The aim of this study was to assess the effect of milk source and of cheese production protocol on proteolytic and lipolytic pattern of cheese during ripening. The study involved six dairy factories located in Monti Dauni Meridionali area of Southern Italy; three dairy factories processed the milk produced by their own cow herds, while the other three dairy factories processed the milk collected in other dairy farms located in the neighbouring area. Cow milk processed to cheese had different nutritional parameters and hygienic quality. Caciocavallo cheese showed differences in the evolution of proteolysis during ripening and in the intensity of the lipolytic process detected at the end of ripening. The main factors influencing Caciocavallo cheese features were the quality of the starting milk, differences in technological steps such as milk heating, type of starter cultures and coagulant used.

For its characteristics, donkey milk has been proposed as an alternative to goat or artificial milk to feed allergic infants. Therefore, it is important to increase our knowledge on health and immunological characteristics of donkey milk. Ten donkeys, bred as companion animals, were enrolled in this study and sampled once a month, for eight months. Milk (10 ml) was collected from each half udder for somatic cell count (SCC), bacteriological analysis and total bacteria count (TBC). The major pathogens were tested for antimicrobial susceptibility, and Staphylococcus aureus isolates were further genotyped by nanoarray analysis. Whey lysozyme and NAGase (NAG) activities were also assessed. Overall, 101 half-udder milk samples were taken. They showed very low values of TBC (<250 cfu/ml) and SCC (<50 000 cells/ml) and a minor prevalence of pathogens: Staph. aureus was isolated only from 5 milk samples (3 animals), Streptococcus equi from 2 samples and Str. equisimilis from a single sample. All the isolates were sensitive to all antibiotic classes used in veterinary medicine. None of the Staph. aureus isolates were shown to harbour genes coding for any enterotoxin, toxic-shock syndrome toxin or antibiotic resistance. Lysozyme levels were always very high (4000–5000 U/ml), while NAG values were mostly low (<50 U/ml), out of the last part of lactation. The results of this study confirmed the low prevalence of intramammary infections in donkey and the absence of food-borne pathogens, suggesting that donkey milk could be a safe food, if the mammary gland is healthy and the animals are milked in proper hygienic conditions.

Ninety-nine individual milk samples from 37 cows in lactation week 10–35, selected for producing well or poorly/non-coagulating milk, were compared regarding protein composition, total calcium content, casein micelle size, pH, and coagulating properties after addition of 0·05% CaCl2. The results showed that a low κ-casein concentration in milk was a risk factor for non-coagulation. CaCl2 addition improved coagulating properties (coagulation time, curd firmness) of nearly all samples and eliminated differences between poorly/non-coagulating and well-coagulating milk, particularly regarding curd firmness. A second, independent data set with 18 non-coagulating or well-coagulating milk samples were analysed for protein composition, where indications of a similar association with κ-casein was observed.

Non-specific (innate) immune response plays a major role in defending the udder from bacterial invasion. Moreover, recent investigations suggest that mammary gland epithelial cells (MGEC) could have a large and important role as a source of soluble components of immune defences. Despite many attempts to find other ways to control/prevent mastitis (i.e. vaccine) antimicrobial therapy is still the most used and effective means of curing clinical and subclinical mastitis. However, drug concentrations and therapy durations are far from the optimal in order to reduce costs. Therefore, efficacy of antimicrobial therapy is dependent not only on the substance activity but also on the positive interactions with the host innate immune response. Surprisingly, information on these interactions is rather scarce in the mastitis field. A simple experimental model was developed based on BME-UV cell line, Staphylococcus aureus as a challenge and a macrolide as an antimicrobial to assess the interactions among epithelial cells, Staph. aureus and the potential effects of antimicrobials on the immune system. The results of this study confirmed that tylosin has good antimicrobial activity against both intracellular and extracellular Staph. aureus in bovine MGEC without affecting cell functions. In this study, a significant down-regulation of IL-1 and IL-6 was observed, while TNF and IL-8 expression rate numerically increased, but differences were not significant. To our knowledge, this is the first paper assessing the concentration of two lysosomal enzymes, lysozyme and N-acetyl-β-d-glucosaminidase (NAGase), in Staph. aureus-stimulated MGEC. The results of this study confirmed that tylosin could have a significant effect on the release of these enzymes. Moreover, even if both enzymes have a similar substrate as a target, the results suggest different secretion mechanisms and an influence of antimicrobial treatment on these mechanisms. Successful mastitis cure is the result of achieving the optimal efficiency of both innate immune defences and therapeutical activities, by means of killing bacteria without eliciting an excessive inflammatory response. Therefore, antimicrobials for mastitis therapy should be selected not only on bacterial sensitivity, but also for their positive interactions with the innate immune response of the mammary gland. This study showed that an in-vitro model based on Staph. aureus challenge on MGEC could be helpful in assessing both the intracellular and extracellular activity of antimicrobials and their influence on epithelial cell immune and inflammatory response.

