Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period.

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-ß (IL-1ß), Interleukin 6 (IL-6) and Tumor Necrosis Factor-α (TNF-α), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors.

Bovine innate immune phenotyping via a standardized whole blood stimulation assay.

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system-the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1ß and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1ß, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49-0.61) and between neutrophils and cytokine responses to R848 (0.38-0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.

Effects of an oral hydro-ethanolic purple coneflower extract on performance, clinical health and immune parameters in calves.

The objective of this randomized, placebo-controlled, double-blinded field trial was to investigate the effects of oral administration of purple coneflower (Echinacea purpurea L. (EP)) on performance, health and immune parameters in calves. Calves (n = 27) were enrolled to three groups (9 calves per group): 0.5 g EP/calf per day (ECL), 5 g EP/calf per day (ECH) or placebo. Calves were vaccinated with Bluetongue-Virus (BTV) serotype 4 vaccine to investigate EPs effects on seroconversion. Clinical and performance parameters, inter alia body weight, health and milk intake were recorded for 57 days. Blood samples were analyzed for BTV antibodies and IgG by ELISA, white and red blood cell counts by flow cytometry and mRNA abundance of various inflammatory markers in leukocytes (IL-1ß, IL-8, tumor necrosis factor α (TNFα), cyclooxygenase 2 (Cox-2) and prostaglandin E synthase) was studied. The findings demonstrated no differences between groups regarding performance parameters. In all groups, calves suffered from diarrhea for a minimum of 2 days, but EP reduced the number of diarrhea days by 44% in ECL and increased the body temperature. Interestingly, ECL resulted in an increased number of respiratory disease days during the follow-up period. EP did not change blood cell and IgG counts, whereas eosinophil granulocytes were reduced in ECL. Decreased levels of hemoglobin and hematocrit were found in ECH. Prostaglandin E synthase levels in leukocytes were higher in ECL and ECH, whereas no differences were obtained for IL-1ß, IL-8, TNFα and Cox-2. Due to the unexpected occurrence of BTV seropositive calves before the first vaccination, 13 calves were excluded from the evaluation on seroconversion and no statistical analyses could be performed regarding antibody production. BTV-4 antibodies were not produced in 4 placebo-calves, whereas 4 of 5 and 1 of 6 ECL- and ECH-calves produced antibodies. Further investigations are needed to draw final conclusions on mode of action and efficacy of EP in calves.

Innate and humoral immune parameters at delivery in colostrum and calves from heifers experimentally infected with Neospora caninum.

Neospora caninum is a leading cause of abortion in cattle worldwide. The study of the immune response against N. caninum is critical to understand its epidemiology, pathogenesis, diagnosis and, ultimately, in preventing and controlling bovine neosporosis. Herein, we determined the gene expression of innate immune components endosomal RNA-sensing TLRs, BMAP28 cathelicidin, TNF-α and IL-10 and characterized the variation in both IgG ratio and avidity at delivery in N. caninum-infected heifers challenged at day 210 of gestation, colostrum and their calves. Increased BMAP28 expression was observed not only in colostrum but also in peripheral blood mononuclear cells (PBMC) and umbilical cord of calves from N. caninum-infected heifers in comparison with mock-infected control group. In addition, statistically significant decrease of TLR7 and IL-10 expression levels were observed in umbilical cord, suggesting an attempt to avoid an exacerbated immune response against the parasite. At delivery, serum and colostrum samples from infected group evidenced specific IgG anti-N. caninum. Infected heifers showed IgG1/IgG2 ratios <1 and high avidity specific IgG. As expected, colostrum samples of these animals exhibited a high IgG1 concentration and elevated avidity values. Three out of four calves from N. caninum-infected heifers had specific IgG with IgG1/IgG2 ratios>1 and lower avidity values before colostrum intake. Interestingly, both IgG1/IgG2 ratios and avidity values increased in seropositive calves after colostrum intake. Overall, this study provides novel information on neonatal immunity in congenitally infected calves, which is essential to understand how the immune pathways could be manipulated or immune components could be employed in order to improve protection against neosporosis.

Astragalus root extract improved average daily gain, immunity, antioxidant status and ruminal microbiota of early weaned yak calves.

