A preparation of the bacteriocin lacticin 3147 (prepared from a demineralized whey protein fermentation liquor) was combined as a powder with a bismuth-based intramammary teat seal and evaluated for its potential as an antimicrobial in non-lactating cows. The lacticin/teat seal formulation enabled significant bacteriocin release from the seal without the requirement for a surfactant. Studies in vivo in lactating cows demonstrated that this formulation was effective in reducing bacterial recoveries (~20-fold) from teats deliberately inoculated with Staphylococcus aureus after infusion. Moreover, this formulation also significantly reduced the numbers of Staph. aureus recovered from teats that were exposed to the challenge bacterium before the infusion of the teat seal preparation. The powdered preparation of lacticin 3147 did, however, cause some teat irritation as evidenced by associated rises in somatic cell count (SCC). However, this effect was short-lived and when the mean SCC readings pre-infusion and the final two readings post-infusion were compared, there was no significant difference in the immunological acceptance between treatments.

Ewe milk fat from five different herds was studied to determine the content of conjugated linoleic acid (CLA) isomers. Research was carried out by combining gas chromatography-mass spectrometry (GC-MS) of fatty acid methyl esters (FAME) and 4,4-dimethyloxazolyne derivatives (DMOX) with silver ion-high performance liquid chromatography (Ag+-HPLC). Reconstructed mass spectral profiles of CLA characteristic ions from DMOX were used to identify positional isomers and Ag+-HPLC to quantify them. Total CLA content varied from 0·57 to 0·97 g/100 g of total fatty acids. FAME and DMOX were separated into a complex mixture of minor isomers and major rumenic acid (9-cis 11-trans C18:2) by GC-MS using a 100-m polar capillary column. Rumenic acid would represent more than 75% of total CLA. 11-trans 13-trans, 11–13 cis/trans plus trans/cis and 7–9 cis/trans plus trans/cis were the main CLA isomers after rumenic acid. Minor amounts of 8–10 and 10–12 C18:2 isomers were also found. Although most of the isomers were present in each herd's milk, differences in content were observed for some CLA species.

Transgenic and knockout mouse models are extensively used to study the mechanisms of tumour formation. The availability of mouse models to study metastatic spread of tumours is although quite limited. S100A4(mts1), that belongs to the S100 family of Ca-binding proteins, has been shown to function as a metastasis-promoting protein. We generated strains of mice with modified expression of S100A4 in order to understand the mechanism by which S100A4 protein stimulates metastatic spread of the tumour cells. Transgenic mice over-expressing the S100A4 gene in the mammary gland were crossed with GRS/A mice, characterized by a high incidence of spontaneous non-metastatic mammary tumours. The resulting hybrid mice developed metastatic tumours. Transgenic mice with ubiquitous expression of S100A4 developed vascular tumours, hemangiomas and contained enhanced levels of the S100A4 protein in the blood. Based on these observations we demonstrated that extracellular S100A4 functions as an angiogenic factor. Study of tumour development in the S100A4 – deficient mouse model demonstrated key role of extracellular S100A4 in stimulation of tumour development and metastasis formation.

The thermal resistance of the lactococcal bacteriophage P008 was investigated between 55 and 80 °C. Inactivation kinetics revealed an order of reaction above 1 and could be determined by a non-1st-order regression model. Phage inactivation was influenced by the medium (milk and Ca-M17-broth). Within the investigated temperature range, milk had a protective effect on phage P008. This was reflected in the rate constant and in the activation energy. Thermal phage inactivation studies reported in literature were re-analysed using non-1st-order regression. The obtained kinetic parameters showed that phage P008 belongs to the most heat resistant lactococcal phages investigated so far.

The influence of pH (4·5–6·5), sodium chloride content (125–375 mM), calcium chloride content (10–30 mM), protein concentration (70–90 g/l) and lactose content on the gel hardness of goat whey protein concentrate (GWPC) in relation to the origin of the acid whey (raw or pasteurized milk) was studied using a factorial design. Gels were obtained after heat treatment (90 °C, 30 min). Gel hardness was measured using texture analyser. Only protein concentration and pH were found to have a statistically significant effect on the gel hardness. An increase in the protein concentration resulted in an increase in the gel hardness. GWPC containing 800 g/kg protein formed gels with a hardness maximum at the pHi, whereas GWPC containing 300 g/kg protein did not form true gels. Whey from pasteurized milk formed softer gels than whey from raw milk. A high lactose content (≈360 g/kg) also reduced the gelation performance of GWPC.

