Upregulation of interferon-stimulated genes and interleukin-10 in peripheral blood immune cells during early pregnancy in dairy cows

In cows, interferon-tau (IFNT) regulates maternal recognition around days 15-19 after artificial insemination (AI). The present study hypothesized that if key target genes of IFNT are clearly upregulated in earlier stages of pregnancy, these genes could be use as indices of future pregnancy in cows. Therefore, we determined the expression of these genes in peripheral blood mononuclear leukocytes (PBMCs) and polymorphonuclear granulocytes (PMNs) during the maternal recognition period (MRP). Twenty multiparous Holstein cows were subjected to AI on day 0 and categorized into the following groups: pregnancy (Preg, n = 9), embryonic death (ED, n = 5) and non-pregnancy (NP, n = 6). Progesterone levels in the Preg group were higher than those in the NP group on days 12-21. ISG15 and OAS-1 (IFN-stimulated genes: ISGs) mRNA in PBMCs on day 8 was higher in the Preg group than in the NP group, and these mRNAs in PMNs was higher in the Preg group on day 5 than in the NP and ED groups. Interleukin-10 (IL-10, Th2 cytokine) mRNA expression increased on day 8 in the PBMCs of pregnant cows. Tumor necrosis factor α (TNFα, Th1 cytokine) mRNA expression was stable in all groups. In an in vitro cell culture experiment, IFNT stimulated mRNA expression of ISGs in both PBMCs and PMNs. IFNT stimulated IL-10 mRNA expression in PBMCs, whereas IFNT increased TNFα mRNA levels in PBMCs in vitro. The results suggest that ISGs and IL-10 could be responsive to IFNT before the MRP in peripheral blood immune cells and may be useful target genes for reliable indices of pregnancy before the MRP.     

Equine herpesvirus type-1 modulates CCL2, CCL3, CCL5, CXCL9, and CXCL10 chemokine expression

Equine herpesvirus type 1 (EHV-1) is highly prevalent in horses and causes rhinopneumonitis, abortion, and encephalopathy. Studies on the related human herpes simplex virus and of murine models of EHV-1 suggest that chemokines play important roles in coordinating of innate and adaptive immune responses, and thus effective control of herpesvirus infection and prevention of severe clinical disease. Here, equine peripheral blood mononuclear cells (PBMC) were infected with one of three EHV-1 strains, which differ in pathogenicity (RacL11, NY03=abortogenic, Ab4=neurogenic). Changes in CCL2, CCL3, CCL5, CXCL9 and CXCL10 chemokine gene expression relative to non-infected PBMC were measured by real-time PCR. CXCL9 and CXCL10 gene expression was up-regulated 10h post infection and decreased to the level of non-infected cells after 24h. CCL2 and CCL3 were significantly down-regulated 24h post infection with NY03 and Ab4. CCL5 was up-regulated 24h after infection with RacL11. Ab4 infected PBMC had significantly lower expression of all chemokines except CCL2 24h post infection then RacL11 infected cells. While there was not a significant difference between NY03 and the other strains, there was a trend with each chemokine toward NY03 inducing less expression then RacL11 but more then Ab4. The data suggested that EHV-1 infection of PBMC induced up-regulation of inflammatory chemokines CCL5, CXCL9 and CXCL10, and down-regulation of chemotactic CCL2 and CCL3. The data also implies that different EHV-1 strains have varying effects on all five chemokines, with the nuropathogenic strain, Ab4, having the greatest suppressive potential.     

Oxidative status during late pregnancy and early lactation in dairy cows

In the last few years, the detection of free radical damage and the body's defences against it have become increasingly important in clinical medicine as a complementary tool in the evaluation of metabolic status. The aim of this study was to evaluate, under field conditions, the anti-oxidant status of healthy cows during late pregnancy and lactation onset using two parameters: (1) plasma levels of malondialdehyde (MDA), a degradation product of lipid peroxidation, and (2) total antioxidant status (TAS). Results were compared with those obtained in another group of cows with lesser metabolic demands. We also investigated possible relationships between antioxidant status markers and other relevant blood parameters. Our results confirmed the characteristic metabolic changes associated with late pregnancy and early lactation. MDA and TAS provided an accurate reflection of the internal physiological status of the animal. The data indicated increased lipid peroxidation around parturition, but with wide individual variations that may be attributable not only to the physiological stage but also to unknown factors that will have to be further considered in future studies.     

Apoptosis and necrosis of blood and milk polymorphonuclear leukocytes in early and midlactating healthy cows.