BACKGROUND: Early weaning in yak calves is being attempted to improve yak reproduction rate. However, this has to be done with caution because of the high mortality rate of calves due to the lack of nutrients and the harsh environmental conditions. Twenty-four weaned male yak calves were used in a 60 day feeding trial in which astragalus root extract (ARE) was supplemented. They were assigned randomly to one of four dietary treatments (n = six per treatment) that differed in ARE level: 0 g kg-1 (control), ARE0 ; 20 g kg-1 , ARE20 ; 50 g kg-1 , ARE50; and 80 g kg-1 dry matter intake (DMI), ARE80 . RESULTS: Final bodyweight and average daily gain (ADG) were significantly higher and the DMI/ADG ratio was significantly lower in calves with ARE supplementation than control (ARE0 ) calves. Ruminal concentrations of acetate and propionate and serum concentration of superoxide dismutase in ARE80 calves were higher than in the other groups and serum concentration of insulin was higher in ARE80 calves than in ARE20 calves. Serum immunoglobulin G (IgG) and interleukin-2 (IL-2) concentrations in ARE-fed calves were higher than in controls. Serum tumor necrosis factor (TNF-α) concentration was higher in ARE50 and ARE80 groups than ARE0 calves and serum interleukin-6 (IL-6) concentration was higher in ARE80 than in ARE0 calves. Serum immunoglobulin A (IgA), IgG and immunoglobulin M (IgM) concentrations increased with age in ARE-fed calves. ARE supplementation increased the abundance of fiber degrading bacteria. CONCLUSION: ARE at a dosage of 5% to 8% DMI can be supplemented to early weaned yak calves to improve growth performance, antioxidant capacity and immunity. © 2020 Society of Chemical Industry.

IL1B triggers inflammatory cytokine production in bovine oviduct epithelial cells and induces neutrophil accumulation via CCL2.

PROBLEM: The oviduct is essential for reproduction. We previously showed that oviduct epithelial cells (OECs) isolated from aged cows expressed higher levels of inflammatory cytokines, including interleukin (IL) 1A and IL1B. In addition, aging is associated with tissue dysfunction and cellular senescence via a senescence-associated secretory phenotype (SASP) and immune cell accumulation. We investigated whether IL1A or IL1B causes SASP production, cellular senescence, and inflammatory responses in bovine OECs. METHOD OF STUDY: The OECs were isolated from bovine oviducts from young (mean 50.3 months) and aged cows (mean 157.0 months) and cultured. RESULTS: Treatment with IL1A or IL1B induced SASP production (IL8, IL6, TNFA, and CCL2) and mRNA expression of cell adhesion molecules in bovine OECs, but both IL1s did not induce cellular senescence in OECs and migration of polymorphonuclear neutrophils (PMNs). Cultured medium of OECs treated with IL1s, especially IL1B, dramatically induced PMN migration. Treatment with the CCL2 inhibitor, but not IL8 or its receptor CXCR2 inhibitors, significantly reduced immune cell migration in IL1B-treated OEC-cultured medium. Treatment with IL1B increased PMN adhesion to OECs, resulting in further SASP production in OECs due to a PMN-OEC interaction. CONCLUSION: We suggest that senescence-associated IL1s cause SASP production in bovine OECs and CCL2 induced by IL1B is essential for the migration of immune cells to OECs. Specifically, IL1B regulates PMN migration and adhesion to bovine OECs, and PMNs accelerate inflammatory cytokine production from bovine OECs via a direct interaction. These phenomena may contribute to chronic oviductal inflammation, resulting in subfertility.

Spontaneous poisoning by Cestrum intermedium in dairy cattle / Intoxicação espontânea por Cestrum intermedium em bovinos de leite

Acute hepatotoxicity caused by plants poisoning is responsible for economic losses in farm animals in Brazil. Reports of Cestrum intermedium natural poisoning in cattle are not commonly described in Rio Grande do Sul (RS). This study aimed to document an outbreak of spontaneous C. intermedium poisoning in dairy cattle in the Central-Eastern Mesoregion of RS. Three nine-month-old Holstein and Jersey heifers were affected after they were placed in a small paddock with shortage forage. In this area, specimens of C. intermedium Sendtn with signs of consumption were observed. Morbidity and lethality rates were 100% and clinical courses ranged from 9 to 12 hours. At post mortem examination of the three heifers, there was predominance of acute liver lesions. The liver was moderately enlarged and on the cut surface there was a marked accentuation of the lobular pattern and hemorrhage. Inside the rumen, partially digested C. intermedium Sendtn leaves were observed. The histological aspects of the liver were mostly centrilobular coagulative necrosis and hemorrhage, frequently extended to the midzonal region. The immunohistochemistry technique was performed, in which the polyclonal antibody caspase 3 was used in liver fragments. Moderate to marked immunolabeling was observed in the cytoplasm and nucleus of hepatocytes, predominantly on the periphery of areas of hepatic necrosis indicating cell apoptosis. The diagnosis of C. intermedium Sendtn poisoning in dairy cattle in this study was based on epidemiological, clinical and anatomopathological findings. Since the C. intermedium poisoning is uncommon in dairy cattle, we are describing it for the first time in the Central-Eastern Mesoregion of RS, and represents a differential diagnosis of other acute toxic liver diseases in cattle.(AU)

