In this research communication we exploited the potential use of milk microRNAs (miRs) as biomarkers for bovine tuberculosis (bTB). bTB is a zoonotic disease caused by Mycobacterium bovis which affects animal health, influencing herd economic sustainability. Diagnosis is based on skin delayed-type hypersensitivity reaction and quantification of interferon gamma but both techniques are influenced by several confounding factors. Thus, new methods for early diagnosis are required. In this context, microRNAs have been used as promising biomarkers for both infectious and non-infectious diseases. To determine the possible involvement of microRNAs in bTB, we analysed the expression of four immune-related miRs in 200 cows grouped in cases and controls with respect to positivity to tuberculosis. The analysis showed a different magnitude of expression in the groups indicating that active tuberculosis could influence miRs expression. We used expression values of miR-146a, the highest differentially expressed miR, for Receiver operating characteristic (ROC) curve analysis. In order to determine a test cut-off value for miR-146a expression that would differentiate cases and controls, a value for the miR-146a expression higher than 8 was selected as this gave a test specificity and sensitivity of 80·0% and 86·0% respectively. These values confirm the possibility of using miR-146a as a milk prognostic biomarker for bovine tuberculosis.

The purpose of this study was to determine tetracycline and other antimicrobial residues in milk from dairy cows treated for clinical mastitis. Data on cow health and drugs used were obtained from the farm veterinarians. A milk sample from each affected udder quarter of each cow was taken for bacterial identification before treatment. All 35 cows in the study were treated with an intramammary product containing tetracycline, neomycin, bacitracin, and prednisolone (tetracycline injector) and also simultaneously with other drugs via the intramammary or parenteral route. The withhold period of the ‘tetracycline product’ was eight milkings but, in all cases, longer withhold periods were prescribed due to off-label application of additional combinations of drugs. Milk samples from treated udder quarters, taken two milkings before and two after the prescribed withhold period, were analysed for antimicrobial residues. Additionally, milk samples were taken from untreated healthy udder quarters to check if any crossover of drugs had occurred. Three screening tests were used for antimicrobial detection. In 15 (42·9%) cows the milk samples from the treated infected quarters contained tetracycline residues above the maximum residue limit (MRL) after the prescribed withhold period and, in two cases (5·7%) the same was true for neomycin and cefquinome residues. Cephalexin above the MRL was detected in only one case (2·9%). Beta-lactams did not exceed the MRL after the prescribed withhold period. Antimicrobial residues were not detected in milk samples from untreated quarters. Cow condition including parity, decreased milk production and severity of mastitis significantly influenced the excretion of antimicrobials in milk (P ≤ 0·05). No significant differences were found between the tetracycline positive and tetracycline negative cows regarding the bacteria involved, combination antimicrobial therapy, or treatment interval and duration.

In this Research Communication we report the results of a controlled study conducted under field conditions in which we analysed milk ejection curves in cows with chronic mastitis, and assessed the influence of antinflammatory treatment with ketoprofen. Total milking time was reduced in chronic mastitis cows, irrespective of ketoprofen treatment, and the proportion of bimodal flow curves was increased. This latter effect was partially reversed by ketoprofen. To our knowledge, this is the first study showing that chronic mastitis has a significant effect on the milk ejection curve. Antinflammatory treatment with ketoprofen was shown to be efficacious in reducing these negative effects, re-establishing a pattern close to the one observed in healthy cows.

Optimization of dairy fermentation processes often requires multiplexed pH measurements over several hours. The method developed here measures up to 90 samples simultaneously, where traditional electrode-based methods require a lot more time for handing the same number of samples. Moreover, the new method employs commonly used materials and can be used with a wider range of fluorescence readers than commercial 96-well plates with optical pH sensors. For this application, a milk-like transparent medium is developed that shows acidification properties with dairy starters that are similar to milk. Combination of this milk-like medium and 3 fluorescent indicators allow precise measurements of pH in a range of 4·0–7·0. The new method showed much higher throughput compared to the benchmark electrode systems while being as accurate, as shown by successful application for a comparison of various dairy starter cultures and for optimizing the inoculation rate.