Increased milk somatic cell counts (SCC) are used as an indicator for bovine mastitis. During mastitis, polymorphonuclear leukocytes (PMN) become the predominant cell type. Shortly after parturition, the severity of mastitis is increased and several PMN functions are downregulated. Apoptotic and necrotic processes of PMN could influence SCC and PMN functions. In this study, the percentages of apoptotic and necrotic PMN in blood and milk from early and midlactating healthy cows were compared. Apoptosis and necrosis of PMN were quantified using a dual-color flow cytometric procedure with fluorescein labeled annexin-V (green) and propidium iodide (red). Using this technique three different subpopulations of bovine PMN could be detected: apoptotic cells (high intensive green fluorescence), necrotic cells (high intensive green and high intensive red fluorescence) and viable cells (low intensive green and low intensive red fluorescence). Following a 4 h incubation of blood from both groups of cows at 37 degrees C to induce apoptosis, the mean percentage of apoptotic blood PMN was significantly higher (P < 0.01) in early lactating cows (15.1%, n = 9) compared with midlactating cows (5.3%, n = 10). The mean percentage of necrotic PMN remained lower than 5% in all cows. In contrast to blood, no significant difference was found between the percentage of apoptotic PMN in milk from early (41.2%, n = 7) and midlactating cows (34.0%, n = 8). The percentage of necrotic PMN in milk from early lactating cows (25.9%, n = 7) was significantly higher than that in midlactating cows (14.2%, n = 8) (P < 0.05). Higher percentages of apoptotic as well as necrotic PMN were consistently found in milk compared to blood in all cows. From these results, it can be concluded that spontaneously induced apoptosis was higher in blood PMN from early lactating cows than in blood PMN from midlactating cows. The higher percentage of necrotic milk PMN in early lactating cows than in midlactating cows could be explained by the induction of secondary necrosis.     

荷斯坦奶牛妊娠早期血浆差异蛋白质组学研究

采用二维凝胶电泳(2-DE)结合MADIL-TOF-TOF串联质谱法对荷斯坦奶牛妊娠早期血浆进行蛋白质组学分析鉴定,发现了6种差异蛋白在妊娠后表达量上调,分别是凝集素1前体、转甲状腺素蛋白前体、3,5-二碘水杨酸络合的牛血清白蛋白的晶体结构的A链、白蛋白、未命名蛋白产物及结合珠蛋白-25同源物1前体。对这6种蛋白在不同时期含量的变化趋势及可能引起每种蛋白含量变化的原因和在妊娠过程中的功能进行了分析。     

Variations in blood composition in dairy cows during pregnancy and after calving

The effects of pregnancy and of the onset of lactation on blood composition was studied in 21 Friesian cows. Among the 23 components studied only seven showed significant variations. Serum iron decreased at the end of pregnancy while creatinine increased throughout the last six months. There were decreases in blood glucose, cholesterol and alanine aminotransferase at the end of pregnancy. Triglycerides increased rapidly after drying-off and serum urea increased in the first month after calving.     

Comparison of commercial ELISA blood tests for early pregnancy detection in dairy cows

The objective of the present study was to compare two commercially available blood-based pregnancy tests, namely BioPRYN, an ELISA for pregnancy-specific protein B (PSPB), and an ELISA for pregnancy-associated glycoprotein (PAG), for early pregnancy diagnosis in dairy cattle using transrectal ultrasonography as a gold standard. Transrectal ultrasonography was conducted 26-58 days after artificial insemination (AI) in 197 cattle from 19 farms. Concurrently, a blood sample was collected for determination of serum PSPB and PAG. Transrectal palpation was performed approximately 120 days after AI to verify that pregnancy was maintained. For PSPB and PAG, there were no significant differences (P>0.05) in sensitivity (98.0 and 97.8%), specificity (97.1 and 91.2%), positive predictive values (99.3 and 97.8%), negative predictive values (91.9 and 91.2%) and accuracy (97.8 and 96.4%). In conclusion, the two blood pregnancy assays were equally efficacious and were highly accurate (based on transrectal ultrasonography as the gold standard).     

A decrease in blood beta-carotene in dairy cows during late pregnancy period

In four successive seasons of green and winter feeding in the years 1980-1984, beta-carotene concentrations were determined in the plasma of venous blood of 3441 cows in the first phase of lactation, in the other phase of lactation, in the eighth month of pregnancy and in the 9th-9.5th months of pregnancy. The cows in the other phase of lactation had the significantly highest beta-carotene concentrations in the two feeding seasons. In the eighth month of pregnancy there occurs a significant drop that continues also in the 9th-9.5th months of pregnancy. In the first phase of lactation beta-carotene concentrations remained at the level of the values from the 9th-9.5th months of pregnancy. In the period of winter feeding, the mean concentrations of beta-carotene in all groups of cows were deep below the limit value of 7.44 mumol . l-1, which indicated that it was necessary to fortify feeds with vitamin A; in the cows in the 9th-9.5th months of pregnancy and in the cows in the first phase of lactation they were lower than the critical value of 5.58 mumol.l-1, which signaled the disorders of reproduction processes. In the course of the years of investigation beta-carotene concentrations varied in the same manner in all groups of cows in keeping with the vegetation period of the feed, feed kind and quality.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene expression profiling of peripheral mononuclear cells in lame dairy cows with foot lesions

