The COST Programme

The Cooperation in Science and Technology Programme (COST) is an inter-governmental framework for European Co-operation in the field of Scientific and Technical Research. COST is managed by the European Science Foundation, and in May 2005 supported almost 200 Actions involving nearly 30000 scientists from 32 European member countries and more than 50 participating institutions from 11 non-member countries. These Actions are networks of coordinated national research project in fields which are of interest to a minimum number of participants (at least 5) from different member states. COST Actions cover basic and pre-competitive research as well as activities of public utility. The members of each Action are drawn from member countries which have ratified the Action, and their activities are informed by a programme accepted by the ESF management at time of application and reviewed on an annual basis throughout the Action. Each Action comprises three principal activities: the workshops where the science or technology matters of the Action are presented and discussed; the Action's short-term scientific missions, which allow contacts made during workshop networking to be developed; and, thirdly, the dissemination of the Action's science through publication or electronic communication.

The extent of primary and secondary proteolysis of cheeses made from raw (RA), pasteurized (PA, 72 °C, 15 s) or pressure-treated (PR, 500 MPa, 15 min, 20 °C) goats' milk was assessed. Modifications in cheese-making technology were introduced to obtain cheeses with the same moisture content, and thus studied per se the effect of milk treatment on cheese proteolysis.

The PR milk cheese samples were differentiated from RA and PA milk cheeses by their elevated β-lg content, and by the faster degradation of αs1-, αs2- and β-CN throughout ripening. Non-significant differences were found in either pH 4·6 soluble-nitrogen or trichloracetic acid soluble-nitrogen contents of cheeses. However, the pasteurization of milk decreased the free amino acid production in cheese. The RA milk cheeses had the highest amount of proline and the lowest concentrations of serine, tyrosine, arginine and α-aminobutyric acid, whereas PR milk cheese showed higher levels of arginine.

Cholesterol and phospholipid concentrations in serum lipoproteins, plasma activities of lecithin:cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP) and lipoprotein lipase (LPL) activity in adipose tissue biopsies were measured ante and post partum in dairy cows given either free or restricted access to feed during the dry period. After parturition, all cows were fed ad libitum. The purpose of this study was to try to understand the earlier observed marked drop post partum in plasma triacylglycerol (TAG) in terms of lipoprotein metabolism in cows developing fatty liver post partum. As would be expected, free access to feed during the dry period induced a rise of hepatic TAG concentrations post partum associated with a decrease in plasma TAG levels. Total and free cholesterol concentrations in the VLDL, IDL, LDL and HDL2 fractions fell immediately after parturition. VLDL and IDL cholesterol concentrations remained at a constant, low level during the entire sampling period post partum, whereas the drop in LDL and HDL2 cholesterol post partum was followed by a rebound rise. Plasma LCAT and PLTP activities decreased by on average 19% and 33%, respectively, after parturition and then rose to values seen before parturition, but there was no effect of feeding regimen during the dry period. Activities of LCAT and PLTP were significantly correlated with cholesterol and phospholipid concentrations in LDL and HDL2. Plasma LCAT activity, as measured with exogenous substrate, and PLTP activity were both positively correlated with HDL3 phospholipid levels. LPL activity in adipose tissue dropped after parturition, the drop being smaller after feeding ad libitum during the dry period. It is concluded that the drop in adipose tissue LPL activity post partum is at variance with the simultaneous fall in plasma TAG. Possibly, the decrease in adipose tissue LPL activity helps to channel fatty acids away from adipose tissue into the udder. The post-partum changes in lipid transfer proteins in the blood are in line with the changes observed in the levels of the lipoproteins.

Sorting of normal and abnormal milk at time of milking is done visually for conventional milking systems, but more concrete standards are needed when milking is done in automatic milking systems (AMS). Several panel tests were carried out to find out how different consumer groups, milkers and advisors look at and respond to the visual appearance of milk mixed with blood, in order to set a limit for what they think is acceptable. It is concluded from the test panel results that milk samples with 0·4% or more of blood all will be scored as pink and samples with 0·1% blood (about 6 μM-haemoglobin or 100 mg/l) can be visually detected if they are compared with milk samples without blood. The consumer group scored fewer of the samples with 0·1% blood as normal than did the professional groups. The test panel scored 65% of the samples with 1% blood as normal when milk was presented in a black strip cup, which is the reference method when foremilking takes place in a conventional parlour. Only 2% of the milk samples with 2% blood (about 120 μM-haemoglobin or 2000 mg/l) were scored as normal in a black strip cup and should consequently be detected by conventional as well as automatic systems. One model of AMS was tested for its ability to detect and separate milk coloured by blood. The model separated milk with [ges ]6 μM-haemoglobin. Milk mixed with blood injected into the milk stream for a short time at the beginning of milking was not separated. We lack data on how blood is naturally expelled into milk and in what amount. We propose that cow composite milk with >6 μM-haemoglobin should be separated because at this level milk will have a red tinge.