Intoxicações por plantas que causam hepatotoxicidade aguda são responsáveis por prejuízos econômicos em animais de produção no Brasil. Relatos de intoxicações naturais por Cestrum intermedium em bovinos não são comumente descritos no Rio Grande do Sul (RS). Este trabalho teve como objetivo documentar um surto de intoxicação espontânea por C. intermedium em bovinos leiteiros ocorrido na região Centro-Oriental do RS. Três bovinos com nove meses de idade, das raças Holandês e Jersey, foram acometidos depois de serem introduzidos em um pequeno piquete com escassez de forragem. Nessa área foram observados exemplares de Cestrum intermedium Sendtn com sinais de consumo. As taxas de morbidade e letalidade foram de 100% e os cursos clínicos variaram de 9 a 12 horas. Na necropsia dos três bovinos, observou-se predomínio de lesões hepáticas agudas. O fígado dos animais acometidos estava moderadamente aumentado de tamanho e na superfície de corte havia marcada acentuação do padrão lobular e hemorragia. Na abertura do rúmen, foram observadas folhas de Cestrum intermedium Sendtn parcialmente digeridas. Histologicamente, o fígado apresentava necrose coagulativa centrolobular e hemorragia. Frequentemente, essas alterações se estendiam para a região mediozonal. Foi realizada a técnica de imuno-histoquímica, na qual utilizou-se o anticorpo policlonal caspase 3 em fragmentos de fígado. Notou-se marcação moderada a acentuada em citoplasma e núcleo de hepatócitos, predominantemente na periferia das áreas de necrose hepática indicando apoptose celular. O diagnóstico de intoxicação por Cestrum intermedium Sendtn nos bovinos leiteiros desse estudo, foi realizado com base nos achados epidemiológicos, clínicos e anatomopatológicos. A intoxicação por C. intermedium é incomum em bovinos leiteiros, sendo descrita pela primeira vez na região centro oriental do RS e representa um diagnóstico diferencial de outras hepatopatias tóxicas agudas.(AU)

Intramammary inoculation with lactic acid bacteria at dry-off triggers an immunomodulatory response in dairy cows.

The use of antibiotics to prevent bovine mastitis is responsible for the emergence and selection of resistant strains. Lactic acid bacteria (LAB) could be introduced into animal feed as an alternative prevention method that would bypass the risk of resistance development. In previous research, we demonstrated that two probiotic LAB strains isolated from bovine milk were capable of stimulating the production of antibodies and the host's immune cellular response in the udder. The present study aimed to elucidate whether the antibodies of animals inoculated with these strains were able to increase phagocytosis by neutrophils and inhibit the growth of different mastitis-causing pathogens. Moreover, the effect of LAB on the expression of pro-inflammatory cytokines was assessed. Ten animals were inoculated intramammarily with 106 cells of the two strains at dry-off. The blood serum was tested for its ability to opsonize bovine mastitis pathogens, the in vitro bactericidal activity of bovine blood and milk against these pathogens was determined, and cytokine mRNA expression was quantified in milk somatic cells. The inoculated animals did not show abnormal signs of sensitivity to the LAB. Their blood serum significantly enhanced the phagocytosis of Staphylococcus spp. and the LAB. Escherichia coli and Streptococcus uberis were inhibited by the milk serum but not the blood serum, whereas Staphylococcus aureus and Staphylococcus haemolyticus were inhibited by both. In regard to cytokine expression, interleukin (IL)-1ß increased markedly for up to 4 h post-inoculation, and an increase in IL-8 was observed 4, 12 and 24 h after inoculation. Tumour necrosis factor-α mRNA increased 1 and 2 h after inoculation and a significant difference was registered at 6 h for interferon-γ. This rapid immunomodulatory response shows that inoculating animals with LAB at dry-off, when they are especially susceptible, could be a useful strategy for the prevention of bovine mastitis.

Effect of Lonicera japonica extract on lactation performance, antioxidant status, and endocrine and immune function in heat-stressed mid-lactation dairy cows.

