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Dairy cattle face a variety of stressful events on a daily basis. More specifically, climate change has resulted in more frequent heat stress events that increase the incidence of chronic bacterial infections by inducing conditions like leaky gut syndrome, whereby the integrity of the intestinal epithelium is compromised allowing for luminal bacterial lipopolysaccharide (LPS) endotoxin to infiltrate the host's bloodstream resulting in acute or chronic systemic stimulation of the innate immune system. Repeated exposure to LPS over a short period of time is reported to induce immunotolerance within the host. This LPS tolerance is an essential immunohomeostatic response that can protect against over activation of the inflammatory response during subsequent exposure to LPS. In the present study, Holstein calves (n = 20) were initially stress challenged with either saline, or 100, 200 or 400 ng/kg of LPS administered intramuscular, and again re-challenged with 200 ng/kg of LPS 2-weeks later. Serum was collected every 2 hr for 6 hr to profile changes in circulatory stress biomarkers after the repeated LPS exposures. Heifers that were initially challenged with 100, 200 and 400 ng/kg of LPS demonstrated significantly attenuated cortisol responses in the second challenge (p < 0.01, 0.01 and 0.05, respectively), whereas control animals who previously received saline demonstrated a strong cortisol response at 2 hr after receiving 200 ng/kg of LPS (p < 0.05). The cytokine/chemokine (IL-6, CCL2, CCL3 and CCL4) responses were also attenuated during the LPS rechallenge (p < 0.05). Finally, microRNA expression profiles were determined to assess the epigenetic response to repeated LPS exposure. Interestingly, miR-31 and miR-223 were downregulated in response to the second LPS challenge. The present study demonstrates the dynamic nature of the stress response in dairy cattle as it relates to the development of LPS tolerance. Understanding the roles of various stress biomarkers in the context of innate immune cell tolerance is essential for evaluating their impact on immune system homeostasis., Competing Interests: Conflict of Interest Statement All authors declare that they have no conflicts of interest., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

