Milk Leukocytes Research Articles

The Use of Flow Cytometry in the Analysis of Sows’ Colostrum and Milk

Abstract The flow cytometry method is used in many fields, not only scientific, but also clinical. In science, flow cytometry is used in immunology, molecular biology, microbiology or plant biology. In medicine, has its use, in the diagnosis of tumours, in reproductive and prenatal diagnosis, in transplants or in haematology. In our experimental work, we used this method to analyse colostrum and milk of sows. The aim of this study was to determine the number of somatic cells, the number of leukocytes and the number of T-lymphocytes. Colostrum samples were collected within 24 hours of delivery and then milk samples were collected at weekly intervals during 3 weeks. Statistical analysis revealed significant differences in the number of leukocytes (CD45+) in colostrum and milk. The most significant difference was noted between colostrum and milk in weeks 2 and 3 of the experiment (P < 0.0001), with the highest values found in colostrum. On the other hand, we found that the levels of helper T-cells (CD4+) and cytotoxic T-cells (CD8+) gradually increased over time (P > 0.05), with the highest values observed in the third week after farrowing. We can conclude that flow cytometry can be successfully used for the examination of somatic cells in sows’ milk.

Effects of parity and week after calving on the metabolic, redox, and immune status of dairy cows

At the onset of lactation in dairy cows, inflammation and oxidative stress may occur and result in a risk of pathologies and lower milk yield. To propose an innovative management strategy for cows during this period, it is essential to better understand these physiological variations. Our objective was to evaluate the metabolic, redox and immune status of 7 primiparous and 8 multiparous Holstein cows during late gestation and the first months of lactation. Blood samples were collected between 3 weeks before calving until 12 weeks postpartum. Milk samples were also collected, but only at the time points after calving. The metabolic (nonesterified fatty acids (NEFA), BHB, glucose, urea, calcium) and redox (reactive oxygen metabolites (ROM), oxidative stress index (OSI), glutathione peroxidase activity, vitamin E) statuses were analyzed in plasma or erythrocytes. The expression of genes related to antioxidant functions was determined in leukocytes collected from milk. For immune status, plasma cytokine levels and the production of reactive oxygen species (ROS) in classical and regulatory neutrophils were measured in 2 whole blood ex vivo challenges. The data were analyzed using a mixed model that included the fixed effects of parity and week and their interaction. Milk yield, plasma NEFA and BHB in wk 2 and 4 after calving were higher in multiparous cows than in primiparous cows, whereas glucose and calcium tended to be lower. Plasma ROM and OSI levels in wk 8 were higher in multiparous than in primiparous cows. Multiparous cows also displayed higher glutathione peroxidase activity in erythrocytes, and antioxidant transcription factor and superoxide dismutase-1 expression levels in milk leukocytes. Moreover, multiparous cows had higher plasma concentrations of vitamin E but lower plasma levels of cytokines CXCL10, CCL2, IL1Rα and IFNγ. Following ex vivo whole blood stimulation with Escherichia coli, lower IL1α and TNFα levels were measured in multiparous than in primiparous cows. Intracellular ROS production by neutrophils was lower in multiparous than in primiparous cows. These results thus indicated marked physiological changes in wk 8 compared with wk 2 and 4 of lactation. These differences in the physiological status of primiparous and multiparous cows offer interesting perspectives for potential dietary strategies to prevent pathologies which take account of parity and week relative to calving.

Strategic administration of antioxidant multiminerals and vitamins to transitional buffaloes augments antioxidant and udder defense mechanisms in early lactation