Here, we examined the effects of Lonicera japonica extract (LJE) on lactation performance, antioxidant status, and endocrine and immune function in heat-stressed mid-lactation dairy cows. Twenty-four healthy Chinese Holstein mid-lactation dairy cows, all with similar milk yield (30.0 ± 1.0 kg/d), parity (2.5 ± 0.3), and days in milk (105 ± 5 d) were allocated to 4 groups using a randomized complete block design: a negative control group (without LJE supplementation; CON) and groups that received LJE at 14, 28, and 56 g/d. The experiment lasted 10 wk over a hot summer, with a pre-feeding period of 2 wk. Cows were exposed to heat stress, as the average temperature-humidity index was greater than 72. The results showed that LJE had no effect on respiration rate; however, it reduced the rectal temperature of dairy cows experiencing heat stress in both a linear and quadratic manner; the lowest (39.03°C) was recorded for the LJE-28 group, lower than the CON group. Supplementation with LJE did not affect dry matter intake, milk yield, or milk composition. The majority of biochemical parameters in serum were unaffected by supplementation with different amounts of LJE; the exception was creatinine, which was reduced quadratically. Compared with the CON group, serum triiodothyronine concentrations increased significantly in the LJE-28 group. Addition of LJE to the diet increased thyroxine concentrations quadratically; values peaked at 18.62 ng/mL in the LJE-28 group. Furthermore, supplementation with increasing amounts of LJE quadratically increased the activity of glutathione peroxidase and total antioxidant capacity in serum but decreased concentration of malondialdehyde. Although we detected no differences in the concentrations of IgA, IgM, or cytokines, dairy cows in the LJE-28 group had higher IgG and IL-4 concentrations than did cows in the CON group. Supplementation with LJE increased concentrations of IgG and IL-4 in the serum quadratically but decreased that of IL-2. Finally, heat shock protein 72 concentrations in the serum tended to fall quadratically as the amount of LJE increased. In summary, LJE had no negative effects on lactation performance but helped to alleviate heat stress by improving antioxidant status and promoting endocrine and immune functions. Supplementation with LJE at 28 g/d is recommended for lactating dairy cows experiencing heat stress during hot summers.

The Freemartin Cattle and Clinical Transplantation: From the Ancients to Modern Day.

This historical retrospective explores the study of the freemartin condition and its impact on the discovery of immunologic tolerance and the field of transplant surgery-from the ancient Romans, to early modern anatomists Valsalva, Scarpa, and Hunter, to contemporary immunologists Owen, Medawar, and Billingham, and to legendary transplant surgeon Joseph Murray. The legacy of freemartin cattle in the understanding of acquired tolerance and transplant immunology represents generations of scientific inquiry guided by careful observation and occasional serendipity, and the present-day immunologists and surgeons exploring immune transplant tolerance owe much to the history of the freemartin, several millennia in the making.

Effects of lipopolysaccharide exposure in primary bovine ruminal epithelial cells.

The objective of this study was to investigate whether cultured ruminal epithelial cells (REC) responded to lipopolysaccharide (LPS) stimulation and determine whether LPS induced a proinflammatory response. Primary bovine REC were isolated and grown in culture for 2 studies. In study 1, REC were isolated from Holstein bull calves (n = 8) and grown in culture for 10 to 12 d. Cells were then exposed to 0, 10,000, 50,000, or 200,000 endotoxin (E)U/mL of LPS (Escherichia coli O55:B5) for either 6 or 24 h. The effect of LPS exposure on cell viability was analyzed by flow cytometry using a propidium iodide stain. In study 2, cells were isolated from Holstein bull calves (n = 5) and yearling beef heifers (n = 4). Cells were exposed to either 1,000 or 50,000 EU/mL of LPS using the following conditions: (1) medium alone time-matched controls, (2) 12-h LPS exposure, (3) 24 h of LPS exposure, (4) 36 h of LPS exposure, (5) 12 h of LPS exposure followed by LPS removal for 24 h before restimulating with LPS for an additional 12 h (RPT), and (6) 12 h of LPS exposure followed by LPS removal for 36 (RVY). For both experiments, total RNA was extracted from REC and real-time quantitative PCR was performed to determine relative expression of genes for toll-like receptors (TLR2 and TLR4), proinflammatory cytokines (TNF and IL1B), chemokines (CXCL2 and CXCL8), a lipid mediator (PTGS2), and growth factor-like cytokines (CSF2 and IL7). In study 1, LPS exposure did not negatively affect cell viability. Treatment of cells with LPS resulted in increased transcript abundance for all genes analyzed. The TLR2, IL7, and TLR4 had a greater magnitude of change at 6 h compared with 24 h. Quadratic expression patterns were detected for TNF, IL1B, CXCL2, CXCL8, and CSF2. These results suggested that REC increase expression of proinflammatory genes following exposure to LPS. In study 2, all genes analyzed were upregulated in a quadratic manner following exposure to LPS for different time intervals. The TLR4, TNF, CXCL2, CXCL8, CSF2, and IL7 gene expression was significantly greater after a single 12 h of LPS exposure than after RPT exposure, suggesting repeated exposure of REC to LPS may induce a tolerogenic effect. When LPS was removed from the medium (RVY), transcript abundance for all genes analyzed decreased and expression of TLR2, TLR4, and IL7 returned to baseline levels, suggesting REC recovered following exposure to LPS. Overall, the data suggest cultured REC respond to LPS stimulation by increasing transcription of proinflammatory genes and this transcriptional response was influenced by the dose, duration, and frequency of LPS exposure.

