Pre-clinical safety and toxicity assessment of Limosilactobacillus fermentum NCDC 400 in murine model.

Comprehensive safety assessment of potential probiotic strains is crucial in the selection of risk-free strains for clinical translation. This study aimed to evaluate the biosafety of Limosilactobacillus fermentum NCDC 400, a potential probiotic strain, using oral toxicity tests in a Swiss albino mouse model. Mice were orally gavaged with low (108 CFU/mouse/day) and high (1010 CFU/mouse/day) doses of NCDC 400 for 14 (acute), 28 (subacute), and 90 (subchronic) days to assess behavioral, hematological, biochemical, immunological, and histological effects. The administration of NCDC 400 did not result in any observable adverse effects on general health parameters, including body weight, feed and water intake, and organ indices. Hematological and biochemical parameters, such as glucose, serum enzymes, urea, creatinine, serum minerals, total serum proteins, and lipid profile, remained largely unaffected by the test strain. Notably, NCDC 400 administration led to a significant reduction in harmful intestinal enzymes and improvement in gut health indices, as indicated by fecal pH, lactate, ammonia, and short-chain fatty acids. There were no instances of bacterial translocation of NCDC 400 to blood or extra-intestinal organs. Immune homeostasis was not adversely affected by repeated exposure to NCDC 400 in all three oral toxicity studies. Histopathological examination revealed no strain-related changes in various tissues. Based on these findings, a dose of 1010 CFU/mouse/day was considered as the No Observable Effect Level (NOEL) in healthy mice. In conclusion, this study demonstrates the safe and non-toxic behavior of L. fermentum NCDC 400. The results support and ensure the safety and suitability for clinical trials and eventual translation into clinical practice as potential probiotic.

Reliability of udder infrared thermography as a non-invasive technology for early detection of sub-clinical mastitis in Sahiwal (Bos indicus) cows under semi-intensive production system.

The present study focused on Sahiwal cows, a prominent milch breed in tropical India, to correlate udder temperature with physiological markers of stress and inflammation during subclinical mastitis (SCM). The primary goal was to assess the potential of udder infrared thermography for the early detection of SCM under the semi-intensive production. Cows were categorized based on milk somatic cell counts (SCC), with healthy (H) cows having SCC <2 × 105 cells/mL and no history of mastitis, and cows with subclinical mastitis (SCM) and initial stages of clinical mastitis (CM) having quarter milk SCC of 2-5 × 105 and >5 × 105 cells/mL, respectively. Firstly, udder thermograms were analysed for udder skin surface temperature (USST), teat skin surface temperature (TSST), and teat apex temperature (TAT) using Fluke software to determine the optimal site for temperature measurement during intramammary infection. Secondly, milk samples were collected for automatic estimation of compositional changes, electrical conductivity, and pH. Thirdly, milk whey was separated for quantifying stress and inflammatory indicators, including cortisol, prolactin, and acute-phase proteins (APPs): milk amyloid A and milk haptoglobin using bovine-specific ELISA kits. Significant increases (p < 0.01) in USST, TSST, TAT, cortisol, and APPs were observed in SCM and CM compared to healthy cows, while prolactin levels decreased (p < 0.01). The correlation matrix revealed strong positive correlations of SCC with USST (r = 0.84, p < 0.01). In ROC analysis, USST demonstrated cut-off values of 37.74 and 39.58 °C, with accuracy (p < 0.05) of 98% for SCM and 95% for CM, surpassing both TAT and TSST. Therefore, the combination of these non-invasive methods increases the reliability and accuracy of infrared thermography for early detection of SCM, providing valuable insights for the development of a protocol for routine screening and udder health monitoring in indigenous dairy cows.

Injection of antioxidant trace minerals/vitamins into peripartum crossbred cows improves the nutritional and immunological properties of colostrum/milk and the health of their calves under heat stress conditions.