Addition of methylated β-lactoglobulin (Met-BLG) in the medium of MDCK cell lines infected with influenza virus subtype H1N1 reduced hemagglutination activity (HA) in a concentration dependent manner. Antiviral activity of Met-BLG depended on its concentration, viral load, and duration of infection. Using 17 μg/ml of Met-BLG inhibited 50% of HA of H1N1 grown in MDCK cells at 1 MOI after 24 h incubation at 37°C and in 5% CO2. Extension of incubation time enhanced antiviral action since the same concentration of Met-BLG inhibited about 61% of viral activity after 48 h. This viral inhibition was accompanied by a protection of MDCK cells as observed by using neutral red or by direct microscope examination. Reduction of viral RNA replication upon the addition of Met-BLG (50 μg/ml) was observed by real time-PCR showing a reduction of viral log value of about 0·9. When viral stock solution was mixed with 25 μg/ml Met-BLG in absence of cell lines, the morphology and viability of virus particles were significantly affected as observed by electron microscopy, and the number of intact virus particles was reduced by roughly 65%.

Donkey's milk is well tolerated by human infants with cow's milk allergy and is useful in the treatment of human immune-related diseases and in the prevention of atherosclerosis. Thyroid hormones (TH) stimulate lactation and active triiodothyronine (T3) in colostrum and milk could take paracrine action supporting lactogenesis in the mother, and play physiological roles for the suckling offspring (systemic or within the gastrointestinal tract). The aims were to measure TH concentrations in donkey blood and milk, validate ELISA methods, evaluate the effects of sample collection and post-collection handling and the stability of TH in milk and blood serum and plasma samples. In milk and blood samples obtained from lactating jennies total concentrations of TH were assayed using competitive-type ELISA kits. Good validation results were obtained for both TH concentrations in blood serum and plasma and T3 in milk samples extracted with cold (−20°C) ethanol alkalinized (pH 9·0) with NH4OH. In most of the milk extract samples, thyroxine (T4) concentrations resulted below the sensitivity threshold. Intra- and inter-assay coefficients of variations of TH concentrations in different blood and milk samples were below 10%. Parallelism tests gave displacement lines parallel to those of the calibrators for both TH in blood serum and plasma and for T3 in milk extracts. Mean recovery rates were between 95% and 123%, but the concentration values approaching the highest calibrators were overestimated. Therefore, serum and plasma samples for T3 assay must be previously diluted with buffer. Both TH concentrations in blood serum and plasma and T3 in milk did not change during storage for up to 6 months at −20°C. In conclusion, the ELISA methods tested in the present study are suitable for determination of both TH concentrations in donkey blood samples, and for T3 measurement in milk, after extraction with cold alkaline ethanol.

Microscopic conformation, growth behaviour and freezing sensitivity of seven strains of Geotrichum candidum, a ripening starter, were studied and compared according to their macroscopic morphotypes. It has been shown that the thallus forming units (TFU)×ml−1/OD600nm ratio as a function of time is an interesting parameter to follow G. candidum sporulation through the growth behaviour. Microscopic conformation, growth behaviour and freezing sensitivity are clearly strain specific and mostly related to their corresponding morphotypes “yeast”, “mould” or “intermediate”. The two “mould” strains that sporulate weakly (UCMA103, UCMA499) showed a low survival rate to freezing stress whereas the “yeast” strains expressed a significant resistance owing to the arthrospore abundance. Interestingly, one strain (UCMA96) which appeared on solid medium in accord with the “mould” morphotype respond similarly to freezing stress.

