PubMed Health⌕ Search

Biomedical subjects

A J Bramley

Publications and source records attributed to A J Bramley.

At least 19 recordsLinked to original sources
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

The coagulase of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site-specific coagulase-deficient mutants.

The sequence of the coagulase gene (coa) from Staphylococcus aureus strain 8325-4 is reported. The deduced amino acid sequence of the coagulase protein is compared with previously reported sequences of coagulases from strains 213 and BB. The secreted mature forms of coagulase proteins are composed of three distinct segments: (i) the N-terminal 150-270 residues, which are c. 50% identical, (ii) a central region with high (greater than 90%) residue identities, and (iii) a C-terminal region composed of repeated 27-amino-acid residue sequences. The variable N-terminal sequences are probably responsible for antigenic differences among coagulases of different serotype. The region of coagulase which binds to prothrombin and activates it to form staphylothrombin is also located in the N-terminal half of the protein. A site-specific substitution mutation in the coa gene, which abolished plasma clotting activity, was isolated by recombinational allele-replacement in strains 8325-4 and M60. The Coa- mutants did not show diminished virulence in subcutaneous and intramammary infections of mice. No evidence for a role for coagulase in virulence of toxigenic or nontoxigenic strains was obtained. This contradicts findings of several groups using Coa- mutants generated by chemical mutagenesis and suggests that the earlier results were obtained with strains that had suffered additional mutations in virulence-related genes.

Alleles↗
PubMed1990

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.

Animals↗
PubMed1989

Effect of udder preparation on transmission of Staphylococcus aureus while milking with a multi-valved cluster.

With two groups of 12 cows, a procedure involving foremilking, teat washing and drying before milking was compared with no premilking preparation for its effects on mastitis and teat contamination. Half the cows in each group were deliberately infected in a single udder quarter with Staphylococcus aureus and milked alternately with uninfected cows for 9 weeks. To minimize transfer during milking all cows were milked with the use of a multi-valved claw. Five new staphylococcal infections developed in the 12 animals in the teat preparation group compared to one in the control group. A significantly higher incidence of teat orifice colonization developed in the prepared cows compared to the control cows (17 v. 7). Foremilking and teat preparation led to transfer of staphylococci between quarters (within cow) but this was effectively prevented by the multi-valve claw in the absence of teat preparation.

Animals↗
PubMed1989

Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus aureus for the mouse mammary gland.

Mutants of Staphylococcus aureus which fail to express alpha-toxin (Hly), beta-toxin (Hlb), or both have been constructed by site-specific mutagenesis. The virulence of the mutants was compared with that of wild-type toxigenic strains by intramammary inoculation of lactating mice. A bovine strain, M60, and a laboratory strain, 8325-4, caused acute mastitis and death within 48 h for 60% of the mice inoculated. Animals inoculated with Hly mutants also developed acute mastitis, but no deaths occurred. Comparisons of Hly- or Hlb-positive strains with the double mutation Hly Hlb showed that both toxins led to a significantly higher recovery of S. aureus from the gland 48 h postinfection. Histopathological examination of mammary glands showed that phagocytosis of bacteria occurred irrespective of toxigenicity, but toxigenic strains, particularly those which were Hly+, continued to multiply, invaded the interalveolar tissues, and produced severe lesions. Stimulation of an inflammatory response by inoculation of the mammary gland with endotoxin prior to challenge with S. aureus reduced recovery of the bacteria 10- to 100-fold and, under these conditions, neither alpha-toxin nor beta-toxin contributed significantly to growth and survival.

Animals↗
PubMed1988

Histochemical localization and possible antibacterial role of xanthine oxidase in the bovine mammary gland.

Xanthine oxidase (XO) was demonstrated to be present in the teat canal and secretory tissue of the bovine mammary gland by histochemical techniques. Homogenates of these tissues were able to replace XO in an antibacterial assay with Streptococcus uberis. The action of XO on its substrate hypoxanthine was shown to provide an essential component for anti-streptococcal activity mediated by lactoperoxidase. A mechanism is proposed whereby the interaction of XO, lactoperoxidase and thiocyanate may provide antibacterial activity in the teat canal.

Animals↗
PubMed1988

An in-line sampling technique to measure the bacterial contamination of milk during milking.

The development of devices to sample milk at the clawpiece and at the end of the pipeline during milking are described. The results of tests to compare the bacteriological and compositional quality of milk collected from the sampling devices or from the recorder jar or milk cans were similar, demonstrating that the samples provided accurate and representative values. The use of the in-line sampling technique, whereby samples of the milk are taken at three points during its passage through the milking plant, to assess accurately the sources of bacterial contamination of bulk tank milk on farms is discussed.

Animals↗
PubMed1988

A multi-valved milking machine cluster to control intramammary infection in dairy cows.

A milking machine claw incorporating valves to prevent movement of milk between teats during milking, and its contribution to the prevention of udder infection under experimental and field conditions, is described. Under experimental conditions a suspension of Streptococcus agalactiae and Str. dysgalactiae was introduced into the milking cluster during milking; 11 of 40 quarters became infected using a conventional claw piece whereas none of 40 quarters milked with the multi-valve claw developed intramammary infection. In a 12-month experiment in ten commercial herds, each split into two equal susceptibility groups, the multi-valved cluster reduced the incidence of new infection with coliforms and Str. uberis by 17% (P less than 0.1). The incidence of all new infections and new clinical cases was 14 and 25% lower compared to conventional clusters but these reductions were not significant.

Animals↗
PubMed1986

Influence of the lactoperoxidase system on susceptibility of the udder to Streptococcus uberis infection.

Lactoperoxidase (LP), thiocyanate (SCN-), pH and somatic cell counts (SCC) were measured in mammary secretions from 20 cows collected 14 d before drying-off, 7 and 21 d after drying-off, and 3-18 d postcalving. The inhibitory activity of the secretions on Streptococcus uberis was determined and the susceptibility of the udder to infection by this organism was tested by intramammary infusion of 250 colony forming units at the above stages. LP, SCN-, pH and SCC increased during involution and fell postcalving. The secretions collected before drying-off, 7 d after drying-off and postcalving inhibited growth of Str. uberis.; those collected 21 d after drying-off did not. Inhibitory activity in pre-drying-off secretions was destroyed by heating and restored by addition of LP, glucose and glucose oxidase, but addition of these substances to secretion 21 d after drying-off did not provide a full inhibitory system. The growth of Str. uberis in the secretions was correlated with intramammary susceptibility, since challenges with Str. uberis at 14 d before drying-off, at 7 and 21 d after drying-off and postcalving led to 43.8, 25.0, 81.3 and 37.5% of quarters becoming infected. It is suggested that the LP/SCN-/H2O2 system plays a role in protecting the lactating mammary gland from infection with Str. uberis but becomes ineffective as involution progresses.

Animals↗
PubMed1985

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.

Animals↗