Lameness is a major health issue and likely the single most common cause of pain and discomfort in dairy cattle. Appropriate treatment is delayed or neglected due, in part, to lack of reliable detection. Assessment of cows with lameness is currently limited to subjective visual scoring systems based on locomotion and posture abnormalities. These systems are unreliable to detect lameness, and therefore, a large number of cows remain undiagnosed. The objective of this research was to search for potential biomarkers for lameness-associated painful inflammatory foot lesions in dairy cattle using microarray-based gene expression profiling of peripheral blood mononuclear cells (PBMC). BOTL5 microarrays spotted in duplicate with cDNA representing bovine immune response genes were interrogated with cDNA samples in an eight-array, balanced complete block design with dye swap. Samples from eight lame cows with inflammatory foot lesions and from eight sound cows were pair-matched by age, weight, days in lactation, and pregnancy status at time of PBMC collection and directly compared with each other on individual arrays. Statistical analysis of resulting fluorescence intensity data revealed 31 genes that were putatively differentially expressed in lame versus sound cows (P < 0.05). Of these, BLASTn analysis and gene ontology information showed that 28 genes had high similarity or homology to known human and/or rodent genes. Validation of 15 of these genes known to be important in inflammation and pain was carried out using relative quantitative real-time RT-PCR, which confirmed the up-regulation of interleukin (IL)-2 (12.68 ± 1.47-fold increase) and IL-10 (2.39 ± 0.55-fold increase), matrix metalloproteinase-13 (MMP-13) (10.44 ± 1.14-fold increase), and chemokine C–C motif receptor-5 (CCR5) (5.26 ± 1.05-fold increase), in lame relative to sound cows (P ≤ 0.05). Similarly, granulocyte-macrophage colony-stimulating factor receptor alpha chain precursor (GM-CSF-R-alpha) (2.30 ± 0.63-fold increase) and IL-4 (2.06 ± 0.59-fold increase) showed a tendency (P = 0.10) for up-regulation in lame compared to sound cows. PBMC co-expression of IL-2, MMP-13, CCR5 and IL-10, and potentially IL-4 and GM-CSF-R-alpha appears to be a promising, objective sign of lameness-related inflammatory foot lesions in dairy cattle. In conclusion, this study revealed potential biomarkers of the presence of foot lesions that could boost diagnostic accuracy of lameness and, ultimately, help identify animals in need of pain relief.     

Relationship between plasma progesterone and pregnancy-associated glycoprotein concentrations during early pregnancy in dairy cows

The relationship between the concentration of plasma progesterone (P4) during embryo attachment or at recognition of pregnancy, and that of pregnancy-associated glycoprotein (PAG) was assessed in dairy cows. The outcome of artificial insemination (AI) was classified as positive (AI+), negative (AI?), or late embryonic mortality (EM) by measuring circulating PAG concentrations and by ultrasonography. Based on P4 concentrations at either day 21 or day 15, AI+ and EM cows were classified into ‘low’ (P4 concentrations < mean) and ‘high’ (P4 concentrations > mean) P4 groups. In both experiments, the threshold of P4 concentration between the ‘low’ and ‘high’ groups was approximately 6 ng/mL. PAG concentrations were lower in the ‘low’ group only when P4 concentrations were below the threshold. The study findings suggest that a possible P4 threshold exists below which PAG secretion may be impaired.     

Collateral uterine blood flow in Holstein cows during the third trimester of pregnancy

Blood flow to the gravid and nongravid uterine horns of four multiparous Holstein cows (mean +/- SD, BW=641.8 +/- 95.4 kg; age=4.8 +/- 1.2 years; parity=3.0 +/- 1.2) was measured on days 225, 248, and 266 of gestation. Surgery was conducted on day 214.5 +/- 4.0 of gestation through the flank of the standing cows. Transit-time ultrasonic flow probes (diameter 12 or 14 mm) were fitted surgically around the uterine arteries of each cow. Surgery was completed within two hours of anesthesia, and the animals recovered rapidly following surgery. Uterine blood flow (UBF, l/min) was recorded at 10 sec intervals for approximately 23.5 hours; these values were averaged to determine UBF. The mean gravid UBF was significantly (P<0.05) greater than the nongravid UBF in this study. The range of the gravid and nongravid UBFs varied from 3.61 to 14.05 and 0.72 to 6.54 l/min, respectively. There were no changes (P>0.1) in the mean gravid and nongravid UBFs from day 225 to 266 of gestation.     

