Milk Lymphocytes Research Articles

Factors influencing somatic cell count and leukocyte composition in cow milk: a field study

In recent years, there has been a proliferation of studies investigating the composition of somatic cell count (SCC) of milk, focusing on neutrophils (NEU), lymphocytes (LYM), and macrophages (MAC). These 3 components are indeed crucial for the animal's immune response to mastitis-causing pathogens. The study examined various factors influencing somatic cell count and leucocyte components in cow milk, including lactation stage, parity, and milk electrical conductivity, using data from 179 dairy cows across 6 farms throughout the entire lactation. Statistical analyses, including mixed models and logistic regression, were employed to investigate the relationships between these variables and identify risk factors for high SCC levels. Results showed that factors such as parity and lactation stage were significantly associated with somatic cell composition. In particular, the highest milk NEU values (>60% of the total leucocytic fraction) and lowest MAC values (<20%) were found at the beginning and the end of lactation, that are the critical periods for udder health. High milk electrical conductivity, low milk production, number of parity, and poor hygiene scores were identified as contributing to increased SCC. Additionally, elevated percentages of NEU and LYM in milk were associated with increased risk of high SCC values, indicating potential udder health issues.

Heat stress affects milk yield, milk quality, and gene expression profiles in mammary cells of Girolando cows

Heat stress during lactation affects the physiological responses, hormonal release, health, and productivity of dairy cows. However, the adverse effects of heat stress on milk synthesis, immune response, and cellular apoptosis in mammary cells remains unknown in Bos indicus cows. This study aimed to understand the relationship between milk yield, milk quality, and the expression of genes related to milk synthesis, cell apoptosis, and immune response in mammary cells of Girolando cows. Twenty-four Girolando cows (3/4 Holstein and 1/4 Gir) were subjected to control (CT, with a temperature-humidity index ranging from 60 to 74, n = 12) or heat stress treatments (HS, with a temperature- humidity index ranging from 60 to 85, n = 12), from 111 to 120 d of lactation. Heat stress significantly increased the expression of heat shock proteins (HSPD1 and HSPD90AA1), insulin receptors (INSR), and prolactin receptors (PRLRsf) genes, and decreased the expression of glucocorticoid receptor (NR3C1) gene in mammary cells of the HS cows when compared with the CT cows. The HS cows exhibited significantly higher vaginal temperatures and cortisol release compared with the CT cows. Moreover, the HS cows had significantly lower dry matter intake and milk yield than CT cows. Although, HS cows showed higher percentage of lymphocytes in milk when compared with that from CT cows. There was no effect of heat stress on other leukocyte counts, somatic cell counts, bacterial counts in milk, or milk composition. Finally, this study demonstrated that Girolando cows are susceptible to heat stress, which decreases milk yield and affects the expression of genes linked to milk synthesis in the mammary cells.

102 Circadian patterns of behavior, respiration, rectal temperature, saliva melatonin, cytokine concentrations, and milk immune cells are altered by cooling heat-stressed lactating sows