Multimineral and vitamin injections can provide better nutrient availability at the cellular level, which is essential for mitigating transition period stress and improving the wellbeing and productivity of dairy cows. The present study was conducted to assess the colostrum quality and calf health after intramuscular injection of multi-minerals (MM) and multi-vitamins (MV) to peripartum cows during winter (THI = 58 to 66) and summer (THI = 78 to 82) months. In each season, twenty-four pregnant crossbred Karan Fries cows were grouped into four, each consisting of six cows. Group I, referred to as the Control, received solely the basal diet, without any additional supplements. Groups II, III, and IV were administered additional MM (T1), MV (T2), and a combined MM and MV (T3) along with their basal diet, starting 30 days before calving and continuing for 30 days after calving. Blood samples were collected from the calves, while colostrum/milk samples were obtained from the cows on days 1, 3, 7, and 15 after calving. The somatic cell counts (SCC) in the milk were determined using a cell counter. Cortisol, IgG, IGF1 and total immunoglobulins (TIG) in whey and plasma from cow colostrum/milk or calf blood samples were estimated by ELISA. Cows that calved in the summer exhibited notably reduced levels (P < 0.05) of IgG, milk, and plasma IGF1, along with lower calf body weights, in comparison to those calving in the winter season. Furthermore, the summer months saw significant increases (P < 0.05) in plasma and milk cortisol levels, as well as total somatic cell counts (SCC) in both colostrum and milk samples. Maximum beneficial effect was observed in T3 group. Results indicate that injections to peripartum cows could be an important strategy for improving colostrum quality and calf health during the summer seasons.

Allelic diversity at BoLA DRB3 locus and association with predisposition to clinical mastitis in indicus and crossbred cattle.

Bovine lymphocyte antigen (BoLA) DRB3 locus in healthy and mastitis affected cattle has been genotyped by a polymerase chain reaction and restriction fragment length polymorphisms (PCR-RLFP) using RsaI restriction enzyme, followed by sequencing. In 130 farm animals, 25 BoLA DRB3 alleles have been detected by PCR-RFLP. Three distinct allelic patterns significantly associated with mastitis in Karan Fries crossbred and Sahiwal indicus cattle have been identified, whereas, four other allelic patterns were significantly high in frequency among healthy animals. Sequencing of RFLP genotypes revealed 25 and 47 alleles among healthy Sahiwal and Karan Fries, respectively, while 17 and 38 patterns observed in mastitis affected Sahiwal and Karan Fries animals, respectively. From Tajima's D-test of neutrality, it was concluded that alleles associated with mastitis were expanding in the population, whereas those of healthy were under contraction. Phylogenetic analysis carried out to delineate the evolutionary relationship of the farm and field animals at DRB3 locus, differentiating allelic patterns into six different clusters. Among the phylogenetic lineages, five patterns DRB3*028:01, DRB3*011:03, DRB3*031:01, DRB3*001:01 and DRB3*043:01, were previously reported, whereas one novel allelic variant was observed in indicus and crossbred cattle. This information will help in further exploring the association between BoLA-DRB3 genetic diversity and disease resistance in distinct cattle breeds, important in designing breeding strategies for increasing the distribution of favorable alleles.

JAK3 and PI3K mediate bovine Interferon-tau stimulated gene expression in the blood neutrophils.

Interferon tau, a 23 kDa trophoblast derived protein diffuses out from the uterus into the circulation and leads to the expression of IFNτ stimulated genes viz. ISG15 and OAS1 in blood neutrophils. The IFNτ pathway is species as well as tissue specific. To unsnarl the IFNτ downstream signaling pathway, the blood neutrophils were incubated simultaneously with 10 ng/ml of recombinant bovine interferon tau and the inhibitors of JAK2 (AG490), JAK3 (CP690550), p38 (SB202190), PI3K/Akt (LY294002), and MAPK/Erk (U0126) at specific doses for 4-hr duration. The IFNτ pathway was determined through real-time gene expression of ISG15 and OAS1; immunocytochemistry of ISG15; and Western blotting of ISG15, OAS1, pJAK3 and PI3K. The ISG15 and OAS1 expression decreased significantly (p < 0.001) in the presence of pJAK3 and PI3K inhibitors as compared to a positive control where only interferon tau was used. Immunocytochemistry revealed an attenuated ISG15 response while stimulating blood neutrophils with pJAK3 inhibitor (CP690550) and PI3K inhibitor (LY294002). Similarly, Western blot analysis of neutrophil protein fraction showed weak signals of ISG15, OAS1, pJAK3 and PI3K in the presence of pJAK3 and PI3K inhibitors. The expression profile, immunocytochemistry and western blot analysis revealed a JAK3 and PI3K mediated interferon-tau stimulated gene expression in blood neutrophils.