The effect of an added adjunct culture consisting of facultatively heterofermentative lactobacilli (FHL) on the volatile compounds and sensory characteristics of a Spanish ewes'-milk cheese was examined. Three cheese batches were prepared using a commercial starter, one from raw milk, another from pasteurized milk, and a third from pasteurized milk with an added culture of wild Lactobacillus. paracasei+Lb. plantarum. Analysis of the volatile compounds was carried out by the purge and trap method and gas chromatography with a mass spectrometer and disclosed a total of 86 compounds belonging to the chemical families hydrocarbons, fatty acids, esters, ketones, aldehydes, and alcohols. After ageing for 120 and 240 days, the cheese samples underwent sensory analysis by a panel of expert assessors. The attributes evaluated were characteristic odour and odour intensity and characteristic aroma and aroma intensity. Pasteurization of the milk had an effect on the formation of certain volatile compounds, adversely affecting the characteristic flavour of the cheese. Use of the adjunct culture in addition to the commercial starter improved the flavour of the cheese made from the pasteurized milk, which earned sensory scores similar to those awarded to the cheese made from the raw milk. Use of adjunct cultures consisting of indigenous FHL strains could help to conserve the traditional characteristics of Roncal cheese made from pasteurized milk, although some technical adjustments to the Regulations would be needed.

The decline of mammary epithelial cell (MEC) number during mammary gland involution in the cow is due to inhibition of proliferation and induction of apoptosis. Transforming growth factor-beta 1 (TGF-β1) belongs to a group of intramammary auto/paracrine inhibitors of bovine MEC growth and inducers of apoptosis. However, the mechanism responsible for the regulation of TGF-β1 expression in MEC is not known. The present study examined the effect of the hormones, growth hormone (GH), somatostatin (STS), 17-β oestradiol (E2), progesterone (P4), as well as the growth factors, insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF), on TGF-β1 expression in the bovine MEC lines, BME-UV1 and MAC-T. The model of apoptosis in bovine mammary gland in vitro was applied by reduction of fetal bovine serum (FBS) (from 10% to 2% or 0·5% FBS) in the cell environment to show the relationship between TGF-β1 expression and apoptosis in bovine MEC. RT-PCR, Western blot and laser scanning cytometry (LSC) were used for analysis of TGF-β1 transcript and protein level as well as apoptosis and cell cycle in examined MEC. In this model of apoptosis, FBS deficiency (mimicking the naturally occurring decline in the access of bioactive compounds and nutrients at the end of lactation and dry period) was associated with increased TGF-β1 expression at the level of transcript and protein, induction of apoptosis and inhibition of cell cycle. Exogenous TGF-β1, IGF-I, EGF and GH inhibited FBS-deficiency-stimulated TGF-β1 expression. The suppressive effect of GH was reversed when cells were maintained longer in FBS-deficient medium. In general, STS, E2 and P4 increased TGF-β1 expression. However, this effect was dependent on hormone concentration and cell line. BME-UV1 cells were much more responsive to the peptides, GH, STS, IGF-I and EGF, whereas MAC-T cells were more responsive to the steroid sex hormones: E2 and P4.

Accurately assessing the economic impacts of diseases and other factors that affect milk production requires that the demand for milk be taken into account. Because demand for milk in the USA is relatively inelastic (i.e., consumers generally purchase a somewhat fixed amount over a given time frame, regardless of fluctuations in price), consumers tend to reap much of the benefit of enhanced production. An examination of the economic impacts of Johne's disease indicated that reduced milk production, associated with the determination of dairy operations as Johne's-positive, reduced consumer surplus by $770 million ± $690 million, and resulted in a total loss of $200 million ± $160 million to the US economy in 1996. Most of the economic surplus lost by consumers was transferred to producers, whose economic surplus increased by $570 million ± $550 million as a result of the reduced milk production associated with Johne's disease. Uncertainty analysis showed that the estimated reduction in milk production on Johne's-positive dairy operations accounted for most of the uncertainty in the economic-impact estimates. If Johne's disease had not been present on US dairy operations, then an additional 580 million ±460 million kg of milk would have been produced, but the price would have fallen by 1·1±1·0 cents/kg, and the total value of the milk would have decreased by $580 million ± $560 million.