The aim of the study was to examine the effects of repeated administrations of antioxidant multiminerals and vitamins in transition buffaloes on udder defense mechanism, antioxidant activity and occurrence of intramammary infection (IMI) in early lactation period. Forty clinically healthy pregnant buffaloes were enrolled 45 days before expected date of calving and randomly allocated into five different supplementation groups (n = 8): only basal ration (control), vitamin E and selenium (VES), multiminerals (MM), ascorbic acid (AA) and chromium (Cr) picolinate in basal diet. The udder defense mechanism was monitored by measuring phagocytic activity (PA), myeloperoxidase (MPO) and nitric oxide (NO) productions in milk leukocytes, antioxidant activity was evaluated by measuring total antioxidant capacity (TAC) in plasma and occurrence of IMI was assessed by milk cytology, bacterial count in milk and visible clinical signs of udder until day 28 post-calving. The results showed that the VES and MM supplementations exhibited significantly higher PA, MPO and NO productions of milk leukocytes till first week of lactation whereas, elevated mean TAC in plasma was maintained from day −7 to 1 of calving in MM supplementation group as compared to control group. Statistically, no significant difference in occurrences of subclinical or clinical IMI was noted across the groups until four weeks of lactation. Taken together, it is concluded that repeated administrations of VES and MM to transition buffaloes could be an effective strategy to maintain good udder health by augmenting milk leukocyte functions and antioxidant status and preventing incidence of IMI in early lactation.

The Effects of Different Storage Conditions on Leukocytes in Human Breast Milk.

Breast milk is the best baby food because it contains various nutrients and important factors for the baby's immune system, including leukocytes. This study aimed to determine the effects on morphology, number of cells and breast milk leukocytes count of various ways of storing breast milk based on different temperatures and storage durations. This study was conducted at the Biochemistry Laboratory, Integrated Laboratory and the Histology Laboratory, Universitas Indonesia, Jakarta, Indonesia from September 2022 to February 2023. Transitional breast milk samples from 7 breastfeeding mothers were utilised in the study. A total of 50 mL was divided into 4 tubes of 12.5 mL each and treated based on temperature, storage time and method of thawing frozen breast milk based on the Centers for Disease Control and Prevention's (CDC) recommendations for breast milk storage. The breast milk cells were isolated to calculate the cell number and leukocyte population. Subsequently, the breast milk cells were stained with haematoxylin and eosin to analyse the number and morphology of leukocytes. The findings showed a significant decrease in the breast milk's total number and population and changes in the morphology of breast milk leukocytes after storage. This study indicates that CDC storage recommendations do not affect the quantity of the CD45+ leukocyte population; however, there is a decrease in the total number of leukocytes and alterations in their microscopic morphology. Thus, additional research is recommended to determine whether these modifications influence the function of the breast milk cells.

Frequencies of activated T cell populations increase in breast milk of HCMV-seropositive mothers during local HCMV reactivation.

Human cytomegalovirus (HCMV) can reactivate in the mammary gland during lactation and is shed into breast milk of nearly every HCMV-IgG-seropositive mother of a preterm infant. Dynamics of breast milk leukocytes during lactation, as well as blood leukocytes and the comparison between both in the context of HCMV reactivation is not well understood. Here, we present the BlooMil study that aimed at comparing changes of immune cells in blood and breast milk from HCMV-seropositive- vs -seronegative mothers, collected at four time ranges up to two months post-partum. Viral load was monitored by qPCR and nested PCR. Multiparameter flow cytometry was used to identify leukocyte subsets. CD3+ T cell frequencies were found to increase rapidly in HCMV-seropositive mothers' milk, while they remained unchanged in matched blood samples, and in both blood and breast milk of HCMV-seronegatives. The activation marker HLA-DR was more strongly expressed on CD4+ and CD8+ T cells in all breast milk samples than matched blood samples, but HCMV-seropositive mothers displayed a significant increase of HLA-DR+ CD4+ and HLA-DR+ CD8+ T cells during lactation. The CD4+/CD8+ T cell ratio was lower in breast milk of HCMV-seropositive mothers than in the blood. HCMV-specific CD8+ T cell frequencies (recognizing pp65 or IE1) were elevated in breast milk relative to blood, which might be due to clonal expansion of these cells during local HCMV reactivation. Breast milk contained very low frequencies of naïve T cells with no significant differences depending on serostatus. Taken together, we conclude that the distribution of breast milk leukocyte populations is different from blood leukocytes and may contribute to the decrease of breast milk viral load in the late phase of HCMV reactivation in the mammary gland.