Dairy cattle routinely face a variety of stressors. For example, climate change has resulted in more frequent heat stress events that increase the incidence of bacterial infections by inducing conditions like leaky gut syndrome, whereby the integrity of the intestinal epithelium is compromised allowing for luminal bacteria and their membrane components to infiltrate the host's bloodstream resulting in systemic inflammation. Lipopolysaccharide (LPS) is a well-characterized and biologically relevant microbe-associated molecular pattern (MAMP) that makes up the outer membrane of pathogenic and commensal Gram-negative bacteria and is known to contribute to inflammatory disorders including mastitis, acidosis and septicemia. In the present study, Holstein heifers (n = 20) were randomly allocated into different treatment groups receiving saline, 100, 200 or 400 ng/kg of LPS intramuscularly to create an experimentally induced endotoxemic state. Serum was collected hourly for 8 hr and then again at 24 hr to profile changes in circulatory stress biomarkers. All LPS -challenged animals demonstrated distinct cortisol responses 2 hr post-LPS challenge, and in the 200 ng/kg and 400 ng/kg of LPS treatments cortisol concentrations remained significantly induced for up to 4 hr. Rectal temperature was significantly increased for heifers challenged with 100 and 200 ng/kg of LPS at 2 and 4 hr as compared to their pre-challenge temperature. All LPS-challenged animals demonstrated marked leukopenia and thrombocytopenia as compared to the saline control animals. A total of 8 cytokines, including TNFα, and IL-10, were found to be induced between 2 and 4 hr. Finally, we report that miR-1246, miR-223, miR-29 and miR-31 were significantly induced in animals challenged with LPS as compared to the saline controls. The present study demonstrated that the stress response in dairy heifers is dynamic and there are peak windows of time when cortisol, cytokines and also miRNA are induced, and blood cells are sequestered as part of the systemic inflammatory response. Variability in the response to LPS warrants further investigation in dairy cattle to better understand the contribution of genetics and associations between LPS-induced stress and health and performance., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The frequency of heat waves and hot days are increasing due to climate change, which leads to an increase in the occurrence of heat stress in dairy cattle. Previous studies have shown that dairy cattle identified as high immune responders have a reduced incidence of disease and improved vaccine response compared to average and low responders. Additionally, it has been observed that when cells from immune phenotyped cattle are exposed to in-vitro heat challenge, high immune responders exhibit increased heat tolerance compared to average and low immune responders. Therefore, the objective of this study was to evaluate physiological parameters and the function of blood mononuclear cells of immune phenotyped dairy cattle exposed to in-vivo heat challenge. A total of 24 immune phenotyped lactating dairy cattle (8 high, 8 average and 8 low) were housed in the tie-stall area of the barn and exposed to an in-vivo heat challenge for 4 h on 2 subsequent days, where the temperature was set at 29 ℃. Blood samples were taken both pre- and post-challenge each day and manual respiration rates and rectal temperatures were recorded pre challenge and every 30 min during the challenge. Temperature and humidity measurements were taken in correspondence with all respiration rate and rectal temperature measurements to calculate the temperature humidity index pre heat challenge and at 30-minute intervals during the heat challenge. Blood mononuclear cells were isolated from blood collected pre and post challenge and the concentration of heat shock protein 70 and cell proliferation were assessed. Results showed that average and low responders had significantly greater respiration rates compared to high responders at a temperature humidity index of 77 and above. No significant difference was observed between phenotypes for rectal temperature. High responders had a higher heat shock protein 70 concentration and greater cell proliferation after in-vivo heat challenges compared to average and low responders. These results paralleled those found during in-vitro heat challenge adding further credence to the concept that high responders may be more resilient to heat stress compared average and low responders., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The peripartum period is a period of high stress, transition and management changes for dairy cows. It is associated with higher incidence of both metabolic and pathogenic disease. Both antibody-(AMIR) and cell-(CMIR) mediated immune responses play a key role in the maintenance of health in mammals protecting against extracellular and intracellular pathogens, respectively. Generally, interferon gamma (IFN-γ) has been associated with CMIR, whereas interleukin 4 (IL-4) has been associated with AMIR bias, and interleukin 17 (IL-17A) is associated with pro-inflammatory. It has been previously demonstrated that cows can be classified as high (H), average (A), and low (L) immune responders based upon their AMIR and CMIR to test antigens, and that this classification is associated with disease occurrence throughout lactation. The mechanisms behind these differences in phenotype and the effects of the peripartum period have not been fully investigated. The aim of this study was to determine the effects of the peripartum period on cytokine production of CD4+ T-cells or T helper (Th) cells, key mediators of the adaptive immune response. Immune response phenotyped cows were selected based on H-AMIR/L-CMIR (H-AMIR cows; n = 10) and H-CMIR/L-AMIR (H-CMIR cows; n = 11) response to test antigens. Isolated CD4+ T-cells collected at 28 days before calving (prepartum samples), 4 days after calving (early postpartum samples), and 21 days after calving (late postpartum samples) from these groups were stimulated with Concanavalin-A (ConA) with unstimulated controls. Subsequently, IL-4, IFN-γ, and IL-17A concentrations were quantified by ELISA. Overall, there was no obvious decline in IL-4, IFN-γ or IL-17A close to calving observed from CD4+ T-cells from each of these phenotypically distinct groups of cows. However, CD4+ T-cells isolated from H-CMIR secreted higher amounts of IL-4 (746.43 ± 428 pg/mL), IL-17A (446IL ± 62 pg/mL), and IFN-γ (7755.79 ± 4449 pg/mL) than H-AMIR cows (IL-4 (212.15 ± 121 pg/mL), IL-17A (163.15 ± 87 pg/mL), and IFN-γ (2909.771 ± 1671 pg/mL)) on day 21 after calving, late postpartum. This study indicates a genetic predisposition based on immune response phenotype of cytokine production from CD4+ T-cells around calving., (Copyright © 2018. Published by Elsevier B.V.)