Lactoperoxidase (LPO) is an antimicrobial protein present in milk that plays an important role in natural defence mechanisms during neonatal and adult life. The antimicrobial activity of LPO has been commercially adapted for increasing the shelf life of dairy products. Immobilization of LPO on silver nanoparticles (AgNPs) is a promising way to enhance the antimicrobial activity of LPO. In the current study, LPO was immobilized on AgNPs to form LPO/AgNP conjugate. The immobilized LPO/AgNP conjugate was characterized by various biophysical techniques. The enhanced antibacterial activity of the conjugate was tested against E. coli in culture at 2 h intervals for 10 h. The results showed successful synthesis of spherical AgNPs. LPO was immobilized on AgNPs with agglomerate sizes averaging approximately 50 nm. The immobilized conjugate exhibited stronger antibacterial activity against E. coli in comparison to free LPO. This study may help in increasing the efficiency of lactoperoxidase system and will assist in identifying novel avenues to enhance the stability and antimicrobial function of LPO system in dairy and other industries.

In this Research Communication we describe the effect of temperature and humidity index (THI) on various physiological traits, the plasma heat shock protein 70 (HSP70), heat shock protein 90 (HSP90) and cortisol levels and other blood parameters in crossbred buffalo (Nili-Ravi × Murrah) and Mediterranean buffalo to compare their tolerance to heat stress. As expected, crossbred buffalo had a significantly higher rectal temperature (RT), body surface temperature (BT), respiratory rate (RR), HSP70 and HSP90 levels in summer compared to spring and winter. RT and BT were also significantly higher in spring compared to winter. A significant correlation existed between THI and RT (r = 0·81) and RR (r = 0·84). Importantly, in summer the crossbred buffalo had a significantly lower RT, BT and RR and higher HSP70, HSP90 and cortisol levels than the Mediterranean buffalo. In conclusion, higher THI was associated with significant increase in RT, RR, BT, HSP70, HSP90 and cortisol levels, and the crossbred buffalo were more heat tolerant than Mediterranean buffalo.

Heat stress (HS) negatively affects milk production and has been associated with decreased immune function, and increased rate of intramammary infections (IMI). Research has shown that HS affects gene expression, cell cycle, and cell metabolism in bovine mammary epithelial cells (BMEC). Since BMEC are an initial target of mastitis pathogens, we studied adherence to and internalisation of S. uberis into HS-BMEC, as well as the effect that this interaction has on host cells by measuring HS-BMEC viability and membrane integrity. Results reported in this Research Communication showed that HS reduced cell viability and induced membrane damage. However, these pathological changes, as well as the rate of adherence and internalisation of S. uberis into BMEC, were augmented when S. uberis was cocultured with HS-BMEC. These results may help to understand the pathogenesis of S. uberis IMI as well as the increased susceptibility of mammary glands to IMI in cows subjected to HS.

The work described in this Research Communication addressed the hypothesis that it is possible to produce cheese from donkey milk by appropriate adjusting of the cheesemaking parameters. A series of coagulation trials were performed on donkey milk, alone or fortified with goat milk (85/15 and 70/30, v/v), using calf rennet under different technological conditions. The parameters that changed were pH and concentration of soluble calcium, amount of rennet added and temperature of coagulation. Donkey milk gave rise to sufficiently firm curd only at ‘extreme’ technological conditions and, as expected, addition of goat milk improved coagulation. A cheesemaking protocol was developed for producing fresh cheese prototypes, which were checked for microbiological safety, chemical composition and sensory characteristics. Pure donkey milk gave 5·9% yield, cheese having 6·12 pH, 32·4% dry matter, 2·1% fat and 18·5% protein. The electrophoretic analysis ascertained that β-casein was the most abundant compound in donkey cheese, but whey proteins were also present in non-negligible amounts. Finally, the sensory evaluation demonstrated that all cheeses were acceptable and provided detailed description of their flavour characteristics.

