Milk somatic cell count (SCC) is the gold standard in diagnosis of subclinical mastitis, and is also an important parameter in quality programmes of dairy cooperatives. As routine SCC analysis is usually restricted to central laboratories, much effort has been invested in the search for alternative biomarkers of mastitis and milk quality, including the presence in the milk of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid A (SAA). The aim of this study was to investigate relationships between Hp, SAA and SCC in quarter, cow composite, and bulk tank milk samples. Cows (n=165), without any clinical signs of disease or abnormalities in the milk or udder, from three different dairy farms, were used. Cow composite milk samples from all cows delivering milk at the sampling occasion were taken once in each herd. In one of the farms, representative quarter milk samples (n=103) from 26 cows were also collected. In addition, bulk tank milk samples from 96 dairy farms were included in the study. Samples were analysed for Hp, SAA and SCC, and relationships between the parameters were evaluated at quarter, cow and tank milk levels using Chi-square analysis. Milk samples were categorized according to their SCC, and the presence, or no presence, of SAA and Hp, based on the detection limits of the screening methods (0·3 mg/l and 1·0 mg/l for SAA and Hp, respectively). Hp and SAA were found in milk at quarter, cow composite and bulk tank levels. A large proportion (53%) of the animals had detectable milk concentrations of APP, and SAA was detected more frequently, and at higher concentrations than Hp, regardless of sample type. SAA was detected in as many as 82% of the bulk tank milk samples. Significant relationships were found between Hp, SAA and SCC at quarter and cow composite milk levels, but only between SAA and SCC at bulk tank milk level. Detectable levels of APP were more common at high SCC.

Propolis is a natural product collected by honeybee workers. The product was tested for its antifungal effect against Aspergillus versicolor ATCC 12996 as well as biosynthesis of sterigmatocystin during ripening of Egyptian Ras cheese. The use of different concentrations of aqueous propolis extract 250, 500 and 1000 part per million (ppm) on the cheese surface was investigated. Mould growth and toxin production were completely inhibited at the highest concentration 1000 ppm, while the lower concentrations exhibited definite fungistatic activity during 90 days of ripening. Control cheese demonstrated that the amount of sterigmatocystin produced was proportional to the growth of Asp. versicolor during three months of ripening. It could be concluded that propolis concentration of 1000 ppm could prevent mould growth and sterigmatocystin production in Ras cheese. The economic as well as the public health importance of propolis as a natural preservative in cheese manufacture is discussed.

Ectoine is a compatible solute accumulated in halophilic bacteria in response to high salt concentrations and offers protection from osmotic stress. The occurrence of compatible solutes is widespread among bacteria, yet ectoine has never been detected in foods. The use of an ectoine producing microorganism (Brevibacterium linens) in the surface ripening of red smear cheeses led to the question whether ectoine can be found in cheese. Therefore we examined samples from a variety of cheese manufacturers and different types of red smear cheeses for the presence of ectoine using HPLC and HPLC/MS analysis. Ectoine solely appears in the rind and was detected up to 178 mg/200 g red smear cheese, depending on several factors like ripening status and conditions throughout the cheese production process (e.g. salt concentrations of used brine baths).

