Prolonged Milk Research Articles

Precision Agriculture: MXene-Based 3D Biosensors for Early Detection of Subclinical Mastitis in Dairy Cows

INTRODUCTION Mastitis, inflammation of mammary glands, is caused by pathogens like Streptococcus and Staphylococci, posing an economic challenge in the US dairy industry. Subclinical mastitis (SCM), asymptomatic and hard to detect, leads to prolonged milk yield declines. SCM costs the US over $2 billion annually. Current tests include somatic cell count, California Test, and Wisconsin Mastitis Test, but they offer periodic, qualitative measurements with moderate sensitivities (less than 90%) and specificities (85%). Consequently, there is a pressing need for reliable, early, continuous, and on-site detection of SCM to enhance milk yield, control the spread of infection, and health management, and improve the well-being of dairy cows.Herein, we developed a 3D-printed MXene sensor for the detection of mastitis, providing a high sensitivity and a ≤1 min response time. This tool can aid US livestock farmers in dairy herd management. SENSOR FABRICATION Device manufacturing comprises three steps. First, the CAD design of the sensor (Figure 2) guides printing using a 25 μm resolution extrusion-based 3D printer. A UV-curable epoxy resin forms the sensor base, with unique microstructures enhancing surface area (Figure 3). The sensor geometry incorporates a ~280 μm periodic lateral pattern for increased surface area and a 3D geometric diffusion interface. Uncontrollably, a random microfold pattern emerges due to the epoxy resin's photo-sensitivity. These microstructures, acting as microelectrodes, amplify the sensor's surface area, boosting analytical sensitivity.Next, a 100nm gold layer is applied to the sensor base using an e-beam evaporator with a shadow mask. Sensing materials are then drop-casted onto each electrode. The NAGase sensing electrode is coated with Ti3C2 nanosheets (MXene) decorated with anti-NAGase. The pH sensing electrode modified with poly(3-octyl-thiophene) and is covered by a hydrogen ion-selective membrane (H-ISM). The reference electrode (RE) is constructed by applying Ag/AgCl paste onto grooves around each working electrode (Figure 2). RESULTS AND DISCUSSION First, surface morphologies and electrochemical characterizations were conducted. FE-SEM images depict the lateral pattern and micro-folds of the sensor base (Figure 3). EDS and mapping analyses confirm uniform MXene distribution on the gold surface. Moreover, cyclic voltammetry (CV) explores electrode electrochemical behavior in different media. CV of MXene with ferro/ferricyanide shows distinctive peaks for mediator oxidation and reduction. CV without a mediator reveals characteristic MXene oxidation peaks, highlighting the sensor's mediator-free capability for direct applications in dairy farms without need for mediator solution.For pH sensing, open circuit potentiometry (OCP) was used. Figure 4 shows OCP response rising with increasing pH values. The calibration graph in Figure 4 establishes a linear correlation between pH values and OCP, aligning with the Nernstian equation for ion-selective electrodes. The pH sensor successfully measures milk sample pH, demonstrating remarkable selectivity in the complex milk matrix.The NAGase biosensor employs two sensing modalities for laboratory and field applications. Electrochemical impedance spectroscopy with a mediator generates Nyquist plots for calculating charge transfer resistance (Rct) in the presence of NAGase. Figure 5 displays a calibration graph with a linear correlation between Rct and the logarithmic concentration of NAGase. The NAGase-anti-NAGase interaction forms immunocomplexes on the MXene surface, increasing resistance to the mediator's redox reaction. This feature serves as the sensing mechanism for NAGase, a potential biomarker in mastitis.Due to MXene's self-oxidation tendency without a mediator, CV technique used as a mediator-free alternative for practical field applications. In Figure 6, voltammograms show decreasing oxidative and reductive currents with increasing NAGase concentration. Oxidative peak currents used as the signal, exhibiting a negative correlation with NAGase concentration. The immunocomplexes formation acts as an insulative shield, impeding redox reactions. This innovative sensor aims to reduce time and effort in detecting mastitis in dairy cows, controlling pathogen spread. Figure 1

