Effect of teat skin disinfection on the rate of infection and interval to infection in cows exposed to high levels of Staphylococcus aureus

Richard F. Sheldrake and Roderic J. T. Hoare

Journal of Dairy Research (1981), 48, 1–5

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 the 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 the 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.

The effect of subclinical mastitis on the citric acid concentration of cow's milk was studied by comparing milk from the mastitic quarter with that from a healthy quarter of the same udder. Forty-two pairs of fore-milk from the morning milking of 17 cows were examined and the citric acid content was measured by an automated carboxylic acid analyzer. The citric acid concentration decreased in subclinical mastitic milk and the extent of decrease was proportional to the degree of mastitis as expressed by the quarter difference value in concentration of Na+Cl in mm/1, i.e. the increase in the Na and Cl concentrations in the abnormal milk over that found in normal milk from the same udder. The correlation coefficient between the quarter difference value of Na+Cl and the value for citric acid was –0·77, (d.f. = 46, P <0·001) and a linear regression equation was obtained. The result showed clearly that subclinical mastitis is one of the causes of variation in the citric acid content of cow's milk. The quantitative change in citric acid concentration in subclinical mastitic milk is discussed with reference to the admixture theory for the formation of abnormal milk.

The prevalence of intramammary infection in 4 commercial goat herds was studied in conjunction with electronic somatic cell count and volume analysis, determined using a Coulter Counter and volume analyser.

Neither streptococci nor mycoplasma were isolated from any half and the prevalence of intramammary infection with Staphylococcus aureus ranged from 0 to 3% between herds. For coagulase-negative staphylococci the range for infected halves was 36–71%. There was no significant difference between the mean total microscopic somatic cell count for halves infected with coagulase-negative staphylococci and those free from infection. A similar trend was observed for electronic somatic cell counts although the mean electronic cell count was greater than the mean total microscopic count on the 2 occasions that they were compared. The correlation coefficients between the 2 cell counting methods were 0·86 and 0·94. Between herds there were significant differences in mean electronic somatic cell count, with herd means ranging from 438×103 to 1684×103 cells/ml. In 2 of the 4 herds studied, milk samples from halves infected with coagulase-negative staphylococci had a significantly higher prevalence of cell volume distributions with a modal cell volume between 65 µ3 and 100 µ3. This was attributed to a higher proportion of polymorphonuclear neutrophils.

Use of electronic somatic cell count and cell volume analysis were considered of little value in predicting infection caused by coagulase-negative staphylococci as there was a high proportion of false negative and false positive predictions.

Changes in the ethanol (EtOH) stability/pH profile of skim-milk induced by forewarming have been investigated as a function of temperature and duration of forewarming and of milk pH prior to heating. Forewarming increased the EtOH stability of a milk in the mid-pH range, shifting the profile laterally to more acid pH values, with little effect being observed on the maximum and minimum EtOH stabilities. No significant changes in forewarming effects were obtained on adding the sulphydryl-blocking agent, N-ethylmaleimide, indicating the lack of involvement of serum protein interactions in the stabilizing effects of forewarming. It is suggested that the observed effects of forewarming are due to a heat-induced precipitation of colloidal calcium phosphate. This suggestion is supported by measurements of Ca2+ levels in forewarmed and untreated skim-milk as a function of pH and is in agreement with the previously suggested mechanism for the EtOH-induced coagulation of skim-milk. The effects of permitted additives (citrate, phosphate and Ca) on the EtOH stability of forewarmed milk paralleled their effect on untreated milk. A comparison is made of the effects of forewarming on the heat and EtOH stabilities of skim-milk.

