Small volumes of milk each containing an average of less than 10 colonyforming units of Staphylococcus aureus were injected into the teat cisterns of 39 quarters of 10 cows.

Typical staphylococci were recovered from only 14 quarters following overnight incubation of milk stripped manually from each quarter 10 min after injection. There was an inverse relationship between the total leucocyte count of the milk taken from the quarter before infusion and recovery of staphylococci.

From 4 of 8 quarters from which Staph. aureus could not be recovered by enrichment it was possible to recover the organism by centrifuging the milk and culturing the deposit of leucocytes and bacteria.

It is suggested that release of ingested staphylococci from defunct phagocytes may be important in initiation of infection, failure of antibiotic therapy and recurrence of infection.

Eleven Shorthorn cows were milked for 14 days before their expected calving date. The total amount of prepartum secretion varied, between cows, from 1 to 961.

Determinations of total solids, fat, solids-not-fat, lactose, total protein, ash, calcium, phosphorus, chloride, casein, albumin, globulin, proteose-peptone, N.P.N., vitamin A and carotenoids were made regularly on the secretions of eight of these cows during the prepartum period, and for 4 days postpartum; and, for comparison, on five samples of Short-horn colostrum each representing the total secretion obtained from the two milkings within the first 24 hr. after calving.

Samples of bulk silo milk from 5 creameries in the south west of Scotland were taken over a 16-month period. The average radii of casein micelles were determined by measuring the wavelength dependence of the turbidity of skim-milk diluted with its own ultrafiltrate. In addition, the concentrations of casein, soluble and colloidal Ca and P1 were measured. The average size of casein micelles followed a pronounced seasonal trend with smaller average sizes in the summer compared to the winter period, confirming earlier observations made on milks from individual cows. The average size of casein micelles correlated positively (r = +0·777, P < 0·001) with the amount of colloidal P1 per unit weight of casein and negatively (r = −0·77, P < 0·001) with casein-bound Ca. Average micelle radius was negatively correlated with heat stability at the natural pH of milk (r = −0·61, P < 0·001), but there did not appear to be any causal relation between these 2 variables.

Unsupplemented acid goat whey containing 0·96% protein and 2·76% lactose was fermented aerobically with 32 microflora extracted from various raw milk cheeses and dairy products. These microflora were screened for their ability to hydrolyse whey proteins (α-lactalbumin and/or β-lactoglobulin) and to generate peptides inhibitors of Angiotensin I Converting Enzyme. Five microflora were able to degrade whey protein. The most efficient microflora was able to fully hydrolyse α-lactalbumin and to a lesser extend β-lactoglobulin. It was extracted from Bamalou des Pyrenées cheese. Micro-organisms involved consisted of yeast Kluyveromyces marxianus and lactobacillus Lactobacillus rhamnosus. Both were able to produce ACE inhibitory peptides after whey fermentation.

Changes in the composition of the microflora in Cheddar cheese ripened at elevated temperatures were examined. Cheeses were stored under the following ripening conditions: (i) control, 8°C for 32 weeks; (ii) 15°C, (iii) 17·5°C or (iv) 20°C, all for 8 weeks, then followed by 8°C for 24 weeks; (v) 15°C, (vi) 17·5°C or (vii) 20°C, all for 32 weeks. Generally, the total bacteria, lactic acid bacteria, lactobacilli and ‘undesirable lactobacilli’ (producing off flavours and CO2) counts in cheeses stored above 8°C were higher than in the control cheese. This effect seemed to be dependent on continued storage at the elevated temperature. Streptococcal counts rose slightly during ripening at elevated temperatures and fell slightly during ripening at the control temperature. Numbers of non-lactic acid bacteria were unaffected by elevated storage temperatures. In the only treatments where off flavours developed (17·5°C for 32 weeks, 20°C for 16 weeks or more) there was no apparent relationship between any bacterial group and the off flavours.

The self-association of SH-κ-casein at pH 7·0 in 0·01 m-EDTA, 0·001 m-dithiothreitol buffer, containing 0·1, 0·2, 0·5 or 1·0 m-NaCl is of a monomer–polymer type. The polymer is a spherical particle, diam. 23 nm and mol. wt 570000 (30 monomers). These parameters are not greatly influenced by variations in ionic strength above 0·1. The critical micelle concentration, which is a property of a monomer–polymer equilibrium, decreases with increasing ionic strength. The standard free energy of association is about – 36 kJ/mol. monomer at 20 °C.

