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Diagnostic Evaluation of the IS1081-Targeted Real-Time PCR for Detection of Mycobacterium bovis DNA in Bovine Milk Samples
The ability of Mycobacterium bovis (M. bovis) to survive in bovine milk has emerged as a serious public health concern. The first objective of this study was to evaluate the diagnostic utility of IS1081-targeted real-time PCR for the detection of M. bovis DNA in different fractions of bovine milk. In a model study, bovine milk samples were spiked with serially diluted M. bovis BCG to investigate the detection limit of M. bovis DNA in whole milk and milk fractions (cream, pellet, and pellet + cream combined) using IS1081 real-time PCR. The assay was then used to detect M. bovis DNA in whole milk and milk fractions from naturally infected animals. The results showed that the IS1081 real-time PCR was more sensitive when detecting M. bovis DNA in the cream layer alone and cream + pellet combined compared to whole milk or the pellet alone. While PCR-based diagnostic assays for the detection of M. bovis in milk samples provide a quicker diagnostic tool for bovine tuberculosis, safe processing, and handling of M. bovis-infected milk samples remain a challenge and pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to rapidly inactivate infected specimens while preserving nucleic acid for subsequent Molecular analysis. Therefore, the secondary objective of this study was to evaluate the ability of MTM to inactivate M. bovis BCG in spiked milk samples as well as its ability to preserve BCG DNA for the PCR assay. The results showed that MTM can successfully inactivate BCG alone or in spiked milk samples while preserving DNA for the PCR assay. The CT values of M. bovis BCG alone and spiked milk samples aliquoted in MTM and without MTM were similar at various dilutions. Taken together, our results indicate that using DNA extracted from the milk cream fraction alone or combined milk cream and pellet improved the recovery rate of M. bovis DNA in bovine milk samples. MTM has the potential to provide a safe and rapid sample processing tool for M. bovis inactivation in milk samples and preserve DNA for molecular diagnostics.
Prevalence of Listeria monocytogenes , Salmonella spp., Shiga toxin-producing Escherichia coli , and Campylobacter spp. in raw milk in the United States between 2000 and 2019: A systematic review and meta-analysis
Raw (unpasteurized) milk is available for sale and direct human consumption within some states in the United States (US); it cannot be sold or distributed in interstate commerce. Raw milk may contain pathogenic microorganisms that, when consumed, may cause illness and sometimes may result in death. No comprehensive review for prevalence and levels of the major bacterial pathogens in raw milk in the US exists. The objective of the present research was to systematically review the scientific literature published from 2000 to 2019 to estimate the prevalence and levels of Listeria monocytogenes, Salmonella spp., Shiga toxin-producing Escherichia coli (STEC), and Campylobacter spp. in raw milk in the US. Peer-reviewed studies were retrieved systematically from PubMed®, Embase®, and Web of ScienceTM. The unique complete nonduplicate references were uploaded into the Health Assessment Work Collaborative (HAWC). Based on the selection criteria, twenty studies were included in the systematic review and meta-analysis. Comprehensive Meta-Analysis (CMA) was used for statistical analyses, specifically, random effects meta-analyses were used to synthesize raw bulk tank milk (BTM) and milk filters (MF) data. Data from studies using culture and non–culture-based detection methods were included. Forest plots generated in CMA (Biostat, Englewood, NJ) were used to visualize the results. The average prevalence (event rate) of L. monocytogenes, Salmonella spp., STEC, and Campylobacter spp. in raw BTM in the US was estimated at 4.3% (95% confidence intervals [CIs], 2.8–6.5%), 3.6% (95% CIs, 2.0–6.2%), 4.3% (95% CIs, 2.4–7.4%), and 6.0% (95% CIs, 3.2–10.9%), respectively. Estimated prevalence was generally larger in MF than in BTM. There was not enough data to perform a meta-analysis for the prevalence or levels of pathogens in raw milk from retail establishments or other milk categories.