EFFECTS OF A SACCHAROMYCES CEREVISIAE FERMENTATION PRODUCT ON GROWTH PERFORMANCE OF LITTERS, MILK COMPOSITION AND HEMATOLOGY PROFILE OF PRIMIPAROUS SOWS

A total of 16 gilts (158,158±3.5 kg) were assigned to one of two treatments to determine the effect of a Saccharomyces cerevisiae fermentation product (SCFP) supplementation during lactation on performance of sows and litters. The treatments were: CT) formulated to meet the nutritional requirements according to the National Research Council, NRC (2012); SCFP) similar to CT, plus 0.88% SCFP. Feed intake (FI) was recorded daily from farrowing until day 21 of lactation. At birth and on days 7, 14, and 21 of lactation, the weight of the litters was recorded to determine body weight gain (BWG), and milk samples were taken to determine the nutritional composition of the milk. The FI of the sows and the BWG of the litters were stratified into three phases with 7 days per phase [phase 1 (P1: d1-7); phase 2 (P2: d8-14); phase 3 (P3: d15-21)]. Blood samples were collected from the sows on days 7, 14, and 21 of lactation to determine the hematological profile. A treatment per day interaction was obtained in the FI, with a higher FI in sows fed SCFP during P1 and P2, with no differences in P3 (p<0.05). No differences were found in the BWG of the litters during lactation (p<0.05). Furthermore, there were significant differences in the treatment per day interaction for leukocyte concentration (p<0.05). Sows supplemented with SCFP had a higher percentage of milk fat compared to CT sows (p<0.05). In conclusion, SCFP supplementation stimulated sow feed intake, milk fat content and leukocyte profile of primiparous sows during lactation, without exerting productive improvements in litter performance.

Risk factors of high somatic cell count and differential somatic cells in early lactation associated with selective dry cow therapy.

The routine use of intramammary antimicrobial products in all dairy cows at the beginning of the dry period is no longer allowed in European Union (EU) countries due to the new Regulation (EU) 2019/6 to reduce antimicrobial resistance. This study investigated the application of a selective dry cow therapy scheme and the risk factors of high individual milk somatic cell count (SCC) and individual neutrophil count in early lactation, as a response to the application of a selective dry cow therapy (SDCT) protocol. The study was carried out on three commercial farms, and a total of 243 lactating cows were monitored at the end of lactation and at the beginning of the next one, 91 of which were dried off without the use of antimicrobials (NoT) based on milk SCC, differential somatic cell count (DSCC), and the response of Vetscan DC-Q milk analyser, using a secret algorithm. The remaining 152 cows received antimicrobials (T). After calving, similar means were observed between the two treatment groups for SCC (4.8 vs 4.9 log10 cells/ml for T and NoT, respectively, P=0.5) and total milk leucocyte count (TLC) (5 vs 5.1 log10 cells/ml for T and NoT, respectively, P=0.7) in milk. However, the use of antimicrobials led to a lower DSCC (58 vs 64% for T and NoT, respectively, P=0.01) and lower percentage of neutrophils (59 vs 64% for T and NoT, respectively, P=0.05), although the levels of DSCC and percentage of neutrophils in cows dried off without antimicrobials remained lower than the risk threshold suggested by the international literature. A logistic regression was computed after the application of selective dry cow therapy to identify risk factors of high milk SCC (≥100000 cells/mL) at the beginning of lactation. Increased milk SCC after calving was related to high SCC at the end of lactation and abandonment of antimicrobial therapy at dry-off. Moreover, the length of the dry period, milk protein content, and flank cleanliness in the last test day before dry-off were other factors in the logistic regression. Neutrophil counts at the beginning of the next lactation were affected by the same factors that influenced SCC, together with milk production, TLC, and macrophages on the last test day. The results obtained in these studied farms showed that selective dry cow therapy may be applied without adversely affecting the next lactation.

