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The objectives of this study were to evaluate (1) natural antibodies (NAb) in Canadian Holstein cows, (2) genetic parameters and associations between NAb and specific antibody (SpAb), and (3) the association of NAb with clinical mastitis and differences in incidence rates of clinical mastitis (IRCM) among cows classified as high, average, or low responders for NAb. Natural antibodies (IgG and IgM) to keyhole limpet hemocyanin and SpAb to a type 2 test antigen were measured on 451 Holsteins from 41 herds across Canada. A series of uni- and tri-variate linear animal models were used to estimate genetic parameters and breeding values for NAb and SpAb. The models included the fixed effects of parity and stage of lactation and the random effects of herd-technician, animal, and residual. Using estimated breeding values for NAb, cows were classified as high, average, or low responders and phenotypic associations with the IRCM were investigated and a logistic regression performed. The estimated heritability was 0.27 for SpAb, and was 0.32 and 0.18 for NAb of the IgG and IgM isotypes, respectively. No significant genetic correlations were found between SpAb and NAb. Although no significant differences in the IRCM were found when cows were classified based on NAb IgG, cows classified as high responders for NAb IgM tended to have a lower IRCM compared with other cows. Immunoglobulin-M was associated with a decreased risk of clinical mastitis (odds ratio = 0.958). Results of this study suggest the potential to use NAb IgM as an additional tool to select for disease resistance in cattle, but results need to be validated with a larger sample size. [ABSTRACT FROM AUTHOR]

Data collected from 328 Canadian Holsteins in a research herd at the University of Guelph were used to study associations among expression of bovine leukocyte antigen (BoLA) DRB3.2 alleles, immune response, mastitis resistance via somatic cell counts (SCC), and clinical mastitis, as well as to extend these results to production and type traits. Accordingly, groups of cows were evaluated in vivo for both the antibody-mediated immune response (AMIR) and the cell-mediated immune response (CMIR), which generally predominate in responses to extracellular and intracellular pathogens, respectively. Of note was that associations between BoLA DRB3.2 alleles and immune responses tended to be in the opposite sign for the 2 AMIR and CMIR traits examined. For example, alleles DRB3.2*3 and *24 were associated with higher AMIR but lower CMIR, whereas allele *22 was associated with lower AMIR but higher CMIR. This finding is in agreement with the hypothesis that both traits are genetically independent and represent opposing type 1 and type 2 immune responses. Additionally, BoLA DRB3.2*3 and *11 were associated with lower SCC, whereas alleles *22 and *23 were associated with higher SCC. Finally, allele DRB3.2*3 was also associated with less clinical mastitis, whereas allele *8 was associated with higher mastitis risk. Allele *3 was of particular relevance because it was associated with increased antibodies, as well as reduced mastitis and SCC. This could be due to an indirect relationship between the ability to produce a high antibody response and enhanced defense against intrammamary infections caused by extracellular pathogens. Consequently, the BoLA DRB3.2*3 allele should be investigated further as a candidate for resistance to some types of intramammary infections, the important caveat being its association with lower CMIR, particularly with one of the test antigens used to evaluate delayed-type hypersensitivity. The results of associations between BoLA DRB3.2 and production traits were, in some cases, antagonistic in that BoLA DRB3.2 alleles *11 and *23, which are associated with increased production traits, were associated with lower and higher SCC, respectively. Collectively, these findings advocate the use of alleles *3, *23, and *22 as reference points for more detailed mechanistic studies. This does not imply that genetic selection for mastitis resistance should be based on BoLA alleles, but that information on a variety of genes may aid in identification and selection for improved health. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dairy cattle evaluated for immune responses and identified as high responders are known to have a lower occurrence of economically important diseases, including mastitis, metritis, ketosis, and retained placenta. These high immune responders have also been shown to make more antibody following vaccination and to have improved milk and colostrum quality. Therefore, breeding for improved immune response is expected to have several benefits in the dairy industry. However, a concern of such an approach to improve animal health is the potential cost of lost production due to an allocation of host resources to mount a robust immune response. The objective of this study was to evaluate early- and late-lactation production parameters in cattle classified as having high, average, or low estimated breeding values (EBV) for cell-mediated (CMIR), antibody- mediated (AMIR), and overall immune responses. A total of 561 cows from 6 herds were phenotyped for immune response and ranked based on EBV for CMIR and AMIR. A linear animal model was used to evaluate differences in milk, fat, and protein yields among immune response groups, and a regression analysis was conducted based on immune response EBV. Overall, no difference in production parameters was found based on immune response rank; however, some positive relationships with immune response EBV were found, suggesting that breeding for enhanced immune responsiveness as a prophylactic approach to improve animal health would not come at the cost of lost production. [ABSTRACT FROM AUTHOR]