To study pathway specific gene expression within the immune-endocrine axis of dairy cows with Staphylococcus aureus mastitis, mRNA was collected from blood mononuclear cells (BMCs) and milk somatic cells (MSCs) of cows (n=7) identified as culture positive for S. aureus and their matched negative control cows (n=7) with no evidence of S. aureus mastitis. Labeled cDNA probes derived from BMCs and MSCs of infected and healthy cows were applied to a bovine immune-endocrine cDNA array containing 167 genes. Genes with a log(2) ratio> or =0.5 were considered to be up-regulated and genes with a log(2) ratio< or =-0.5 to be down-regulated. In total, 22 genes were differentially displayed in BMCs and 16 genes in MSCs of case versus controls. Expression of selected genes in BMCs and MSCs were confirmed by real-time PCR. The RT-PCR results were highly correlated with microarray measurements. Some of these genes, such as interleukin (IL)-8 have been previously implicated in other bacterial diseases, and are known to regulate immune responses; whereas, others may reflect novel pathways or genes involved in progressive mammary gland disease. For example, IL-18 was up-regulated in BMCs but not MSCs of mastitic quarters, while IL-17 was more highly expressed in MSCs compared to BMCs. This study identified a number of differentially expressed genes associated with bovine S. aureus mastitis and demonstrates the intricacy of the patterns of gene expression that influence host response to a complex pathogen of significant relevance to both human and veterinary medicine.

Immunoglobulin (Ig) function varies by isotype and antibody activity is best mediated by isotypes most able to control the inciting infection. In pigs, a high ratio of IgG1:IgG2 is associated with resistance to disease caused by the extra-cellular bacterium Actinobacillus pleuropneumoniae. This ratio is controlled by type 1/type 2 cytokines in vitro, reflecting cell- (CMI) or antibody-mediated immune (AMI) responses, respectively. Animals were used which had been previously selectively bred for high (HIR) or low (LIR) combined AMI and CMI and had been immunized with hen eggwhite lysozyme (HEWL) in Quil A (days 0 and 14) while Bacillus Calmette Guérin was given on day 9. To test the hypothesis that lines do not differ in IgG isotype expression as antibody to HEWL, the ratio of anti-HEWL associated with IgG1 and IgG2 was determined at days 0, 9, 14 and 21. The ratio of IgG1:IgG2-associated antibody was always <1.0 indicating a type 1 response and differed significantly over time in HIR and LIR animals. After primary and secondary immunizations, the HIR animals' IgG1:IgG2-associated antibody ratio increased and approached 1 while for LIR animals the ratio decreased. Thus anti-HEWL antibody in HIR, but not LIR, approached balance in type 2:type 1 expression. Individual variation in immune response was frequently significant within each immune response group. Thus, proportional production of anti-HEWL antibody associated with IgG isotypes varies by individual and differs over time as a function of genotype in pigs selectively bred for HIR or LIR.