67 Infant Health Status Impacts the Neutrophil Phenotypes and Polyunsaturated Fatty Acids Composition in Human Milk

Abstract Background The abundance of human milk leukocytes, including neutrophils, changes in response to the health status of mothers and their infants. Elevated leukocyte counts occur during infant infection and return to baseline levels upon recovery. However, it is unclear how infant health status can affect the neutrophil phenotypes and the concentration of fatty acids in their mothers’ human milk. Objectives This study investigates the association between infant health status and human milk neutrophil counts and activation state, and fatty acid levels, in human milk. Design/Methods This is a prospective cohort study of 50 healthy breastfeeding mothers recruited from St. Michael’s hospital, in Toronto, Ontario, who were followed up from 2-4 weeks until 4 months postpartum. Human milk samples were collected from participants and data regarding infant health status were collected from self-reported questionnaires completed by participants at both timepoints, then imputed based on whether infants had taken antibiotics, had been hospitalized, or had been diagnosed with a medical condition, at least 2 weeks prior to samples collection. Neutrophils were quantified using flow cytometry and labelled with a panel of antibodies to detect specific cluster of differentiation (CD) biomarkers. Fatty acids were identified and quantified using a gas chromatography-flame ionization detector (GC-FID). Linear mixed-effects models were used to evaluate the correlation between infant health status and changes in neutrophil counts, along with their expressed CD markers, and fatty acids composition in human milk during lactation. Results The CD markers were classified into 4 categories based on their function: degranulation/activation markers (CD63, CD64, and CD66a), an immunoregulation marker (CD16), adhesion markers (CD11b, CD18), and a lipopolysaccharide receptor (CD14). Human milk from mothers whose infants had a health condition had neutrophils which significantly expressed elevated levels of the CD64 biomarker (β: 85.65, p=0.009 in adjusted models), compared to human milk from mothers of healthy infants. However, there were no significant associations between infant health status, absolute hmPMN counts, andother CD biomarkers (p>0.05). There were also elevated levels of arachidonic acid (β: 0.12, p=0.013) and C22:5n-6 (β: 0.06, p=0.015) fatty acids in the human milk of mothers whose infants had a health condition, compared with human milk from mothers of healthy infants, after adjusting for maternal age, post-pregnancy body mass index, infant sex, and infant feeding pattern. Conclusion This study demonstrates that infant health status is associated with changes in human milk immunological components, suggesting an inflammatory protective response mechanism in the mammary glands triggered by infants infection. How these alterations can affect infant health outcomes in the short and longer terms need critical consideration.

Effect of Saccharomyces cerevisiae byproducts on milk phagocyte function and milk production in mid-lactation cows

Saccharomyces cerevisiae is a supplement option for ruminants due to its ability to stimulate the immune system and productivity; however, there are few studies that demonstrate the effectiveness of this yeast in dairy cattle, especially regarding its effect on milk phagocyte function. Thus, this study examined the effect of two presentations of autolyzed S. cerevisiae on milk phagocyte function and milk production in healthy Holstein cows from the third to the fifth months of lactation with somatic cell count (SCC) less than 200,000 cells mL-1. Ten animals received cell wall-rich S. cerevisiae autolysate (WC 15 g animal day-1); 8 received the cytoplasm-rich extract (CYT 5 g animal day-1) and 7 received a diet without supplementation (C, control) for 60 days. Weekly oxidative metabolism analysis of milk leukocytes, production and milk constituents was carried out. The oxidative metabolism of milk leukocytes was higher in the WC group than in the C group between D32 and D48 (P≤ 0.05) and in the CYT group than in the C group between D24 and D40 (P≤ 0.05). The production and percentage of milk fat increased in CYT at D48 and D56. It is concluded that the CYT group had a greater effect on productivity, while on immunity the effect was intermediate, compared to the WC group, which was efficient in improving the immunity of the mammary gland.

Regenerative Potential of Human Breast Milk: A Natural Reservoir of Nutrients, Bioactive Components and Stem cells.