Commonly measured metabolite and hormone concentrations used to describe the metabolic status of lactating cows undergo diurnal variation resulting in distinct patterns. Studies have shown that feeding events can modulate these diurnal patterns as cows respond to the nutrient intake. What is less clear is the extent to which cow genetics and diet interact to modify the diurnal patterns of specific nutritionally related metabolites and hormones. The objective of this study was to investigate diurnal patterns in circulating metabolite and hormone concentrations in divergent strains of Holstein-Friesian cows (North American, NA; and New Zealand, NZ) offered either fresh pasture (FP) or a total mixed ration (TMR). Plasma concentrations of growth hormone (GH), insulin-like growth factor-1 (IGF-1), glucose, insulin, β-hydroxybutyrate (BHBA), and non-esterified fatty acids (NEFA) were determined at 4-h intervals for a minimum of three consecutive days. All of the above metabolites and hormones exhibited within-day variability. Main effects of genetic strain and diet were observed for plasma IGF-1, and a strain by diet interaction was observed for GH. Time of day×diet interactions were observed for both glucose and insulin. Three-way interactions (time of day×diet×strain) were observed for BHBA and NEFA. These data indicate different levels of diurnal variation, with glucose, insulin, NEFA and BHBA having the largest daily variation. These diurnal patterns need to be considered in future investigations of these metabolites and hormones.

The primary objective of this study is to validate a new fast method for determination of uric acid in milk. The method is based on an enzymatic-fluorometric technique that requires minimal pre-treatment of milk samples. The present determination of uric acid is based on the enzymatic oxidation of uric acid to 5-hydroxyisourate via uricase where the liberated hydrogen peroxide reacts with 10-acetyl-3,7-dihydroxyphenoxazine via peroxidase and the fluorescent product, resorufin, is measured fluorometrically. Fresh composite milk samples (n=1,072) were collected from both Jersey (n=38) and Danish Holstein (n=106) cows from one local herd. The average inter- and intra-assay variations were 7·1% and 3·0%, respectively. Percent recovery averaged 103·4, 107·0 and 107·5% for samples spiked with 20, 40 or 60 μm of standard, respectively, with a correlation (r=0·98; P<0·001) observed between the observed and expected uric acid concentrations. A positive correlation (r=0·96; P<0·001) was observed between uric acid concentrations using the present method and a reference assay. Storage at 4°C for 24 h resulted in lower (P<0·01) uric acid concentrations in milk when compared with no storage or samples stored at −18°C for 24 h. Addition of either allopurinol (a xanthine oxidase inhibitor) or dimethylsulfoxide (a solvent for allopurinol) did not affect milk uric acid concentrations (P=0·96) and may indicate that heat treatment before storage and analysis was sufficient to degrade xanthine oxidase activity in milk. No relationship was observed between milk uric acid and milk yield and milk components. Authors recommend a single heat treatment (82°C for 10 min) followed by either an immediate analysis of fresh milk samples or storage at −18°C until further analysis.

Emulsions were prepared with 20% soy oil and different concentrations of lactoferrin, and tested at pH values from 3 to 7·5. The stability of the emulsions decreased as the pH got closer to the isoelectric point of the protein. A concentration of 1% lactoferrin was determined to be sufficient to provide full coverage of the emulsion droplets. Lactoferrin-stabilized emulsions were then prepared in water at pH 6·6 and their behaviour when added to reconstituted milk was studied. It was observed that lactoferrin emulsions were stable when reconstituted in milk, but they showed aggregation when diluted in milk serum alone. The destabilization was caused by shielding of the charges on the surface of the oil droplets. Stabilization in milk occurred due to interactions at the interface with other soluble proteins. In fact, when β-lactoglobulin or sodium caseinate were added to the serum, stability of the emulsion droplets was restored, indicating that these proteins were able to adsorb at the interface and aid in the stabilization. Since ζ-potential measurements did not show significant overall charge on the emulsion droplets, this suggests that the stabilization forces are not only electrostatic in nature, but that there are other mechanisms at play.