Pathogen-dependent modulation of milk neutrophils competence, plasma inflammatory cytokines and milk quality during intramammary infection of Sahiwal (Bos indicus) cows.

The aim of the current study was to investigate the responses of milk neutrophils and plasma inflammatory cytokines to various mastitis pathogens and subsequently on milk composition. Milk was collected from healthy (n = 10) and clinical mastitis indigenous Sahiwal cows naturally infected either with gram-positive bacteria mainly S. aureus (n = 10) and Strep. agalactiae (n = 10) or with gram-negative bacteria, E. coli (n = 10). Phagocytic activity of milk neutrophils decreased in all mastitis cows with the lowest values recorded during gram-positive bacterial infections. Maximum plasma cortisol levels were observed in cows infected with gram-positive bacteria and were positively correlated with the milk neutrophils percentage and negatively correlated with the phagocytic activity of neutrophils and expression of glucocorticoid receptor. The plasma concentrations of IL-2 and IL-8 increased in all mastitis groups with maximum values recorded during E. coli infections. Unlike gram-negative bacterial infections, gram-positive bacterial infections evoked a minimal tumor necrosis factor-α (TNF-α), and IL-6 response. Milk somatic cell counts, fat, protein, pH and electrical conductivity increased in mastitis cows with the highest values exhibited by Strep. agalactiae infection. The expression of chemokine receptors (CXCR1, CXCR2), IL-8 and CD11b was maximum in mastitis neutrophils infected with E. coli. The expression of glucocorticoid receptor (GRα) decreased in all mastitis groups with the lowest values were found in S. aureus infection. Among the various mastitis pathogens, Strep. agalactiae showed maximum adverse effect on milk quality. Attenuated neutrophils, TNF-α and IL-6 response in cows infected by gram-positive bacteria may contribute to the establishment of chronic mastitis.

Impact of different seasons on the milk somatic and differential cell counts, milk cortisol and neutrophils functionality of three Indian native breeds of cattle.

The present study was undertaken to compare the effect of different seasons on the mammary immunity of three Indian native breeds of cows (Tharparkar, Gir and Sahiwal) well adapted to the tropical region. For this milk samples were collected from cows in winter (THI=57, comfortable zone), hot-dry (HD; THI=76, heat stressful zone) and hot-humid (HH; THI = 82, severe heat stress) and estimated for milk somatic cell counts (SCC), phagocytic activity (PA) of milk neutrophils, milk cortisol and heat shock proteins and function associated genes in milk neutrophils. Milk SCC was evaluated using a cell counter and differential cell counts measured microscopically. Cortisol was quantified in skimmed milk by competitive ELISA. Milk PA was estimated using nitro blue tetrazolium assay, and for gene expression studies, milk neutrophils were isolated and studied for heat shock proteins (HSP40, HSP70, HSP90α) and cell adhesion molecules (CD11b, CD25, CD44) using real-time polymerase chain reaction. All the studied parameters increased in HD and HH seasons with highest values observed in Sahiwal cows. However, PA of neutrophil was highest in Tharparkar cows in winter and decreased gradually at higher THI values during hot seasons. Milk cortisol was positively correlated with expression of various CD molecules and HSPs (p < 0.05) in milk neutrophils but negatively correlated (p < 0.05) with PA during HH season in all breeds. The study revealed that Indian native cows were at considerable risk in HH season and Sahiwal cows were more heat stressed followed by Gir and Tharparkar cows, respectively, and thus may require managemental interventions. Also, the higher expression of HSP70 and CD25 with increasing THI levels in hot seasons makes them suitable biological markers for quantifying heat stress.

Functional characterization of Mammary Gland Protein-40, a chitinase-like glycoprotein expressed during mammary gland apoptosis.