The interaction of β-lactoglobulin and κ-casein at high temperature was studied polarimetrically. The rate of interaction was related to the genetic variant of β-lactoglobulin present. β-Lactoglobulin B, whose thermodenaturation was faster than that of A variant, interacted more rapidly with κ-casein than did the A variant. Velocity sedimentation studies indicated that characteristics of the complex were determined more by the β-lactoglobulin than by the κ-casein. The presence of κ-casein during the thermodenaturation of β-lactoglobulin appeared to prevent the aggregation of β-lactoglobulin from proceeding to completion, the κ-casein complexing with intermediate species and restricting the aggregation process.

The interaction of sheep, horse, pig, human and guinea-pig whey proteins with fatty acids has been studied. Using gel filtration and autoradiography, it was found that sheep β-lactoglobulin and serum albumin from all species had the ability to bind fatty acids in vitro. Sheep β-lactoglobulin, isolated from milk, had ˜ 0·5 mol fatty acids bound per mol monomer protein, and albumin from sheep, horse and pig contained ˜ 4·5, 2·9 and 4·7 mol fatty acids/mol protein respectively. However, β-lactoglobulin from horse and pig milk had neither fatty acids physiologically bound nor the ability to bind them in vitro. Albumin was the only whey protein detected with bound fatty acids in these species as well as in human and guinea pig. This suggests that the ability of ruminant β-lactoglobulin to bind fatty acids was not shared by the same protein of non-ruminants.

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0·1 m-phosphate buffer, pH 6·6, being completely denatured above 70 °C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 °C (minimum at 55 °C), but stability was very high above 80 °C in this buffer. D-values were determined at 10 °C intervals in the range 70–150 °C in SMS from which a Z value of 31·9 °C and an Ea of 8·82 × 104 J mol-1 were calculated; the half-life at 150 °C was 9 s.

Instability at 55 °C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 °C was inversely related to the rate of heating to 80 °C, i.e. length of time spent in the neighbourhood of 55 °C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 °C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 °C by addition of EDTA or by adjusting the reaction pH to 4·2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented.

Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 °C from 80, 100 or 150 °C, but did regain autolytic activity on cooling to below 50 °C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to ∼ 55 °C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 °C.

The changes in the yield and composition of the mammary gland secretion in the first 16 days of the dry period have been investigated in 6 cows. The quantity of secretion, lactose, potassium, casein, α-lactalbumin, β-lactoglobulin and fat present in the gland decreased rapidly. A decrease was also observed for sodium, chloride, residual albumin and proteose-peptone + globulin but was much less marked. Changes in composition were qualitatively similar to those normally occurring at the end of lactation but they were much more pronounced. Within 8 days after drying-off the concentrations in the secretion of sodium, chloride and potassium were similar to those of blood. There were differences between, animals in the rate at which the changes developed. As the secretory activity of the gland ceased, the constituents of the mammary gland secretion appeared to be resorbed.

The objective was to evaluate the efficacy of a single dose of danofloxacin (6 mg/kg bodyweight) given by the intravenous route for the treatment of acute bovine mastitis induced by intra-cisternal infusion of an Escherichia coli strain (26 cfu into one rear quarter of each cow). Twenty-three Prim'Holstein lactating cows were inoculated. To be challenged, the mammary glands had to be productive, free of pathogenic bacteria, and with somatic cell counts (SCC) of <200 000 cells/ml. The cows were treated on an individual basis when predetermined criteria involving both systemic and local clinical signs were satisfied. Allocation to treatment, danofloxacin or negative saline control, was performed according to a randomized treatment allocation plan. Monitoring during a 21-d period after inoculation included individual clinical examination, bacteriological examination and determination of SCC. Esch. coli was isolated from the milk of all inoculated quarters at the first milking post-inoculation and, together with reference to the clinical scores; the challenge was considered to be successful in 20 of the 23 cows. On study day 7 bacteriological cure rates with danofloxacin and saline control were 89% (8/9) and 44% (4/9) respectively. On days 14 and 21 all milk samples that could be collected were negative for Esch. coli in both groups of animals. Beneficial statistically significant differences were found at the end of the observation period (days 19–21 post treatment) between cows treated with danofloxacin and saline for SCC (P=0·0091) and earlier in the study for milk production (P=0·0003) and udder inflammation (P=0·004). Obvious beneficial trends were recorded in the danofloxacin group for rectal temperature, milk quality, general behaviour and appetite. Danofloxacin-treated cows showed statistically significant lower local clinical scores and a more rapid return to pre-inoculation values. It was concluded that systemically administered danofloxacin is effective in terms of bacteriological results, milk production and both systemic and local signs when used in the treatment of induced acute Esch. coli mastitis. Danofloxacin hastens recovery and return to productivity compared with potential self cure.