Abstract Lactating sows are particularly vulnerable to heat stress (HS) due to the increased metabolism from milk production. A greater understanding of HS response could assist in mitigating its detrimental effects on animal production. Two core circadian clock genes were pinpointed in a recent genome study as potential mediators of HS response, indicating the possible alteration of circadian clocks by HS exposure. This study aimed to assess how cooling of lactating sows under natural heat stress conditions in the summer influenced circadian patterns of respiration rate (RR), rectal temperature (RT), behaviors, salivary concentration of cortisol and melatonin, milk cell populations and relative abundance of multiple cytokines in saliva. Lactating sows of mixed parities were divided into two treatment groups: treated with electronic cooling pad (C; n=9) and heat-stressed control (H; n=9). Experiments were conducted during two 48 h periods of increased environmental temperatures from summer heatwaves. During the initial 48 h period, rectal temperature (RT) was monitored every 60 min, rectal temperature (RT) every 30 min, and behaviors of eating, standing, sitting, laying, sleeping, drinking, and nursing every 5 min. Morning milk samples were collected following the completion of the first phase and utilized to categorize and count somatic cells. The second trial was approximately 1 wk later, and during this period saliva samples were collected every 4 h over the 48-h period. Results were analyzed for significant effect of treatments and interaction using periodic regression while circadian patterns were further detected by incorporating a Fourier series model utilizing sine-cosine variables that denote a fit and/or shift in circadian pattern. The analysis revealed that cooling decreased RT and RR while also modifying circadian patterns (P &amp;lt; 0.05). Cooling had no impact on the percentage of time spent in any activity over the 48-h period (P &amp;gt; 0.05). Nevertheless, the daily patterns of eating, standing, and sitting were different between the treatments (P &amp;lt; 0.05). A shift in eating time to later in the day in H sows relative to C sows related to a second rise in RT during the dark phase of the light-dark cycle (P &amp;lt; 0.05). Saliva melatonin concentrations were increased in C sows and circadian pattern altered relative to H (P &amp;lt; 0.05), but there was no effect on salivary cortisol concentrations (P &amp;gt; 0.05). Cooling reduced milk lymphocytes (P &amp;lt; 0.05) and influenced diurnal pattern of saliva cytokines including interleukins (IL) IL-1 alpha, IL-1 beta, IL-10, and IL-15 and transforming growth factor-beta 1. In conclusion, the cooling of heat-stressed sows altered the circadian patterns of physiology, behavior and melatonin, suggesting the need for additional research to investigate whether circadian disruption contributes to reduced production efficiency observed in HS animals.

Immunomodulatory Effect of Infectious Disease of a Breastfed Child on the Cellular Composition of Breast Milk.

Recent studies suggest that the content of immune components in milk is influenced by the mother's health and also by the infant she feeds. We aimed to evaluate the effect of a child's respiratory tract infection on the cellular composition of breast milk (neutrophils, monocytes, eosinophils, lymphocytes, and their subpopulations). Twenty-six breastfeeding mothers whose children were hospitalized for respiratory tract infections were enrolled in the study. The control group consisted of 23 mothers of healthy children. Regarding the children, baseline laboratory blood tests were performed, and nasal swabs were taken for the presence of RS virus. In the next step, milk samples were collected from the mothers to assess the cellular composition of the milk, including neutrophils, monocytes, eosinophils, lymphocytes, and their subpopulations. Significantly higher percentages of T lymphocytes (helper and cytotoxic lymphocytes) were observed in the milk of the studied mothers. There was a significantly higher percentage of milk lymphocytes in the group of affected children with confirmed RSV etiology than in children with excluded RSV etiology. A significant positive correlation was observed between the duration of infection and the percentage of milk NK cells and between milk CD19 lymphocytes and the child's serum leukocytosis. This study may provide evidence of a link between cells in breast milk and disease in the breastfed infant. The severity of the infection, its duration, and the etiological agent of the infection may affect the cellular composition of milk.

Expression of Bovine Leukaemia Virus (BLV) Gp51 Protein in Blood and Milk Cells of Cows with Leukosis.

Bovine leukaemia virus (BLV) is the retroviral causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle and a serious problem worldwide. Its diagnosis is commonly by tests for antibodies recognising the p24 capsid protein and structural glycoprotein (gp) 51. With flow cytometry recently having come to veterinary immunology, applications for it may now include BLV. The study determined BLV gp51 expression in blood and milk lymphocytes of naturally infected cows by flow cytometry. Nineteen Polish Black and White Lowland breed cows aged 4-9 years and naturally infected with BLV and ten uninfected counterparts had blood and milk sampled and cultured. The immunological status of the animals was confirmed with ELISA and PCR. Dual-colour flow cytometry analysis was performed with specific monoclonal antibodies for lymphocyte cluster of differentiation (CD) markers and gp51 viral envelope protein and conjugates labelled with fluorescein isothiocyanate or phycoerythrin. Bovine leukaemia virus gp51 was confirmed in lymphocytes by immunofluorescence with anti-gp51 monoclonal antibodies. The gp51 antigen was detected in blood and milk lymphocytes of infected cows, but the percentage of cells expressing it in milk was much lower than in blood. A depleted number of CD4+ lymphocytes, an augmented number of CD8+ lymphocytes, a lower ratio of CD4+ to CD8+ and a proliferation of CD19+ immunoglobulin M+ cells were also found. These proliferated cells were immature, gave no sign of a tendency to differentiation and were characterised by prolonged vitality.