Effects of prepartum zinc-methionine supplementation on feed digestibility, rumen fermentation patterns, immunity status, and passive transfer of immunity in dairy cows.

The aim of this study was to determine the effects of prepartum supplementation of zinc-methionine (Zn-Met) on feed digestibility, rumen fermentation patterns, and immunity status in dams and passive immunity transfer in their calves. A randomized complete design was used in this study. Forty multiparous Holstein dairy cows in late pregnancy (60 d before the expected calving date) were blocked by parity (2.1 ± 0.3), body weight (651 ± 52 kg), and expected calving date, and randomly assigned to 1 of 4 treatments. Cows were supplemented with Zn as Zn-Met at 0, 20, 40, or 60 mg/kg of dry matter (DM) from 60 d before expected calving date to the calving day. Though the nutrient digestibility was not affected by Zn supplementation, DM intake, Zn digestibility, and Zn deposition increased linearly with increasing Zn-Met supplementation. Ruminal pH and molar proportion of individual volatile fatty acids were similar, whereas a linear decrease and increase were observed in ruminal ammonia and microbial crude protein concentration, respectively, with increasing Zn-Met supplementation. Maternal serum concentration of alkaline phosphatase, carboxypeptidase, Cu and Zn superoxide dismutase, and total antioxidant capacity were greater in cows supplemented with >40 mg of Zn/kg of DM compared with the control group. With increasing Zn-Met supplementation, maternal blood concentration of IL-1 decreased linearly, whereas IL-2 and IL-6 increased linearly, and no differences were observed in IL-4. Concentration of nonesterified fatty acids and ß-hydroxybutyric acids in maternal blood was similar between treatments. No difference was observed in colostrum composition with increasing Zn-Met supplementation. Concentration of Zn and immunoglobulins (including IgA, IgG, and IgM) in maternal blood did not differ among treatments. However, Zn concentration in colostrum and blood of calves increased linearly. The concentration of IgA and IgM in colostrum increased linearly with increasing Zn-Met supplementation, whereas no differences in immunoglobulins were observed in calf blood. In conclusion, Zn supplementation as Zn-Met at 40 of mg/kg of DM may improve antioxidant activity of dam and potentially increase passive immunity transfer in calves.

Intrabreed and interbreed variation of the BOLA-DRB3.2 gene in the Kostroma and Yaroslavl indigenous Russian cattle breeds.

Intrabreed and interbreed variation of BOLA-DRB3 exon 2 (BOLA-DRB3.2) was for the first time studied in the Kostroma and Yaroslavl cattle breeds by PCR-RFLP. These breeds are among the best Russian breeds and were developed as dairy-beef and dairy cattle, respectively. Twenty-nine alleles were observed in five Kostroma samples, and 14 of them proved unique in comparison with two Yaroslavl samples, in which 25 alleles were detected, and 10 of them were unique. The total frequency of bovine leukemia virus (BLV) resistance alleles (*11, *23, and *28) was 23.2% in the Kostroma, while the total frequency of BLV susceptibility alleles (*8, *16, *22, *24) was low, 8.4%. The frequencies were 25.8 and 30.1%, respectively, in Yaroslavl cattle. Testing Hardy-Weinberg equilibrium revealed a significant deficit of heterozygotes: the observed (Ho) and expected (He) heterozygosities were, respectively, 0.734 and 0.859 in Kostroma cattle and 0.613 and 0.886 in Yaroslavl cattle. The intrabreed differentiation (FST) in the Kostroma (4.5%, P = 0.001) was substantially higher than in the Yaroslavl (0.5%, P = 0.158), between the two breeds was 8.2% (P = 0.001). The Bayesian clustering approach showed an intrabreed structure for each of the breeds, with the most probable number of clusters being 2 in the Kostroma and 3 in the Yaroslavl. The structure observed in the Kostroma remained the same when the breed was analyzed together with six additional breeds. Our data provide important clues toward the understanding of the genetic structure of indigenous breeds.