A variety of commercial DNA arrays specific for humans and rodents are widely available; however, microarrays containing well-characterized genes to study pathway-specific gene expression are not as accessible for domestic animals, such as cattle, sheep and pigs. Therefore, a small-scale application-targeted bovine immune-endocrine cDNA array was developed to evaluate genetic pathways involved in the immune-endocrine axis of cattle during periods of altered homeostasis provoked by physiological or environmental stressors, such as infection, vaccination or disease. For this purpose, 167 cDNA sequences corresponding to immune, endocrine and inflammatory response genes were collected and categorized. Positive controls included 5 housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, ribosomal protein L19, beta-actin, beta2-microglobulin) and bovine genomic DNA. Negative controls were a bacterial gene (Rhodococcus equi 17-kDa virulence-associated protein) and a partial sequence of the plasmid pACYC177. In addition, RNA extracted from un-stimulated, as well as superantigen (Staphylococcus aureus enterotoxin-A, S. aureus Cowan Pansorbin Cells) and mitogen-stimulated (LPS, ConA) bovine blood leukocytes was mixed, reverse transcribed and PCR amplified using gene-specific primers. The endocrine-associated genes were amplified from cDNA derived from un-stimulated bovine hypothalamus, pituitary, adrenal and thyroid gland tissues. The array was constructed in 4 repeating grids of 180 duplicated spots by coupling the PCR amplified 213-630 bp gene fragments onto poly-l-lysine coated glass slides. The bovine immune-endocrine arrays were standardized and preliminary gene expression profiles generated using Cy3 and Cy5 labelled cDNA from un-stimulated and ConA (5 microg/ml) stimulated PBMC of 4 healthy Holstein cows (2-4 replicate arrays/cow) in a time course study. Mononuclear cell-derived cytokine and chemokine (IL-2, IL-1alpha, TNFalpha, IFN-gamma, TGFbeta-1, MCP-1, MCP-2 and MIP-3alpha) mRNA exhibited a repeatable and consistently low expression in un-stimulated cells and at least a two-fold increased expression following 6 and 24 h ConA stimulation as compared to 0 h un-stimulated controls. In contrast, expression of antigen presenting molecules, MHC-DR, MHC-DQ and MHC-DY, were consistently at least two-fold lower following 6 and 24 h ConA stimulation. The only endocrine gene with differential expression following ConA stimulation was prolactin. Additionally, due to the high level of genetic homology between ovine, swine and bovine genes, RNA similarly acquired from sheep and pigs was evaluated and similar gene expression patterns were noted. These data demonstrate that this application-targeted array containing a set of well characterized genes can be used to determine the relative gene expression corresponding to immune-endocrine responses of cattle and related species, sheep and pigs.

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.

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.

Potential relationships between amino acid motifs in the antigen binding groove of various alleles of the bovine major histocompatibility complex DR (BoLA-DR) molecule and occurrence of clinical mastitis caused by Staphylococcus species (non-Staphylococcus aureus) were investigated in a case-control study. A significant association (P< or =0.05) was detected between the presence of glutamic acid at position beta 74 and occurrence of mastitis caused by Staphylococcus spp. with a relative risk of 11. This motif is present in BoLA-DRB3.2*22, *23 and *24 alleles. Presence of a positively charged residue (arginine or lysine) at position 13 also showed a tendency (P< o r=0.1) towards an association with a higher risk of clinical mastitis caused by the same bacteria. This motif is present in BoLA-DRB3.2*23 and *8 alleles. Similarly, presence of arginine at position beta 71 (present in alleles *23 and *22) was associated with occurrence of this disease. These positions (beta 13, beta 71 and beta 74) form pocket 4 of the antigen binding groove, which plays an instrumental role in antigen binding and recognition by T-lymphocytes. Thus, it can be concluded that pocket 4 of the BoLA-DR molecule is involved in conferring susceptibility to clinical mastitis caused by Staphylococcus spp.

To test the hypothesis that variation in ability to respond immunologically correlates with health, Yorkshire pigs were bred for high (HIR) and low (LIR) antibody (Ab) and cell-mediated immune response (CMI). Selection was based on standardized measures of Ab (secondary response to hen egg white lysozyme, serum IgG concentration) and CMI (cutaneous delayed-type hypersenstivity to purified protein derivative of tuberculin after immunization with bacillus Calmette-Guérin and in vitro lymphocyte response to Con-A). Differences in Ab and CMI by line were not restricted to the antigens used in the selection. Antibody response to vaccines was highest in HIR and non-responders were restricted to LIR pigs. The HIR pigs had the best rate of weight gain. After infection with Mycoplasma hyorhinis, HIR developed more severe arthritis and less polyserositis. Differences were associated with variation in cytokine message in joint-related cells. Following exposure to attenuated transmissible gastroenteritis virus, natural killer cells of the LIR pigs but not of HIR or control lines, were unresponsive. Genetic selection for Ab and CMI may provide health and productivity advantages and complement traditional health-maintenance methods.