Food allergy to cow’s milk proteins and meat

Introduction. Cow’s milk and dairy products are important sources of high-quality animal proteins, calcium, vitamins, and minerals. They are widely used in the diet of preschool and school-age children and contribute to the normal development and resilience of children against the effects of adverse external factors. However, cow’s milk can cause serious allergic reactions in children, even leading to anaphylaxis. Allergy to cow’s milk proteins is one of the common allergic reactions among preschool-age children. Through conducted research, patterns of the natural course of milk allergy over time have been established. In most cases, by the age of 5 years, children develop tolerance to cow’s milk proteins. However, some school-age children may retain heightened sensitivity to them. Sustaining such prolonged milk allergy in a school-age child requires identifying the cause and conducting in-depth examinations for each specific patient. In such cases, valuable information about elevated sensitivity to whole cow’s milk protein and its components is provided by molecular (component-based) diagnostics. This enables the identification of causally significant components (α-lactalbumin, β-lactoglobulin, bovine serum albumin) of milk protein for the purpose of rational diet therapy, considering cross-reactions with beef meat. The presented article summarizes and systematizes literary and proprietary data on allergy to cow’s milk proteins and its components, as well as to beef meat. Understanding the characteristics of allergy to cow’s milk proteins and its components, cross-reacting with beef, will help avoid serious consequences of irrational elimination diets.

Isolation and Identification of Bacteria and Fungi Correlated with Mastitis in Breastfeeding Women in AI-Anbar Governorate/West of Iraq

Mastitis is a common phenomenon experienced by a lot of breastfeeding women. Lactational mastitis, commonly occurs because of prolonged milk duct engorgement, which allows bacteria to enter through breaks in the skin, resulting in an infection. Mastitis can occur at various stages of lactation, although it is most observed within the second and third weeks following childbirth. For isolation and identification of bacteria associated with mastitis, Blood agar medium, MacConkey agar, and mannitol salt agar were used, whereas Sabouraud Dextrose Agar (SDA) was used for fungal isolation. Different biochemical test has been performed for bacterial isolates including gram stain, catalase, coagulase, and Vitec 2 compact. For fungal isolates, KOH treatment and lactophenol cotton blue test were performed. Fungal DNA was extracted then PCR was used for the detection ITS Gene of candida spp. Using gel electrophoresis of PCR product on 2% agarose. The study revealed Staphylococcus aureus, Klebsiella pneumoniae, and Candida spp. as significant bacteria that are commonly related to mastitis in women. The prevalence of Staphylococcus aureus was seen to be high, with a notable manifestation of lesions. Additionally, both Staphylococcus aureus and Klebsiella pneumoniae were shown to be associated with the presence of redness. Candida albicans had a notable prominence, displaying a heightened prevalence in the regions of the nipple. Furthermore, it demonstrated a correlation with the presence of lesions and redness. The presence of redness was observed as a notable sign in cases linked with Candida tropicalis. This study provides insights into the microbial diversity linked to mastitis mainly in breastfeeding women, highlighting the prevalence of Staphylococcus aureus, Klebsiella pneumoniae, and Candida spp.

Population physiologically based modeling of pirlimycin milk concentrations in dairy cows

Predictions of drug residues in milk are critical in food protection and are a major consideration in the economics of treatment of mastitis in dairy cows. Nonlinear mixed-effects modeling (NLME) has been advocated as a suitable pharmaco-statistical method for the study of drug residues in milk. Recent developments in physiologically based pharmacokinetic (PBPK) modeling of intramammary drugs allow the combination of a mechanistic description of milk pharmacokinetics with NLME methods. The PBPK model was applied to NLME analysis of a data set consisting of milk drug concentrations from 78 healthy cows and 117 with clinical mastitis. Pirlimycin milk pharmacokinetics were adequately described by the model across the range of observed concentrations. Mastitis was characterized by increased variance in milk production volume. Udder residual volume was larger in cows with 1, or 2 or greater diseased mammary glands than in the healthy cows. Low-producing cows had a greater risk of prolonged milk residues. With the exclusion of the low-production cows, the model predicted that healthy cows required a milk discard time 12 h longer than that indicated by the label, and the diseased cows 36 h longer than indicated by the label. More pirlimycin was systemically absorbed in the gram-positive infected compared with the gram-negative infected or healthy cows, suggesting a greater risk of violative meat residues in gram-positive infected cows. Using NLME and PBPK models, we identified factors associated with changes in pirlimycin milk residues that may affect food safety. This model extends the verification of a simple physiologically based framework for the study of intramammary drugs.