Casein genes are expressed in a tissue-specific and highly coordinated manner. The main goals of casein gene promoter studies are to unravel cis- and trans-acting factors involved in the complex signalling pathway controlling milk production, and to explore the possibility of using these promoters for tissue-specific production of heterologous proteins in the mammary gland. Here we present a comparative study of the equine β-casein and κ-casein gene proximal promoters. In order to confirm the assumption that in the horse, as in other mammalian species, casein genes are organized in a cluster located on a single chromosome, we performed in situ hybridization of pro-metaphase chromosomes with two BAC clones containing different equine casein genes. Sequence analysis of the β-casein and κ-casein gene proximal promoters revealed binding sites for activators (STAT5, GRE, NF1, MAF) and repressors (YY1, PMF), characteristic for casein genes. The alignments of casein gene promoters revealed the highest sequence identity in the proximal promoter region between the equine and human β-casein gene promoters. We directly compared the activity of equine β-casein and κ-casein gene promoters in vitro using bovine mammary gland cell line BME-UV1. In this system, the κ-casein gene proximal promoter activated the reporter gene expression more efficiently than the β-casein gene promoter of approximately the same length. The 810 bp of β-casein promoter activated the reporter gene expression more efficiently than the long fragment (1920 bp) and the 1206 bp fragment of the same promoter, which included also 396 bp of 5′ UTR.

Changes in nitrogen compounds during ripening of 40 batches of Manchego cheese made from raw milk (24 batches) or pasteurized milk (16 batches) at five different dairies throughout the year were investigated. After ripening for six months, degradation of p-κ- and β-caseins was more intense in raw milk cheese and degradation of αs2-casein in pasteurized milk cheese. Milk pasteurization had no significant effect on breakdown of αs1-casein. Hydrophobic peptide content did not differ between raw and pasteurized milk cheese, whereas hydrophilic peptide content was higher in raw milk cheese. There were no significant differences between seasons for residual caseins, but hydrophobic peptides were at a higher level in cheese made in autumn and winter and hydrophilic peptides in cheese made in winter and spring. Raw milk cheese had a higher content of total free amino acids and of most individual free amino acids than pasteurized milk cheese. The relative percentages of the individual free amino acids were significantly different for raw milk and pasteurized milk cheeses. The relative percentages of Lys and Ile increased, while those of Val, Leu and Phe decreased during ripening. There were also seasonal variations within the relative percentages of free amino acids. In raw milk cheeses, Asp and Cys were relatively more abundant in those made in autumn, Glu and Arg in cheeses made in winter, and Lys and Ile in cheeses made in spring and summer. Biogenic amines were detected only in raw milk cheese, with the highest levels of histamine, tryptamine and tyramine in cheeses made in spring, winter and spring, respectively.

By its numerous properties and importance in cheese technology (production of colour, flavour, bacteriocins and resistance to salt) Brevibacterium linens is a major cheese ripening bacteria. However, the genetic approach of such biological functions has been hindered, up to now, by the lack of tools necessary to realise genetic modifications in this species. Our objective was to demonstrate that it is possible to genetically modify several strains exhibiting interesting technological properties, especially the production of sulphur compounds. We worked with a phenotypically and genetically diverse collection of 11 strains. We genetically transformed several Brevi. linens with acceptable rates with plasmids classically used to transform lactic acid bacteria and other Gram+ bacteria. These results open up new prospects to investigate the most interesting Brevi. linens metabolic pathways both at the biochemical and genetic level.

The objectives of the study were: to evaluate the interaction between visual appearance and California mastitis test (CMT) score of the foremilk in relation to the cell count of the milk; to evaluate the consequences of sorting milk according to these criteria; and to explore whether visual appearance and CMT score of foremilk depended on the time interval between milkings. Measuring somatic cell count (SCC) in composite milk only and discarding milk above certain thresholds will not ensure that milk from all cows with visually abnormal foremilk is withheld from delivery. Low thresholds of SCC will reduce the frequency of cows with abnormal milk but increase the discarding of milk from cows with visually normal foremilk. CMT score of foremilk differentiated better between cows with high and low SCC in composite milk than visual inspection of foremilk. CMT scores of foremilk decreased with increasing interval between milkings within cow, whereas the visual appearance was independent of the interval. We propose that visual appearance of the foremilk should be kept as a criterion for sorting milk at time of milking. For test purposes, the use of visual appearance of foremilk for differentiation between normal and abnormal milk has to be done on multiple milkings. Additionally, CMT scoring of foremilk improves correct classification of normal and abnormal quarters and especially when including data from the previous milking.