Associations between SARS-CoV-2 Infection or COVID-19 Vaccination and Human Milk Composition: A Multi-Omics Approach
Background: The risk of contracting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via human milk-feeding is virtually nonexistent. Adverse effects of coronavirus disease 2019 (COVID-19) vaccination for lactating individuals are not different from the general population, and no evidence has been found that their infants exhibit adverse effects. Yet, there remains substantial hesitation among this population globally regarding the safety of these vaccines. Objectives: Herein, we aimed to determine if compositional changes in milk occur following SARS-CoV-2 infection or COVID-19 vaccination, including any evidence of vaccine components. Methods: An extensive multiomics approach was taken using a subset of milk samples obtained as part of our broad studies examining the effects on milk of SARS-CoV-2 infection and COVID-19 vaccination. Results: We found that compared with unvaccinated individuals, SARS-CoV-2 infection was associated with significant compositional differences in 67 proteins, 385 lipids, and 13 metabolites. In contrast, COVID-19 vaccination was not associated with any changes in lipids or metabolites, although it was associated with changes in 13 or fewer proteins. Compositional changes in milk differed by vaccine. Changes following vaccination were greatest after 1–6 h for the mRNA-based Moderna vaccine (8 changed proteins), 3 d for the mRNA-based Pfizer (4 changed proteins), and adenovirus-based Johnson and Johnson (13 changed proteins) vaccines. Proteins that changed after both natural infection and Johnson and Johnson vaccine were associated mainly with systemic inflammatory responses. In addition, no vaccine components were detected in any milk sample. Conclusions: Together, our data provide evidence of only minimal changes in milk composition because of COVID-19 vaccination, with much greater changes after natural SARS-CoV-2 infection.
Campylobacter jejuni resistance to human milk involves the acyl carrier protein AcpP
Campylobacter jejuni is a common foodborne pathogen worldwide that is associated with high rates of morbidity and mortality among infants in low- to middle-income countries (LMICs). Human milk provides infants with an important source of nutrients and contains antimicrobial components for protection against infection. However, recent studies, including our own, have found significantly higher levels of Campylobacter in diarrheal stool samples collected from breastfed infants compared to non-breastfed infants in LMICs. We hypothesized that C. jejuni has unique strategies to resist the antimicrobial properties of human milk. Transcriptional profiling found human milk exposure induces genes associated with ribosomal function, iron acquisition, and amino acid utilization in C. jejuni strains 81–176 and 11168. However, unidentified proteinaceous components of human milk prevent bacterial growth. Evolving both C. jejuni isolates to survive in human milk resulted in mutations in genes encoding the acyl carrier protein (AcpP) and the major outer membrane porin (PorA). Introduction of the PorA/AcpP amino acid changes into the parental backgrounds followed by electron microscopy showed distinct membrane architectures, and the AcpP changes not only significantly improved growth in human milk, but also yielded cells surrounded with outer membrane vesicles. Analyses of the phospholipid and lipooligosaccharide (LOS) compositions suggest an imbalance in acyl chain distributions. For strain 11168, these changes protect both evolved and 11168ΔacpP G33R strains from bacteriophage infection and polymyxin killing. Taken together, this study provides insights into how C. jejuni may evolve to resist the bactericidal activity of human milk and flourish in the hostile environment of the gastrointestinal tract.