Roadmap to pregnancy in the first 7 days post-insemination in the cow: Immune crosstalk in the corpus luteum, oviduct, and uterus.

The first 7 days post-insemination are critical for establishment of pregnancy. The pre-ovulatory luteinizing hormone (LH) surge induces ovulation through disruption of the follicle structure that elucidates pro-inflammatory (Th1) responses. Various types of immune cells are recruited into the corpus luteum (CL) to regulate luteal angiogenesis and progesterone (P4) secretion into the circulation to establish pregnancy. The active sperm-uterine crosstalk also induces Th1 responses, mainly via Toll-like receptor (TLR) 2/4 signaling pathway in vitro. The endometrial glands serve as sensors for sperm signals, which trigger Th1 responses. Conversely, the sperm-oviduct binding generates anti-inflammatory (Th2) responses to support sperm survival until fertilization. It is well-established that embryo-maternal crosstalk starts after the embryo hatches out from the zona pellucida (ZP). However most recently, it was shown that the 16-cell stage bovine embryo starts to secrete interferon-tau (IFNT) that induces Th2 immune responses in the oviduct. Once developing embryos descend into the uterine horn, they induce Th2 responses with interferon-stimulated genes (ISGs) expression in the uterine epithelium and local immune cells mainly via IFNT release. Likewise, multiple embryos in the uterus of superovulated donor cows on D7 post-insemination induce Th2 immune responses with ISGs expressions in circulating immune cells. These findings strongly suggest that the maternal immune system reacts to the embryo during the first 7 days post-insemination to induce fetal tolerance. It became evident that the innate immunity of the developing CL, oviduct, and uterus works together to provide optimal conditions for fertilization and early embryonic development during the first 7 days post-insemination.

Effects of n-3 fatty acids on growth, antioxidant status, and immunity of preweaned dairy calves.

This study aimed to estimate the influences of flax oil and a mixture of ethyl esters of fatty acids from flax oil added to a milk replacer (MR) formulation on the biochemical, immunological, and antioxidant status of blood, production parameters, and health of calves. Twenty-seven Holstein-Frisian calves were assigned to 3 groups: the control group without fat added to the diet (CON, n = 9), a group receiving ethyl esters of flax oil (10 g/d) with lyophilized apples (25 g; MRE), or a group receiving flax oil (10 g/d) with lyophilized apples (25 g; MRL). The study was conducted from d 14 to d 42 of life. Intake of MR and feed were recorded daily, and fecal scores, rectal temperature, and body weight were recorded weekly. Laboratory blood tests were conducted every 7 d from d 14 to 42. Supplementation with the formulation containing ethyl esters of flax oil (MRE) positively affected health, average daily weight gain, growth rate, and feed efficiency. Intake of the starter feed was lower in the calves receiving the formulation with flax oil (MRL). The MRE formulations lowered the concentrations of triglycerides, total cholesterol, and LDL fraction in blood serum and decreased the total antioxidant capacity (TAS) and levels of glutathione peroxidase (GPx). The MRL formulation increased TAS and GPx levels in blood serum. The MRE treatment limited the circulating levels of tumor necrosis factor. The MRE and MRL preparations lowered the concentration of SFA (C16:0, C18:0, C18:1) in blood serum, and MRE significantly increased levels of α-linolenic acid. The MRE and MRL preparations beneficially affected production and physiological parameters. Our data support the idea that supplementation with n-3 fatty acids improves calf growth and metabolic and oxidative functions in young calves.

Effect of prepartum energy intake and supplementation with ruminally protected choline on innate and adaptive immunity of multiparous Holstein cows.