Human milk is a complex fluid that contains carbohydrates, lipids, proteins, and other bioactive molecules (immunoglobulins, lactoferrin, human milk oligosaccharides, lysozyme, leukocytes, cytokines, hormones, and microbiome) which provide nutritional, immunological, and developmental benefits to the infant. In addition to their involvement in the development, these bioactive compounds have a key role in anti-oncogenicity, neuro-cognitive development, cellular communication, and differentiation. As a result of technological advancements, it has been discovered that human breast milk contains cells that display many of the characteristics of stem cells with multilineage differentiation potentials. Do these cells have any specific properties or roles? Research efforts on breast milk cells have been mainly focused on leukocytes based on their immunological perspective in the early postpartum period. This review summarizes the nutritional components in human milk, i.e., the macro and micronutrients required for the growth and development of infants. Further, it discusses the research work reported concerning the purification, propagation, and differentiation of breast milk progenitor cells and highlights the advancements made in this newly emerging field of stem cell biology and regenerative medicine.

Alterations in haemato-biochemical and milk leukocyte count in mastitis affected cattle in North Karnataka

Alterations in haemato-biochemical and milk leukocyte count in mastitis affected cattle in North Karnataka

58 Effect of Autolyzed Yeast Feed Additives on the Function of Dairy Leukocytes and on the Milk Constituents of Holstein Cows

Abstract Mastitis is one of the leading causes of losses in dairy farming due to the cost of treatment and disposal rates. Yeast-based products can improve milk production and quality by stimulating immunity and ruminal efficiency. The different yeast derivative products and components can result in different effects. However, there are doubts about their real impact on the mammary gland. The yeast cell wall rich in β-glucans, considered an immunomodulator, acts mainly on phagocytes cells. The yeast cytoplasm is composed of metabolites, mainly amino acids, nucleotides, and vitamins, which impact in increasing the number of ruminal microorganisms and improve the feed conversion, resulting in an increase in milk production. The objective of this study was to evaluate two different autolyzed Saccharomyces cerevisiae feed additives supplemented in dairy cows and their effects on mammary gland phagocyte function, milk somatic cells count (SCC), and production. For this, 25 multiparous Holstein cows, in the middle third of lactation (average production of 25 L/day, and SCC below 200,000 cells/mL) were distributed into 3 treatments: Control group [CG] (n=8); Autolyzed Yeast [AY] (15 g/animal/day, RumenYeast, ICC Brazil Company, n=10); Yeast Metabolites [YM] (5 g/animal/day, MaxiDigest, ICC Brazil Company, n=7). Milk production, CSS, reactive oxygen species (ROS), and cytology of milk leukocytes were measured weakly for 56 days. AY increased phagocytes ROS from d40 and 42 compared to CG (P< 0.05); increased macrophages and decreased neutrophils percentage in milk, and decreased SCC at d48 and d56, compared to CG and YM (P< 0.05). YM increased ROS from d40 until D48 compared with CG (P< 0.05), with no change in the cellular profile of milk. There was an increase in milk production from d32 only in the YM versus CG and AY. In conclusion, AY demonstrated greater activation of the mammary gland cellular immune system, while YM promoted increased milk production and intermediate immune system stimulation.

Abrupt and gradual drying-off procedure and intramammary dry treatment: Impact on udder health status of Chios breed dairy sheep