Lipopolysaccharide and lipoteichoic acid influence milk production ability via different early responses in bovine mammary epithelial cells
Highlights: • LPS and LTA affect on the milk production of lactating BMECs in different manners. • LPS/TLR4 and LTA/TLR2 signaling induce different innate immune responses in BMECs. • Inhibition of p38 and NF-κB may help recover decrease in milk production in mastitis. Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are cell wall components of Escherichia coli and Staphylococcus aureus, which cause clinical and subclinical mastitis, respectively. However, the reason of the difference in symptoms by pathogen type remains unclear. In this study, the influence of LPS and LTA on early response and milk production in lactating bovine mammary epithelial cells (BMECs) was comparatively investigated. The results showed that LPS decreased the secretion of β-casein, lactose, and triglycerides, whereas LTA decreased the secretion of lactose and triglycerides but increased lactoferrin production without any influence on β-casein secretion. In addition, the influence of milk lipid droplet size in BMECs and gene expression related to milk fat synthesis was different between LPS and LTA. LPS increased the gene expression of interleukin (IL)-1β, tumor necrosis factor-α, and IL-8 through the activation of the nuclear factor-κB (NF-κB), p38, and c-Jun N-terminal kinase pathways, whereas LTA increased IL-1β and CC chemokine ligand 5 expression through the activation of the NF-κB pathway. Moreover, these cytokines and chemokines differently affected the milk production ability of BMECs. These results suggested that the pathogen-specific symptoms may be related to the differences in the early response of BMECs to bacterial toxins.
Foodborne illness outbreaks linked to unpasteurised milk and relationship to changes in state laws – United States, 1998–2018
Consumption of unpasteurised milk in the United States has presented a public health challenge for decades because of the increased risk of pathogen transmission causing illness outbreaks. We analysed Foodborne Disease Outbreak Surveillance System data to characterise unpasteurised milk outbreaks. Using Poisson and negative binomial regression, we compared the number of outbreaks and outbreak-associated illnesses between jurisdictions grouped by legal status of unpasteurised milk sale based on a May 2019 survey of state laws. During 2013–2018, 75 outbreaks with 675 illnesses occurred that were linked to unpasteurised milk; of these, 325 illnesses (48%) were among people aged 0–19 years. Of 74 single-state outbreaks, 58 (78%) occurred in states where the sale of unpasteurised milk was expressly allowed. Compared with jurisdictions where retail sales were prohibited (n = 24), those where sales were expressly allowed (n = 27) were estimated to have 3.2 (95% CI 1.4–7.6) times greater number of outbreaks; of these, jurisdictions where sale was allowed in retail stores (n = 14) had 3.6 (95% CI 1.3–9.6) times greater number of outbreaks compared with those where sale was allowed on-farm only (n = 13). This study supports findings of previously published reports indicating that state laws resulting in increased availability of unpasteurised milk are associated with more outbreak-associated illnesses and outbreaks.
Interrogating the role of the milk microbiome in mastitis in the multi-omics era
There is growing interest in a functional understanding of milk-associated microbiota as there is ample evidence that host-associated microbial communities play an active role in host health and phenotype. Mastitis, characterized by painful inflammation of the mammary gland, is prevalent among lactating humans and agricultural animals and is associated with significant clinical and economic consequences. The etiology of mastitis is complex and polymicrobial and correlative studies have indicated alterations in milk microbial community composition. Recent evidence is beginning to suggest that a causal relationship may exist between the milk microbiota and host phenotype in mastitis. Multi-omic approaches can be leveraged to gain a mechanistic, molecular level understanding of how the milk microbiome might modulate host physiology, thereby informing strategies to prevent and ameliorate mastitis. In this paper, we review existing studies that have utilized omics approaches to investigate the role of the milk microbiome in mastitis. We also summarize the strengths and challenges associated with the different omics techniques including metagenomics, metatranscriptomics, metaproteomics, metabolomics and lipidomics and provide perspective on the integration of multiple omics technologies for a better functional understanding of the milk microbiome.
Profile of Twenty-Three Human Milk Oligosaccharides in Han Chinese Mothers throughout Postpartum 1 year
Human milk oligosaccharides (HMOs) are multifunctional carbohydrates in breast milk, which are composed by a variety of structures. This study aimed to identified HMOs concentration profile, milk microbiota composition, and the associations with major maternal characteristics in Han Chinese mothers in the one-year lactation period. Seventeen healthy mothers aged from 28 to 36 years, who gave birth to healthy term infants, were recruited. Carbohydrates were detected using the MIRIS human milk analyzer (HMA), and twenty-three HMOs were quantified using ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS). Results showed that carbohydrates were relatively stable, while total HMO concentrations ranged from 1.74 to 9.72 g/L and decreased gradually over lactation in breast milk. Based on the structure, seven sialylated HMOs concentration showed the significant decline ( p < 0.05 ) after three months in lactation. In addition, the relationships between maternal factors, containing the lactation period, genetic status, delivery mode, parity, and milk microbiota profile, and the HMO composition in healthy women, which still need further investigations, were observed.