Objectives were to evaluate the effect of prepartum energy intake and peripartal supplementation of ruminally protected choline (RPC) on select indicators of immune status in blood plasma and on lipopolysaccharide-stimulated blood cells ex vivo. At 47 ± 6 d before the expected calving date, 93 multiparous Holstein cows were assigned randomly to 1 of 4 dietary treatments in a 2 × 2 factorial arrangement. Cows were fed energy to excess [EXE; 1.63 Mcal of net energy for lactation (NEL)/kg of dietary dry matter (DM)] or to maintenance (MNE; 1.40 Mcal of NEL/kg of dietary DM) ad libitum throughout the nonlactating period. The RPC was fed at 0 or 60 g/d to supply 0 or 12.9 g/d of choline ions top-dressed for 17 ± 4.6 d prepartum through 21 d postpartum. After calving, cows were fed the same methionine-supplemented diet, apart from RPC supplementation. During the last 2 wk before calving and during the first 5 wk postpartum, blood was sampled repeatedly and analyzed for cell types, acute-phase proteins, tumor necrosis factor-α (TNFα), and neutrophil function. Samples of whole blood were collected at 3 and 14 DIM and stimulated with 1 µg/mL lipopolysaccharide (LPS) in vitro for 6 and 24 h. After 6 h of LPS exposure, peripheral blood leucocytes (PBL) were harvested, and relative transcript abundance for select cytokines were measured. Supernatant was analyzed for TNFα after 24 h of LPS exposure. The PBL from cows fed EXE diets during the whole dry period had increased transcripts for the proinflammatory cytokines CXCL8 and TNF, although the plasma concentrations of the acute-phase proteins haptoglobin and fibrinogen, and the killing activity of the blood neutrophils in the postpartum period, were not affected by feeding different energy levels prepartum. Feeding RPC to cows overfed energy prepartum modulated their inflammatory state, as evidenced by decreased IL6 in PBL and reduced mean fluorescence intensity of CD14 during the postpartum period, compared with cows not fed RPC. Feeding RPC also decreased TNFα protein production, abundances of IL1B, CXCL8, and TNF transcripts, and mean fluorescence intensity of CD80 of PBL stimulated by LPS, regardless of prepartum energy intake. In contrast, proportions of blood neutrophils undergoing phagocytosis and oxidative burst were increased at 17 d postpartum in cows supplemented with RPC. Collectively, these data indicate that transition cows supplemented with RPC experienced less inflammation, which may partially explain increased milk production in cows supplemented with RPC.

Effects of feed restriction and supplementary folic acid and vitamin B12 on immune cell functions and blood cell populations in dairy cows.

Cows undergoing a negative energy balance (NEB) often experience a state of immunosuppression and are at greater risk of infectious diseases. The present study aimed to evaluate the impact of a folic acid and vitamin B12 supplement and feed restriction on several immune parameters. Sixteen cows at 45 ± 3 days in milk were assigned to 8 blocks of 2 cows each according to each cow's milk production in the previous week, and within each block, the cows randomly received weekly intramuscular injections of either saline or 320 mg of folic acid and 10 mg of vitamin B12 for 5 weeks. During week 5, the cows were fed 75% of their ad libitum intake for 4 days. Blood samples were taken before the beginning of the experiment, just before feed restriction and after 3 days of feed restriction, in order to evaluate blood cell populations, the phagocytosis capacity and oxidative burst of polymorphonuclear leukocytes (PMNs), the proliferation of peripheral blood mononuclear cells (PBMCs) and concentrations of non-esterified fatty acids (NEFAs) and ß-hydroxybutyrate. The vitamin supplement did not affect any of the tested variables except milk fat and lactose content. Feed restriction reduced milk production and increased the concentration of NEFAs. Feed restriction did not affect blood cell populations but did reduce the percentage of PMN positive for oxidative burst after stimulation with phorbol 12-myristate 13-acetate. The proliferation of PBMCs was reduced when the cell culture medium was supplemented with sera collected during the feed restriction. In conclusion, feed restriction affected the functions of PMN and PBMC and this effect was not prevented by the folic acid and vitamin B12 supplement. These results support the hypothesis that the greater risk of infectious diseases in cows experiencing a NEB is related to impaired immune cell functions by high circulating concentration of NEFAs.

Effect of Holstein genotype on innate immune and metabolic responses of heifers to lipopolysaccharide (LPS) administration.