The study reported in this Research Communication was conducted to characterise Staphylococcus aureus isolates recovered from mastitic bovine milk from dairy herds in Argentina. A total of 829 mastitic milk samples, both clinical and subclinical, were collected from 21 farms by veterinarians and submitted to the laboratory for testing from which 229 S. aureus isolates were recovered, an isolation rate of 28·1%. These isolates were tested for susceptibility to the antibiotics penicillin, erythromycin and clindamycin. Of the 229 isolates, 53 (23·1%) were resistant to penicillin, 31 (13·5%) to erythromycin and 28 (12·2%) to clindamycin. All isolates were negative for the mecA, mecC and pvl genes by PCR. Southernblot hybridisation revealed that the ermC gene was located on plasmid bands. Eighty isolates were randomly selected from the 229 for further characterisation. Restriction analysis of chromosomal DNA with Cf9I followed by PFGE of the 80 isolates revealed 23 distinct pulsotypes at 80% similarity. Seven major types (A, B, N, P, S, T, U and V) accounted for 68·7% of these isolates and 12 pulsotypes (A, B, F, G, J, K, M, N, P, S, T and U) occurred on more than one farm indicating genetic diversity within the farms. MLST of a representative isolate from dominant types identified the STs 97 705, 746, 2102 and 2187 with ST97 being the most predominant. Antibiotic susceptibility testing showed that 53·7% of the 80 randomly selected isolates were resistant to at least one of the three antibiotics tested. To our knowledge, this study represents the first large scale molecular studies on S. aureus isolates from dairy farms in Argentina.

Inflammation of the mammary gland following bacterial infection, commonly known as mastitis, affects all mammalian species. Although the aetiology and epidemiology of mastitis in the dairy cow are well described, the genetic factors mediating resistance to mammary gland infection are not well known, due in part to the difficulty in obtaining robust phenotypic information from sufficiently large numbers of individuals. To address this problem, an experimental mammary gland infection experiment was undertaken, using a Friesian-Jersey cross breed F2 herd. A total of 604 animals received an intramammary infusion of Streptococcus uberis in one gland, and the clinical response over 13 milkings was used for linkage mapping and genome-wide association analysis. A quantitative trait locus (QTL) was detected on bovine chromosome 11 for clinical mastitis status using micro-satellite and Affymetrix 10 K SNP markers, and then exome and genome sequence data used from the six F1 sires of the experimental animals to examine this region in more detail. A total of 485 sequence variants were typed in the QTL interval, and association mapping using these and an additional 37 986 genome-wide markers from the Illumina SNP50 bovine SNP panel revealed association with markers encompassing the interleukin-1 gene cluster locus. This study highlights a region on bovine chromosome 11, consistent with earlier studies, as conferring resistance to experimentally induced mammary gland infection, and newly prioritises the IL1 gene cluster for further analysis in genetic resistance to mastitis.

Goaty flavor and poor consistency may impact consumer acceptance of fermented goat milk. The undesirable characteristics can mainly be attributed to the presence of short-medium chain free fatty acid (SM-FFA) especially C6-C10 fatty acids and low αs1-casein content in goat milk. This study aimed to investigate the effects of polymerized whey protein (PWP) on goaty flavor as well as the texture properties of fermented goat milk in comparison with β-cyclodextrin (β-CD). Samples were evaluated on sensory properties, SM-FFA contents, texture, and apparent viscosity. Compared with control, the fatty acids contents (C6, C8, C10) decreased significantly in fermented goat milk with 0·5% β-CD (22, 71, 54%, respectively) and with 0·7% PWP (45, 58, 71%, respectively). There was a synergistic effect of 0·3% β-CD and 0·6% PWP in decreasing the contents of SM-FFA (C6, C8, C10) sharply by 89, 90, 79%. Under the same percentage of addition, yogurts made with β-CD showed a higher (P < 0·05) apparent viscosity than those with PWP. However, the addition of PWP could increase the texture parameters of fermented goat milk (P < 0·05). Combination of PWP and β-CD presented a more desirable texture and consistency in fermented goat milk. Results indicated that polymerized whey protein can be used to reduce the goaty flavor and improve the texture of fermented goat milk.