The use of artisan-produced lamb rennet pastes, but not any of the other commercial animal rennets, imparts a characteristic flavour to the cheese, so most Mediterranean ewes' milk cheeses are coagulated with this kind of rennet paste. In contrast to the advantages of using lamb or kid rennet pastes from the sensory point of view, questions are still raised as to their hygienic quality. The goal was to examine the microbiological and enzymic quality of lamb rennet pastes prepared by cheese manufacturers for their own use, and evaluate the hygienic quality of raw sheeps' milk cheeses made with them, using Idiazabal cheese as a model. Lamb rennet pastes prepared by artisan cheese makers from the Basque region of Spain (27), and Italy (8) were evaluated. For cheese making experiments 5 different lamb rennet pastes were selected among the 27 samples from the Basque Country region of Spain. Microbiological analyses were carried out on samples from rennet pastes, rennet extracts, milks and cheeses during ripening. Enzymic activities studied in rennet paste were: total coagulating strength and lipase. Analysis of variance and Student's t-tests was performed. The results show that the artisan-produced rennet pastes contain high levels of a variety of microorganisms. After 60 ripening days, which is the minimum ripening period required for Idiazabal cheese prior to its commercialization, no Eschericia coli, Clostridium, Salmonella spp. or Listeria monocytogenes were detected, and levels for the rest of the microorganisms were below the limits of the European legislative standards for cheese manufactured with raw milk. We can conclude that the use of artisan-produced lamb rennet pastes of questionable hygienic quality for the manufacture of raw milk hard cheeses yields products of good hygienic quality.

We investigated effects of subclinical intramammary infection (IMI) on milk somatic cell count (SCC) and milk composition in udder halves of dairy goats. A total of 35 mixed-age Alpine does (70 udder halves; approximately 55 kg body weight) were rotationally grazed on a mixture of vegetative forages (wheat/berseem clover, sudan grass and cowpeas). Milk samples for bacterial analysis and SCC were collected monthly from both halves from April to September, 2001. Across stages of lactation, 19–31% of udder halves became infected. The prevalence of IMI exhibited quadratic patterns through multi-peaked responses within each stage of lactation. Higher rates of IMI were observed during the early stage of lactation (19% in May) and in the late stage of lactation (31% in September). Coagulase negative Staphylococcus (CNS, 43·7%), Staph. aureus (35·4%), and Pseudomonas aeruginosa (12·4%) were the most prevalent pathogens. Within single-strain IMI, log SCC (6·24) was lower (P<0·01) for CNS than those derived from IMI by Staph. aureus (6·49), Ps. aeruginosa (6·53) or Serratia spp. (6·90). Infected udder halves had a higher average SCC (4761 v. 2259×103 cells/ml; P<0·01) than uninfected halves, but uninfected halves often had similar levels of SCC to infected halves. Daily average milk production was not significantly different between infected and non-infected goats and the relationship between IMI and SCC was not always correlated. Effective mastitis screening requires bacteriological culture since SCC was not highly correlated.

In this study, the antihypertensive activity in spontaneously hypertensive rats of two peptides isolated from β-lactoglobulin hydrolysates with thermolysin was evaluated. These peptides, with sequences LLF [β-lg f(103–105)] and LQKW [β-lg f(58–61)], showed potent in vitro ACE-inhibitory activity. Two hours after administration, both sequences caused a clear and significant decrease in the blood pressure of these rats. The impact of a simulated gastrointestinal digestion on ACE-inhibitory and antihypertensive activities of these peptides was also studied. The results showed that both fragments were susceptible to proteolytic degradation after incubation with pepsin and Corolase PP®. In addition, their in vitro ACE-inhibitory activity decreased after the simulated digestion. It is likely that fragment LQK was the active end product of the gastrointestinal digestion of peptide LQKW. The fragment LL, observed after digestion of peptide LLF, probably exert its antihypertensive effect through a mechanism of action different than ACE-inhibition.

High hydrostatic pressure disruption of casein micelle isolates was studied by analytical ultracentrifugation and transmission electron microscopy. Casein micelles were isolated from skim milk and subjected to combinations of thermal treatment (85°C, 20 min) and high hydrostatic pressure (up to 676 MPa) with and without whey protein added. High hydrostatic pressure promoted extensive disruption of the casein micelles in the 250 to 310 MPa pressure range. At pressures greater than 310 MPa no further disruption was observed. The addition of whey protein to casein micelle isolates protected the micelles from high hydrostatic pressure induced disruption only when the mix was thermally processed before pressure treatment. The more whey protein was added (up to 5 g/l) the more the protection against high hydrostatic pressure induced micelle disruption was observed in thermally treated samples subjected to 310 MPa.