Heifers (n = 4/genotype) from unselected (stable genotype since 1964, UH) and contemporary (CH) Holsteins that differed in milk yield (6,200 and 11,100 kg milk/305 d) were used to assess the impact of selection on innate immune and acute-phase response to an endotoxin (lipopolysaccharide; LPS). Jugular catheters were implanted 24 h before LPS administration. Blood samples were collected at -1, -0.5, 0, 1, 2, 3, 4, 6, 8, and 24 h relative to iv administration of 0.5 µg LPS/kg BW. Rectal body temperature (BT) was determined at these sampling times and at 5 and 7 h. Dermal biopsies were collected after the 24 h blood sample and processed to isolate fibroblasts. Plasma was analyzed for tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), serum amyloid A (SAA), xanthine oxidase (XO), and nitrate + nitrite (NOx), cortisol, glucose, and IGF-1 content. Isolated fibroblasts were exposed to IL-1ß or LPS and IL-6 and IL-8 content of culture media determined. Exposure to LPS increased BTs and plasma concentrations of TNF-α, IL-6 SAA, XO, cortisol, and glucose (P < 0.05) in both genotypes. Plasma concentrations of TNF-α, XO, NOx, and glucose did not differ (P > 0.25) between the genotypes, but IL-6 and SAA concentrations were reduced (P < 0.05) in CH relative to UH heifers while cortisol and IGF-1 concentrations tended (P < 0.08) to be reduced in CH heifers. After 36 h exposure to LPS, concentrations of IL-6 were greater (P < 0.05) in culture media from incubations of CH than UH fibroblasts but concentrations of IL-8 did not differ between genotypes. There was a trend (P = 0.08) for IL-8 concentrations to be reduced in media from CH fibroblasts exposed to IL-1ß for 24 h but IL-6 concentrations did not differ between genotypes. Results indicate 50 yr of selection has reduced the robustness of the innate immune and acute-phase response to LPS in the contemporary Holstein heifer.

Effect of a trace mineral injection at weaning on growth, antioxidant enzymes activity, and immune system in Nellore calves.

The objective of this study was to evaluate the effects of injectable trace minerals (ITM) at the time of weaning on growth, antioxidant, and immune response of Nellore (Bos indicus) calves. Weaned calves (n = 159; 213 ± 32 kg) were stratified by body weight (BW) and randomly assigned to 1 of 2 treatments: injection (1 mL/45 kg of BW) of saline or ITM. Saline solution consisted of 0.9% NaCl, whereas ITM had 60, 10, 5, and 15 mg/mL of Zn, Mn, Se, and Cu, respectively. The application of ITM increased (P ≤ 0.04) the plasma concentrations of superoxide dismutase on day 7 and plasma glutathione peroxidase on day 7 and day 21. The ITM calves had greater leukocyte concentration on day 64 (P = 0.04), whereas neutrophil and mast cell concentrations did not differ (P ≥ 0.67) between treatments. The ITM calves tended (P ≤ 0.08) to have greater concentrations of lymphocytes on day 64 and eosinophils on day 21, but not (P ≥ 0.15) monocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin compared to saline calves. The ITM calves had less mean corpuscular hemoglobin concentration (MCHC; P = 0.02) and tended to have increased platelet concentrations on day 21 (P = 0.08). Growth performance did not differ between treatments (P ≥ 0.78). Thus, ITM at weaning did not impact growth performance, but increased plasma concentrations of antioxidant enzymes and blood platelets, improved components of the immune system, and reduced MCHC of Nellore calves.

Characterization of the NLRP1 inflammasome response in bovine species.

Inflammasomes act as sensors of infection or damage to initiate immune responses. While extensively studied in rodents, understanding of livestock inflammasomes is limited. The NLRP1 inflammasome sensor in rodents is activated by Toxoplasma gondii, Bacillus anthracis lethal toxin (LT), and potentially other zoonotic pathogens. LT activates NLRP1 by N-terminal proteolysis, inducing macrophage pyroptosis and a pro-inflammatory cytokine response. In contrast, NLRP1 in macrophages from humans and certain rodent strains is resistant to LT cleavage, and pyroptosis is not induced. Evolution of NLRP1 sequences towards those leading to pyroptosis is of interest in understanding innate immune responses in different hosts. We characterized NLRP1 in cattle (Bos taurus) and American bison (Bison bison). Bovine NLRP1 is not cleaved by LT, and cattle and bison macrophages do not undergo toxin-induced pyroptosis. Additionally, we found a predicted Nlrp1 splicing isoform in cattle macrophages lacking the N-terminal domain. Resistance to LT in bovine and human NLRP1 correlates with evolutionary sequence similarity to rodents. Consistent with LT-resistant rodents, bovine macrophages undergo a slower non-pyroptotic death in the presence of LPS and LT. Overall, our findings support the model that NLRP1 activation by LT requires N-terminal cleavage, and provide novel information on mechanisms underlying immune response diversity.