Milk lymphocyte profile and macrophage functions: new insights into the immunity of the mammary gland in quarters infected with Corynebacterium bovis

BackgroundsThe present study explored the viability of bovine milk macrophages, their intracellular production of reactive oxygen and nitrogen species (RONS), and their phagocytosis of Staphylococcus aureus, as well as the profile of lymphocytes, from healthy udder quarters and udder quarters infected by Corynebacterium bovis. The study included 28 healthy udder quarters from 12 dairy cows and 20 udder quarters infected by C. bovis from 10 dairy cows. The percentages of macrophages and lymphocytes were identified by flow cytometry using monoclonal antibodies. Macrophage viability, RONS production, and S. aureus phagocytosis were evaluated by flow cytometry.ResultsMilk samples from quarters infected with C. bovis showed a lower percentage of macrophages but an increased number of milk macrophages per mL and a higher percentage of macrophages that produced intracellular RONS and phagocytosed S. aureus. No effect of C. bovis infection on macrophage viability was found. Udder quarters infected by C. bovis showed a higher percentage of T cells and CD4+ T lymphocytes, but no effect was found on the percentage of CD8+ CD4− T, CD8− CD4− T, or B lymphocytes.ConclusionsThus, our results corroborate, at least in part, the finding that intramammary infections by C. bovis may offer protection against intramammary infections by major pathogens.

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep.

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV-positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.

New Insights into the Significance of PARP-1 Activation: Flow Cytometric Detection of Poly(ADP-Ribose) as a Marker of Bovine Intramammary Infection.

Bovine intramammary infections are common diseases affecting dairy cattle worldwide and represent a major focus of veterinary research due to financial losses and food safety concerns. The identification of new biomarkers of intramammary infection, useful for monitoring the health of dairy cows and wellness verification, represents a key advancement having potential beneficial effects on public health. In vitro experiments using bovine peripheral blood mononuclear cells (PBMC), stimulated with the bacterial endotoxin lipopolysaccharide (LPS) enabled a flow cytometric assay in order to evaluate in vivo poly-ADP-ribose (PAR) levels. Results showed a significant increase of PAR after 1 h of treatment, which is consistent with the involvement of PARP activity in the inflammatory response. This study investigated PARP-1 activation in leukocyte subpopulations from bovine milk samples during udder infection. A flow cytometric assay was, therefore, performed to evaluate the PAR content in milk leukocyte subsets of cows with and without intramammary infection (IMI). Results showed that milk lymphocytes and macrophages isolated from cows with IMI had a significant increase of PAR content compared to uninfected samples. These results suggest mastitis as a new model for the study of the role of PARP in zoonotic inflammatory diseases, opening a new perspective to the “One Health” approach.

Lymphocyte proliferation Response of blood and milk lymphocytes of crossbred cows during peripartum period

Lymphocyte proliferation Response of blood and milk lymphocytes of crossbred cows during peripartum period

Surfaces that Adhesively Discriminate Breast Epithelial Cell Lines and Lymphocytes in Buffer and Human Breast Milk.