Bovine Somatotropin (bST) is a peptide hormone secreted by the anterior pituitary gland and its recombinant form (rbST) is used for artificially boosting milk yield in cows. Identification of rbST is difficult in that there is little difference from the pituitary bST (pbST). In this work, we further studied the possibility of immunologically discriminating between rbST and pbST. With this purpose, we produced mouse monoclonal antibodies using, as antigen, a peptide mimicking the N-terminus of rbST from Monsanto (rbST-M) conjugated to keyhole limpet haemocyanin (KLH) and polyclonal antibodies in rabbits immunized with the whole bST or rbST-M. Hence, we developed a sandwich ELISA with the obtained antibodies for detection and quantification of bST in serum and compared its performance on the two worldwide commercialized rbSTs: rbST-M and rbST from LG Life Science (rbST-LG). The lowest detection limit of the assay was 0·05 ng/ml for rbST-M, 0·10 ng/ml for rbST-LG and 0·15 ng/ml for pbST. Furthermore, the assay showed the capability to amplify the signal in the presence of rbSTs, recognizing more efficiently rbST-M and rbST-LG than pbST (ECn pbST/ECn rbST: 3 and 1·6 respectively). Its employment for measuring bST levels in sera from bovines administered with rbST LG allowed us to detect exceptional values due to the treatment itself and probably further increased as a consequence of the higher affinity for rbSTs of our monoclonal antibody.

A study was conducted to determine whether intramammary antibiotic treatment of heifer mammary glands following the first milking after calving was effective for reducing the percentage of mammary quarters infected during early lactation. Jersey and Holstein heifers from two research herds were assigned to one of three treatment groups: (1) no intramammary infusion following the first milking after parturition, (2) intramammary infusion of all quarters with pirlimycin hydrochloride following the first milking after parturition and (3) intramammary infusion of all quarters with novobiocin sodium plus penicillin G procaine following the first milking after parturition. Almost 93% of Jersey heifers (40/43) and 73·1% of quarters (125/171) were infected at the first milking. Almost 77% of quarters (33/43) were cured following treatment with pirlimycin, 61·8% (21/34) were cured following treatment with penicillin-novobiocin and 39·6% (19/48) of infections were eliminated spontaneously in the untreated control group. Significantly fewer infections were observed in pirlimycin or penicillin-novobiocin treated mammary glands of Jersey heifers during early lactation than in untreated control mammary glands. Almost 89% of Holstein heifers (32/36) and 52·8% of quarters (76/144) were infected at the first milking. About 57% (12/21) of quarters were cured following treatment with pirlimycin, 41·4% (12/29) were cured following treatment with penicillin-novobiocin and 23·1% (6/26) of infections were eliminated spontaneously in the untreated negative control group. Significantly fewer infections were observed in pirlimycin treated mammary glands of Holstein heifers during early lactation than in untreated control mammary glands. However, no significant differences were observed following penicillin-novobiocin treatment of Holstein heifers after the first milking of lactation compared with untreated control quarters. Coagulase-negative staphylococci, Streptococcus uberis and Streptococcus dysgalactiae subsp dysgalactiae were isolated most frequently in heifers from both herds.

The objective was to compare the prevalence of subclinical mastitis (SM) and of udder pathogens in 60 Swiss organic (OP) and 60 conventional production systems (CP). Cows (n=970) were studied for SM prevalence and udder pathogens at median 31 d and 102 d post partum. Cows showing a [ges ]1+ positive California Mastitis Test (CMT) in at least one quarter were considered to have SM. Cow-level prevalences of SM for visits at 31 d and 102 d post partum (39% and 40% in OP and 34% and 35% in CP) were similar, but quarter-level prevalences of SM were higher (P<0·02) in OP than CP (15% and 18% in OP and 12% and 15% in CP). Median somatic cell counts in milk at 31 d post partum were higher (P<0·05) in OP than CP cows (43000 and 28000 cells/ml, respectively), but were similar at 102 d post partum in OP and CP cows (45000 and 38000 cells/ml, respectively). In milk samples from quarters showing a CMT reaction [ges ]2+ the prevalences of coagulase negative staphylococci were lower (P<0·05) at 102 d post partum, whereas prevalences of non-agalactiae streptococci were higher (P<0·05) in OP than in CP cows at 31 d and 102 d post partum. In conclusion, under Swiss conditions, subclinical mastitis is a greater problem in organic than in conventional production systems, but differences are not marked.