Short communication: Persistent contamination by Listeria monocytogenes of bovine raw milk investigated by whole-genome sequencing

Following the persistent detection of Listeria monocytogenes in raw bovine milk sold through a vending machine, the 120 lactating cows of the herd producing the milk were subjected to bacteriological investigation. A single cow with subclinical mastitis (1.2-1.3 × 105 somatic cells/mL) and persistent L. monocytogenes excretion was detected. The cow was subjected to antimicrobial therapy, but L. monocytogenes excretion remained high (>3.0 × 102 cfu/mL). Following culling of the infected cow, L. monocytogenes disappeared from the tank milk, and further isolates were recovered from the mammary parenchyma and lymph nodes of the infected cow. To investigate the clonal nature of the contamination, all isolates recovered in the study (n = 13) were analyzed by serogroup PCR, pulsed-field gel electrophoresis, and whole-genome sequencing. Our results demonstrated the clonal nature of the contamination. All isolates belonged to lineage II, serogroup IIa, sequence type 37, clonal complex 37 and harbored some virulence determinants. This case showed that, although relatively rare, prolonged milk contamination by L. monocytogenes can originate from subclinical and persistently infected cows, posing a health risk to consumers.

Excretion of Dietary Cow's Milk Derived Peptides Into Breast Milk.

Nanoflow-HPLC-tandem mass spectrometry (MS/MS) was used to analyze the peptide fraction of breast milk samples collected from a single non-atopic donor on different days (10 samples) after receiving an oral load of cow's milk (by drinking 200 mL of bovine milk). In addition, breast milk was sampled from the same lactating mother over a 6-h period at five time points after drinking cow's milk. We aimed to trace the intra-individual variability and to define a time profile of the excretion of dietary peptides into breast milk. Overall, 21 peptides exclusively originating from both bovine caseins and whey proteins with no match within the human milk proteome were identified in the breast milk samples. These peptides were missing in the breast milk obtained from the mother after a prolonged milk- and dairy-free diet (three samples). The time course of cow's milk-derived β-Lg f(125–135) and β-casein f(81–92) in breast milk was determined from the MS ion intensity of the peptide signals. No intact cow's milk gene products were detected by HPLC-MS/MS analysis and Western blotting with anti-β-Lg antibody, but dot-blot analysis confirmed the occurrence of β-Lg fragments in the enriched peptide fraction of breast milk. These data suggest shifting the analytical perspective for the detection of dietary food allergens in breast milk from intact proteins to digested peptide fragments. The possible sensitization and elicitation potential or the tolerogenic properties of such low amounts of dietary peptides for the breastfed newborns remain to be explored.

Prolonged milk provisioning and extended maternal care in the milking spider Toxeus magnus: biological implications and questions unresolved

Prolonged milk provisioning and extended parental care for nutritionally independent offspring, previously considered to only co-occur in long-lived mammals (Clutton-Brock, 1991; Royle et al., 2012), were recently reported in the reproduction of the milking spider, Toxeus magnus (Chen et al. 2018). Newly hatched T. magnus spiderlings require 53 days to develop to maturity, with an average adult body length of 6.6 mm. The mother provides milk droplets to her newly hatched spiderlings until they develop into subadults (~38 days old), during which their body lengths increase from 0.9 mm at birth to 5.3 mm at weaning. Although spiderlings can forage for themselves at around 20 days old, they remain in the breeding nest for weeks after maturity.

Prolonged milk provisioning in a jumping spider.

Lactation is a mammalian attribute, and the few known nonmammal examples have distinctly different modalities. We document here milk provisioning in a jumping spider, which compares functionally and behaviorally to lactation in mammals. The spiderlings ingest nutritious milk droplets secreted from the mother's epigastric furrow until the subadult stage. Milk is indispensable for offspring survival in the early stages and complements their foraging in later stages. Maternal care, as for some long-lived vertebrates, continues after the offspring reach maturity. Furthermore, a female-biased adult sex ratio is acquired only when the mother is present. These findings demonstrate that mammal-like milk provisioning and parental care for sexually mature offspring have also evolved in invertebrates, encouraging a reevaluation of their occurrence across the animal kingdom, especially in invertebrates.

Bactérias psicrotróficas e atividade proteolítica no leite cru refrigerado

The prolonged milk refrigeration in the dairy farm and its transport in insulated containers on trucks, has given products with lower quality standards since...

Insulin sensitivity in calves decreases substantially during the first 3 months of life and is unaffected by weaning or fructo-oligosaccharide supplementation