We report new surface coatings that adhesively distinguish three breast epithelial cell lines (MCF-10A, MCF-7, and TMX2-28) when cell suspensions in buffer or breast milk are flowed over the coatings. We also report the selective capture of epithelial cells and rejection of Jurkat lymphocytes, with average selectivities exceeding 60 and captured cell purities often exceeding >99%. The surfaces achieve the dual goals of selective cell capture and resistance to fouling by proteins and other components of breast milk. The coatings do not rely on antibody targeting of cell surface markers but instead contain polycation chains embedded within a layer of end-tethered poly(ethylene glycol) (PEG) chains. The PEG, somewhat shielding the polycations, prevents surface fouling by proteins, nondesired cells, and other milk components, while the polycations produce electrostatic attractions that are heterogeneous on nanoscopic length scales. These electrostatic heterogeneities on the engineered coating, shown to produce curvature-selective particle capture in other studies, produce cell selectivity here. The ability of the engineered surfaces to discriminate these cell lines via an electrostatic driving force is remarkable, as the cells are of very similar surface charge as evidenced by their nearly identical ζ-potentials. The current surfaces, which likely distinguish cells based on their electrostatic surface landscape combined with other factors, adhesively distinguish cell lines that may differ only slightly in their expression of a surface marker, or cancer cells that minimally express EpCAM but which have different distributions of electrostatic charge on their surfaces. These surfaces are among the first to be documented for the compatibility of a polymer brush with human breast milk and may find use in technologies that capture cells from human breast milk or other complex fluids for cancer risk assessment.

MAIT cells, TCR γδ+ cells and ILCs cells in human breast milk and blood from HIV infected and uninfected women.

Human breast milk cells remain poorly characterized for the presence of unconventional T lymphocytes and innate lymphoid cells (ILCs). Early breast milk was collected from eight HIV-uninfected and 11 HIV-infected women 3-12days after delivery. Mucosal-associatedinvariant T cells (MAIT cells), TCR γδ cells, and innate lymphoid cells (ILCs) were analyzed in breast milk and paired blood samples. CD161+/TRAV1-2+MAIT cells were detected in breast milk, accounting for a median (IQR) of 0.08% (0.06-0.16) and 0.17% (0.16-0.31) of CD45+ breast milk cells in HIV-uninfected and HIV-infected women, respectively. A selective compartmentalization of γδ T lymphocytes was observed in breast milk. Median (IQR) frequency of γδ T lymphocytes was 8.95% (8.64-12.14) among breast milk lymphocyte cells compared to 2.54% (1.81-4.10) in blood (P=0.03) in HIV-uninfected women, and 7.26% (4.22-10.54) in breast milk versus 3.31% (2.54-3.80) in blood (P=0.004) from HIV-infected women. The proportion of group 1 ILC (ILC1) among total ILCs was higher in breast milk compared to blood in HIV-uninfected women (P=0.03) and HIV-infected women (P=0.001). The frequency of ILC2 among total ILCs tends to be lower in breast milk compared to blood in HIV-uninfected women (P=0.06) and HIV-infected women (P=0.03). Unconventional T cells and ILCs that may be involved in both the protection against infection of the lactating mammary gland and maturation of infant's gut and microbiomes account for a detectable fraction of breast milk cells.

Effect of peripartum micronutrient supplementation on postpartum udder health in high-yielding crossbred cattle

The study was aimed to evaluate the effect of peripartum micronutrient supplementation on postpartum udder health in high-yielding crossbred cows. High-yielding crossbred cows (12) of late pregnancy were selected and divided into two groups, viz. supplemented (6) and unsupplemented (6). Supplemented animals were supplied with a mineral mixture (@25 g daily) fortified with copper (Cu), selenium (Se), manganese (Mn) and zinc (Zn) as per the recommendation of National Research Council (NRC) over and above the normal ration starting from 45 days precalving and continued till 45 days postcalving (based on their expected date of calving). Milk samples from each animal were collected and different attributes of udder immunity were evaluated. The supplemented group exhibited higher udder immunity as depicted by lower milk somatic cell count and higher activity of milk lymphocytes.

Diurnal variation of milk somatic and differential leukocyte counts of Murrah buffaloes as influenced by different milk fractions, seasons and parities