Pooled milk samples from 115 Bergamasca meat sheep were collected aseptically five times from lambing to weaning to determine the prevalence of intramammary infection, somatic cell counts and milk quality parameters (protein, fat and lactose), and effects of infection on lamb weight gain. The global prevalence of subclinical intramammary infection was 51·2%. The Staphylococcus genus was responsible for the greatest prevalence (53·3% among infected udders). Staphylococcus aureus was isolated in 8·4% of infected milk samples. Infection status had significant effects on fat and protein percentage and on somatic cell count. Lamb growth was greatest for lambs of ewes with no infection and decreased as the number of infected samples increased. No significant differences were detected in the growth of lambs with dams infected by different bacterial species.

Chronic subclinical mastitis is usually not treated during the lactation. However, some veterinarians regard treatment of some types of subclinical mastitis to be effective. The goal of this research was to develop a stochastic Monte Carlo simulation model to support decisions around treatment of chronic subclinical mastitis caused by Streptococcus uberis. Factors in the model included the probability of cure after treatment, probability of the cow becoming clinically diseased, transmission of infection to other cows, and physiological effects of the infection. Using basic input parameters for Dutch circumstances, the average economic costs per cow of an untreated chronic subclinical mastitis case caused by Str. uberis in a single quarter from day of diagnosis onwards was €109. With treatment, the average costs were higher (€120). Thus, for the average cow, treatment was not efficient economically. However, the risk of high costs was much higher when cows with chronic subclinical mastitis were not treated. A sensitivity analysis showed that profitability of treatment of chronic subclinical Str. uberis mastitis depended on farm-specific factors (such as economic value of discarded milk) and cow-specific factors (such as day of diagnosis, duration of infection, amount of transmission to other cows and cure rate). Therefore, herd level protocols are not sufficient and decision support should be cow specific. Given the importance of cow-specific factors, information from the current model could be applied to automatic decision support systems.

Four semi-hard Italian goats' milk cheeses, Flor di Capra (FC), Caprino di Cavalese (CC), Caprino di Valsassina (CV) and Capritilla (C), were compared for compositional, microbiological, biochemical, volatile profile and sensory characteristics. Mean values for the gross composition in part differed between cheeses. At the end of ripening, cheeses contained 7·98−8·51 log10 cfu/g of non-starter lactic acid bacteria. Lactobacillus paracasei, Lb. casei and Lb. plantarum were dominant in almost all cheeses. As shown by the Principal Component Analysis of RP-FPLC data for the pH 4·6-soluble fractions and by the determination of free amino acids, secondary proteolysis of CC and CV mainly differed from the other two cheeses. A total of 72 volatile components were identified by steam distillation-extraction followed by gas chromatography-mass spectrometry. Free fatty acids and esters qualitatively and quantitatively differentiated the profile of CV and CC, respectively. The lowest concentrations of volatile components characterized FC. Descriptive sensory analysis using 17 flavour attributes was carried out by a trained panel. Different flavour attributes distinguished the four goats' cheeses and relationships were found with volatile components, biochemical characteristics and technology.

