Developmental adaptations of γδ T cells and B cells in blood and intestinal mucosa from birth until weaning in Holstein bull calves.

This study aimed to characterize the development of systemic and colon tissue resident B and γδ T cells in newborn calves from birth until weaning. At birth, calves have limited capacity to initiate immune responses, and the immune system gradually matures over time. Gamma delta (γδ) T cells are an important lymphocyte subset in neonatal calves that confer protection and promote immune tolerance. A total of 36 newborn calves were enrolled in a longitudinal study to characterize how systemic and colon tissue resident B and γδ T cells develop from birth until weaning. Blood and colon biopsy samples were collected on d 2, 28, and 42 to determine the proportions of various B and γδ T cell subsets by flow cytometry. We classified γδ T cells into different functional subsets according to the level of expression intensity of the coreceptors WC1.1 (effector function) and WC1.2 (regulatory function). Furthermore, naive B cells were classified based on the expression IgM receptor, and activation state was determined based on expression of CD21 and CD32, 2 receptors with opposing signals involved in B cell activation in early life. Additional colon biopsy samples were used for 16S sequencing, and microbial diversity data are reported. At birth, γδ T cells were the most abundant lymphocyte population in blood, accounting for 58.5% of the lymphocyte pool, after which the proportions of these cells declined to 38.2% after weaning. The proportion of γδ T cells expressing WC1.1 decreased by 50% from d 2 to d 28, whereas no change was observed in the expression of WC1.2. In the colon, there was a 50% increase of γδ T cells after weaning and the proportion of WC1.2+ γδ T cells doubled from d 28 to 42. The proportion of IgM+ B lymphocytes in blood increased from 23.6% at birth to 30% after weaning, were the proportion of B cells expressing CD21 increased by 25%, while the proportion of B cells expressing CD32 decreased by 30%. While no changes were observed for the overall proportion of IgM+ B lymphocytes in the colon, there was a 6-fold increase in the proportion of CD21+ B cells from pre- (d 28) to postweaning (d 42). Microbial diversity increased from d 2 of life to 28 and declined abruptly after weaning. The reduction in microbial diversity during weaning was negatively correlated with the increase in all γδ T cell subsets and CD21+ B cells. These data suggest that developmental adaptations after birth coordinate expansion of γδ T cells to provide early systemic protection, as well as to steer immune tolerance, while B cells mature over time. Additionally, the increase of colonic γδ T cells on d 42 suggests a protective role of these cells during weaning.

Short communication: Characterizing ovine serum stress biomarkers during endotoxemia.

Breeding stress-resilient livestock is a potential strategy to help mitigate the negative effect of environmental and pathogenic stressors. The hypothalamic-pituitary-adrenal axis and immune system are activated during stress events and release mediators into the circulation that help restore physiological homeostasis. The purpose of this study was to assess a comprehensive set of circulatory mediators released in response to an acute immune stress challenge to identify candidate biomarkers that can be used for the selection of stress-resilient animals. Fifteen female lambs were stress challenged with an intravenous bolus of lipopolysaccharide (LPS; 400 ng/kg), and blood was collected from the jugular vein at 0, 2, 4, and 6 h after LPS challenge to identify and monitor candidate stress biomarkers; temperature was also recorded over time. Biomarker responses were evaluated with a repeated-measures model to compare time points with baseline values. As expected, all sheep had a monophasic febrile response to LPS challenge, and cortisol increased and returned to baseline by 6 h. The cytokines tumor necrosis factor-α, IL-6, IFN-γ (proinflammatory), and IL-10 (anti-inflammatory) increased, but only tumor necrosis factor-α returned to baseline during the monitoring period. The cytokines IL-1α, IL-1ß, IL-17α (proinflammatory), and IL-4 (anti-inflammatory) did not respond to LPS challenge. All chemokines (CCL2, CCL3, CCL4, CXCL10, and IL-8) responded to LPS challenge; however, only CCL2, CCL3, CCL4, and CXCL10 increased over time, and only CCL3, CCL4, and CXCL10 returned to baseline during the monitoring period. MicroRNA (miR-145, miR-233, and miR-1246) also increased and remained elevated during the study. In summary, the LPS challenge induced a strong stress response in Rideau-Dorset sheep that could be monitored with a distinct profile of circulatory biomarkers.

Short communication: Prevalence of digital dermatitis in Canadian dairy cattle classified as high, average, or low antibody- and cell-mediated immune responders.