MILK : a Python scripting interface to MAUD for automation of Rietveld analysis
Modern diffraction experiments ( e.g. in situ parametric studies) present scientists with many diffraction patterns to analyze. Interactive analyses via graphical user interfaces tend to slow down obtaining quantitative results such as lattice parameters and phase fractions. Furthermore, Rietveld refinement strategies ( i.e. the parameter turn-on-off sequences) tend to be instrument specific or even specific to a given dataset, such that selection of strategies can become a bottleneck for efficient data analysis. Managing multi-histogram datasets such as from multi-bank neutron diffractometers or caked 2D synchrotron data presents additional challenges due to the large number of histogram-specific parameters. To overcome these challenges in the Rietveld software Material Analysis Using Diffraction ( MAUD ), the MAUD Interface Language Kit ( MILK ) is developed along with an updated text batch interface for MAUD . The open-source software MILK is computer-platform independent and is packaged as a Python library that interfaces with MAUD . Using MILK , model selection ( e.g. various texture or peak-broadening models), Rietveld parameter manipulation and distributed parallel batch computing can be performed through a high-level Python interface. A high-level interface enables analysis workflows to be easily programmed, shared and applied to large datasets, and external tools to be integrated with MAUD . Through modification to the MAUD batch interface, plot and data exports have been improved. The resulting hierarchical folders from Rietveld refinements with MILK are compatible with Cinema: Debye–Scherrer , a tool for visualizing and inspecting the results of multi-parameter analyses of large quantities of diffraction data. In this manuscript, the combined Python scripting and visualization capability of MILK is demonstrated with a quantitative texture and phase analysis of data collected at the HIPPO neutron diffractometer.
Cooling and dietary crude protein affected milk production on heat-stressed dairy cows
Environmental factors, such as temperature and humidity, exert dramatic effects on the production, health, and well-being of lactating dairy cows. Heat-stressed cows undergo a series of homeorhetic adaptations that alters cellular homeostasis and nutrient utilization. As such, the objective of this study was to determine the effects of cooling and dietary crude protein (CP) content on fermentability of rumen inoculum, milk production, and health of heat-stressed cows. It was hypothesized that cooled but not heat-stressed cows would support productivity in response to an increase in dietary CP content. Thirty-six multiparous Holstein cows were randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement (9 cows/treatment). Treatments consisted of cooling (CO) or moderate long-term heat stress (HS) and diets containing low CP content (LCP; 12.5% CP) or moderate CP content (MCP; 16.1% CP) of dry matter. The CO treatment provided heat abatement with sprinklers and fans that came on at > 20.0°C, whereas the HS treatment did not provide sprinklers and fans. Cows were housed in pens in a freestall barn, and treatments were imposed for 21 d during July and August. Compared with the HS treatment, CO decreased afternoon rectal and vaginal temperatures (0.60 and 0.70°C) and afternoon respiration rate (27.6 breaths/min). Treatment interactions showed that compared with the LCP treatment, MCP increased milk fat yield (23%) in CO but not in HS cows. Compared with the LCP treatment, MCP decreased milk protein percent (0.14 units) in CO but increased milk protein percent (0.09 units) in HS cows. Rumen inoculum collected on d 21 showed a ~3-fold increase in total gas production in vitro in CO compared with the HS treatment. Relative to the LCP treatment, MCP increased plasma glucose and insulin and decreased total free fatty acids concentrations. In summary, under the conditions of this study CO but not HS cows responded to a CP stimulus by increasing productivity.