The behaviour of Streptococcus thermophilus in combination with Lactococcus lactis subsp. cremoris or subsp. lactis mesophilic starters in experimental Cheddar cheese is reported. In a standard manufacturing procedure employing a 38 °C cook temperature, even very low levels (0·007%) of Str. thermophilus combined with normal levels of the mesophilic starter (1·7%) resulted in increased rates of acid production, the formation of significant amounts of galactose (∼ 13 mmol/kg cheese), and populations nearly equivalent to those of the mesophilic lactic starter in the curd before salting. At a 41 °C cook temperature, the Str. thermophilus attained a higher maximum population (∼ log 8·2 colony forming units (cfu)/g) than the Lc. lactis subsp. cremoris (∼ log 6·8 cfu/g) and formed more galactose (∼ 28 mmol/kg). Lactobacillus rhamnosus, deliberately added to a cheese made using Str. thermophilus starter and which contained 24 mmol galactose/kg at day one, utilized all the galactose during the first 3 months of cheese ripening. Adventitious non-starter lactic acid bacteria had the potential to utilize this substrate too, and a close relationship was demonstrated between the increase in this flora and the disappearance of the galactose. Some possible consequences for cheese quality of using Str. thermophilus as a starter component are discussed.

An immunoassay to quantify αS1-casein (αS1-CN) in milk using an optical biosensor, based on surface plasmon resonance (SPR) measurement, has been developed. The assay consists of a two-step sandwich strategy, with two anti-αS1-CN antibodies directed against each extremity of the molecule. This strategy permits only intact αS1-CN to be quantified and not its degradation products. The calibration curve was obtained using a reference milk powder with a known αS1-CN concentration. Analysis time per sample was less than ten minutes. The antibody-coated surface could be used for more than 150 determinations. Detection limit was established at 0·87 μg/ml and the intra- and inter-assay variation coefficients were 2·86 and 5·31%, respectively. The method was applied to raw milk to quantify intact αS1-CN, with no pre-treatment of the sample. An initial analysis of 48 milk samples permitted αS1-CN concentrations ranging from 8·8 to 12·06 mg/ml to be obtained.

Eighteen cows with a wide range of quarter peak flow rates (0·35–2·22 kg/min) were inoculated with Streptococcus agalactiae and Str. dysgalactiae 4 mm into each streak canal every 3 d for 12 d. Thirty of the 72 quarters developed intramammary infection. Mean peak flow rate and length of streak canal of those quarters that became infected were 1·26 ± 0·08 kg/min (mean ± SEM) and 11·47 ± 0·41 mm respectively, compared with 1·01 ± 0·05 kg/min and 12·05 ± 0·35 mm for those that remained uninfected. Logistic regression analysis showed that the probability of infection increased significantly with the increase in peak flow rate (P = O·O1). The influence of streak canal length on new infection, after allowing for the effect of peak flow rate, was not significant at the 5% level (P = 0·07), suggesting that there may be an inverse relationship between teat duct length and infection, but that it is of secondary importance to peak flow rate. If increased mass of milk distends the teat duct by raising intramammary pressure, then quarter susceptibility to infection is further compromised. These results strongly suggest that the benefits of reduced infection from mastitis control, achieved despite dramatic increases in milk flow rate and milk yield, are significantly underestimated.

This study aimed to describe the expression profiles of microRNAs (miRNAs) from mammary gland tissues collected from dairy cows with Streptococcus agalactiae-induced mastitis and to identify differentially expressed miRNAs related to mastitis. The mammary glands of Chinese Holstein cows were challenged with Streptococcus agalactiae to induce mastitis. Small RNAs were isolated from the mammary tissues of the test and control groups and then sequenced using the Solexa sequencing technology to construct two small RNA libraries. Potential target genes of these differentially expressed miRNAs were predicted using the RNAhybrid software, and KEGG pathways associated with these genes were analysed. A total of 18 555 913 and 20 847 000 effective reads were obtained from the test and control groups, respectively. In total, 373 known and 399 novel miRNAs were detected in the test group, and 358 known and 232 novel miRNAs were uncovered in the control group. A total of 35 differentially expressed miRNAs were identified in the test group compared to the control group, including 10 up-regulated miRNAs and 25 down-regulated miRNAs. Of these miRNAs, miR-223 exhibited the highest degree of up-regulation with an approximately 3-fold increase in expression, whereas miR-26a exhibited the most decreased expression level (more than 2-fold). The RNAhybrid software predicted 18 801 genes as potential targets of these 35 miRNAs. Furthermore, several immune response and signal transduction pathways, including the RIG-I-like receptor signalling pathway, cytosolic DNA sensing pathway and Notch signal pathway, were enriched in these predicted targets. In summary, this study provided experimental evidence for the mechanism underlying the regulation of bovine mastitis by miRNAs and showed that miRNAs might be involved in signal pathways during S. agalactiae-induced mastitis.