The aim of this study was to identify the main Enterobacteriaceae species responsible for early gas blowing during curdling and the first week of ripening in raw goats’ milk cheese. Two batches of raw goats’ milk cheese were selected. One of them showed early blowing within the first 24 h of cheese ripening while the other showed no alteration. Although initial levels of Enterobacteriaceae were similar in defective and non-defective cheese, their dynamics (growth and disappearance rates of the species detected) were different. Klebsiella oxytoca and Enterobacter cloacae were the main species in the defective curd, whereas Buttiauxela spp. was predominant in normal curd. Hafnia alvei was the prevailing isolated species for both normal and defective cheese throughout the ripening process. The highest gas production was rendered by K. oxytoca and H. alvei, mainly isolated from curd and cheese. However, other species relevant in milk or curd, like Pantoea ssp. or Buttiauxela spp. were considered as low gas producers. The analysis of digitalized images of cheese showed that most of the cheese eyes were formed before the first week of ripening, although this process continued during maturation.

According to the species found in the defective and non-defective cheese, their proportions at different ripening stages, their ability to produce gas and eye formation, K. oxytoca might be considered the most likely responsible for early blowing in raw goats’ milk cheeses; while H. alvei increased the eyes number in the later stages of the ripening period.

The enzymatic hydrolysis of milk proteins yield final products with improved properties and reduced allergenicity. The degree of hydrolysis (DH) influences both technological (e.g., solubility, water binding capacity) and biological (e.g., angiotensin-converting enzyme (ACE) inhibition, antioxidation) properties of the resulting hydrolysate. Phenomenological models are unable to reproduce the complexity of enzymatic reactions in dairy systems. However, empirical approaches offer high predictability and can be easily transposed to different substrates and enzymes. In this work, the DH of goat milk protein by subtilisin and trypsin was modelled by feedforward artificial neural networks (ANN). To this end, we produced a set of protein hydrolysates, employing various reaction temperatures and enzyme/substrate ratios, based on an experimental design. The time evolution of the DH was monitored and processed to generate the ANN models. Extensive hydrolysis is desirable because a high DH enhances some bioactivities in the final hydrolysate, such as antioxidant or antihypertensive. The optimization of both ANN models led to a maximal DH of 23·47% at 56·4 °C and enzyme–substrate ratio of 5% for subtilisin, while hydrolysis with trypsin reached a maximum of 21·3% at 35 °C and an enzyme–substrate ratio of 4%.

The objective of the study reported in this Research Communication was to investigate the effect of fermentation temperature (37 and 45 °C) and different ratios of Streptococcus thermophilus to Lactobacillus bulgaricus (3 : 1, 1 : 1 and 1 : 3) on Kermanshahi roghan and yoghurt fatty acid profiles (FAP) in order to obtain a product with optimized fatty acid profiles. Kermanshahi roghan is a yoghurt by-product in western Iran (Kermanshah). The results revealed that incubation temperature at 37 °C as compared to 45 °C had a better effect on fatty acid profiles of roghan and yoghurt. Furthermore, the results showed that fatty acid profile of roghan is better than yoghurt at two experimental temperatures. On the other hand, the roghan products made by equal ratio of S. thermophilus and L. bulgaricus (1 : 1) had the best quality of fatty acid profiles. Although a lower incubation temperature increases incubation time, our finding suggests that inoculation ratio 1 : 1 at 37 °C as compared to 45 °C can affect the quality of roghan and yoghurt fatty acid profiles.