Postbiotic Gamma-Aminobutyric Acid and Camel Milk Intervention as Innovative Trends Against Hyperglycemia and Hyperlipidemia in Streptozotocin-Induced C57BL/6J Diabetic Mice
Diabetes is a serious disease that threatens human health worldwide. The study hypothesis is to investigate the novel trends that may aid in the prevention of diabetic complications. Camel milk was presented as traditional functional food, and Lactobacillus brevis KLDS 1.0727 and KLDS 1.0373 strains were shown to synthesize postbiotic Gamma-aminobutyric acid as a potential food additive, which can therapeutically intervene against hyperglycemia and hyperlipidemia in streptozotocin-induced C 57 BL/6J mice. During a four-week timeframe, body weight and postprandial blood glucose levels were monitored. Post-euthanasia, blood plasma was obtained to investigate hyperlipidemia, insulin concentrations, liver, and renal functions. The liver, pancreas, kidney, and spleen underwent histopathological examinations. The results demonstrated that KLDS 1.0727 and KLDS 1.0373 ( LAC S1 , LAC S2 ) and camel milk treatments all had a significant influence on hypoglycemic activity, as evidenced by reduced postprandial blood glucose levels. LAC S1 , LAC S2 , and camel milk therapy significantly reduced blood hypolipidemic, and some liver enzymes such as (alanine aminotransferase and aspartate transaminase) levels. Therefore, we recommend consuming camel milk regularly and expanding its use with fermented foods containing L. brevis , one of the probiotics capable of producing gamma-aminobutyric acid (GABA) as future food additives that can improve human health and reduce the prevalence of several diseases disorders.
Pharmacokinetics and relative bioavailability study of two cefquinome sulfate intramammary infusions in cow milk
In this study, two intramammary infusions of cefquinome sulfate were investigated for pharmacokinetics and bioavailability. Twelve lactating cows for each group were administered an effective dose of 75 mg/gland for cefquinome, with milk samples collected at various time intervals. The concentrations of cefquinome in milk at different times were determined by the UPLC-MS/MS method. Analyses of noncompartmental pharmacokinetics were conducted on the concentration of cefquinome in milk. Mean pharmacokinetic parameters of group A and group B following intramammary administration were as follows: AUC last 300558.57 ± 25052.78 ng/mL and 266551.3 ± 50654.85 ng/mL, C max 51786.35 ± 11948.4 ng/mL and 59763.7 ± 8403.2 ng/mL, T 1/2 5.69 ± 0.62 h and 5.25 ± 1.62 h, MRT 7.43 ± 0.79 h and 4.8 ± 0.78 h, respectively. Pharmacokinetic experiments showed that the relative bioavailability of group B was 88.69% that of group A. From our findings, group B (3 g: 75 mg) shows a quicker drug elimination process than group A (8 g: 75 mg), which suggests that the withdrawal period for the new formulation may be shorter.
Structure and Interdigitation of Chain-Asymmetric Phosphatidylcholines and Milk Sphingomyelin in the Fluid Phase
We addressed the frequent occurrence of mixed-chain lipids in biological membranes and their impact on membrane structure by studying several chain-asymmetric phosphatidylcholines and the highly asymmetric milk sphingomyelin. Specifically, we report trans-membrane structures of the corresponding fluid lamellar phases using small-angle X-ray and neutron scattering, which were jointly analyzed in terms of a membrane composition-specific model, including a headgroup hydration shell. Focusing on terminal methyl groups at the bilayer center, we found a linear relation between hydrocarbon chain length mismatch and the methyl-overlap for phosphatidylcholines, and a non-negligible impact of the glycerol backbone-tilting, letting the sn1-chain penetrate deeper into the opposing leaflet by half a CH 2 group. That is, penetration-depth differences due to the ester-linked hydrocarbons at the glycerol backbone, previously reported for gel phase structures, also extend to the more relevant physiological fluid phase, but are significantly reduced. Moreover, milk sphingomyelin was found to follow the same linear relationship suggesting a similar tilt of the sphingosine backbone. Complementarily performed molecular dynamics simulations revealed that there is always a part of the lipid tails bending back, even if there is a high interdigitation with the opposing chains. The extent of this back-bending was similar to that in chain symmetric bilayers. For both cases of adaptation to chain length mismatch, chain-asymmetry has a large impact on hydrocarbon chain ordering, inducing disorder in the longer of the two hydrocarbons.