Lameness is a major animal welfare issue affecting Canadian dairy producers, and it can lead to production, reproduction, and health problems in dairy cattle herds. Although several different lesions affect dairy cattle hooves, studies show that digital dermatitis is the most common lesion identified in Canadian dairy herds. It has also been shown that dairy cattle classified as having high immune response (IR) have lower incidence of disease compared with those animals with average and low IR; therefore, it has been hypothesized that IR plays a role in preventing infectious hoof lesions. The objective of this study was to compare the prevalence of digital dermatitis in Canadian dairy cattle that were classified for antibody-mediated (AMIR) and cell-mediated (CMIR) immune response. Cattle (n = 329) from 5 commercial dairy farms in Ontario were evaluated for IR using a patented test protocol that captures both AMIR and CMIR. Individuals were classified as high, average, or low responders based on standardized residuals for AMIR and CMIR. Residuals were calculated using a general linear model that included the effects of herd, parity, stage of lactation, and stage of pregnancy. Hoof health data were collected from 2011 to 2013 by the farm's hoof trimmer using Hoof Supervisor software (KS Dairy Consulting Inc., Dresser, WI). All trim events were included for each animal, and lesions were assessed as a binary trait at each trim event. Hoof health data were analyzed using a mixed model that included the effects of herd, stage of lactation (at trim date), parity (at trim date), IR category (high, average, and low), and the random effect of animal. All data were presented as prevalence within IR category. Results showed that cows with high AMIR had significantly lower prevalence of digital dermatitis than cattle with average and low AMIR. No significant difference in prevalence of digital dermatitis was observed between high, average, and low CMIR cows. These results indicate that having more robust AMIR is associated with lower prevalence of digital dermatitis hoof lesions.

Differential effects of lactobacilli on activation and maturation of mouse dendritic cells.

Lactic acid bacteria (LAB) are of interest because of their potential to modulate immune responses. The effects of LAB range from regulation to stimulation of the immune system. A series of studies were performed in vitro to study the effects of six lactic acid bacteria (LAB), Lactobacillus helveticus LH-2, Lactobacillus acidophilus La-5, La-115, La-116 and La-14, and Lactobacillus salivarius, on maturation and activation of mouse dendritic cells. Production of tumour necrosis factor (TNF)-?, interleukin (IL)-6 and IL-10 by dendritic cells (DCs) was determined after treating cells with live LAB. The expression of DC maturation markers, CD80 and CD40, was also measured using flow cytometry after stimulation with LAB. In addition, the expression of Toll-like receptors (TLRs) 2, 4 and 9 by DCs stimulated with LAB was measured. Our results revealed that LAB act differentially on pro-inflammatory and anti-inflammatory cytokine production and induction of co-stimulatory molecules by DCs. Specifically, L. salivarius was found to be the most effective LAB to induce pro-inflammatory cytokine production and expression of co-stimulatory molecules. Moreover, La-14, La-116 and La-5 induced moderate maturation and activation of DCs. On the other hand, LH-2 and La-115 were the least effective lactobacilli to induce DC responses. The present study also revealed that L. salivarius was able to induce the expression of TLR2, 4 and 9 by DCs. In conclusion, various strains and species of LAB can differentially regulate DC activation and maturation, providing further evidence that these bacteria may have the ability to influence and steer immune responses in vivo.

Characterization of naturally processed and presented peptides associated with bovine major histocompatibility complex (BoLA) class II DR molecules.

Differential regulation of genetic resistance to infectious disease may partially be explained by variation in the binding affinity and the repertoire of pathogen-derived antigenic peptides associated with major histocompatibility complex (MHC) molecules. In this study, we investigated characteristics of peptides that bind to the bovine MHC allele BoLA-DRB3*2703, which is associated with occurrence of clinical mastitis in Holstein dairy cattle, and assigned a putative peptide-binding motif to this allele. This was achieved by in vitro expression of allele *2703 as well as a control allele, BoLA-DRB3*1201 which is present at high frequency in Holsteins. Transfected cell lines alone (for allele *1201) or in combination with blood mononuclear cells from an animal homozygous for allele *2703 were used as the source of naturally processed and presented peptides. Subsequent to elution of peptides from BoLA-DR+ cells, their sequences were determined by electrospray ionization mass spectrometry. Eluted peptides were between 13 and 20 amino acids long and the majority were in sets of overlapping sequences. These peptides were derived from intra- and extracellular proteins, as well as foreign proteins present in the culture medium. Some peptides had originated from molecular chaperones present in the endoplasmic reticulum, such as ER-60 and GRP78, pointing to some degree of overlap and cross-sampling between MHC class I and class II antigen presentation pathways. Consistent with reports of human and mouse MHC class II-associated peptides, putative peptide-binding motifs could be assigned to alleles *2703 and *1201, comprising a hydrophobic or an aromatic residue at relative position 1, a hydrophobic residue at position 4 and a small residue at position 6 of the eluted peptides. These findings provide the foundation for future studies of molecular mechanisms of MHC-disease associations of cattle.