Migration of leukocytes into the mammary gland is an essential element of resistance to infection which is likely influenced by expression of adhesion molecules. The contribution of subsets to mammary gland resistance remains unclear. Mononuclear cells from milk and blood of dairy cows were examined for variation in CD4+, CD8+, and WC1+ (Workshop Cluster 1; marker for gammadelta T cells) lymphocyte phenotypes and expression of LFA-1 and L-selectin at several time points during the periparturient period and at Week 16 of lactation. Proportions of CD4+ T cells were higher (p < or = 10.05) in blood than milk at all times between Week 0 and Week 16 relative to calving; the inverse was true of CD8+ cells. Expression of L-selectin was lower (p < or = 0.05) on CD4+ cells and higher on CD8+ cells from milk. The WC1+ subset was more frequent in blood than in milk except at calving when the opposite was true. After calving, proportions of L-selectin+ WC1+ cells decreased steadily to Week 16. Expression of LFA-1 was examined on mononuclear cell populations and found to be lower on milk cells and did not vary over time. We conclude that proportions of T cells subsets differ significantly between blood and milk, particularly around calving. Corresponding variations in L-selectin expression may indicate a role for this molecule in regulating the movement of CD8+ and WC1+ T cells into the bovine mammary gland.

Pigs selected for high (H) or low (L) combined antibody and cell-mediated immune response were infected with Mycoplasma hyorhinis. Following the infection, arthritis was more severe in the H pigs, while pleuritis and peritonitis were more severe in the L pigs. Since Mycoplasma infections in pigs often cause just mild signs, indicators of the inflammatory response may aid diagnosis of such infections. In addition, data about the genetic influence on inflammatory response indicators are scanty in the pig. The objectives of the study were therefore: firstly, to determine interferon-alpha (IFNalpha) and haptoglobin in M. hyorhinis infected pigs and, secondly, to investigate if the inflammatory response as determined by these indicators was influenced by genetic selection. There was no consistent increase of IFN-alpha in serum following infection. The serum haptoglobin concentration started to increase 3 days post-infection and there was no difference between the two breeding lines. Hence, M. hyorhinis infection in pigs is reflected in increased serum haptoglobin concentration, but no effect of the magnitude of the inflammatory response on this indicator by selection for high or low immune response was observed.

Pigs have been selected for high (H) or low (L) combined antibody and cell-mediated immune response to test the high immune response phenotype as a candidate for an indirect approach to improving health and productivity in livestock. Mycoplasma hyorhinis infection was induced in H and L pigs of the 4th generation of selection to test the hypothesis that immune response lines differ in response to infection. The major disease sign, arthritis, was more severe in the H pigs both clinically and at necropsy. M. hyorhinis was isolated at higher colony counts from synovial fluids of the H pigs. In contrast, pleuritis and peritonitis were less severe in pigs of the H than those of the L line. Pericarditis, although less in H than L pigs, did not differ significantly by line. Synovial fluid antibody to M. hyorhinis did not differ by line but H pigs produced serum antibody earlier and to a higher titre than did L pigs. Selection for H or L immune response therefore alters response to M. hyorhinis, however there is no indication of a consistent line-related health advantage.

The objectives of this study were to determine hormone and antibody response profiles from the prepartum period to peak lactation, and evaluate potential immunomodulatory effects of the classic endocrine hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and cortisol. Specifically, 33 Holstein cows were immunized with ovalbumin (OVA) and Escherichia coli J5 at weeks -8 and -3 prior to parturition. At parturition (week 0), cows received an additional immunization of OVA. Blood was collected at weeks -8, -3, 0, 3 and 6 relative to parturition and various samples were used to determine plasma hormone concentration, serum immunoglobulin (Ig), and specific antibody response to OVA and E. coli. Colostrum and milk samples were also collected post-parturition to monitor local immunoglobulin and antibody responses. Results indicated that not all periparturient cows exhibited depressed immune response, and that antibody response to OVA could be used to partition cows into 3 groups recognizing animals with sustained measurable antibody response before and after parturition (Group 1), animals which responded poorly to immunization at parturition (Group 2), and animals which did not respond to immunizations at week -3 or parturition (Group 3). Cows with the highest antibody response to OVA (Group 1) also tended (P < or = 0.10) to have the highest response to E. coli J5 at parturition and had the lowest incidence of disease, particularly mastitis. Antibody response to OVA measured in milk tended to be higher in Group 1 cows, particularly at week 0 (P < or = 0.06) compared to cows of Group 3. IGF-I was higher (P < or = 0.05) in cows of Group 1 than Group 3 at peak lactation (week 6).

