A.J. Bramley

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 (>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.

Taxonomic Studies on Streptococcus bovis and Streptococcus equinus: Description of Streptococcus alactolyticus sp. nov. and Streptococcus saccharolyticus sp. nov.

Summary Genetical and biochemical studies were performed on strains of Streptococcus bovis, S. equines and possibly related taxa. Strains presently designated S. bovis and S. equines were found to be very heterogeneous. The type strains of S. bovis and S. equines were found to be genetically closely related (> 70 % DNA homology) and represent a single species. Strains of S. equines from pigs and chickens were found to be sufficiently distinct from the other taxa examined to warrant a new species, S. alactolyticus sp. nov. Some atypical S. bovis strains from straw and cattle also formed a distinct group on the basis of DNA/DNA homologies. These strains were biochemically quite distinct from the other taxa examined and are considered to represent a new species, for which the name S. saccharolyticus sp. nov. is proposed.

Variations in the susceptibility of lactating and non-lactating bovine udders to infection when infused with Escherichia coli.

Small numbers of Escherichia coli were infused into both lactating and non-lactating udders. Twelve of the 17 lactating quarters infused became infected, and all 12 showed clinical symptoms of udder disease. The 5 lactating quarters which did not become infected all had pre-infusion somatic cell counts greater than 300000 cells/ml milk, whilst all the quarters which became infected had cell counts less than 300000 cells/ml milk. E. coli was subsequently recovered from only 6 of the 16 non-lactating quarters infused. In only 2 of these quarters did clinical infection follow, both quarters being in a cow infused 2 d before calving. The remaining 4 quarters from which E. coli was recovered were all negative within 5 d of infusion. These differences in susceptibility are discussed, particularly with reference to the frequent occurrence of coliform mastitis at and shortly after calving.

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.

Studies on the control of coliform mastitis in dairy cows.

SUMMARY A series of experiments are briefly reported which have been carried out to study the pathogenesis of coliform mastitis in dairy cows, and thereby gain information of use in preventing the disease. A relationship between levels of coliform contamination of bedding and the new infection rate is indicated. It is shown that certain milking machine conditions can induce a high rate of coliform infection, but attempts to prevent infection by minimizing cyclic and irregular vacuum fluctuations during machine milking were unsuccessful. It is suggested that maintenance of low levels of coliform contamination of bedding is the only method of control which can be effective.

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.

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.

The carriage of summer mastitis pathogens by muscid flies

Over two successive summers, on six dairy farms in southern England, 2764 muscid flies∗ were netted from dairy heifers and subjected to bacteriological examination for the presence of the pathogens associated with summer mastitis. Six species of flies were commonly represented in the catches but of these the females of Hydrotaea irritans were the flies most strongly implicated in carriage. A total of 27 isolations of one or more pathogens was made from 825 Hydrotaea irritans examined (3·3%). Actinomyces pyogenes and Streptococcus dysgalactiae (14 and 16 isolations respectively) were the most common pathogens detected, but isolations of Peptococcus indolicus (7), Staphylococcus aureus (6) and Streptococcus uberis (2) were also made from Hydrotaea spp. The isolation of more than one pathogen from the same fly occurred very significantly more frequently than would be expected by chance alone (P < 0·001) suggesting a common source of contamination despite the fact that several isolations occurred from flies caught from cattle free of summer mastitis. Hydrotaea meteorica were found to be potential carriers of the pathogens in June.

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.

Evidence of penetration of the bovine teat duct by Escherichia coli in the interval between milkings.

In 3 consecutive experiments, each using 20 cows, the application of Escherichia coli to teat ends after milking led to high rates of intramammary infection. These infections were not prevented by disinfection of the teats before milking, by the installation of shields in the short milk tubes of th milking cluster or by the use of an individual quarter milking cluster. Rates of infection were significantly lower when teat contamination was applied 1 h before milking compared to contamination applied immediately after milking. These data suggest that penetration of te teat duct by the E. coli occurred in the period between contamination and milking. Seventy four percent of infections occurred in hindquarters and there were variations in the susceptibility of cows to infection.