Veal calves at the age of 4 to 6 mo often experience problems with glucose homeostasis, as indicated by postprandial hyperglycemia, hyperinsulinemia, and insulin resistance. It is not clear to what extent the ontogenetic development of calves or the feeding strategy [e.g., prolonged milk replacer (MR) feeding] contribute to this pathology. The objective of this study was therefore to analyze effects of MR feeding, weaning, and supplementation of short-chain fructo-oligosaccharides (FOS) on the development of glucose homeostasis and insulin sensitivity in calves during the first 3 mo of life. Thirty male Holstein-Friesian calves (18±0.7 d of age) were assigned to 1 of 3 dietary treatments: the control (CON) group received MR only, the FOS group received MR with the addition of short-chain FOS, and the solid feed (SF) group was progressively weaned to SF. The CON and FOS calves received an amount of MR, which gradually increased (from 400 to 1,400 g/d) during the 71-d trial period. For the SF calves, the amount of MR increased from 400 to 850 g/d at d 30, and then gradually decreased, until completely weaned to only SF at d 63. The change in whole body insulin sensitivity was assessed by intravenous glucose tolerance tests. Milk tolerance tests were performed twice to assess changes in postprandial blood glucose, insulin, and nonesterified fatty acid responses. Whole-body insulin sensitivity was high at the start (16.7±1.6×10−4 [μU/mL]−1), but decreased with age to 4.2±0.6×10−4 [μU/mL]−1 at the end of the trial. The decrease in insulin sensitivity was most pronounced (~70%) between d 8 and 29 of the trial. Dietary treatments did not affect the decrease in insulin sensitivity. For CON and FOS calves, the postprandial insulin response was 3-fold higher at the end of the trial than at the start, whereas the glucose response remained similar. The SF calves, however, showed pronounced hyperglycemia and hyperinsulinemia at the end of the trial, although weaning did not affect insulin sensitivity. We conclude that whole body insulin sensitivity decreases by 75% in calves during the first 3 mo of life. Weaning or supplementation of short-chain FOS does not affect this age-related decline in insulin sensitivity. Glucose homeostasis is not affected by supplementation of short-chain FOS in young calves, whereas postprandial responses of glucose and insulin to a MR meal strongly increase after weaning.

Iron Requirements for Infants with Cow Milk Protein Allergy

H istorically, cow milk has been used as a major source of protein and balanced nutrition for infants and children in resource-rich and resource-constrained countries. It is a rich source of energy, protein, and calcium. It is also an important source of fat; many of its fatty acids are readily metabolized to essential omega-3 fatty acids. Regular consumption of milk during childhood has been shown to enhance bone density later in life. However, reliance upon whole pasteurized cow milk as the sole or major source of nutrition for infants during the first year of life can increase the risk for iron deficiency and, in extreme cases, iron-deficient anemia (IDA) for a number of reasons. In 1972, Woodruff et al showed that 7 of 12 infants (ages 7-17 months) with IDA who ingested between 720 and 1920 mL whole milk daily had higher albumin turnover rates than a group of 5 normal infants. Parenteral iron had no effect on albumin turnover, but replacing whole milk with reconstituted evaporated milk or soy formula decreased the high albumin turnover rates to normal levels, suggesting that large amounts of cow milk protein could result in iron and protein loss in the gastrointestinal tract. Cow milk contains low levels of ascorbic acid, a factor that increases iron absorption, and certain proteins that negatively impact iron absorption. Cow milk contains about 4 times more calcium than human milk, and calcium negatively impacts iron absorption. Furthermore, cow milk has a higher protein concentration than human milk, and ingestion of large amounts can lead to metabolic and fluid imbalances, particularly in children with febrile illnesses. Thus, several entities have recommended against whole cow milk ingestion until 9-12 months and then, only when given in moderate amounts in conjunction with appropriate complementary foods.

Two-year changes in bone and body composition in young children with a history of prolonged milk avoidance

No previous longitudinal studies of calcium intake, anthropometry and bone health in young children with a history of avoiding cow's milk have been undertaken. We report the 2-year changes of a group of 46 Caucasian children (28 girls, l8 boys) aged 8.1+/-2.0 years (mean +/- SD) who had low calcium intakes at baseline and were short in stature, with elevated body mass index, poor skeletons and lower Z scores for both areal bone mineral density (BMD, in grams per square centimeter) and volumetric density (bone mineral apparent density, BMAD, in grams per cubic centimeter), compared with a reference population of milk drinkers. At follow-up, adverse symptoms to milk had diminished and modest increases in milk consumption and calcium intake had occurred. Total body bone mineral content (BMC) and bone area assessed by dual energy X-ray absorptiometry had increased (P<0.05), and calcium intake from all sources was associated with both these measures (P<0.05). However, although some catch-up in height had taken place, the group remained significantly shorter than the reference population (Z scores -0.39+/-1.14), with elevated body mass index (Z scores 0.46+/-1.0). The ultradistal radius BMC Z scores remained low (-0.31+/-0.98). The Z scores for BMD had improved to lie within the normal range at predominantly cortical sites (33% radius, neck of femur and hip trochanter) but had worsened at predominantly trabecular sites (ultradistal radius and lumbar spine), where values lay below those of the reference group (P<0.05). Similarly, although volumetric BMAD Z scores at the 33% radius had normalized, BMAD Z scores at the lumbar spine remained below the reference population at follow-up (-0.67+/-1.12, P<0.001). Our results demonstrate persisting height reduction, overweight and osteopenia at the ultradistal radius and lumbar spine in young milk avoiders over 2 years of follow-up.

Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling.

Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.

Prolonged milk residue in two cows after subcutaneous injections of penicillin at an extra-label dose

Prolonged milk residue in two cows after subcutaneous injections of penicillin at an extra-label dose

In vitro culture of cryopreserved bovine mammary cells on collagen gels: Synthesis and secretion of casein and lactoferrin

The preparation, cryopreservation, and culture on type I collagen gels of lactating bovine mammary cells with prolonged milk protein synthesis and secretion in vitro is described. Cryopreserved cells prepared as acinar fragments from either lactating or developing mammary glands attached to the collagen substratum within 24–48 hr after plating in serum and hormone supplemented medium. During continued culture in hormone-supplemented (insulin, cortisol, and prolactin) serum-free medium outgrowth of cells from the attached acinar fragments was observed beginning on day 2, with continued outgrowth to near confluence by day 6. Two morphologically distinct cell types were evident; initial outgrowth was by large polygonal cells that were subsequently overlain by spindle-shaped cells. Cells from both lactating and developing mammary glands sustained substantial milk protein secretion for at least 14 days in culture. Alpha S1-casein synthesis and secretion in cultures of lactating mammary cells was dependent on a critical minimum cell population density, below which alpha S1-casein was not secreted. In contrast, lactoferrin (LF) secretion into the medium increased linearly with the increase in cell population density. Cells Cryopreserved up to 16 months secreted LF at levels comparable to fresh cultures of the same cells.

The milk of the fur‐seal, Arctocephalus tropicatis gazella; in particular the composition of the aqueous phase

Milk and plasma samples from the Southern fur‐seal (Arctocephalus tropicalis gazella), collected from animals shot between 0 and 26 days post‐partum, have been analysed. The aqueous phase of milk (ultracentrifugate) was hyperosmotic to plasma by about 35 m‐osmol/kg water but contained no lactose. The osmolality of milk could be accounted for by the ionic content, of which sodium and potassium (in approximately equal amounts) were the main cations. The fat content of the milk was very high (approximately 40%), except in very early lactation (0 and 1 day post‐partum). The concentrations of chloride, calcium, magnesium, phosphate and citrate in the aqueous phase are given. Major differences between the composition of fur‐seal milk and that of other mammals are pointed out, and discussed in relation to water conservation, energy and fat supply, and prolonged milk storage in this snecies.

Evidence against nutritional adaption of tolerance to lactose.

Controversy exists over the etiology of primary lactose intolerance with intestinal lactase deficiency. The low activity of lactase may be either due to a genetically controled reduction of enzyme activity or to an adaptive response to a lack of dietary lactose. Lactose tolerance tests in 24 subjects (9 persons who are offspring of marriages between Asians and Europeans, 6 lactose tolerant individuals from populations with a high incidence of lactose intolerance, and 9 lactose intolerant subjects with a history of prolonged milk consumption) are more easily interpreted by assuming genetic control of lactase activity as the main determinant of lactose tolerance. The findings in these 24 subjects contradict some of the commonly cited arguments supporting an adaptive origin of lactose intolerance.

COMPARATIVE STUDIES OF THE INFLUENCE OF ACID-FORMING AND BASE-FORMING DIETS ON THE METABOLISM OF RATS

Infant feeding regimens formulated by pediatricians of representative institutions have been calculated by us to be acid-forming; that is, the dietaries contain an excess of acid-forming elements (chlorine, sulphur and phosphorus) over basic elements (sodium, potassium, calcium, magnesium and iron) (table 1). Prolonged milk mixtures¹used in the feeding of very young infants, however, are all base-forming. The daily requirements of inorganic elements during growth indicate a preponderance of basic over acid-forming elements²(table 2). This is actually reflected in the inorganic composition of the child's body, (table 3). The growing young animal requires these inorganic constituents—acid-forming and base-forming—for the maintenance of a labile acid-base mechanism, for the normal contractility of the muscular system, the normal irritability of the nervous system, for the physiologic osmotic pressure relations, for the establishment of membrane equilibria (Donnan), acids and alkalis of digestive secretions, for the synthesis of tissues (bone, blood,