Milk cell counts are good indicators of a mammary infection and milk quality. The present study was done to record diurnal rhythmicity in the milk somatic and differential cell counts during different seasons, milk strips and parity in Murrah buffaloes. Milk somatic cell counts (SCC) were measured by SCC counter and milk differential cell counts were measured microscopically after making a milk smear and staining it to identify neutrophils, lymphocytes and macrophages. Maximum milk SCC was observed in the summer season. Milk neutrophils were lowest during thermoneutral (TN), intermediate during the winter season and highest during the summer season. Milk lymphocytes were highest during the winter season, intermediate in the TN and lowest in the summer season. Diurnal rhythm in the milk SCC and neutrophil percentage is noticed in the summer season only. Maximum milk SCC values were observed in the late strip but neutrophils were highest in the early strip. Diurnal rhythm was observed in the late strip for neutrophils and in mid strip for the lymphocytes. Milk SCC and milk neutrophils were found to be the highest in the multiparous buffaloes and diurnal rhythm was observed only in the lymphocytes of primiparous buffaloes. Milk macrophages were higher in the morning samples of primiparous as compared to the multiparous buffaloes. In this pioneer study, diurnal rhythms in the milk cell counts of buffaloes have been studied extensively. This will help in maintaining low milk cell counts in buffalo and thus help in getting more milk per buffalo during stress periods.

Correlation between in vitro activity of milk leukocytes with milk constituents in high yielding crossbred cows

The relationship between the in vitro activity of milk leukocytes and milk constituents were studied in 48 high yielding clinically healthy crossbred cows. Milk somatic cell counts (SCC) was negatively correlated with milk lactose, total soid and solid not fat (SNF) content. Milk neutrophils were positively correlated with protein and Cu content and negatively correlated with fat content in milk. However milk lymphocytes were positively correlated with milk fat content and negatively correlated with protein content. Milk macrophages were positively correlated with milk SNF and total solid content. Milk lymphocyte blastogenic response (SI) was significantly positively correlated with milk protein, fat percentage and copper content and negatively correlated with milk lactose, milk total solids, SNF and zinc content. PI of milk neutrophils was positively correlated with zinc content and milk fat percentage and negatively correlated with milk total solid and SNF content. PI of milk macrophages was positively correlated with milk fat content. In the present investigation, immuno-potentiative role of various milk constituents such as milk protein, copper and zinc content on in vitro leukocyte activity was established.

Transcriptome Analysis in Human Breast Milk and Blood after Inactivated or Attenuated Influenza Immunization

BackgroundThe goal of this study was to identify transcriptomic signatures (RNA-Seq) in human peripheral blood mononuclear cells (PBMCs) and breast milk lymphocyte cells (BMLCs) in response to trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV).MethodsWe performed a randomized, double-blind study in breastfeeding women who received either LAIV and intramuscular placebo, or TIV and intranasal placebo. A subset of subjects with available samples (LAIV, n = 10 and TIV, n = 6) was used for this study. Human milk was collected on days 0, 2, 8, and 28, and blood samples were collected on days 0 and 28. PBMC and BMLC RNA was extracted for RNA-Seq and differentially expressed (DE) gene analysis.ResultsWe identified a total of 382 DE BMLC genes in the LAIV group, most of which were up-regulated at day 28. DE genes were preferentially involved in innate immune signaling pathways including cytokine-cytokine receptor interaction, TNF signaling, and NF-kappa B signaling. For TIV, 3 DE genes were identified of which 2 (IL1A and IL1B) overlapped with LAIV. Response time trends for co-expressed gene clusters by vaccine group showed that LAIV generally induced an early (day 2) up-regulation of innate immune signaling pathway genes, while TIV induced peak innate immune signaling gene responses ahead of LAIV (day 8 vs. day 28). A group of known interferon-alpha/β-inducible genes (IFIT3, OAS3, IFI44L, MX1, OAS2, IFIT1, IFI6) showed higher responses at day 2 for TIV but stronger peak levels by day 28 for the LAIV group (Fig 1). While no such innate immune signaling responses were observed in PBMCs at day 28, we identified an up-regulation of IgG gene (IGHG1 and IGHG3) expression in the TIV group (Fig 2).ConclusionWe observed increased innate immune signaling responses in BMLC but not in PBMC at day 28 for the LAIV group. We hypothesize that breastfeeding extends the innate response to LAIV via mucosal immunity. Gene cluster time trends indicated an earlier innate immune signaling response for TIV. The day 28 increase in IGHG3 gene expression levels in TIV group PBMCs was correlated with corresponding increases in serum ELISA IgG titers for the influenza B antigen (Fig 3). Additional studies are required to investigate the differences in innate response signaling seen for BMLC and PBMC in this study.Disclosures All authors: No reported disclosures.