Regulation of bovine E-selectin expression by recombinant tumor necrosis factor alpha and lipopolysaccharide.

Induction of adhesion molecules by cytokines and LPS is an important mechanism of regulating leukocyte migration into tissue. Expression and regulation of E-selectin may be differentially influenced by the stimuli involved with effects on mRNA or surface protein kinetics. Surface protein and mRNA expression kinetics of bovine E-selectin were measured and compared in primary cultures of bovine aortic endothelial cells (BAEC) stimulated for various periods of time with recombinant bovine tumor necrosis factor alpha (rbTNF-alpha) or Escherichia coli lipopolysaccharide (LPS). E-selectin mRNA expression was measured via quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) using a construct that contained multiple synthetic oligonucleotides for several bovine adhesion molecules and cytokines. Surface expression of E-selectin was measured by flow cytometry. Unstimulated BAECs expressed minimum or no E-selectin on the surface. A low number of endothelial cells expressed surface E-selectin as early as 1h post-stimulation and surface expression was sustained after both stimuli for 24-72h. Mean fluorescence intensity (MFI) indicated peak surface concentration of E-selectin at 6 h post-stimulation after LPS followed by a gradual decrease to 72h without returning to baseline values. Mean fluorescence intensity following stimulation with TNF-alpha increased slightly between 0 and 72h. The pattern of mRNA expression differed between stimuli. LPS-stimulated BAECs expressed peak amounts of E-selectin mRNA at 6 h, followed by a decline to baseline by 24 h. Conversely, BAECs stimulated with rbTNF-alpha expressed significantly (p pound 0.05) higher amounts of mRNA at 1h than compared to unstimulated controls (0 h), but this decreased to below baseline levels by 6h; followed by a gradual increase and eventually a sharp increase between 18 and 72 h. To account for the lack of correlation between mRNA and protein expression, it was hypothesized that shedding of surface E-selectin accounted at least in part, for the large increase in mRNA expression seen at 18-72h. Culture supernatants from rbTNF-alpha-treated BAECs were harvested, and tested for the presence of shed E-selectin using ELISA. Unstimulated culture supernatants contained little or no E-selectin. Between 6 and 48 h, the concentration of E-selectin in culture supernatants from rbTNF-alpha-stimulated BAECs increased approximately two-fold, suggesting that the sharp increase in E-selectin mRNA expression around 18 h may be related to significant loss of surface E-selectin during this period.

Regulation of bovine intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on cultured aortic endothelial cells.

Endothelial cell adhesion molecules (AM) intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) are important mediators of cell migration from blood into tissue. The kinetics of ICAM-1 mRNA and VCAM-1 protein expression in bovine aortic endothelial cells (BAEC) were determined using quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) and flow cytometric analysis, respectively. Stimulation of BAEC with recombinant bovine tumor necrosis factor alpha (rbTNF-alpha) resulted in protein expression of VCAM-1 on less than 5% of all cultured BAECs at 1h post-stimulation, followed by a significant increase at 3h that was maintained until 48h when the proportion of VCAM-1 positive (+) cells decreased significantly, but not to baseline proportions. The expression kinetics for VCAM-1 were similar on cells stimulated with lipopolysaccharide (LPS) except at 24h, when there was a significantly higher proportion of BAEC expressing VCAM-1 than at any other time. The expression of ICAM-1 mRNA differed significantly between stimuli. Expression of ICAM-1 mRNA peaked at 12-18h and then diminished but remained at amounts above baseline up to 72h after stimulation. Stimulation with LPS induced a significant increase in ICAM-1 mRNA expression between 1 and 12h after which the amounts rapidly decreased to baseline. In summary, different stimuli produced similar expression kinetics of VCAM-1 surface protein but different kinetics of ICAM-1 mRNA expression.