Changes in some blood micronutrients, leukocytes and neutrophil expression of adhesion molecules in periparturient dairy cows.

Dairy cows are highly susceptible to infectious diseases, like mastitis, during the period around calving. Although factors contributing to increased susceptibility to infection have not been fully elucidated, impaired neutrophil recruitment to the site of infection and changes in the concentrations of some micronutrients related with the function of the immune defence has been implicated. Most of the current information is based on studies outside the Nordic countries where the conditions for dairy cows are different. Therefore, the aim of the study was to evaluate changes in blood concentrations of the vitamins A and E, the minerals calcium (Ca), phosphorous (P), and magnesium (Mg), the electrolytes potassium (K) and sodium (Na) and the trace elements selenium (Se), copper (Cu) and zinc (Zn), as well as changes in total and differential white blood cell counts (WBC) and expression of the adhesion molecules CD62L and CD18 on blood neutrophils in Swedish dairy cows during the period around calving. Blood samples were taken from 10 cows one month before expected calving, at calving and one month after calving. The results were mainly in line with reports from other countries. The concentrations of vitamins A and E, and of Zn, Ca and P decreased significantly at calving, while Se, Cu, and Na increased. Leukocytosis was detected at calving, mainly explained by neutrophilia, but also by monocytosis. The numbers of lymphocytes tended to decrease at the same time. The mean fluorescent intensity (MFI) of CD62L and CD18 molecules on blood neutrophils remained constant over time. The proportion of CD62L+ neutrophils decreased significantly at calving. The animals were fed according to, or above, their requirements. Therefore, changes in blood levels of vitamins, minerals and trace elements were mainly in response to colostrum formation, changes in dry matter intake, and ruminal metabolism around calving. Decreased levels of vitamins A and E, and of Zn at calving might have negative implications for the functions of the immune defence. The lower proportion of CD62L+ neutrophils at calving may result in less migration of blood neutrophils into the tissues, and might contribute to the increased susceptibility to infections at this time.     

Long‐term impact of puerperal metritis on the profiles of peripheral blood leukocytes in peripartum dairy cows

To determine the effects of puerperal metritis on the immune response, changes in the differential peripheral blood leukocyte counts were analyzed during the peripartum period in cows with or without metritis. Multiparous Holstein cows were examined for uterine health disorders and classified into two groups: healthy (n = 11) or metritis (n = 5) cows. The lymphocyte and monocyte counts and the proportion of CD8⁺ lymphocytes were higher in cows with metritis compared to healthy cows. Moreover, the effects of puerperal metritis on the lymphocyte counts and CD4⁺/CD8⁺ ratio persisted weeks after the uterine inflammation had self‐resolved. Taken together, the findings of the present study indicate the possible long‐term alterations of systemic immune responses in cows with puerperal uterine inflammation. © 2015 Japanese Society of Animal Science     

Gene expression profiles of CD4/CD8 double‐positive T cells in porcine peripheral blood

A characteristic subset of T cells, known as double positive T cells (DPTC) and expressing both cluster of differentiation 4 (CD4) and CD8, is observed in porcine peripheral blood. Previous studies suggested that DPTC might be memory cells. However, detailed phenotypes and functions of DPTC are yet to be fully elucidated and thus, the relatedness of DPTC with memory phenotypes remains unclear. In this study, DPTC gene expression profiles in peripheral blood were analyzed by DNA microarray in Experiment 1 and compared with those of CD4 single positive T cells (4SPTC) and CD8 single positive T cells (8SPTC). Expressions of IFNG, CCL5, NCK2, CCR2 and ITGB1 were higher than that of 4SPTC and 8SPTC. In contrast, expressions of CCR7 and SELL were lower than that of 4SPTC and 8SPTC. These results suggested that DPTC were either effector T cells or effector memory T cells (T_EM). Next, to determine whether DPTC were effector T cells or T_EM, differences in the response of DPTC and 8SPTC against immunized/primed antigens were compared (Experiment 2). While DPTC showed quick elevation of IL2 and CD25 gene expressions against in vitro stimulation of primed/immunized antigens, 8SPTC did not. These results suggest that at least some DPTC likely belong to T_EM.     

Cytochemical demonstration of esterases in peripheral blood leukocytes.

Cytochemical methods for alpha naphthyl acetate esterase and chloroacetate esterase have been used to identify human monocytes and granulocytes. In this study, a standard procedure for staining alpha naphthyl acetate and chloroacetate esterase activities was modified by extending the range of pH of the incubation mixture and the duration of staining and was applied to cat, dog, goat, guinea pig, hamster, human, pig, rabbit, rat, and sheep leukocytes. The results for both enzymes showed (1) incubation time and pH had discrete effects on staining and (2) species differences for in vitro conditions to demonstrate esterase activity were pronounced.