The study provides the first comprehensive information on the variability of milk protein genes of Bos indicus and Bos taurus cattle breeds in Cameroon and Nigeria. The investigations indicate a high diversity of milk protein genes for the zebu populations. Of the investigated alleles, 21 out of 29 were observed. The method of single strand conformation polymorphism (SSCP) was a particularly useful technique because it allowed discrimination of alleles, including zebu-specific alleles at the CSN2 (I) and CSN3 (AI and H) loci, not separated by protein electrophoretic techniques and also made possible the detection of a further CSN1S1 5′ promoter allele (CSN1S1Prom5), which is also zebu-specific. Characterization of CSN1S1Prom5 showed that it was the most variable of all described CSN1S1 promoter alleles. A potential GATA consensus motif is created by mutations in CSN1S1Prom5. Intra-breed diversity measured as mean effective number of alleles was higher in the zebu populations than in the taurine breeds. Of the expected casein haplotypes, 96 out of 320 were present in the studied breeds. 2-C-A-A2-H (CSN1S1Prom2-CSN1S1C-CSN1S2A-CSN2A2-CSN3H) and 5-C-A-A2-H were zebu-specific while 1-B-A-A2-B was specific to the taurines. Overall distribution of alleles and haplotypes clearly separated the zebu populations from the taurine breeds. Zebu influence on the taurine breed Namchi was detected through the occurrence of zebu alleles and haplotypes. High variability of milk proteins also means availability of resources for breed development, phylogenetic studies, and conservation and management decisions.

To study the effect of growth hormone (GH) on the functions of mammary epithelia, we examined the effect of GH on the synthesis and secretion of α-casein in a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-d-pregnant Holstein heifer. GH receptors (GHR) were observed in the BMEC on the membrane as well as in the cytoplasm. After BMEC were plated onto cell culture inserts, GH stimulated α-casein release in both the presence and absence of the lactogenic hormone complex, which included dexamethasone, insulin and prolactin (DIP). DIP enhanced the effect of GH on α-casein release. Although αs1-casein mRNA expression was not detected in untreated control cells, its expression was observed in BMEC in response to the GH, DIP and GH+DIP treatments. Expression was greater for GH and GH+DIP than for just DIP. Expression of GHR mRNA was increased by DIP treatment, while the mRNA expression was little changed by GH treatment. We conclude that GH acts on BMEC and induces the expression of both the α-casein gene and protein. GHR gene expression was shown to be regulated by DIP and GHR. GHR may be involved in a synergic effect between GH and DIP on casein secretion. These results suggest that GH, in addition to its widely accepted homeorhetic role in vivo, also can act on the mammary parenchyma, and that the effects of GH on mammary epithelial cells could partly account for the clear galactopoietic effect of recombinant bovine GH seen in lactating dairy cows.

Cancer develops from overlapping events that tend to deregulate the metabolism and unbalance the homeostasis of cells. Sphingolipids, major components of biological membranes, are also mediators of intracellular signalling. Their metabolism can be influenced by diverse stimuli and the accumulation or deficiency of intermediates may trigger proliferation and/or impair the ability of damaged cells to undergo apoptosis. Many sphingolipid-regulated functions are implicated in tumour initiation, promotion, progression and responsiveness to chemotherapy. In this review, evidence of the alteration of sphingolipids metabolism and signalling will be discussed in breast cancer prevention and therapy.