Cytokines in Mycoplasma hyorhinis-induced arthritis in pigs bred selectively for high and low immune responses.

Yorkshire pigs were bred selectively for high and low immune responses (H and L pigs, respectively) based on multiple antibody (Ab) and cell-mediated immune response traits. In a previous experiment, generation 4 (G4) pigs of each line were infected with Mycoplasma hyorhinis. High responders had a more rapid and higher Ab response and less polyserositis, but arthritis was more severe in H pigs than in L pigs. To test the hypothesis that line differences were attributable to differential expression of cytokines, M. hyorhinis infection was induced in pigs of G8. Arthritis was more severe clinically (P,

Associations of the bovine major histocompatibility complex DRB3 (BoLA-DRB3) alleles with occurrence of disease and milk somatic cell score in Canadian dairy cattle.

Potential associations were investigated between bovine leucocyte antigen (BoLA) alleles and occurrence of disease. Cows (Holstein n = 835; Jersey n = 66) were examined for polymorphisms of the second exon of the BoLA-DRB3 gene, using the polymerase chain reaction (PCR), followed by digestion of the amplified fragments with three restriction endonucleases. Disease occurrences were recorded for each cow throughout one lactation. Milk somatic cell count data were retrieved through the Dairy Herd Improvement records and converted to somatic cell score (SCS). There were no effects of BoLA alleles on SCS in Jersey cows, but BoLA-DRB3.2*16 was significantly associated (P < or = 0.05) with lower SCS in Holsteins. Since the number of Jerseys was relatively small and prevalence of diseases in this population was low, health records of Jerseys were not analyzed further. BoLA associations with occurrence of disease in Holsteins were investigated using a log-linear model. There was a significant (P < or = 0.05) association between BoLA-DRB3.2*23 and occurrence of severe mastitis, from which coliforms were the most commonly isolated bacteria. The BoLA allele *3 was associated with a lower risk of retained placenta (P < or = 0.05) and alleles *16 (P < or = 0.05) and *22 (P < or = 0.05) with a lower risk of cystic ovarian disease. Although more studies are required to confirm the present findings, it can be concluded that BoLA alleles may have potential usefulness as genetic markers of higher or lower risk of disease occurrence in cows.

Effects of a core antigen vaccine against gram-negative bacteria on physiologic and yield parameters of dairy cows during late lactation and the dry period.

The objective of the study was to assess the effects of a core antigen vaccine against Gram-negative bacteria on feed consumption, milk yield, somatic cell count, hematologic parameters, and milk progesterone concentrations for dairy cows in late lactation and the dry period. Sixty-eight multiparous Holstein cows from two farms were paired by days in milk and were randomly selected to receive either the vaccine or placebo. Cows received a secondary immunization with the same product (vaccine or placebo) 3 wk following the primary immunization. The physiologic and yield outcomes were measured prior to each immunization, at the time of each immunization, and for one or more periods following each immunization. No significant differences between vaccinated and placebo groups were detected for daily milk weight, daily feed intake, somatic cell score, rectal body temperature, or milk progesterone concentration (pregnant cows). Cows in the vaccinated group had significant elevations in total blood leukocyte counts following the secondary immunization, which was due to an increase in the neutrophil fraction 24 h postimmunization.

Effects of supplemental chromium on production of cytokines by mitogen-stimulated bovine peripheral blood mononuclear cells.

This study determined whether supplementing the diets of dairy cows during the peripartum period with organic trivalent Cr influenced the capacity of their peripheral blood mononuclear cells to produce activation cytokines in response to stimulation with mitogens in vitro. Nine cows were fed 0.5 ppm of Cr/d per cow from 6 wk prepartum to 16 wk postpartum; 10 other periparturient cows served as unsupplemented controls. Mononuclear leukocytes, enriched from peripheral blood during wk 0, 2, 4, and 6 of lactation, were cultured with or without the T-lymphocyte mitogen, concanavalin A. Culture supernatants, harvested at 24, 48, or 72 h, were assayed for interleukin-2, interferon-gamma, and tumor necrosis factor-alpha. The cytokines were barely detectable in the supernatants from the unstimulated cultures, but supernatants from mitogen-stimulated cultures contained higher concentrations of each cytokine. For cows fed Cr, concentrations of all three cytokines in the culture supernatants of the mitogen-stimulated mononuclear cells decreased significantly relative to values for unsupplemented cows, particularly around peak lactation for the 24- and 48-h cultures. Theses results extended our previous observations and supported the hypothesis that organic Cr is immunomodulatory in high producing cows.