MGP-40 is a chitinase-like protein which is over expressed during mammary gland involution. However, its physiological function in the mammary gland is poorly understood. In the present investigation, we have reported the functional significance of buffalo specific MGP-40 in the mammary gland by using an in vitro model of the buffalo mammary epithelial cell (BuMEC) line. MGP-40 was highly up regulated in BuMECs in serum starved condition as well as after treatment with prolactin suggesting its role in the stress response. Subsequently, to study the effect of MGP-40 on BuMECs, the cells were transfected with a mammalian expression construct of pCI neo harboring MGP-40 gene. It was observed that over expression of MGP-40 enhanced proliferation of BuMECs and protected the cells from apoptosis under serum free condition. In contrast, MGP-40 attenuated the mitogenic effect of insulin in BuMECs. Besides, over expression of the MGP-40 reduced dome formation, acinar polarization and casein synthesis in BuMECs in the presence of lactogenic hormones, it also induced Stat3 phosphorylation and epithelial to mesenchymal transition (EMT) -like features. Together, our data suggest that MGP-40 is involved in protection of BuMECs under stress conditions, inhibits cellular differentiation and induces EMT-like features. A schematic diagram depicting possible association of MGP-40 in various molecular pathways has been presented.

Effect of micronutrient supplementation on the immune function of crossbred dairy cows under semi-arid tropical environment.

This study assessed the effect of micronutrient supplementation around peripartum period on immune function, reproductive performance, milk yield and milk quality of crossbred cows. Thirty pregnant crossbred cows in their late gestation were selected and randomly divided into five groups for study. Six cows in each group were supplemented with vitamin E (VE) (2000 IU/cow/day), vitamin A (VA) (100,000 IU/cow/day), copper (Cu) (20 ppm/cow/day), zinc (Zn) (80 ppm/cow/day) individually from 45 days pre-calving to 45 days post-calving and one group without any supplementation served as control. Immune function was studied by in vitro phagocytic activity (PA) of blood neutrophils, lymphocyte proliferation response (LPR) and plasma interleukin-8 (IL-8) concentration. Supplementation of VA significantly (P < 0.05) increased the in vitro PA of blood neutrophils and decreased milk somatic cell counts (SCC). Zn supplementation significantly (P < 0.05) increased the T lymphocyte proliferation response, whereas B lymphocyte LPR was significantly (P < 0.05) increased with both VA and Zn supplementation as compared to the control cows. Plasma IL-8 concentration was significantly (P < 0.05) higher in all supplemented cows. Supplementation of VE, VA and Zn significantly (P < 0.05) reduces days open, whereas VA significantly (P < 0.05) reduced the service per conception. In this study, it is concluded that VE, VA and Zn supplementation around peripartum period can boost the immunity and improve the reproductive performance of crossbred cows in a semi-arid tropical environment.

Comparative Profiling of Milk Somatic Cells Proteomes Revealed Key Players in Mammary Immune Mechanisms During Mastitis in Tropical Sahiwal (Bos indicus) Cows.

PURPOSE: Bovine mastitis poses a significant economic burden on the dairy industry worldwide. This pioneering proteomic study conducted a comparative profiling of milk somatic cell (SC) proteins contributing to mammary immune defense during subclinical and clinical mastitis (CM) in Sahiwal (Bos indicus) cows. EXPERIMENTAL DESIGN: Based on California mastitis test (CMT) scores, milk SC counts, differential leukocyte counts (DLCs), and bacteriological culture results, quarter milk SC samples were categorized into healthy (H), subclinical mastitis (SCM), and CM groups. Comparative proteome profiling of milk SCs was done using a label-free liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic approach. RESULTS: The identified upregulated proteins in mastitis groups such as Vanin 2, Thioredoxin reductase-like selenoprotein T, Ceramidase, Lymphocyte antigen 75, Misshapen-like kinase 1 (MINK1), Thrombospondin 1, Macrophage scavenger receptor 1, Leupaxin, and Lipoamide acyltransferase, involved in immune responses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed immune functions and pathways like antigen processing, complement cascades, extracellular matrix receptor interaction, efferocytosis, leukocyte migration, chemokine, peroxisome proliferator-activated receptors (PPARs), and transforming growth factor (TGF)-beta signaling. CONCLUSIONS AND CLINICAL RELEVANCE: These findings provide essential information on proteomic profiling in milk SCs and contribute valuable insights into immune-related proteins regulated during mastitis in dairy cows. Further, validated proteins (Vanin 2, MINK1, and Thrombospondin 1) offer potential inflammatory biomarkers for early mastitis detection in dairy cows.

Correlation of udder thermogram and somatic cell counts as a tool for detection of subclinical mastitis in buffaloes.