In order to evaluate the impact of drying-off procedure on udder health status and the effect of intramammary dry treatment on prevention of new intramammary infections and improving cure rate of mammary abnormalities, a randomized single blinded controlled trial was performed in which 80 Chios breed dairy ewes were randomly allocated into 2 groups. Ewes of Group A (n=40) dried off gradually during a 15-day period, whereas in ewes of Group B (n=40), udder drying-off took place abruptly. Half of the ewes of each group received intramammary infusion (1 syringe/teat) of benzathine cloxacilline (IDT subgroup, n=20). The rest of the ewes of each group received no treatment at all (control subgroup, n=20). Representative samples of teat duct material and milk were aseptically collected from each mammary half for cytological and microbiological examination. Samples were collected using both conventional and aseptic techniques at the time of enrollment until the final milking before dry off (7 to 14 days before the expected dry period), at dry period (approximately 65 days) and continued at lambing until the end of the ongoing milking period. 61.8% of bacterial isolates obtained from teat duct and milk were identified as coagulase-negative staphylococci (CNS). No significant differences were noticed between the two groups in the frequency of mammary gland infection peri-partum (p=0.466), in the risk of new mammary infections during dry period (p=0.750) and in the cure rate of any subsequent mammary infection (p=0.131). Drying-off procedure had no significant impact on somatic cell counts (p=0.760) or milk leucocyte subpopulations (p>0.05) but had a significant effect on milk production of the next lactation period (p<0.001). Ewes treated with antibiotic agent presented a significantly higher cure rate of subsequent mammary infections (p=0.036) and a significantly lower risk of new mammary infections (p=0.039) during dry period, compared to the control group. No statistically significant differences were noticed concerning the impact of treatment on cytological profiles (p>0.5), somatic cell counts (p=0.581) and milk production (p=0.705). The results strengthen the hypothesis that drying off procedure does not affect cure rate neither reduce the risk of new infections during dry period and has no effect on development of mastitis around dry period whereas the use of intramammary dry treatment provides a better bacteriological cure rate of the mammary gland and decreases the possibility of new intramammary infections during dry period.

Microbial antigen in human milk: a natural vaccine?

Microbial antigen in human milk: a natural vaccine?

Impact of IgG Isotype on the Induction of Antibody-Dependent Cellular Phagocytosis of HIV by Human Milk Leukocytes.

Approximately 100,000 mother-to-child transmission (MTCT) events of HIV via human milk feeding occur each year. However, only about 15% of infants milk-fed by untreated HIV+ mothers become infected, suggesting a protective effect of the milk itself. Infants ingest 105-108 maternal leukocytes daily via milk, which remain functional beyond ingestion. Such function may be elicited by maternal milk antibody (Ab). Though IgA is dominant in milk, most HIV-specific milk Abs are of the IgG subclass, highlighting the importance of investigating the function of each IgG isotype in the milk context. Though Ab effector function mediated by the constant (Fc) domain via interaction with Fc Receptors (FcRs), such as Ab-dependent cellular phagocytosis (ADCP), are critical in protecting against HIV infection, ADCP is largely unexplored as it relates to mitigation of MTCT. Presently we report the ADCP activity of milk leukocytes against HIV particles and immune complexes (ICs), using 57 unique samples from 34 women, elicited by IgG1/2/3/4 of monoclonal (m)Ab 246-D. Granulocyte ADCP of HIV was most potent compared to other phagocytes when elicited by IgG1/3/4. IgG1/3 activated granulocytes similarly, exhibiting 1.6x-4.4x greater activity compared to IgG2/4, and a preference for virus compared to ICs. Notably, CD16- monocyte ADCP of a given target were unaffected by isotype, and CD16+ monocytes were poorly stimulated by IgG1. IgG2/4 elicited potent IC ADCP, and in terms of total leukocyte IC ADCP, IgG4 and IgG3 exhibited similar function, with IgG4 eliciting 1.6x-2.1x greater activity compared to IgG1/IgG2, and CD16+ monocytes most stimulated by IgG2. These data contribute to a more comprehensive understanding of Fc-mediated functionality of milk leukocytes, which is critical in order to develop therapeutic approaches to eliminating this route of MTCT, including mucosal administration of mAbs and/or a maternal vaccination aimed to elicit a potent milk Ab response.