Low somatic cell count (SCC) is a reliable indicator of high-quality milk free of pathogenic microorganisms. Thus, an important goal in dairy practice is to produce milk with low SCC. Selection for cows with low SCC can sometimes lead to extremely low SCC in single quarters. The cells in milk are, however, predominantly immune cells with important immune functions. To investigate the mammary immune competence of quarters with very low SCC, healthy udder quarters of cows with normal SCC of (40–100)×103 cells/ml and very low SCC of <20×103 cells/ml were challenged with lipopolysaccharide (LPS) from Escherichia coli. In the first experiment, SCC and cell viability after a challenge with 50 ng of LPS/quarter was investigated. In the second experiment, tumour necrosis factor α (TNF-α) concentration and lactate dehydrogenase (LDH) activity in milk, and mRNA expression of various innate immune factors in milk cells were measured after a challenge with 100 μg LPS/quarter. LPS challenge induced an increase of SCC. SCC levels reached were higher in quarters with normal SCC and maximum SCC was reached 1 h earlier than in very low SCC quarters. The increase of TNF-α concentrations in milk in response to LPS challenge was lower in quarters with very low SCC than in quarters with normal SCC. The viability of cells and the LDH activity in milk increased in response to LPS challenge, however, without a difference between the groups. The mRNA expression of IL-1β and IL-8 was increased in milk cells at 12 h after LPS challenge, whereas that of TNF-α and lactoferrin was not increased at the measured time points (12, 24 and 36 h after LPS challenge). No differences of mRNA expression of measured immune factors between normal and very low SCC samples were detected. The study showed that udder quarters with very low SCC responded with a less marked increase of SCC compared with quarters with normal SCC. This difference corresponded with simultaneously lower TNF-α concentrations in milk. However, the immune competence of the cells themselves based on mRNA expression of TNF-α, IL-8, IL-1β, and lactoferrin, did not differ. The results may indicate that very low SCC can impair the immune competence of udder quarters, because the immune response in udder quarters with lower SCC is less efficient as fewer cells contribute to the production of immunoregulators.

The objective of this clinical control trial was to examine the effectiveness of the classical homeopathic treatment strategy in cases of mild and moderate bovine clinical mastitis in comparison with antibiotic and placebo treatments. Owing to characteristics of the selected herds, only cases of clinical mastitis caused by environmental pathogens and clinical cases with negative bacteriological result in the pre-treatment milk sample were included in the trial. A total of 136 lactating dairy cows with 147 affected quarters from four herds in Germany were randomly allocated to three treatment groups. The cows were examined on days 0, 1, 2 and on days 7, 14, 28 and 56 post initial infection to assess clinical signs. Simultaneously, with the exception of days 1 and 2, quarter milk samples for laboratory examinations (bacteriology, somatic cell count) were collected to assess bacteriological and cytological cure rates. On days 28 and 56, treatment strategies did not differ significantly with respect to the clinical outcomes and the total cure rate in cases of bacteriological negative mastitis (n=56). In cases of pathogen-positive mastitis (n=91), the cure rate after 4 and 8 weeks was similar between the two treatment strategies, homeopathy and antibiotic treatment, but the difference between the homeopathic and the placebo treatment at day 56 was significant (P<0·05). The results indicate a therapeutic effect of homeopathic treatment in cases of mild and moderate clinical mastitis. However, independent of treatment strategy and bacteriological status, the total cure rate was on a low level, revealing limitations in the effectiveness of both antibiotic and homeopathic treatment strategies.

The present study was conducted to assess the interrelation between teat anatomy and machine milking in dairy buffaloes raised in Switzerland. A 3-min pre-stimulation induced milk ejection before cluster attachment in most cases and caused an optimal milk removal during machine milking. In an additional experiment, longitudinal cross-section ultrasound was obtained before and after a 3-min pre-stimulation. Teat wall thickness, teat diameter, cisternal diameter and teat canal length were evaluated. It was observed that 3-min pre-stimulation dramatically reduced teat canal length whereas all the other anatomical parameters remained unchanged. The vacuum needed to open the teat canal was also measured before and after a 3-min pre-stimulation by using a special teat cup with only the mouthpiece of the liner remaining on the top of the teat cup (no liner, no pulsation). Without pre-stimulation but after wetting the teat canal by stripping one squirt of milk out of the teat, no milk could be withdrawn with a vacuum up to 39 kPa. However, after pre-stimulation, milk flow occurred in all buffaloes at a vacuum between 16 and 38 kPa. In the last experiment, the teat tissue was examined in slaughtered buffaloes and compared with teat tissue of cows. No difference was noted in histological sections and teat canal length was similar in cows and buffaloes. Proximal to the teat canal, the teat did not pass into an open cistern but the lumen was collapsed. In conclusion, buffaloes need to be well pre-stimulated because the tissue above the teat canal provides additional teat closure before milk ejection. Therefore, milk can only be obtained after pre-stimulation.