Mechanisms controlling exchange and partition of minerals and β-casein between micellar and soluble phases during cooling have been studied using suspensions of milk proteins of varying concentrations in the presence of aqueous phases having varying mineral compositions. In these studies the final equilibrium between Ca and phosphate was found to be regulated by formation of Ca phosphates of varying solubilities. The effect of the total casein level on mineral and protein equilibria was also studied. In milk, part of the β-casein content is bound to micelles by hydrophobic bonds; the proportion present in this form was increased by spontaneous micellar demineralization during cooling as well as by addition of a complexant to milk. At low temperatures, an equilibrium between the micellar and the monomeric states was reached which depends upon the total casein level and on the hydrophobic-bonded β-casein content. Not all of these hydrophobic bonds were broken when milk was cooled.

Different fractions of human κ-casein characterized by their sensitivity to chymosin were separated, after reduction, by affinity chromatography on thiol-Sepharose in a 0·1 m-Tris-HCl buffer, pH 7·0, containing 7 M-urea, 0·3 m-NaCl and 1 mm-EDTA. The fraction retained on the column and bearing the SH-groups was then chromatographed on hydroxyapatite in a 5 mm-phosphate buffer, pH 6·8, containing 0·2 m-KC1, 4·5 m-urea and 2 mm 2-mercaptoethanol. The fraction not retained on this column was purified by chromatography on DEAE-cellulose in a 6·6 m-urea, 0·02 m-imidazole, 0·02 m 2-mercaptoethanol buffer, pH 7·0. Two chymosinsensitive fractions were identified by starch-gel electrophoresis at pH 8·6; one of low electrophoretic mobility, the other a smeared band covering the whole distance of migration, in which several bands with mobilities greater than those of β-caseins could be detected.

Whole casein preparations obtained from paired quarters (one healthy and one subclinically infected) of each of 6 individual cows were fractionated quantitatively by column chromatography on hydroxyapatite. Infection was associated with an apparent decrease in the αs- and β-casein fractions and increase in the γ- and κ-casein fractions. The increase in the κ-casein fraction was attributable mainly to enrichment with chymosin-resistant minor components of casein.

The profile produced by column chromatography on DEAE-cellulose of casein isolated from a healthy quarter of a single cow differed significantly from that from a subclinically infected quarter of the same cow. These differences were increased by incubation of the quarter milks at 37 °C for 8 h. Extensive fragmentation of [14C]methylated αs2- and β-casein, and less extensive fragmentation of [14C]methylated αs1-casein, occurred during incubation of these proteins in milk from an infected quarter. Fragmentation was almost entirely inhibited by soybean trypsin inhibitor. From a consideration of the chromatographic behaviour of the radiolabeled fragments on DEAE-cellulose, and other data presented, it was concluded that proteolysis of caseins by a plasmin-like enzyme was sufficient to account, in qualitative terms, for most of the compositional changes exhibited by casein from subclinically infected quarters.

A method for determining chymosin and bovine pepsin A in commercial extracts of bovine veils, based on the use of the synthetic hexapeptide (Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe) as reference substrate, is reported. Chymosin and bovine pepsin A were separated chromatographically from extracts and assayed for clotting activity on a reconstituted skim-milk standardized with reference chymosin and bovine pepsin A, themselves standardized in relation to the hexapeptide. The effect of pH on the absorbance difference between the hexapeptide and the Leu-Ser-Phe(NO2) tripeptide resulting from its hydrolysis was studied. It was found that the ‘optimal’ pH for determining the activities of the reference enzyme solutions was 4·7.

Six chymosin and 3 bovine pepsin A preparations were assayed on the hexapeptide to define the relation between the proteolytic activity and the amount of active enzyme. At pH 4·7 and 30 °C 1 mg chymosin and 1 mg bovine pepsin A hydrolysed 100 and 2700 μm-peptide/s respectively. The clotting activity of these preparations was assayed on a reconstituted skim-milk to standardize it and thus define the relation between the clotting time and the amount of active enzyme. Chymosin had a specific clotting activity twice that of bovine pepsin A. At equal clotting activities, bovine pepsin A was 55 times more active than chymosin on the hexapeptide at pH 4·7.