Cellular detection of the chemokine receptor CXCR4 in bovine mammary glands and its distribution and regulation on bovine leukocytes

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

Immunomonitoring of Human Breast Milk Cells During HCMV-Reactivation

BackgroundBreast milk leukocytes may play a role in protecting the infant from pathogens. The dynamics and the role of lymphocytes in human cytomegalovirus (HCMV)-seropositive mothers shedding HCMV into breast milk during the first months postpartum (p.p.) are mostly unclear.MethodsBreast milk cells were analyzed by Pappenheim panoptic and alpha-naphthyl acetate esterase staining as well as by imaging and polychromatic flow cytometry to simultaneously establish their morphological and phenotypic properties. The latter were characterized in HCMV-seropositive and seronegative mothers´ breast milk cells at different time points p.p.ResultsPanoptic staining of breast milk cells revealed the presence of monocytes/macrophages, granulocytes and lymphocytes. Imaging flow cytometry data combining phenotypic and morphological analysis identified NKT-like cells, NK cells, epithelial cells, T cells and monocytes/macrophages. HCMV-seropositive but not -seronegative mothers had significantly higher T cell frequencies in mature milk.ConclusionsThe presence of lymphocyte subsets in breast milk may be more influenced by the HCMV-seropositivity of the mother than previously recognized.

Characterization of Microbiome on Feces, Blood and Milk in Dairy Cows with Different Milk Leucocyte Pattern.

Simple SummaryMastitis is an inflammation of the mammary gland caused by microorganisms and associated with an altered immune response. Recently, several studies hypothesized that a translocation of some bacteria from the gastrointestinal tract to the mammary gland can occur and that this bacterial crossing could be the cause of certain mastitis. The aim of this research is to investigate the bacteria translocation from the gut to the mammary gland, the so-called entero-mammary pathway, through the study of the fecal, blood and milk microbiome. Cows were recruited on the basis of their mammary gland health status and classified as healthy, at risk of mastitis and with mastitis. The microbial composition of feces, blood and milk were analyzed through high-throughput sequencing technique and the results were checked through a quantitative real-time PCR analysis. Although small differences were found in the microbiome of these three specimens between the groups of animals, beta biodiversity, that is, the ratio between whole and individual species diversity, highlighted a microbial community change in the milk of cows with different udder health conditions. The three matrices shared a high number of taxa; however, our results do not confirm a bacterial crossing from gut to milk, that still remains hypothetical.Mastitis is an inflammatory disease of the mammary gland, caused by the invasion of microorganism on this site, associated with an altered immune response. Recent studies in this field hypothesize that the origin of these pathogens can also be from the gastrointestinal tract, through the entero-mammary pathway in relation to an increase in gut permeability. In this study, we wanted to investigate if inflammatory status of the mammary gland is related to an alteration of gut permeability. The microbiome of feces, blood and milk of lactating cows, recruited on the basis of the total somatic cell count and of the percentage of polymorphonuclear neutrophils and lymphocytes, was studied. Cows were divided into healthy (G), at risk of mastitis (Y) and with mastitis (R) classifications. The bacterial DNA was extracted and the V3 and V4 regions of 16S rRNA sequenced. Moreover, the quantification of total bacteria was performed with quantitative real-time PCR. A non-parametric Kruskal–Wallis test was applied at the phylum, family and genera levels and beta biodiversity was evaluated with the unweighted UniFrac distance metric. Significant differences between groups were found for the microbial composition of feces (Clostridiaceae, Turicibacteriaceae for family level and Clostridium, Dorea, SMB53 and Turicibacter for genus level), blood (Tenericutes for phylum level and Mycoplasma for genus level) and milk (OD1 and Proteobacteria for phylum level, Enterobacteriaceae and Moraxallaceae for family level and Olsenella and Rhodococcus for genus level). The beta biodiversity of feces and blood did not change between groups. Significant differences (p < 0.05) were observed between the beta diversity in milk of G group and Y group and between Y group and R group. The number of taxa in common between feces, blood and milk were 8 at a phylum, 19 at a family and 15 at a genus level. From these results, the bacterial crossing from gut to milk in cows was not confirmed but remained hypothetical and deserves further investigation.