Lactoferrin (Lf) is a molecule naturally present in bovine milk that affects the availability and transport systems of iron. Lf also binds endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria and modulates the immunological response. In the present study, concentrations of bovine Lf (bLf) and citrate in milk were determined in early (EL) and late (LL) lactating dairy cows, using an experimentally induced endotoxin mastitis model and a crossover design. Nine clinically healthy Finnish Ayrshire cows were challenged twice with 100 μg endotoxin infused into one udder quarter. Milk samples were collected from the challenged and control quarters of each cow before and after endotoxin infusion during 3 d, and bLf and citrate concentrations were measured. In all cows, clinical signs of mastitis were seen at both times of challenge, but the response was more severe in EL than in LL. Concentration of bLf in the milk started to rise approximately 8 h after endotoxin infusion and was still higher than normal on the third day, especially in the late-lactating cows. In milk of the LL group, concentrations of bLf were significantly higher than in the EL group. In contrast, concentrations of citrate were higher in milk of the EL cows compared with the LL cows. Concentration of bLf and citrate varied substantially among cows. The molar ratio of citrate to bLf before and after challenge was significantly higher during the EL period. The results of this study partly explain why cows in early lactation are more susceptible to intramammary infections and why mastitis is more severe in them.

Concentrations of IgA and IgM in milk from normal lactating cows were measured to determine whether they were affected by lactation number, lactation stage, daily milk production, milk composition and somatic cell score (SCS). A total of 284 Chinese Holstein cows showing no signs of mastitis were selected from more than 1600 cows from one farm. Concentrations of IgA and IgM in normal milk were determined by ELISA. Results revealed that average milk IgA and IgM concentrations were 235±11 and 32±3 μg/ml, respectively. IgA and IgM concentrations in milk were found to have an association with lactation number, stage of lactation, daily milk production and SCS. Both SCS and daily milk production were found to be strong significant (P<0·01) direct factors correlating to IgA concentration. Stage of lactation and SCS were found to be strong significant (P<0·01) direct factors correlating to IgM concentration.

Our previous studies demonstrated prompt elevation of proteinase activity in mammary secretion of drying-off cows and goats. The current study examined the progressive changes in composition of cow mammary secretion following drying-off and, in parallel, characterized the mode of peptide neogenesis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-electrospray-ionization (LC-ESI) MS/MS. The results show that the percentage of casein of total milk protein at time of drying-off was 76%, which dropped to 41%, 24%, and 16%, respectively, 1, 2, and 3 weeks after drying-off. Levels of β-lactoglobulin and α-lactoalbumin in mammary secretions of drying-off cows decreased prominently while levels of lactoferrin, BSA, and casein derived-proteins increased concomitantly compared with regular milk. A fractionation procedure was applied to remove molecules larger than 10 kDa before MALDI-TOF MS and LC-ESI MS/MS and the results show that the MALDI-TOF MS peptide profile of mammary secretion ranging from m/z 600 to 4000 was apparently modified after drying-off for 1 week, whereas species 1590 m/z and 2460 m/z were most obviously enriched compared with regular milk. LC-ESI MS/MS results were used to map peptide sequence with Mascot search server and under no post translational modification to reduce database size and 202 novel β-casein-derived peptides were successfully identified in mammary secretion after drying-off for 1 week in contrast to regular milk. Accordingly at least 48 additional cleavage positions were assigned on β-casein for mammary secretion. Among the 202 novel peptides, 5 are homologous with confirmed opioid agonists, angiotensin 1-converting enzyme inhibitors, or immuno-modulators. In conclusion, peptides are released in situ from milk proteins within short intervals following drying-off in cows. They might play roles in the transition of mammary glands from lactating to non-lactating. With specified post-translational modifications and focused functional screening, novel peptides are yet to be discovered in dry cow mammary secretion.

The potential of ethidium monoazide (EMA) real-time PCR method based on molecular beacon probe for rapid detection of viable bifidobacteria present in probiotic yogurt was evaluated in this work. A real-time PCR with molecular beacon assay was developed to determine genus Bifidobacterium quantitatively in order to increase the sensitivity and specificity of assay. EMA was used to treat probiotic yogurt prior to DNA extraction and real-time PCR detection to allow detection of only viable bacteria. The primer set of Bif-F/Bif-R which is genus-specific for Bifid. was designed. The specificity of the probes ensures that no signal is generated by non-target amplicons. Linear regression analysis demonstrated a good correlation (R2=0·9948) between the EMA real-time PCR results and the plate counting, and real-time quantitative PCR results correlated adequately with enumeration of bifidobacteria by culture for commercial probiotic yogurt. This culture-independent approach is promising for the direct and rapid detection of viable bifidobacteria in commercial probiotic yogurt, and the detection can be carried out within 4 h. The detection limit for this method is about 104 cell/ml. In conclusion, the direct quantitative EMA real-time PCR assay based on molecular beacon described in this research is a rapid and quantitative method.