Quality characteristics of goat's milk yoghurt made from homogenized and non-homogenized concentrates prepared by vacuum evaporation, addition of dried skimmed goat's milk, reverse osmosis or ultrafiltration were observed. Yoghurts made from ultrafiltered milk gave the best flavour and viscosity, whilst homogenization of the concentrates also increased these qualities. Cow's milk yoghurts prepared in the same way were improved to an even greater extent by homogenization. Stirred goat's milk yoghurt had too low a viscosity to be acceptable, unless made from homogenized ultrafiltered milk.

The interactions of 23 bacteriophages on strains of lactic streptococci at 30, 38 and 40 °C were studied in reconstituted skim-milk and broth media. In milk, 3 different temperature mediated responses were observed. Extensive phage replication at 30, 38 and 40 °C with inhibition of acid production by infected cultures was observed for 8 phages; extensive phage replication at 30 and 38 °C, but not at 40 °C, was observed for 4 phages, while 11 phages replicated at 30 °C but not at 38 or 40 °C. Studies in broth media revealed that failure of phage to replicate at 38 and 40 °C was not due to lack of host growth in most cases, but due to a temperature sensitivity on the part of the phage. In one phage–host system, phage replication continued after growth of the host had ceased. The significance of these findings to starter selection for cheese manufacture is discussed.

Carbonyl sulphide (COS) was detected in the headspace of Cheddar cheeses in concentrations ranging from 0·01 ng ml-1 to 0.3 ng ml-1. It was also found in the headspace above certain packaging materials and sealing materials used in the analytical procedures. Evidence that COS was produced within the cheese was given by the findings that experimental cheese containing added cysteine had consistently higher COS levels (up to 1·0 ng ml-1) and that COS could be produced chemically from mixtures of cysteine or methionine with riboflavin and ascorbic acid in laboratory trials.

The hydrolysis of isolated αs1- and β-caseins by Penicillium roqueforti aspartyl proteinase produced comparable quantities of pH 4·6 soluble N. The amount of non-protein nitrogen obtained with β-casein was clearly lower than that obtained with αs1-casein, showing that few low molecular weight peptides were released when this casein was hydrolysed. Electrophoresis of αs1-casein hydrolysates produced by aspartyl proteinase showed 5 bands of mobility close to or higher than that of αs1-casein. β-Casein hydrolysates gave 4 bands, 2 of which (βPrapl and βPrap2) showed low electrophoretic mobility. The products corresponding to βPrapl and βPrap2 were purified from a β-casein hydrolysate and identified as fragments Val98-Val209 and Glu100-Val209 of β-casein respectively. The occurrence of the βPrapl and βPrap2 bands in electrophoretic patterns obtained from sterile curd with aspartyl proteinase and controlled-flora curd, where P. roqueforti was the only micro-organism developing, showed the presence of aspartyl proteinase synthesis and activity in cheese. A band of very low electrophoretic mobility (βPrmpl) was present in electrophoregrams of controlled-flora curd inoculated with P. roqueforti. This band, resulting from the action of the metalloproteinase on β-casein, revealed that this enzyme was both synthesized in and active in cheese.

Four enzymes of citrate metabolism (viz. citrate lyase, acetolactate synthase, diacetyl reductase and acetoin reductase) were constitutively present in cells of several strains of Streptococcus lactis subsp. diacetylactis. In strain DRC1, which was studied in detail, diacetyl reductase and acetoin reductase were partly repressed and acetolactate synthase partly induced by growth on citrate. The stage of growth also affected the formation of each enzyme. The buffer species affected the activity of acetolactate synthase, diacetyl reductase and acetoin reductase.

Citrate stimulated growth, totally induced citrate lyase, partly induced acetolactate synthase activity and partly repressed both diacetyl and acetoin reductases in Leuconostoc lactis NCW1. Similar results were obtained with 2 other leuconostocs and a heterofermentative lactobacillus. In 2 of the 3 leuconostocs tested, diacetyl reductase and acetoin reductase were NADPH specific, while in the 2 heterofermentative lactobacilli, they were NADH specific.