This study aimed to monitor the mammary health of 37 multiparous Murrah buffaloes through infrared thermography (IRT). Based on the California Mastitis Test (CMT) and milk somatic cell counts (SCC), buffaloes were grouped into healthy (H, n = 16), subclinical mastitis (SCM, n = 10), and clinical mastitis (CM, n = 11). Buffaloes were milked twice daily in the morning (5:00-6:00 AM) and evening (5:00-6:00 PM). Rectal temperature and respiratory rates were recorded, CMT was performed and thermal images of the mammary gland of all the buffaloes were taken before and after each milking. Milk samples were analysed after each milking for SCC, fat, Solids-Not-Fat (SNF), density, protein, lactose, salts, conductivity, and pH immediately in the laboratory from fresh milk samples. The surface temperature of the periocular region of both the eyes, muzzle, flank, and vagina were also taken. Thermal images were used to assess the surface temperature of the udder (USST), teat apex (TAT), teat barrel (TB1T), teat base (TB2T), and teat skin surface (TSST). Eye and USST showed significantly higher temperatures (p < 0.05), whereas skin surface temperatures (SST) of different body parts were non-significant in both SCM and CM animals than buffaloes in the H group. Milk SCC showed a positive correlation with conductivity (r > 0.7), salts, and pH (r < 0.6) and a negative correlation with fat, SNF, density, protein, and lactose. TAT, TB1T, TB2T, TSST, and USST were positively correlated with milk SCC. Receiver Operating Characteristic (ROC) analysis of H and SCM groups showed that USST before milking had optimum sensitivity (Se = 0.80) and specificity (Sp = 0.906) among the various skin temperatures recorded. Thermal images captured during the morning showed higher sensitivity compared to images taken in the evening. Results indicate IRT can be used to monitor the mammary health of buffaloes but using IRT in conjunction with milk SCC can help in the accurate prediction of SCM in dairy buffaloes.

Effect of micronutrient supplementation around calving on the plasma cortisol levels of Murrah buffaloes and Sahiwal and Karan Fries cows.

Micronutrients when fed around peripartum may reduce the stress induced by cortisol. With this objective, 24 Sahiwal (SW) and 24 Karan Fries (KF) cows and 24 Murrah (Mu) buffaloes were taken and divided into four groups of six each. Vitamin E (VE), zinc (Zn) and copper were supplemented from 30 days pre- to 30 days postcalving in groups 1, 2 and 3. Animals without supplementation served as control. Blood sampling was done on days 30, 15, 7 and 3 precalving; at calving; and on days 3, 7, 15 and 30 postcalving. Plasma cortisol levels were measured by ELISA. Supplementation of VE and Zn significantly (P<0.05) reduced plasma cortisol levels at calving and at 30 days postcalving. KF cows exhibited best results with VE, whereas SW cows and Mu buffaloes responded best to Zn. Copper increased the peripartum cortisol levels. Reduction in cortisol levels may help in keeping the animal healthy and stronger to fight immuno-suppression generally observed around the period of peripartum.

Repeated injection of multivitamins and multiminerals during the transition period enhances immune response by suppressing inflammation and oxidative stress in cows and their calves.