Alterations in the proportions of peripheral blood lymphocyte subsets was monitored using flow cytometry, before, during and after the peripartum period (week-8 to week (wk) 16) of dairy cows, when pregnancy and lactational stresses prevail and disease incidence is highest. A health code was assigned to each animal at each sampling time, and a subset of non-pregnant, non-lactating (NPNL) cows were sampled for comparisons, and to examine the effect of pregnancy and lactation only, on lymphocyte subset proportions. Results were expressed as the percentage of positive stained cells expressing CD2, CD4, CD8, WC1(gamma delta), IgM and class II major histocompatibility complex (MHC-II) molecules, as determined by flow cytometry. Comparisons of subset proportions were made across time and between health status categories. Mean fluorescence intensity (MFI) values were also examined across the periparturient period for CD2, CD4, CD8 and gamma delta T cell subsets. All T cell subsets varied significantly during the perparturient period in healthy cows, especially around parturition. B cell and MHC-II+ populations remained relatively constant until after calving and then significantly (P < or = 0.06) decreased. Compared to NPNL cows, all T cell subsets were significantly lower in healthy cows at most time points, whereas B cell and MHC-II+ cells were comparable to the NPNL values. CD2, CD4, and CD8 subsets decreased significantly at wk-3 and returned to initial subset proportions by wk 16. Conversely, the WC1 population increased at wk-3 relative to calving but declined to approximate initial proportions at calving. Health status had no significant overall effect on any subset unless it was separated by weeks in which case there were interactions between health and week for CD2 and CD8. Results indicate that variations in T lymphocyte subsets and in the concentrations of surface marker molecules (MFI) occur more as a result of pregnancy and lactation than health status in the dairy cow and that these factors have the least influence upon B cell and MHC-II+ populations during the periparturient period.

The objective of this study was to determine the effects of supplemental dietary chromium (Cr) on health status and mastitis-related parameters, as well as neutrophil phagocytic activity of dairy cows during late pregnancy and early lactation. In addition, possible interactions and involvements of Cr with insulin, cortisol, somatotropin (rBST) and insulin-like growth factor-1 (IGF-1) were directly investigated in vitro based on blastogenic responses using peripheral blood lymphocytes (PBL) of eight cows fed the control diet. Forty pregnant Holsteins, 18 primiparous and 22 multiparous, at week (wk) 6 before the expected calving dates were randomly assigned to treatments: control and supplemental chelated Cr (0.5 ppm) in the diet. All cows were managed in a normal production operation and health status was assessed by recording the incidence of health problems during the experimental period of wks 6 before and 16 after calving (-6 to 16 wks). Mastitis-related parameters included somatic cell counts (SCC), bacterial colony counts of milk samples from each mammary gland quarter of all cows during wk 1 to wk 8 postpartum. Peripheral blood neutrophil phagocytic function of eight cows fed either control or supplemental Cr diet was determined by the ability to take up uniform fluorescent beads measured by flow cytometry. Supplemental Cr had no effect (P > 0.10) on health status of cows during late pregnancy and early lactation, or on SCC and bacterial colonies of quarter milk samples from early lactation (wks 1 to 8). Supplemental Cr also did not affect neutrophil phagocytic function of cows from 6 wks prepartum to 6 wks postpartum. However, in the in vitro study of PBL (from control animals, not fed Cr diet) blastogenesis with addition of insulin or cortisol at two levels (0.05 and 0.5 ng ml-1), insulin and cortisol enhanced or had no effect on PBL proliferations with or without concanavalin A (con A) simulation. However, further Cr addition in the culture medium containing supplemental insulin or cortisol, particularly CrCl3, additively increased (P < 0.05) BPL blastogenic activities with or without con a stimulation. Conversely, addition of rBST or IFG-1 (0.5 and 5.0 ng ml-1) in the culture medium enhanced PBL proliferation, but addition of Cr gave no additional effect. These results indicated that supplemental Cr had no beneficial effect on health status, mastitis-related parameters or neutrophil phagocytic activity of dairy cows. However, in vitro study confirmed and extended our previous observations that Cr has an effect on lymphocyte proliferation and this may associate with insulin or cortisol actions.