Milking-related release of oxytocin, prolactin, and cortisol was studied following three pre-milking treatments. Six Murrah buffaloes were treated with direct application of milking cluster (O), a 1-min pre-stimulation (M), and combined feeding and pre-stimulation (MF). Machine milk yield, stripping yield and milk composition were recorded. Milk ejection occurred significantly earlier with MF than M and O (P<0·05; 2·50, 5·10 and 6·33 min, respectively). In all treatments, milk ejection occurred with small increases >3–5 ng/l in oxytocin concentration. Increase in oxytocin concentration over a threshold level and milk ejection occurred simultaneously and were closely correlated (r=0·83, P<0·05). There was a positive correlation between total time oxytocin concentration remained elevated over threshold levels and machine yield (r=0·86, P<0·05). For treatment O, milk ejection was inhibited during machine milking, while a marked increase in oxytocin occurred during hand stripping (6 and 16 ng/l, respectively). For treatment M, mean oxytocin concentrations remained unchanged during pre-stimulation but increased during subsequent machine milking and hand stripping (6·38, 18·06 and 12·36 ng/l, respectively). For treatment MF, although there was a 3·6-fold increase during pre-stimulation, oxytocin increased by 10-fold and 3-fold during machine milking and hand stripping, and was significant for machine milking (P<0·05, 17·32, 47·86, 18·13 ng/l, respectively). Milk-ejection-related cortisol release was visible only in treatment MF. For treatments O and M, prolactin concentration increased prior to the increase in oxytocin. The stripping yield was higher, and fat content in the stripping yield significantly lower, for treatment O indicating incomplete milking. Thus buffaloes are easily disturbed even by small changes in milking routines.

High pressure homogenisation (HPH) is a novel dairy processing tool, which has many effects on enzymes, microbes, fat globules and proteins in milk. The effects of HPH on milk are due to a combination of shear forces and frictional heating of the milk during processing; the relative importance of these different factors is unclear, and was the focus of this study. The effect of milk inlet temperature (in the range 10–50 °C) on residual plasmin, alkaline phosphatase, lactoperoxidase and lipase activities in raw whole bovine milk homogenised at 200 MPa was investigated. HPH caused significant heating of the milk; outlet temperature increased in a linear fashion (0·5887 °C/°C, R2=0·9994) with increasing inlet temperature. As milk was held for 20 s at the final temperature before cooling, samples of the same milk were heated isothermally in glass capillary tubes for the same time/temperature combinations. Inactivation profiles of alkaline phosphatase in milk were similar for isothermal heating or HPH, indicating that loss of enzyme activity was due to heating alone. Loss of plasmin and lactoperoxidase activity in HPH milk, however, was greater than that in heated milk. Large differences in residual lipase activities in milks subjected to heating or HPH were observed due to the significant increase in lipase activity in homogenised milk. Denaturation of β-lactoglobulin was more extensive following HPH than the equivalent heat treatment. Inactivation of plasmin was correlated with increasing fat/serum interfacial area but was not correlated with denaturation of β-lactoglobulin. Thus, while some effects of HPH on milk are due to thermal effects alone, many are induced by the combination of forces and heating to which the milk is exposed during HPH.

Whey protein hydrolysates were generated at different total solids (TS) levels (50–300 g/l) using the commercially available proteolytic preparation Debitrase™ HYW20, while enzyme to substrate ratio, pH and temperature were maintained constant. Hydrolysis proceeded at a faster rate at lower TS reaching a degree of hydrolysis (DH) of 16·6% at 300 g TS/l, compared with a DH of 22·7% at 50 g TS/l after 6 h hydrolysis. The slower breakdown of intact whey proteins at high TS was quantified by gel-permeation HPLC. Reversed-phase (RP) HPLC of hydrolysate samples of equivalent DH (~15%) generated at different TS levels indicated that certain hydrophobic peptide peaks were present at higher levels in hydrolysates generated at low TS. Sensory evaluation showed that hydrolysates with equivalent DH values were significantly (P<0·0005) less bitter when generated at 300 g TS/l (mean bitterness score=25·4%) than hydrolysates generated at 50 g TS/l (mean bitterness score=39·9%). A specific hydrophobic peptide peak present at higher concentrations in hydrolysates generated at low TS was isolated and identified as β-lactoglobulin f(43–57), a fragment having the physical and chemical characteristics of a bitter peptide.