Periparturient dairy cows undergo major physiological and metabolic changes as well as immunosuppression, associated with decrease in plasma concentrations of various minerals and vitamins. The present study was conducted to investigate effects of repeated injections of vitamins and minerals on oxidative stress, innate and adaptive immune response in periparturient dairy cows and their offspring. Experiment was carried out on 24 peripartum Karan-Fries cows, randomly divided into four groups (n=6): control, Multi-mineral (MM), Multi-vitamin (MV) and Multi-minerals and Multi-vitamin (MMMV). Five ml of MM (Zinc 40 mg/ml, Manganese 10 mg/ml, Copper 15 mg/ml, Selenium 5 mg/ml) and five ml of MV (Vitamin E 5 mg/ml, Vitamin A 1000 IU/ml, B-Complex 5 mg/ml, and Vitamin D3 500 IU/ml) were injected intramuscularly (IM) to the MM and MV groups. MMMV group cows were injected with both. In all treatment groups, injections and blood sampling were carried out on 30th, 15th, 7th days before and after expected date of parturition and at calving. In calves, blood was collected at calving and on 1, 2, 3, 4, 7, 8, 15, 30 and 45 days post-calving. Colostrum/milk were collected at calving and at days 2, 4, and 8 post-calving. A lower percentage of total neutrophils and immature neutrophils, higher percentage of lymphocytes together with increased phagocytic activity of neutrophils and proliferative capacity of lymphocytes found in blood of MMMV cows/calves. Lower relative mRNA expression of TLRs and CXCRs and higher mRNA expression of GR-α, CD62L, CD11b, CD25 and CD44 found in blood neutrophils of MMMV groups. Total antioxidant capacity was higher, activity of antioxidant enzymes (SOD and CAT), TBARS levels were lower in the blood plasma of treated cows/calves. In both cows/calves, plasma pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, IL-8, IL-17A, IFN-γ and TNF-α) increased, whereas anti-inflammatory cytokines (IL-4 and IL-10) decreased in MMMV groups. Total immunoglobulins increased in colostrum/milk of MMMV injected cows and plasma of their calves. Results indicate that repeated injections of multivitamins and multiminerals to peripartum dairy cows could be a major strategy to improve immune response and decrease in inflammation and oxidative stress in transition dairy cows and their calves.

Indoleamine 2, 3-dioxygenase 1 mediated alterations in the functionality of immune cells, deciphers the pregnancy outcomes in crossbred dairy cows.

Pregnancy establishment in bovines requires maternal immune cell modulation. Present study investigated possible role of immunosuppressive indolamine-2, 3-dioxygenase 1 (IDO1) enzyme in the alteration of neutrophil (NEUT) and peripheral blood mononuclear cells (PBMCs) functionality of crossbred cows. Blood was collected from non-pregnant (NP) and pregnant (P) cows, followed by isolation of NEUT and PBMCs. Plasma pro-inflammatory (IFNγ and TNFα) and anti-inflammatory cytokines (IL-4 and IL-10) were estimated by ELISA and analysis of IDO1 gene in NEUT and PBMCs by RT-qPCR. Neutrophil functionality was assessed by chemotaxis, measuring activity of myeloperoxidase and ß-D glucuronidase enzyme and evaluating nitric oxide production. Changes in PBMCs functionality was determined by transcriptional expression of pro-inflammatory (IFNγ, TNFα) and anti-inflammatory cytokine (IL-4, IL-10, TGFß1) genes. Significantly elevated (P < 0.05) anti-inflammatory cytokines, increased IDO1 expression, reduced NEUT velocity, MPO activity and NO production observed only in P cows. Significantly higher (P < 0.05) expression of anti-inflammatory cytokines and TNFα genes were observed in PBMCs. Study highlights possible role of IDO1 in modulating the immune cell and cytokine activity during early pregnancy and may be targeted as early pregnancy biomarkers.

Pregnancy stage-dependent modulation of neutrophil function may impact embryo survivability and pregnancy outcome in crossbred cows.

Pregnancy is a complicated physiological process that involves synchronized coordination between immune and endocrine systems. Neutrophils have been suggested as a critical immune cell for embryo implantation and pregnancy maintenance. The present study was conducted to evaluate the dynamic changes in the mRNA expressions of the cluster of designation (CD11b, CD31, CD44 and CD62L) molecules and interferon-stimulated genes (ISG15, MX1 and OAS1) in blood neutrophils throughout pregnancy in dairy cows and correlate them with the outcome of pregnancy. Blood samples were taken from negative control (NC) group, and non-pregnant (NP) group at the time of artificial insemination (AI, day zero) and on days 10, 14, 16, 18, and 21 post-AI. In pregnant (P) cows, samples were taken as described above and after every 30 days until the time of parturition. In aborted cows, samples were collected until the time of the abortion. Comparison between pregnant, non-pregnant and aborted cows revealed that the expression of CD molecules increased (p < 0.05) on days 14, 16, 18 and 21 post-AI only in NP cows as compared to other groups. Although the expression of CD molecules remained constant throughout the study period in pregnant and aborted cows, the expression of CD11b, CD31 and CD62L increased (p < 0.05) on the day of abortion and parturition. Unlike CD molecules, the expression of CD44 decreased significantly (p < 0.05) at the time of abortion. There was a significant (p < 0.05) increase in the expression of interferon-stimulated genes including MX1, OAS1 and ISG15 during the peri-implantation period in pregnant cows, and at the time of abortion in aborted cows. However, the expression of ISGs was lower (p < 0.05) in non-pregnant cows as compared to the other groups. The results revealed the critical role played by neutrophils during pregnancy and form the basis to unravel the underlying mechanism for neutrophil associated immunological infertility in bovines.