Fast protein liquid chromatography was used to study the κ-casein fraction of casein micelles from bulk milk and from milk from individual animals homozygous for κ-caseins A and B. The extent of glycosylation of the κ-casein appeared to have no effect on its distribution in casein micelles of different sizes, nor did it affect the rate at which κ-casein was destroyed during renneting. The rate of breakdown of κ-casein during renneting was also almost independent of micellar size. The results may indicate a difference between methods which analyse for intact κ-casein or for the product macropeptide during renneting.

The dry period plays an important role in maintenance of udder health. Cows are most susceptible to intramammary infections (IMI) after dry-off and near parturition and drying-off procedures may affect the likelihood of IMI at calving. The objective of this study was to evaluate the association of milk yield and infection status at dry-off with the likelihood of IMI at calving by examining different drying-off methods. Cows (n=112) at the Ohio State University Waterman Dairy Teaching and Research Herd were randomly assigned to either an intermittent or a standard, twice-daily milking group 1 week prior to dry-off. All quarters of all cows in the herd were treated with an antibiotic dry-cow product after the last milking. Milk samples were collected 1 week prior to dry-off (pre-dry), on the day of dry-off, and within 3 d of parturition to determine infection status of the quarters. Association between IMI at calving and cumulative milk yield for the final week of lactation and drying-off method was examined using generalized estimation equations with logic link, accounting for potential confounders, such as pre-dry and dry-off infection status, and for the correlated data structure due to quarters clustered within cows. Intermittent milking significantly reduced milk yield at the end of lactation. Increasing cumulative milk yield during the last week of lactation was significantly associated with a greater probability of IMI at calving for quarters that were uninfected prior to dry-off: uninfected quarters of cows producing more than 115 kg during the last week of lactation were 7·1-times more likely to be infected at calving (P=0·0081) than uninfected quarters of cows producing less than 75 kg. Even though the overall cure rate over the dry period was relatively high at 84%, the odds of a quarter being infected at calving was 7·6- and 3·3-times higher if it was infected at dry-off with major pathogens (P<0·0001) or minor pathogens (P=0·028), respectively, compared with an uninfected quarter at dry-off. The results suggest that decreasing milk yield prior to dry-off may serve as an effective means to maintain good udder health in a herd.

α-Lactalbumin and SDS in addition to β-lactoglobulin introduced pH sensitivity to the heat stability–pH curve of serum protein free casein micelles particularly by increasing stability in the pH range 6·4–6·7. Bovine serum albumin, ovalbumin and lysozyme caused marked destabilization of milk and casein micelle suspensions throughout the pH range 6·4–7·4. Tetramethyl ammonium bromide caused destabilization of milk at pH values > 7·0, but had no effect in the region of maximum stability while the non-ionic detergents Triton X-100 and Tween 80 had no effect on heat stability.

Samples of winter bedding and summer pasture, rinses of teats and milking equipment, and milk samples from bulk tanks from 4 farms were analysed bacteriologically for total count (TC), thermoduric count, total spore count (TSC) and psychrotrophic spore count (PSC).

In bedding and pasture samples the TSC comprised the main component of the thermoduric flora but was less than 0·1 % of the TC. The PSC formed ∼ 1 % of the TSC in winter bedding and ∼23 % of the TSC in pasture. For pasture, the PSC: TSC ratio was highest for the upper layer of soil and lower for the grass and deeper layer of soil. The TSC formed 5% of the thermoduric flora in rinses of milking equipment and only a very low PSC was obtained. The TSC of farm bulk tank milks was higher in the winter than the summer but the PSC was similar in both seasons. This was due to an increase of ∼9 times in the PSC:TSC ratio for milks in summer compared with winter. The PSC in raw and commercially pasteurized milks from a local creamery showed a similar seasonal trend.