Alteration in the in vitro activity of milk leukocytes during different parity in high yielding cross-bred cows

In vitro activity of milk leukocytes (viz. neutrophils, lymphocytes and macrophages) was evaluated in forty-eight (48) clinically healthy high-yielding cross-bred cows of mid-lactation stage (100–200 days of lactation), divided into four groups namely 1st parity (n = 12), 2nd parity (n = 12), 3rd parity (n = 12) and 4th and above parity (n = 12). Milk samples were taken (250 ml/cow) were taken. Milk somatic cell counts (SCC) and differential leukocyte counts (DLC) were performed microscopically. In vitro phagocytic index (PI) of milk neutrophils and macrophages was evaluated by colorimetric nitro blue tetrazolium reductive assay. Mitogen-induced milk lymphocyte blastogenic response was measured by colorimetric MTT (tetrazolium) assay after isolation of the milk leukocytes by density gradient centrifugation. Milk SCC differed significantly (p < 0.01) between different parity. Cows of 4 and above parity showed significantly (p < 0.01) higher milk SCC compared to primiparous cows. There was no significant difference in milk DLC during different parities in high-yielding cross-bred cows. There was a significant (p < 0.01) variation in lymphocyte blastogenesis amongst parity. The highest value of lymphocyte blastogenesis was seen at 3rd parity, whereas lowest value was obtained in the cows of both 1st and 4th or above parity. PI of milk neutrophils did not differ significantly between parity. PI of milk macrophages was significantly (p < 0.01) higher in 3rd parity and lower (p < 0.01) in 1st and 4th parities. The study indicated that depressed activity of milk lymphocytes and macropages was lower and SCC was higher in the cows of 4th and above parity indicating more mammary stress and hence susceptible to udder infection and mastitis. Therefore, better care and managemental interventions should be taken around these periods.

Physiological Threshold of Somatic Cell Count in Milk of Polish Heath Sheep and Polish Lowland Sheep

Abstract The study was conducted on 320 sheep of two breeds: Polish Heath Sheep (PHS) and Polish Lowland Sheep (PLS). Udder health status was assessed based on somatic cell count (SCC) and bacteriological examination of milk. Cells of the immune system (leukocyte populations and lymphocyte subpopulations) were identified in the blood and milk of sheep by flow cytometry, using a panel of specific monoclonal antibodies and fluorescent dyes. The study showed that the highest proportion of neutrophils and a lower percentage of lymphocytes (CD4+, CD8+, CD19+, WC1-N2+) in milk and blood occurred in sheep of both breeds in which milk SCC ranged between 201 and 300×103 cells/ml. In light of existing research, these results suggest that fluctuations in somatic cell count of ewe milk are physiologically determined up to 200×103cells/ml and result from udder health disturbances above this level.

Alteration in the activity of blood and milk leukocytes together with the serum enzyme profile during sub-clinical mastitis in cross-bred cows

In vitro activity of blood and milk leukocytes together with serum enzyme profile during sub-clinical mastitis in crossbred cows were evaluated after collection of blood and milk samples from normal (10) and sub-clinical mastitic (10) cows. Blood total leukocyte counts (TLC) and differential leukocyte counts (DLC) were estimated by standard hematological procedure. Milk somatic cell counts (SCC) was performed microscopically. In vitro phagocytic activity of blood and milk neutrophils was performed by colorimetric nitro blue tetrazolium (NBT) assy and mitogenconcanavalin A (con A) induced blood and milk lymphocyte blastogenic response was evaluated by colorimetric MTT (tetrazolium) assay. Serum total protein and alkaline phosphatase (ALP) were measured by the stand biochemical methods. The alanine amino transferase (ALT) and aspertate amino transferase (AST) activities in serum were estimated by commercially available kit. Milk SCC was significantly higher in sub-clinical mastitic cows. Phagocytic index of both blood and milk neutrophils was significantly lower in sub-clinical mastitic cows than normal animals. Con- A induced blood and milk lymphocyte blastogenic response was significantly loweredin sub-clinical mastitic cows than. Serum albumin, globulin ratio decreased significantly during sub-clinical mastitis. Serum AST and ALP level in sub-clinical mastitic cows was significantly higher. The study indicated decreased blood and milk leukocyte activity and higher AST and ALP during the sub-clinical mastitis which could be used as a diagnostic tool for sub-clinical mastitis.