Our aim was to develop an explant model to define more precisely the early response of bovine mammary epithelial cells to infection. Therefore we investigated the mRNA expression encoding for some soluble immunological factors in lipopolysaccharide (LPS)-treated bovine mammary gland explants. Explants were taken out from the mammary gland of eight lactating cows after slaughter then incubated with LPS (10 μg/ml) for 6 h. The mRNA expression of α-lactalbumin (α-la), various cytokines, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, and two immunoglobulin receptors, the neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIGR), were assessed with qPCR before and after 3 h and 6 h of LPS challenge. Both immunoglobulin receptors and α-la increased at 3 h then recovered their initial level at 6 h whereas IL-1β, IL-6 and IL-8 increased only after 6 h (P<0·05). Surprisingly, TNF-α transcripts did not show any regulation in response to the LPS treatment. We nevertheless concluded that our model was valid to examine the short-term response of mammary epithelial cell challenged with LPS.

Phenomena generating FFAs, important flavour precursors, are significant in cheese ripening. In Camembert-like cheeses, it was intended to establish the relationships between the dynamics of FFA concentrations changes and the succession of ripening microflora during ripening. Experimental Camembert-type cheeses were prepared in duplicate from pasteurised milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti, and Brevibacterium aurantiacum under aseptic conditions. For each cheese and each cheesy medium, concentrations of FFAs with odd-numbered carbons, except for 9[ratio ]0 and 13[ratio ]0, did not change over time. For long-chain FFAs, concentrations varied with the given cheese part (rind or core). K. lactis produced only short or medium-chain FFAs during its growth and had a minor influence on caproic, caprylic, capric, and lauric acids in comparison with G. candidum, the most lipolytic of the strains used here. It generated all short or medium-chain FFAs (4[ratio ]0–12[ratio ]0) during its exponential and slowdown growth periods and only long-chain ones (14[ratio ]0–18[ratio ]0) during its stationary phase. Pen. camemberti produced more long-chain FFAs (14[ratio ]0–18[ratio ]0) during its sporulation. Brev. aurantiacum did not generate any FFAs. The evidence of links between specific FFAs and the growth of a given microorganism is shown.

Milk quality is measured by a variety of parameters and one of these is the number of somatic cells in the milk. Within the bovine dairy industry there are maximum acceptable levels on bulk milk cell counts and these are being introduced into caprine dairy production. The reference method for cell counting in goats is the direct microscope (DM) method using pyronin Y-methyl green stain, which stains cell DNA. The use of the DeLaval cell counter (DCC) on goats' milk was compared with this reference method. Samples from 102 udder halves were cell counted using the direct microscope method and the DeLaval cell counter (DCC). Each sample was counted twice by two different observers by the DM method and once using DCC. DCC showed large coefficients of regression (1·03) and correlation (0·95) when compared with the DM cell count (F1,199=1080·0; P<0·001). Goat, udder half and reader type were significant factors.

Several milk components related to immune defences (lysozyme, lactoferrin and γ-globulins, γ-G) and to inflammation (somatic cell counts, SCC; N-acetyl-β-glucosaminidase, NAGase; albumin) were considered. Forty-one quarters and 685 samples of 24 cows were included in the study; among them 534 samples were defined as negative (78·0%), 93 as diseased (13·5%) and 58 (8·5%) as subclinical. The pattern of each milk component in quarters always negative during the follow-up period was evaluated by a mixed model. Statistical analysis showed that days in milk (DIM), age (primiparous, pluriparous), herd and the interaction between herd and days in milk significantly influenced all the markers, with very few exceptions. A subset of samples including the negative quarters before the first outcome of an infection or a subclinical mastitis and the samples from quarters always negative was also selected. The analysis showed that herd, DIM and health status had a significant influence on most markers. Overall, primiparous cows were confirmed to have higher levels of most of the markers than pluriparous cows. The presence of a herd effect on non specific immune defences in fully negative quarters implies that when the mechanisms behind their release are fully elucidated, it might be possible to modulate them. Udder tissues were confirmed as an important source of some immune components, as supported by the inconsistency between SCC mean values and NAGase, lysozyme and lactoferrin values. Overall, quarters with high levels of NAGase, lysozyme and γ-G, exposed to bacteria, did not develop subclinical mastitis. Hence, invading pathogens could induce the development of subclinical IMI when these components and γG are in low concentration.