Fluorescence resonance energy transfer (FRET) microscopy was used to study interactions between proteins in intact cells. We showed that growth hormone (GH) causes transient homodimerization of GH receptors tagged with yellow or cyan fluorescent proteins. The peak of FRET signaling occurred 2 to 4 min after hormonal stimulation and was followed by a decrease in FRET signal. Repeating those experiments in cells pretreated with the inhibitor of internalization methyl-β-cyclodextrin, or in potassium-depleted cells showed no difference in the kinetics of FRET signaling as compared with the non-treated cells, indicating that the decrease in FRET signal does not result from receptor internalization by the pathways inhibited by methyl-β-cyclodextrin or potassium depleted but might occur by other pathways of internalization. Using a similar methodology, we also demonstrated that ovine placental lactogen (oPL) causes transient heterodimerization of GH and prolactin (PRL) receptors 2·5 to 3 min after oPL application. On the other hand, oGH or oPRL had no effect at all, further substantiating the finding the oPL, which lacks a specific receptor, acts in homologous systems by heterodimerization of GH and PRL receptors. We also demonstrated that both PRL and leptin (LEP) are capable of transactivation of the oncogenic receptors erbB2 and erbB3. Upon PRL or LEP stimulation of HEK-293T cells transfected with LEP or PRL receptors and erbB2 or erbB3, erbB proteins are first phosphorylated and then activate MAPK (erk1/erk2). However, the FRET experiments failed to document any evidence of a direct interaction between erbB2 and the PRL or LEP receptors, suggesting that erbB activation probably occurs via activated JAK2, translocated from the respective receptors to erbB2.

Comlek peyniri is a typical artisanal cheese in Central Anatolia. This type of cheese was made by using the indigenous lactic acid bacteria (LAB) flora of cow or ewes' milk. Majority of the samples were taken from fresh cheese because the aim was to isolate homofermentative LAB. Initially 661 microbial isolates were obtained from 17 cheese samples. Only 107 were found to be homofermentative LAB. These isolates were selected and identified by using both phenotypic and molecular methods. Phenotypic identification included curd formation from skim milk, catalase test, Gram staining and light microscopy, growth at different temperatures and salt concentrations, arginine hydrolysis, gas production from glucose, and carbohydrate fermentation. Molecular identification was based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 16S rRNA gene-ITS (internally transcribed spacer) region. By combining the phenotypic and molecular identification results, isolates belonging to each of the following genera were determined at species or subspecies level: 54 Lactococcus lactis subsp. lactis, 21 Enterococcus faecium, 3 Ec. faecalis, 2 Ec. durans, 10 Ec. sp., 15 Lactobacillus paracasei subsp. paracasei, and 2 Lb. casei strains. Technological characterisation was also performed by culturing each of the strains in UHT skim milk, and by monitoring pH change and lactic acid production at certain time intervals through the 24 h incubation. Results of the technological characterisation indicated that 33% of the isolates (35 strains) were capable of lowering the pH of UHT milk below 5·3 after 6 h incubation at 30 °C. Thirty four of these strains were Lc. lactis subsp. lactis, and only one was an Ec. faecium strain.

Seven variables – electrical conductivity (EC), somatic cell count (SCC), N-acetyl-β-D-glucosaminidase (NAGase), lactose, protein, fat and pH – were compared in four quarter milk fractions (MF1: strict foremilk; MF2: first 12–15 ml foremilk; MF3: subsequent 40–45 ml milk; MF4: strippings) and in one cow composite milk sample (CC) per cow. The study used 142 quarters from 37 lactating cows of the German Black Pied breed. To rule out any possible effect due to management, animal physiology and analytical procedures, the collection and processing of milk samples from each cow was repeated for three consecutive days, and the means of 3-d values were used. All variables were affected significantly by milk fraction and udder health. Compared with foremilk, EC, lactose and protein levels in strippings decreased, while SCC, NAGase and fat increased. The pH of foremilk and strippings did not differ significantly in healthy or in mastitic quarters. The difference between MF1 and MF2 was significant for EC in mastitic quarters, and for SCC in healthy quarters only. In general, mastitis resulted in a significant increase in EC, SCC, NAGase and protein but in a decrease in lactose and fat contents of milk in one or more of the milk fractions studied. Comparison of cow composite milk samples from healthy and mastitic cows revealed the significance (P<0·01) of udder health for EC, SCC and lactose. Of the different parameters that can distinguish between healthy and mastitic quarters or cows, EC could be used to classify 76% of quarters and 73% of cows correctly, while the lactose content permitted correct identification of 81% of quarters and 76% of cows. NAGase and pH could be used to determine the status of 73% and 61% of quarters, respectively. In general, the correlation observed in strippings was higher than in foremilk for almost all the variables studied. Surprisingly, EC, SCC, NAGase and lactose in milk from healthy quarters of mastitic cows (with at least one mastitic quarter) differed significantly (P<0·05) from those from healthy quarters of cows with all four healthy quarters, indicating an inconsistent effect of mastitic quarters on neighbouring healthy quarters (quarter interdependence).