The mammary gland undergoes extensive tissue remodelling during each lactation cycle. During pregnancy, the epithelial compartment of the gland is vastly expanded (Benaud et al. 1998). At the end of lactation the epithelial cells undergo apoptosis and adipocyte differentiation is induced (Lilla et al. 2002). Ductal and alveolar growth during puberty and pregnancy, and the involution process require the action of proteolytic enzymes (including matrix metalloproteinases, plasminogen and membrane-peptidases) and the corresponding genes are activated during these periods (Benaud et al. 1998; Alexander et al. 2001). Matrix metalloproteinases (MMP) are expressed in several cell types of the mammary gland including stromal fibroblasts (e.g., MMP3, MMP2), epithelial cells (e.g., MMP7 or MMP9), adipocytes (e.g., MMP2) and lymphoid cells (e.g., MMP9) (Crawford et al. 1996; Lund et al. 1996; Wiseman et al. 2003). A number of knock-out mice, which are deficient for individual MMP genes (e.g., MMP2, MMP3) or plasminogen, display alterations to mammary gland structure and impairment of lactation (Lund et al. 1999; Wiseman et al. 2003).

The objective was to measure teat canal length and diameter, teat diameter and teat wall thickness by ultrasonographic scanning in order to determine the differences in bovine breeds, and to study the influence of teat canal length and diameter on the occurrence of mastitis. A total of 269 lactating dairy cows of four different breeds (Brown Swiss, Simmental, Simmental crossbred with Red Pied, and Holstein-Friesians) from seven Upper Austrian dairy farms were examined. Average teat canal length of Brown Swiss animals was shortest (15·7 mm) followed by Holstein-Friesians (17·2 mm) and Simmental (18·3 mm). These differences in teat canal length were highly significant (P[les ]0·001). There was no significant difference in teat canal length between pure-bred and crossbred Simmentals. Differences of teat canal diameter between breeds were significant (P[les ]0·05). Brown Swiss animals had the largest diameters (2·0 mm) and Holstein-Friesians the smallest (1·7 mm). Differences in teat diameter between Brown Swiss, Holstein-Friesian and Simmental were also significant. No differences were found between the pure-bred and crossbred Simmental cows. The narrowest teats were in Holstein-Friesians and the widest in Simmental. Holstein-Friesians also exhibited the thinnest teat walls while the Simmental had the thickest ones. Teat canal length and diameter were correlated with udder health. Teat canals of healthy udders tended to be longer (17·4 mm) and narrower (1·8 mm) than teat canals of infected udders (15·8 mm, 2·1 mm; P[les ]0·001). A logistic regression model showed significant effects of teat canal length, teat canal diameter and lactation number on udder health.

Experimental and epidemiological studies indicate that antioxidant food polyphenols could have antimitotic activities, interfering with cancer initiation, progression or mortality. Circulating polyphenols are far lower than the nominal value in foods. In the rare studies dealing with polyphenol bioavailability, it was noted that their active concentrations in the blood are <1% of their food concentration. In the present study we investigated the effect of four polyphenols (resveratrol, and the flavonoids quercetin, catechin and epicatechin, major constituents of wine) in the hormone-sensitive human cancer cell line T47D, at concentrations compatible with their calculated plasma concentrations after ingestion of a moderate quantity of wine (nM or pM). Our results indicate that cell growth was decreased, with cells being arrested at the S phase of the cycle. In addition, we provide evidence of a bimodal modulation of the NO/NOS system, affecting its activity and transcription. We show that modulation of this system is sufficient to explain polyphenol action on this cell line. This result suggests a potential importance of wine ingestion and possibly the consumption of other polyphenol-rich dietary foods and drinks in the control of breast cancer cell growth.