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.

Monocyte function was investigated in second (G2) and third (G3) generation pigs selected for high and low antibody and cell-mediated immune responsiveness. In groups of pigs from the high-and low-immune response lines, monocyte release of superoxide anion (O2-) was assayed in response to phorbol 12-myristate-13-acetate and expression of the Class II-MHC (MHC-II) antigens SLA-DR and SLA-DQ, determined using flow cytometry. Analysis of variance using a linear model demonstrated no significant intergroup differences in O2- production by lymphokine-activated monocytes from G2 pigs. In G3 pigs, there were no significant intergroup differences in the percentage of MHC-II+ cells or in the density of expression of either SLA-DR or SLA-DQ. In individual pigs, monocyte SLA-DR and SLA-DQ expression was similar in terms of the percentage of MHC-II+ cells and in the magnitude of MHC-II expression. Litter contributed significantly to variation in monocyte O2- production in G2 pigs (P < or = 0.005) and SLA-DQ (P < or = 0.01) expression. Although the lines differed significantly in correlates of antibody and cell-mediated immune response, there was no apparent effect of selection for high and low immune responsiveness in swine on monocyte O2- production and MHC-II expression.

Avidity indices of antibody to hen egg-white lysozyme (HEWL) were measured by chaotropic ion (SCN-) elution enzyme-linked immunosorbent assay (ELISA) in pigs grouped as high, control or low for various immune and innate resistance-related traits. The avidity index was the molar concentration of SCN- required to reduce by 50% the ELISA optical density value for a given serum. The index was independent of the amount of antibody. Eight- to ten-week-old Yorkshire pigs were immunized with HEWL and serum antibody measured by ELISA as one of five traits used to assign them to high, low or control response groups. Serum antibody avidity for HEWL was evaluated on Day 14 and Day 30 after primary (Day 0) and secondary (Day 14) immunization. The effects of response group, gender, litter, serum IgG concentration and anti-HEWL antibody on avidity were determined using a linear model. Antibody avidity indices varied amongst individuals. Mean avidity indices for sera collected on Days 14 and 30 were 0.61 +/- 0.43 and 1.22 +/- 0.56, with maximum indices of 2.64 and 2.86 respectively. Avidity of secondary response antibody was significantly higher (P less than or equal to 0.05). Pigs of the high response group had significantly higher secondary antibody avidity than those of the control (P less than or equal to 0.08) and low groups (P less than or equal to 0.01). Avidity index was positively correlated with antibody to HEWL on Days 14 and 30 but not to preimmunization serum IgG concentration or to other measured traits.

Variation in serum IgG and IgM concentration was determined in three homozygous SLA-defined strains of miniature swine (SLAa, SLAc and SLAd) and one recombinant strain SLAg (ABcDd) as part of a study of SLA and other genetic effects on immune response. Data were obtained from 119 8-week-old piglets from 29 litters by 12 sires and analyzed using a SAS linear model for the effects of SLA haplotype, sire, dam, litter, sex, season of birth and sow parity. SLA haplotype (P less than 0.10) and other genetic effects due to sire (P less than or equal to 0.001) and dam (P less than or equal to 0.002) contributed to the variation in serum IgG concentrations. Season of birth and sow parity also affected IgG concentration as did litter effects. Least squares mean comparisons indicated that pigs of the dd, dg and gg haplotypes had significantly higher serum IgG than did pigs of the other haplotypes. Heritability estimates for IgG, calculated by paternal half-sib correlation, ranged from 0.31 to 0.27, indicating that selection for increased serum IgG concentrations would be possible. For serum IgM concentrations, only the effect of litter was significant at P less than or equal to 0.001 and P less than or equal to 0.009 by the radial immunodiffusion test read at 24 or 48 h. Since sire variance components estimates were negative, heritabilities were not calculated for IgM and are assumed to be zero.