The objective was to clarify the association between bulk tank milk somatic cell count (BTSCC) and total bacterial count (BTTBC) and coliform bacteria count (BTCBC) in a large set of data based on the currently accepted legal limit of BTSCC=400000/ml. We analysed the database obtained from one of four laboratories offering routine estimation of microbiological indicators and counts of somatic cells in bulk tank milk samples in the Czech Republic during the year 2003 (74174; 73921 and 33020 records of BTSCC, BTTBC and BTCBC estimations, respectively, in milk from 2769 suppliers). Raw data of BTSCC (with arithmetic mean 220000/ml; 95th percentile=502000/ml; 99th percentile=784000/ml) indicated that the BTSCC limit was exceeded in 12% of samples. BTSCC did not sufficiently reflect the hygiene status of particular producing herds because correlation coefficients between bulk tank milk somatic cell score (BTSCS) and log BTTBC or log BTCBC were low. Categorization of herds according to the percentage of records exceeding the BTSCC limit gave significantly higher correlation coefficients for the association between this characteristic and log BTTBC or log BTCBC (r=0·84 and r=0·68, respectively). The percentage of records exceeding the BTSCC limit was a useful tool to highlight problem herds kept in inadequate hygienic conditions in primary milk production. Likewise, the value of BTSCS>5 seemed to be a useful tool for the discrimination of problem herds.

An outstanding parameter in cheese making is the type of coagulant, which greatly influences the characteristics of the final products. Proteolysis is the most important set of biochemical changes during ripening of most cheeses, and is carried out, in different magnitude, by proteolytic agents originated in milk, rennet (or rennet substitute), and starter and non-starter micro-organisms (Silva & Malcata, 2000). The demand for alternative sources of milk coagulants, to replace the expensive and limited natural rennet supplies, has increased (Esteves et al. 2001). All commercial enzymes employed as milk coagulant are aspartic proteinases, which are most active at acidic pH and preferentially cleave peptide bonds between residues with hydrophobic side-chains (Silva & Malcata, 1999).

To define better the function of Nerve Growth Factor (NGF) in breast cancer progression, we investigated whether this polypeptide was able to induce breast cancer cell invasion. NGF inhibited aggregation of tumour cells through modulation of the E-cadherin/catenin complex function. In addition, NGF induced the breast cancer cells to invade into Matrigel. We focused our attention on how NGF prevents aggregation, in order to discover the signalling pathway that leads tumour cells to acquire the invasive phenotype. Moreover, studies on the identification of signalling pathways that are responsive for NGF-induced invasion will be basically described.

The necessary unequivocal and generally accepted definitions of normal and abnormal milk are not available. A precise definition is needed in order for companies to develop sensors to detect and sort abnormal milk at the time of milking. Experts at a workshop defined abnormal milk to be that from cows whose foremilk had changed in homogeneity or was coloured by blood. The objectives of this paper were: firstly, to explore how different groups of people scored the appearance of foremilk; and secondly, to develop a method suitable as an objective reference for testing of manual and automatic detection systems. Consumers, farmers and advisors did not agree on the visual appearance of normal, watery, clotty milk, or milk with blood, and experience is needed to score the visual appearance of foremilk correctly. It seems reasonable to expect a sensitivity of at least 70% for detection of abnormal milk during foremilking. Filter sizes 0·05, 0·07, 0·1, 0·2, 0·5, 1·0, and 2·0 mm were used to filter milk from cows with visually abnormal foremilk. If clots appeared in the foremilk, clots appeared on all size filters, but the filter with pore size 0·1 mm was the easiest to read and work with. The filter method is not reliable in identifying quarters with watery, yellowish, or bloody milk, whereas the method seems consistent, and at least as good as scoring of visual appearance in finding clots in the milk. Clots should show clearly on the filter to be counted as abnormal milk. All clinical cases with clots in the foremilk can be found on the filter and such cases have high somatic cell count (SCC). Both trained and untrained persons using the filter method can score normal and abnormal foremilk with a high specificity (>90%) and a high sensitivity (>80%). The filter method is recommended as a reference for scoring the homogeneity of foremilk.