JAK3 and PI3K mediates the suppressive effects of interferon tau on neutrophil extracellular traps formation during peri-implantation period.

Interferon tau (IFNτ) is the main maternal signal for pregnancy in ruminants and modulates the functions of various immune cells, including neutrophils. Neutrophil extracellular traps (NETs) are one of the main defence mechanisms of neutrophils. In this study, we observed higher (p < 0.01) ex-vivo NETs extrusion by blood neutrophils from day 16-18 post artificial insemination (AI) in non-inseminated and inseminated non-pregnant cows compared to pregnant cows. In vitro study also showed that IFNτ hampers NETs formation in dose and time dependent manner. The lowest (p < 0.01) NETs formation and the highest (p < 0.01) mRNA expression (RT-PCR) of IFNτ stimulated genes (ISG15, OAS1, MX1) were observed when neutrophil incubated with 9 ng/mL IFNτ for 3.5 h. Signalling cascades mediating IFNτ impairment of NETs formation were identified using inhibitors of JAK2, JAK3, p38, PI3K/Akt and MAPK/Erk. IFNτ reduced (p < 0.01) the mRNA expression (RT-PCR) and concentration (ELISA) of genes and proteins that mediate NETs formation in blood neutrophils including histones (H1, H2), neutrophil elastase (NE) and myeloperoxidase (MPO). However, the effects of IFNτ on these genes and proteins were eliminated in the presence of JAK3 or PI3K inhibitors. Immunocytochemistry study also showed strong MPO signal in the presence of JAK3 or PI3K inhibitors as compared to positive control (PC, IFNτ alone). The results indicate that IFNτ impairs NETs formation using JAK3 and PI3K and thus essential for successful implantation and establishment of pregnancy in cows.

A Comparative Study on Changes in Total and Differential Milk Cell Counts, Activity, and Expression of Milk Phagocytes of Healthy and Mastitic Indigenous Sahiwal Cows.

Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription-polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 103 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.

Peripartum changes in the activity and expression of neutrophils may predispose to the postpartum occurrence of metritis in dairy cows.

Metritis is a postpartum uterine pathology that causes a huge economic loss due to increased culling risk and impaired milk yield and reproduction in cows. The present study was carried out to study the changes in the activity and expression of blood neutrophils in crossbred dairy cows with and without metritis. Collection of blood samples was done at -3, -2 and - 1 weeks before calving, at calving and during the first day of metritis diagnosis in metritis group (n = 8) or at day 8-10 post calving in healthy group (n = 8). Neutrophils were studied for its percentage (microscopically), respiratory burst (nitro blue tetrazolium assay), myeloperoxidase (MPO) concentrations (sandwich ELISA) and expression of CXCR1, CXCR2, TLR2, TLR4, GRα, CD11b, CD14, CD25, CD44, CD47 and CD62L (RT-PCR). Immunocytochemistry was used to investigate MPO concentration and CD14 activity, and western blotting was used for estimating MPO. Although most of these parameters changed in the cows that developed metritis one week before calving, MPO and CD14 got altered much earlier. Myeloperoxidase concentrations and expression of CD14 were considerably lower starting from -2 weeks before calving in cows that developed metritis compared to healthy cows. Further studies are warranted to study the possible use of MPO and CD14 to identify transition cows more vulnerable to develop metritis several weeks before disease occurrence.

Supplementation of antioxidant micronutrients reduces stress and improves immune function/response in periparturient dairy cows and their calves.