Dry and Wet CO 2 Capture from Milk‐Derived Microporous Carbons with Tuned Hydrophobicity
Abstract Pore size distribution and surface chemistry of bio‐derived (milk) microporous dominated carbon “MDC” is synergistically tuned, allowing for promising carbon capture in a dry CO 2 atmosphere and in mixed H 2 O–CO 2 . The capture capacity is attributed to the synergy of a large total surface area with an ultramicroporous and microporous texture (e.g., S tot 1889 m 2 g −1 , S mic 1755 m 2 g −1 , S ultra 1393 m 2 g −1 ), and a high content of nitrogen and oxygen heteroatom moieties (e.g., 5 at% N, 10.5 at% O). Tailored two‐step low‐temperature pyrolysis‐chemical activation is employed to take advantage of the intrinsic properties of the precursor, allowing for this unusual textural properties‐heteroatoms combination. For example, tested at 1 bar and 295 or 273 K, MDCs adsorb up to 22.0 and 29.4 wt% CO 2 , respectively. MDCs are also tailored to be hydrophobic, with CO 2 /H 2 O adsorption selectivity even after prolonged cycling. Maximum working capacities of 10.8 wt% for pure CO 2 and 3.5 wt% for a flue gas simulant (15% CO 2 , 85% N 2 ) are measured using temperature swing adsorption with dynamic purge gases, while being minimally affected by humid conditions. This work is directly aligned with the United Nation’s Sustainable Development Goal 13, take urgent action to combat climate change and its impacts.
Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides
Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs.
Comparative genomics reveals a dynamic genome evolution in the ectomycorrhizal milk-cap ( Lactarius ) mushrooms
Ectomycorrhizal fungi play a key role in forests by establishing mutualistic symbioses with woody plants. Genome analyses have identified conserved symbiosis-related traits among ectomycorrhizal fungal species, but the molecular mechanisms underlying host specificity remain poorly known. We sequenced and compared the genomes of seven species of milk-cap fungi (Lactarius, Russulales) with contrasting host specificity. We also compared these genomes with those of symbiotic and saprotrophic Russulales species, aiming to identify genes involved in their ecology and host specificity. The size of Lactarius genomes is significantly larger than other Russulales species, owing to a massive accumulation of transposable elements and duplication of dispensable genes. As expected, their repertoire of genes coding for plant cell wall-degrading enzymes is restricted, but they retained a substantial set of genes involved in microbial cell wall degradation. Notably, Lactarius species showed a striking expansion of genes encoding proteases, such as secreted ectomycorrhiza-induced sedolisins. A high copy number of genes coding for small secreted LysM proteins and Lactarius-specific lectins were detected, which may be linked to host specificity. This study revealed a large diversity in the genome landscapes and gene repertoires within Russulaceae. The known host specificity of Lactarius symbionts may be related to mycorrhiza-induced species-specific genes, including secreted sedolisins.
Metagenomes and Metagenome-Assembled Genomes from Microbial Communities Fermenting Ultrafiltered Milk Permeate
Fermentative microbial communities can be utilized for the conversion of various agroindustrial residues into valuable chemicals. Here, we report 34 metagenomes from anaerobic bioreactors fed lactose-rich ultrafiltered milk permeate and 278 metagenome-assembled genomes (MAGs). These MAGs can inform future studies aimed at generating renewable chemicals from dairy and other agroindustrial residues.