Psychrotrophs, particularly strains of Pseudomonas fluorescens, dominate the microflora of refrigerated raw milk and secrete heat-stable extracellular enzymes (proteases and lipases) which survive pasteurisation and even UHT heat treatments and degrade the casein and fat components of raw milk causing a reduction in cheese yield, gelation of UHT milk and off flavours in many dairy products. These enzymes are usually produced in the late log/early stationary growth phases when the cell density is high. This fact indicated that induction of these enzymes may be a candidate for quorum sensing, a phenomenon by which bacteria can sense and respond to cell population size by means of chemical signals based on the homoserine lactone molecule. It is assumed that the production of these enzymes contributes to the selective advantage of psychrotrophs and hence have a significant effect on their growth kinetics. In the work reported here nine proprietary homoserine lactone compounds were screened, using water as a control, for their effect on the lag phase duration (LPD) and exponential growth rate (EGR) of three strains of Pseudomonas fluorescens isolated from refrigerated raw milk after 1, 3 and 5 days storage. Two compounds viz. N-benzoyloxycarbonyl-L-homoserine lactone and N-3-oxyhexanoyl-DL-homoserine lactone were found to significantly (P<0·001) reduce the LPD and increase the EGR of the three strains. Further work with these compounds is warranted, monitoring growth in parallel with enzyme production, to determine the extent to which extracellular enzyme production is mediated by the quorum sensing phenomenon in this species.

Studies of the potential of high pressure homogenisation (HPH) for the combined pasteurisation/homogenisation of raw bovine milk were undertaken. Raw milk was preheated to 45 °C and HPH-treated at 150, 200 or 250 MPa; milk outlet temperature at these pressures were 67, 76·8 and 83·6 °C, respectively, with a holding time of ~20 s. Raw and commercially pasteurized and homogenized (CPH) milk samples were analysed as controls. Fat globules in HPH samples were approximately half the size of those in CPH samples, although differences were not significant (P>0·05). β-Lactoglobulin was denatured at pressures [ges ]150 MPa, although little denaturation of α-lactalbumin was observed. Numbers of psychrotrophic bacteria in raw milk were reduced by 2·73 log cycles by HPH at 150 MPa and were uncountable following HPH at 200 or 250 MPa. Mesophilic bacterial counts were reduced by 1·30, 1·83 and 3·06 log cycles by HPH at 150, 200 or 250 MPa, respectively. No viable Staphylococcus aureus nor coliform cells remained in any HPH milk samples. HPH did not affect the colour of milk and HPH samples did not cream during refrigerated storage. The activities of plasmin, alkaline phosphatase and lactoperoxidase in milk were all greatly reduced by HPH. Pseudomonas fluorescens, inoculated into milk (~106 cfu/ml), was reduced to undetectable levels by HPH at 200 MPa (milk inlet temperature, ~10 °C); however, Ps. fluorescens proteinase was quite resistant to HPH under such conditions. Overall, owing to the significant increase in temperature and the possibility of varying the holding time, there may be potential applications for HPH as a novel liquid milk processing technique, combining many advantages of conventional homogenization and pasteurization of milk in a single process.

Differential cell count of milk is a traditional parameter for the evaluation of udder health. The literature shows great variation in differential cell counts of the milk of healthy mammary glands: macrophages range from 0% to 80%, lymphocytes from 1·5% to 79·5%, polymorphonuclear neutrophils from 3% to 95%, and epithelial cells from 1% to 19%. We conducted three studies to seek explanations for such variation. In the first, we evaluated the impact of polyethylene and glass sampling bottles. The aim of the second study was to compare the results of differential cell counts performed by three different technicians. The third study evaluated two methods of smear preparation. When polyethylene plastic bottles were used, the macrophage population was minimized but lymphocytes remained unaffected. This was shown by an exemplary flow cytometric analysis using four monoclonal antibodies against three lymphocyte surface structures. There were significant differences in the differential cell counts of 40 smears made by three technicians despite identical operating procedures. For the sediment smear, milk was centrifuged once and the sediment spread by eye on a glass slide. For the “coffee grinder” smear method, the sample was subjected to four centrifugations and then placed on a cover glass in order to spread the sediment using centrifugal force. The coffee grinder procedure led to a reduction of lymphocytes and an enrichment of polymorphonuclear neutrophils without affecting the macrophage population. Both methods made it possible to distinguish different udder health classes. It can be concluded that differential cell counts are a useful tool for comparing and monitoring udder health only if: samples are taken in a glass bottle; smears are prepared with the identical technique; and the differential cell counts are performed by a single person.