BACKGROUND: Periparturient period induces stress in cows which fluctuates hormonal and metabolic function and causes immune suppression. Apart from impairing the health, production, and reproduction of cows, it also influences the well-being of newborn calves by decreasing the colostrum quality. Micronutrients are known for optimal health and production and their effects on parturition stress, immune response in both cow and its calf need to be explored. AIM: The aim of this study was to see the effect of oral supplementation of micronutrients during the prepartum period on the health status of crossbred dairy cows and subsequently on their newborn calves. METHODS: A total of 42 healthy multiparous cows were selected and randomly divided into five groups with seven cows in each group, i.e. control (Basal Diet, BD), VA group (BD + vitamin A, 105 IU), Zn group (BD + zinc sulphate, 60 ppm), VE group (BD + vitamin E, 2500 IU), and combined supplementation (CS) group (BD + combination of VA, Zn, and VE). The supplements were offered in compounded concentrate DM (100 g) to individual cows once daily before the morning feeding and the remaining portion was incorporated in the TMR. Feeding was started one month before the expected days of calving till calving. Blood samples were collected from cows at days -15, -7, -3, 0, +3, +7, and +15 relative to the day of calving. Blood samples from newborn calves and milk samples of cows were collected at days 0, +3, +7, and +15. Milk somatic cell counts (SCC) were estimated using a cell counter. Cortisol was estimated by ELISA kit in blood and milk plasma of cows and in the blood plasma of their calves. Total immunoglobulins (Ig) were estimated in milk of cows and serum of calves using zinc sulphate turbidity method. Blood neutrophils from cows and calves were studied for phagocytic activity (PA) using nitro blue tetrazolium (NBT) assay.Data were analysed by repeated-measures two-way ANOVA using the mixed procedure of SAS, and the pairwise comparison was performed using a multiple comparison test (Tukey). RESULTS: Combined supplementation of micronutrients decreased (P < 0.05) maternal blood plasma (control vs. CS group, 5.98 ±â€¯0.20 vs. 3.86 ±â€¯0.23 ng/mL) and milk plasma (3.96 ±â€¯0.13 vs. 2.71 ±â€¯0.10 ng/mL) cortisol, milk SCC (3.05 ±â€¯0.11 vs. 2.12 ±â€¯0.10 × 105 cells/mL) and increased (P < 0.05) total milk Ig concentration (18.80 ±â€¯0.11 vs. 23.04 ±â€¯0.57 mg/mL) and the PA of blood neutrophils (0.84 ±â€¯0.03 vs. 1.07 ±â€¯0.03). Similarly, lower blood cortisol concentration (9.69 ±â€¯0.35 vs. 6.02 ±â€¯0.18 ng/mL) and higher (P < 0.05) total Ig (23.26 ±â€¯0.11 vs. 30.34 ±â€¯0.70 mg/mL) and PA of blood neutrophils (0.37 ±â€¯0.02 vs. 0.52 ±â€¯0.02) were observed in the calves born to CS group of cows as compared to the control. Highest (P < 0.05) positive effects (lower stress levels and higher immune response) of treatment were noticed in CS group followed by VE group and then Zn group. However, VA group didn't differ from the control group. CONCLUSION: Our results indicate that micronutrient interventions during the prepartum period can improve the health status of dairy calves and subsequently the well-being of their calves.

Interaction between stress hormones and phagocytic cells and its effect on the health status of dairy cows: A review.

Dairy cows are exposed to various stressors during their production cycle that makes them more susceptible to various diseases. Phagocytes (neutrophils and macrophages) are important soldiers of the innate immune system. Neutrophils are the first responders to an inflammatory response and stress and kill pathogens by generating reactive oxygen species and by the release of various antimicrobial peptides, enzymes, neutrophil extracellular trap formation, etc. Macrophages, the other phagocytes, are also the cleanup crew for the innate immune system that removes debris, pathogens, and dead neutrophils later on after an inflammatory response. The neuroendocrine system along with phagocytes exhibits an immunomodulatory potential during stressful conditions. Neuroendocrine system directly affects the activity of phagocytes by communicating bidirectionally through shared receptors and messenger molecules such as hormones, neurotransmitters, or cytokines. Different immune cells may show variable responses to each hormone. Short time exposure to stress can be beneficial, but repeated or extended exposure to stress may be detrimental to the overall health and well-being of an animal. Although some stresses associated with farming practices in dairy cows are unavoidable, better understanding of the interactions occurring between various stress hormones and phagocytic cells can help to reduce stress, improve productivity and animal welfare. This review highlights the role played by various stress hormones in modulating phagocytic cell performance of dairy cattle under inflammatory conditions.