Dynamics of micro-structural changes in milk during the renneting process were analysed using high-resolution ultrasonic spectroscopy in combination with dynamic rheology and NIR transmission measurements. Two independent ultrasonic parameters, velocity and attenuation were measured in the frequency range 2 to 15 MHz, as a function of time after addition of rennet to milk. The results show an initial decrease of 20 nm for the average diameter of micelles caused by hydrolysis of the κ-casein ‘hairy’ layer followed by an aggregation of the micelles into small clusters (effective aggregation number of 3) and then formation of the gel structure. It was found that evolution of ultrasonic attenuation in the renneting process could well be described by the scattering of the ultrasonic waves on aggregates. The evolution of ultrasonic velocity is well described by the scattering theory but deviates from the predicted curve at the gelation stage of the process, which shows the difference in propagation of ultrasonic waves in a gel structure compared with dispersions. Overall, we found high-resolution ultrasonic spectroscopy to be a powerful tool for analysis of microscopic processes in the formation of milk gel. It allows the characterisation of the pre-gelation processes, such as hydrolysis and aggregation, and the initial stages in the formation of the gel network as well as monitoring of the microscopic evolution in the gel at the post-gelation stage.

The properties of commercial directly and indirectly heated UHT milks, both after heating and during storage at room temperature for 24 weeks, were studied. Thermally induced changes were examined by changes in lactulose, furosine and acid-soluble whey proteins. The results confirmed previous reports that directly heated UHT milks suffer less heat damage than indirectly heated milk. During storage, furosine increased and bovine serum albumin in directly heat-treated milks decreased significantly. The changes in lactulose, α-lactalbumin and β-lactoglobulin were not statistically significant. The data suggest that heat treatment indicators should be measured as soon as possible after processing to avoid any misinterpretations of the intensity of the heat treatment.

Among the species investigated, the rate of spontaneous cancers of mammary glands is highest in humans and dogs (Hamilton, 1974; Owen, 1979). In addition, spontaneous mammary tumours are known to occur in strains of mice (Webster & Muller, 1994), rats (Russo et al. 1990; Sukumar, 1995) and cats (Hamilton, 1974; Kessler & von Bombard, 1997). Although both oestrogen and progesterone are known to be involved in normal mammary development as well as in the proliferative changes that occur during the oestrous or menstrual cycle, pregnancy and lactation (Going et al. 1988), only the role of oestradiol has been extensively investigated. The role of progesterone in mammary cancer is not understood and, although progesterone metabolism is known to occur in mammary tissue, the potential autocrine/paracrine role of the in situ metabolites has only recently begun to be explored in human breast tissues and cell lines (Wiebe et al. 2000).

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.

The involvement of Staphylococcus aureus exosecretions in bovine udder infection (Younis et al. 2003) suggests that four different monomer protein bands appearing between 36 and 31 kDa, are associated with the severity of the cow's infection response. Three out of these four bands have been identified by means of protein sequencing. Band B, with a MW of 35 kDa was identified as Panton-Valentaine leucocidin LukF'-PV chain- Staph. aureus; band C, with a MW of 32 kDa was identified as leucocidin chain LukM precursor- Staph. aureus; and band D was found to be similar, but not identical, to phosphatidylinositol-specific phospholipase-C-X. Bands B and C were purified by gel filtration using FPLC. The ability of these proteins to induce udder inflammation in vivo, and proliferation response in vitro and cytokine secretion were tested for both the crude exosecretions and purified bands. Three cows were inoculated intracisternally, with three quarters receiving either 0·007–0·008 mg (as total proteins) of Staph. aureus FR2449/1 bacterial exosecretion, pooled fraction 39–41 (bands B and C), or culture broth medium. The fourth quarter was left free as a control. Quarters that received fraction 39–41 of Staph. aureus FR2449/1, exhibited induced inflammation, which was indicated by increased somatic cell count and enhanced NAGase activity that was significantly higher than that of the original Staph. aureus FR2449/1 bacterial exosecretion. Proliferation tests of bovine blood lymphocytes in vitro showed that the pooled fraction 39–41 stimulated bovine proliferation of mononuclear cells much more than the original Staph. aureus FR2449/1 bacterial exosecretion. Secretion of TNF-α, IL-1β, IL-6 and IL-8 was in accordance with the contents of LukF'-PV and LukM precursor in the exosecretions. The results suggest that LukM/LukF' induce inflammation into the udder by a mechanism similar to that of LPS or by a unique mechanism(s) which requires further investigation.