Latex immune agglutination method with a multipurpose auto-analyser (the automated latex assay) was validated for determination of bovine lactoferrin (BLF) in various dairy products. Reproducibility-within-laboratory (intermediate precision) due to day for infant formula, UHT milk and yogurt supplemented with BLF at 50 mg/100 g for infant formula and UHT milk, and at 100 mg/100 g for yogurt were 1·62 to 3·10 mg/100 g. Reproducibility-within-laboratory due to analysis (morning, noon, and evening) for raw milk was 1·59 mg/100 g. Trueness, accuracy in determining known amounts added for BLF-supplemented dairy products was −4·7 to −2·0 mg/100 g. BLF concentration in raw milks was 20·3 to 21·8 mg/100 g. Although interference by the matrixes took place in infant formula and raw milk, BLF assays were accurately carried out by 2000-fold dilution or standard-addition method. Automated latex assay for BLF is simple, rapid, precise and accurate enough for a routine method in various dairy products containing BLF.

The main objective was to determine the prevalence of intramammary infections (IMI) in dairy cows in Flanders, Belgium. Data were obtained from quarter milk samples of dairy herds subjected to a mandatory yearly screening of all lactating cows. A total of 178 668 quarter milk samples were collected at 1087 cross-sectional dairy herd screenings performed in three consecutive years. Of the dairy cows, 40% had at least one culture-positive quarter. More than 50% of all IMI were caused by non-aureus staphylococci. Streptococcus agalactiae is almost eradicated in Flanders, whereas Staphylococcus aureus was isolated from 18% of the culture-positive quarters. In addition, the distribution of mastitis pathogens in quarter milk samples from selected dairy cows with an elevated somatic cell count (SCC) is described. From 6390 cows with a geometric mean composite SCC ⩾250 000 cells/ml, nearly 65% had at least one culture-positive quarter. The majority of the IMI were caused by non-aureus staphylococci (41·1%), whereas Staph. aureus and aesculin-positive cocci were found in respectively 25% and 18% of the culture-positive milk samples. We conclude that more efforts are needed in the prevention and control of subclinical mastitis in Flanders. Non-aureus staphylococci are the predominant cause of IMI, warranting more research regarding the epidemiology and pathogenicity of those species.

We used a Mannheimia haemolytica isolate to study differences in susceptibility to experimental mastitis between two breeds of dairy sheep. The isolate was deposited into the teat duct of Karagouniko (K, n=8) or Frisarta (F, n=8) ewes. The animals were monitored by means of clinical, bacteriological, cytological and pathological methods. K ewes did not develop any systemic or mammary clinical signs, whilst F ewes became ill and developed acute clinical mastitis 12 h later (P<0·001). Bacteria were isolated from 34/48 samples from K ewes and from 46/46 samples from F ewes. Positive California mastitis test (CMT) results were 17/24 samples from K ewes and 23/23 samples from F ewes; leucocytes were seen in Giemsa-stained films. Total pathology score summed over all group K ewes was 41 (maximum possible: 128); Man. haemolytica was isolated from 12/24 tissue samples. Total pathology score summed over all group F ewes was 93; Man. haemolytica was isolated from 24/24 tissue samples. Hyperplastic lymphoid nodules consisting of lymphocytes and plasma cells with germinal activity were characteristically present at the border between teat duct–teat cistern of group K ewes; no such structures were observed in teats of group F ewes. The results identified differences in susceptibility/resistance to a mastitis pathogen among animals of the two breeds. Defence mechanisms of the teat appeared to be inadequate against the invading organisms; as lymphoid nodules have been considered important defensive mechanisms of the ovine teat, their observed lack in Frisarta ewes might have predisposed them to development of mastitis.