Immunomodulatory activity of blood serum from chromium-supplemented periparturient dairy cows.

Our previous research showed enhanced immune responses, including mitogen-induced blastogenesis of peripheral blood mononuclear cells from feedlot calves and periparturient dairy cows supplemented with dietary chromium (Cr). The objective of the present study were to test whether blood sera from Cr-supplemented periparturient cows contained immunomodulatory activity for mitogen-stimulated peripheral blood mononuclear cells and, if so, to determine if this activity was explicable by differences in blood profiles of some glucose-regulating hormones (insulin, cortisol, growth hormone, insulin-like growth factor I, and tumor necrosis factor-alpha) between Cr-supplemented and unsupplemented (control) animals. Blood sera from ten unsupplemented cows and nine Cr-supplemented cows (0.5 ppm day-1) were collected weekly from 2 weeks before to 6 weeks after parturition, and were used to supplement (1, 10, and 20% vol/vol) culture medium supporting concanavalin A (Con A) stimulated mononuclear cells enriched from blood of four nulliparous donor cows. Hormone concentrations were determined using radioimmunoassays. Con A-induced blastogenesis was enhanced when 1, 10, and 20% sera from Cr-supplemented cows was added to the mononuclear cell cultures, and this was particularly evident around parturition. Conversely, peripartum sera from unsupplemented cows depressed Con A-induced blastogenesis. Except for a marginal rise in blood cortisol 2-4 weeks after parturition, no significant effects of Cr supplementation on other hormones (insulin, growth hormone, insulin-like growth factor I, tumor necrosis factor-alpha) were observed. These observations suggest that factors in peripheral blood serum from Cr-supplemented cows, other than absolute concentrations of the glucose-regulating hormones studied, modulate Con A-induced blastogenesis of mononuclear leukocytes.

Clinical, biochemical, and hematologic evaluation of normal dogs after administration of rabbit anti-dog thymocyte serum.

Renal allografting is the only long-term alternative to euthanasia in dogs with end-stage kidney disease. The purpose of this study was to determine the clinical, biochemical, and hematologic effects of rabbit anti-dog thymocyte serum (RADTS) in normal dogs and to develop a safe and practical route of administration before its use in an allograft immunosuppressive protocol. Thirteen mongrel dogs were divided into three groups; each received RADTS subcutaneously, intramuscularly, or intravenously. The inflammation and pain associated with subcutaneous administration was unacceptable. A significant (p < or = .05) leukopenia and lymphopenia developed in all dogs, regardless of the route of administration of RADTS. Thrombocytopenia was a consistent finding after intravenous administration and with high doses given intramuscularly. Both the intravenous and intramuscular routes were well tolerated by all dogs with minimal or no discomfort. Serum creatinine was unchanged, whereas serum alanine aminotransferase activity increased in one dog. There were no histologic changes in any of the kidneys examined.

Genetic and other effects on antibody and cell mediated immune response in swine leucocyte antigen (SLA)-defined miniature pigs.

Miniature pigs of eight swine leucocyte antigens (SLA) haplotypes were immunized with sheep erythrocytes (SRBC), hen egg white lysozyme (HEWL) and the synthetic peptide (T, G)-A--L to induce antibody. Bacillus Calmette Geurin (BCG) and dinitrochlorobenzene (DNCB) were used to induce cell mediated immune response (CMI). Analysis of variance by least squares was used to assess the effects of SLA haplotype, sire, dam, litter and sex of pig on the magnitude of the primary and secondary antibody response and on dermal delayed type hypersensitivity induced by purified protein derivative of tuberculin (PPD) and DNCB-induced contact hypersensitivity. The statistical model accounted for 43.50-77.30% of the observed variability in antibody and CMI at various times after immunization or challenge. While SLA had a significant effect on both antibody and CMI to some antigens at some, but not all times, sire, dam and litter were more frequently significant and to a greater degree. Haplotypes dd, dg and gg produced more antibody to SRBC and (T, G)-A--L while dg and gg had higher primary, but not secondary antibody response to HEWL. Delayed hypersensitivity to PPD was most marked in pigs of dd, dg and gg haplotypes while contact hypersensitivity to DNCB was expressed least in the dg and gg haplotype pigs. Heritability estimates were high for response to (T, G)-A--L and HEWL indicating feasibility of selective breeding for these traits.