Sub-clinical mastitis (SCM) affects milk composition. In this study, we hypothesise that large-scale mining of milk composition features by pattern recognition models can identify the best predictors of SCM within the milk composition features. To this end, using data mining algorithms, we conducted a large-scale and longitudinal study to evaluate the ability of various milk production parameters as indicators of SCM. SCM is the most prevalent disease of dairy cattle, causing substantial economic loss for the dairy industry. Developing new techniques to diagnose SCM in its early stages improves herd health and is of great importance. Test-day Somatic Cell Count (SCC) is the most common indicator of SCM and the primary mastitis surveillance approach worldwide. However, test-day SCC fluctuates widely between days, causing major concerns for its reliability. Consequently, there would be great benefit to identifying additional efficient indicators from large-scale and longitudinal studies. With this intent, data was collected at every milking (twice per day) for a period of 2 months from a single farm using in-line electronic equipment (346 248 records in total). The following data were analysed: milk volume, protein concentration, lactose concentration, electrical conductivity (EC), milking time and peak flow. Three SCC cut-offs were used to estimate the prevalence of SCM: Australian ≥ 250 000 cells/ml, European ≥200 000 cells/ml and New Zealand ≥ 150 000 cells/ml. At first, 10 different Attribute Weighting Algorithms (AWM) were applied to the data. In the absence of SCC, lactose concentration featured as the most important variable, followed by EC. For the first time, using attribute weighted modelling, we showed that the concentration of lactose in milk can be used as a strong indicator of SCM. The development of machine-learning expert systems using two or more milk variables (such as lactose concentration and EC) may produce a predictive pattern for early SCM detection.

This Research Paper addresses the hypothesis that using teatcups with automatic valves, without cutting off the vacuum prior to cluster removal, could increases the risk of mastitis and affect other milking variables on goats. A first trial used 46 intramammary infection (IMI)-free goats that had been milked with normal teatcups (without automatic valves) during a pre-experimental period of 8 ± 2 d postpartum. These animals were divided into two groups (n = 23), randomly assigning each group to teatcups with automatic valves (teatcups A) or without automatic valves (teatcups B) for a 20-week experimental period. During this period, several strategies were applied to increase teat exposure to pathogens in both experimental groups. In the first eight weeks of the experimental period, the new IMI rate per gland was significantly higher (P < 0·05) in the group of animals milked with teatcups A (6 of 46; 13%) than in the group milked with teatcups B (1 of 46; 2%). However, throughout the rest of the experimental period the same number of glands appeared with new IMI (n = 7) in both animal groups. SCC was higher in goats milked with teatcups A, but no significant differences were found in the remaining variables (milk production and composition, frequency of liner slips + teatcup fall-off). In a second experiment, in a crossover design (54 goats in fourth month of lactation, 2 treatments – teatcups A and B – in 2 experimental periods each lasting 1 week), no differences were observed in total milk, average milk flow, total milking time or teat thickness changes after milking between both teatcups. However, teatcups A worsened slightly the maximum milk flow. We concluded that the use of teatcups with automatic valves, without cutting off the vacuum prior to cluster removal, increases the risk of mastitis on goat livestock farms.

The objectives of the research reported in this Research Communication were to compare the variation of hind quarter skin surface temperature pre- and post- milking in dairy cows and to determine the optimal time to capture images by infrared thermography for improving the sensitivity and specificity of mastitis detection in dairy cows. Hind quarter infrared images of 102 Holstein dairy cows were captured from the caudal view by an infrared camera pre-milking and post-milking. The udder skin surface temperature was measured with the help of the image processing software. No significant difference was found between the left and right quarter skin surface temperature pre- and post- milking. The hind quarter skin surface temperature pre-milking was not significantly influenced by milk yield, but exhibited a rising trend along with the increase of milk yield. The hind quarter skin surface temperature post-milking was significantly influenced by milk yield. This leads us to conclude that the sensitivity and specificity of IRT in mastitis detection may be influenced by milk yield and it may be better to capture the infrared images of cow udders pre-milking.

