% and streptococcal and coliform counts by 50%. Bacterial counts were significantly lower in cows at pasture during the summer and there was no reduction in count due to teat washing and drying. Bacteriological counts increased at each stage as the milk passed through the milking machine. The milking equipment significantly increased the total colony count by between 2000 and 3000/ml, and the bulk tank added a further 1500 to 2000/ml. The mean rinse bacterial counts of the milking equipment were higher in summer than winter, averaging 4.4 X 10(7) bacteria/m2 compared with 3.5 X 10(7)/m2 respectively. Although this level of bacterial contamination of the equipment is high by current standards, very low bulk milk bacterial counts were nevertheless achieved, particularly in the summer. This confirms that organisms from this source are not a major contaminant of the bulk milk. There was a very poor correlation between rinse counts and the bulk milk bacterial count, but a strong correlation (0.98) between total and streptococcal counts of the bulk milk. The unreliability of the use of rinse techniques to assess the contribution of milking equipment to bacterial counts of raw milk is emphasized.

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.

O controle da "mosca-do-chifre" em novilhos de raça leiteira / Control of the horn fly in heifers of milky race

A eficácia dos brincos inseticidas utilizados no controle das moscas em geral e da "mosca-do-chifre" em particular foi investigada na Inglaterra em novilhas leiteiras. O número de moscas foi reduzido em 90% nos animais portadores de um único brinco e em 99% naqueles que receberam um par, em comparaçao com animais de controle. Em ambos os grupos de tratamento, a mosca hematófaga Haematobia irritans ficou, efetivamente, sob controle absoluto