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows has been studied in 10 high-yielding (milk production: 5000 l per lactation) Karan Fries crossbred (Holstein Friesian × Tharparkar) cows. Milk and blood samples were collected from all the experimental animals. Isolation of milk phagocytes (neutrophils and macrophages) and lymphocytes were done by density gradient centrifugation. In vitro phagocytic index of milk neutrophils and macrophages was performed by colorimetric NBT reductive assay. Mitogen-induced milk lymphocyte blastogenic response was estimated by colorimetric MTT (tetrazolium) assay. Total plasma cortisol and prolactin were estimated by enzyme immune assay. Highest value of plasma cortisol and prolactin was observed at calving which decreased significantly (p < 0.01) on 15th day postpartum for both prolactin and cortisol. Immune activity of milk leukocytes was highest on day 0 colostrum and decreased significantly (p < 0.01) on 7th day postpartum. A significant (p < 0.01) rise of plasma prolactin was observed around 135th and 225th days postpartum, whereas a peak level of plasma cortisol was observed at 105th, 180th, and 270th days postpartum. Phagocytic index of milk neutrophils and macrophages remains almost in a steady state during mid-lactation period (between 100 and 200 days postpartum). A decline in increasing trend of milk phagocytic activity was observed during late lactation. Mitogen-induced milk lymphocyte blastogenic response was highest on day 0 colostrum which decreased significantly (p < 0.01) on 15th day postpartum. Con A-induced milk lymphocyte blastogenic response showed an increasing trend from 120th to 210th days postpartum. Upon correlation study, it showed that the plasma cortisol has a negative effect on milk leukocyte activity, while prolactin has a positive effect, though the effect is lactation stage specific.

The neutrophil function and lymphocyte profile of milk from bovine mammary glands infected with Streptococcus dysgalactiae.

Streptococcus dysgalactiae is a bacterium that accounts for a notable proportion of both clinical and subclinical intramammary infections (IMIs). Thus, the present study explores the function of milk neutrophils and the lymphocyte profile in mammary glands naturally infected with Streptococcus dysgalactiae. Here, we used 32 culture-negative control quarters from eight clinically healthy dairy cows with low somatic cell counts and 13 S. dysgalactiae-infected quarters from six dairy cows. Using flow cytometry, we evaluated the percentage of milk monocytes/macrophages and neutrophils, expression of CD62L, CD11b and CD44 by milk neutrophils, the levels of intracellular reactive oxygen species (ROS) production and phagocytosis of Staphylococcus aureus by milk neutrophils, and neutrophil viability. Furthermore, the percentages of B cell (CD21(+)) and T lymphocyte subsets (CD3(+)/CD4(+)/CD8(-); CD3(+)/CD8(+)/CD4(-); and CD3(+)/CD8(-)/CD4(-)), and the expression of CD25 by T milk lymphocytes (CD3(+)) and T CD4(+) milk cells were also assessed by flow cytometry using monoclonal antibodies. The present study showed a higher SCC and percentage of milk neutrophils, and a decrease in the percentage of milk monocytes/macrophages from S. dysgalactiae-infected quarters when compared to uninfected ones. We also observed a higher expression of CD11b by milk neutrophils and a tendency toward a decrease in neutrophil apoptosis rate in S. dysgalactiae-infected quarters. In addition, the S. dysgalactiae-infected quarters had higher percentages of milk T cells (CD3(+)) and their subset CD3(+)CD8(+)CD4(-) cells. Overall, the present study provided new insights into S. dysgalactiae IMIs, including distinct lymphocyte profiles, and a tendency toward an inhibition of apoptosis in milk neutrophils.