High Levels of Interferon-Alpha Expressing Macrophages in Human Breast Milk During SARS-CoV-2 Infection: A Case Report

Introduction: In addition to hand washing and wearing masks, social distancing and reducing exposure time to <15 minutes are the most effective measures against the spread of COVID-19. Unfortunately, three of these guidelines are very difficult, if not impossible, for nursing babies: they cannot wear masks, stay six feet away from the lactating breasts, nor consistently finish within 15 minutes while nursing. We report a case of a nursing mother with SARS-CoV-2 infection, documenting changes of immune cells and cytokines in breast milk with and without the infection. Case Description: With Institutional Review Board (IRB) approval, we obtained expressed breast milk samples from a lactating mother before and during SARS-CoV-2 infection as documented by reverse transcription-PCR. Using flow cytometry analysis, we measured the immune cell profiles and expression of cytokines such as interferon alpha (IFNα) in milk leukocytes before and during infection. Results: There was an eightfold increase in IFNα+ milk leukocytes, from 1% before SARS-CoV-2 infection to 8% when actively infected. The milk macrophages showed the highest increase in IFNα expression. Both T and B lymphocytes showed mild increase. Innate lymphoid cells, neutrophils, and natural killer cells showed no increase in IFNα expression and the dendritic cells actually showed a reduction. Conclusion: We document the presence and high expression of IFNα in the breast milk macrophages of a lactating mother with confirmed COVID-19, compared with her milk before the infection.

Feasibility and accuracy of using different methods to detect pregnancy by conceptus-stimulated genes in dairy cattle

Development of new methods for early diagnosis of pregnancy can be important to increase the reproductive efficiency and profitability of dairy herds. The bovine conceptus secretes IFN-τ that stimulates the transcription of several genes in circulating immune cells and extrauterine tissues. The aims of this study were to evaluate the mRNA abundance for pregnancy predictability of a classic gene stimulated by IFN-τ (ISG15) and a novel potential pregnancy marker (LGALS3BP) in peripheral blood mononuclear cells (PBMC), total blood leukocytes (TBL) or milk leukocytes (TML), and cervical cells (CC) on d 20 after timed artificial insemination (TAI) in dairy cattle. Eighteen Holstein females (12 cows and 6 heifers) were submitted to an estrous synchronization protocol for TAI (d 0). On d 20 post-TAI, blood samples were collected from coccygeal vessels for isolation of PBMC and in Tempus Blood RNA tubes (Applied Biosystems) for TBL. Samples of CC were collected using a cytological brush, and the TML were isolated from milk samples collected before routine milking. Pregnancy diagnosis was performed on d 30 post-TAI using transrectal ultrasonography, and females were classified as pregnant (n = 8) or nonpregnant (n = 10). Total RNA was extracted and mRNA abundance of target genes (ISG15 and LGALS3BP) was quantified by reverse-transcription quantitative PCR and normalized in relation to reference genes. Data were analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute Inc.). The mRNA abundance of ISG15 was greater in pregnant than in nonpregnant animals for PBMC, TBL, and CC. No difference was detected for TML based on pregnancy status. For LGALS3BP mRNA abundance, no difference was detected between pregnant and nonpregnant animals for PBMC, TBL, and TML, but a tendency for greater abundance in pregnant animals was observed for CC. The fold change for ISG15 in each pregnant cow related to the mean of nonpregnant animals was 2.73 ± 0.31, 3.40 ± 2.17, 1.64 ± 0.29, and 0.005 ± 0.002 for PBMC, CC, TBL, and TML, respectively. The fold change for LGALS3BP in each pregnant cow related to the mean of nonpregnant animals was 0.97 ± 0.38, 1.77 ± 0.39, 0.20 ± 0.08, and 0.70 ± 0.11 for PBMC, CC, TBL, and TML, respectively. The receiver operating characteristic curve analysis indicated that ISG15 abundance predicted pregnancy in PBMC (area under curve, AUC = 0.92) and CC (AUC = 0.77) but not in TBL (AUC = 0.72) or TML (AUC = 0.52). In conclusion, mRNA abundance for ISG15 in PBMC was the best predictor for pregnancy at d 20 post-TAI, whereas TBL and TML were not good predictors of pregnancy on d 20 post-TAI. The mRNA abundance of LGALS3BP was not associated with pregnancy status in any type of cell evaluated.