Evaluation of eleven candidate probiotic Lactobacillus strains isolated from human milk showed that some of the strains were well endowed with desirable cell surface and attachment attributes. The cell surface properties (hydrophobicity, auto-aggregation, attachment to collagen and HT-29 monolayer) of probiotic Lactobacillus species of human milk origin were compared with reference probiotic/ non-probiotic species and pathogenic strains. The bacterial adhesion to hydrocarbons (BATH) was determined using three aliphatic (Chloroform, n-Hexane and n-Octane) and two aromatic (Toluene and Xylene) solvents. Maximum affinity of Lactobacillus strains towards chloroform and toluene indicated the presence of low electron acceptor/ acidic surface components on cell surface of most of the strains. The highest value of per cent hydrophobicity was recorded with chloroform in HM1 (L. casei) (97·10 ± 3·35%) and LGG (98·92 ± 1·24%). A moderate auto-aggregation attribute was observed in all of our Lactobacillus isolates. Only HM10, HM12 and HM13 exhibited comparatively enhanced precipitation rate after 7 h of incubation period. The adhesion potential to collagen matrix was highest in LGG (26·94 ± 5·83%), followed by HM1 (11·07 ± 3·54%) and HM9 (10·85 ± 1·74%) whereas, on HT-29 cells, HM8 (14·99 ± 3·61%), HM3 (13·73 ± 1·14%) and HM1 (11·21 ± 3·18%) could adhere effectively. In this manner, we noticed that although the cell surface properties and adhesion prospective of probiotic bacteria were strain dependent, five of our isolates viz. HM1, HM3, HM8, HM9 and HM10 exhibited promising cell surface properties, which could be further targeted as indigenous probiotic.

Oxytocin release, milking characteristics, and teat condition were investigated with reduced claw vacuum and pulsation settings compared to milking at regular settings with or without pre-stimulation. The reduced vacuum and pulsation settings during low milk flow are expected to protect the teat tissue before the occurrence of milk ejection at the start of milking, and at the end of milking during a potential overmilking period, i.e. at a milk flow <200 g/min. Seven cows were machine-milked either after a 60 s manual pre-stimulation, or without pre-stimulation and reduced vacuum and pulsation settings, or at full vacuum and normal pulsation during the start of milking. Plasma oxytocin (OT) concentration increased similarly in response to manual pre-stimulation and to both milking with reduced, or with full vacuum and pulsation settings, however delayed by 1 min if the cluster was attached without pre-stimulation. In all treatments OT concentrations remained elevated throughout milking. Milk flow curves were mostly non-bimodal at milkings after manual pre-stimulation and bimodal at milkings without pre-stimulation. The main milking time was shorter and average milk flow was higher during milking after pre-stimulation, but did not differ between treatments without pre-stimulation. Milk yields and peak flow rates were not affected by treatments. Either reduced or full vacuum settings were again applied during an intended overmilking from 200 to 100 g/min of milk flow towards the end of milk harvest. Pre-milking teat ultrasound cross sections were recorded one day before the experiment started. Post-milking ultrasound cross sections were performed at 15 min after each experimental milking. Teat wall thickness was increased after milking as compared to pre-milking but did not differ among treatments. In conclusion, OT release and milking performance are similar if milking is performed with pre-stimulation, or without pre-stimulation but reduced claw vacuum and b-phase during low milk flow.

This Research Paper addresses the hypothesis that the type of dulce de leche formula affects formation of 5-hydroxymethylfurfural (HMF) during production and storage. Milk and sweetened condensed milk are products in which the Maillard reaction (MR) defines important characteristics such as colour and taste. There are few studies on the effects of pH, time, concentration, and sugar type on the formation of HMF or other MR markers in DL. Four formulas (varying in the addition of sodium bicarbonate and the type of sugar) were analysed for moisture, lipid, protein, ash, carbohydrate, water activity (Aw), and soluble solids. We found low variability in physicochemical and compositional attributes, but an elevation of HMF indices throughout the manufacturing. We determined that the addition of glucose and the use of relatively high concentrations of sodium bicarbonate caused these HMF indicator increases. These results inform DL research and production by the dairy industries and the scientific community, and highlight the importance of control in manufacturing.