Genetic selection for enhanced immune response has been shown to decrease disease occurrence in dairy cattle. Cows can be classified as high (H), average, or low responders based on antibody-mediated immune response (AMIR), predominated by type-2 cytokine production, and cell-mediated immune response (CMIR) through estimated breeding values for these traits. The purpose of this study was to identify in vitro tests that correlate with in vivo immune response phenotyping in dairy cattle. Blood mononuclear cells (BMC) isolated from cows classified as H-AMIR and H-CMIR through estimated breeding values for immune response traits were stimulated with concanavalin A (ConA; Sigma Aldrich, St. Louis, MO) and gene expression, cytokine production, and cell proliferation was determined at multiple time points. A repeated measures model, which included the effects of immune response group, parity, and stage of lactation, was used to compare differences between immune response phenotype groups. The H-AMIR cows produced more IL-4 protein than H-CMIR cows at 48 h; however, no difference in gene expression of type-2 transcription factor GATA3 or IL4 was noted. The BMC from H-CMIR cows had increased production of IFN-γ protein at 48, 72, and 96 h compared with H-AMIR animals. Further, H-CMIR cows had increased expression of the IFNG gene at 16, 24, and 48 h post-treatment with ConA, although expression of the type-1 transcription factor gene TBX21 did not differ between immune response groups. Although proliferation of BMC increased from 24 to 72 h after ConA stimulation, no differences were found between the immune response groups. Overall, stimulation of HAMIR and H-CMIR bovine BMC with ConA resulted in distinct cytokine production profiles according to genetically defined groups. These distinct cytokine profiles could be used to define disease resistance phenotypes in dairy cows according to stimulation in vitro; however, other immune response phenotypes should be assessed. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate IgG and β-lactoglobulin (β-LG) concentrations in colostrum and milk of Canadian Holsteins (n = 108) classified as high (H), average (A), or low (L) for antibody-mediated (AMIR) or cell-mediated immune responses (CMIR) based on estimated breeding values. It was hypothesized that H-AMIR and H-CMIR cows produce colostrum (first milking) and milk (d 5 postcalving) with higher concentrations of IgG and β-LG. Data for IgG and β-LG in colostrum and milk were analyzed independently using mixed linear models. Least squares means were compared using Tukey's test. Cows classified as H-AMIR had higher IgG and β-LG concentrations in colostrum compared with A- and L-AMIR cows; 84% of H-AMIR, 69% of A-AMIR, and 68% of L-AMIR cows had over 5,000 mg/dL IgG in colostrum. No differences in IgG and β-LG concentrations in colostrum were noted among cows ranked on CMIR or in milk of cows ranked on AMIR. β-Lactoglobulin and IgG concentrations were positively correlated in colostrum. Breeding cows for H-AMIR status may reduce failure of passive transfer of IgG in their calves; β-LG may play a role in bovine immune defenses. Colostrum from H-AMIR cows may serve as a more economical feedstock source for manufacturing natural health products. [ABSTRACT FROM AUTHOR]

The objective of this study was to use previously calculated estimated breeding values for cell- (CMIR) and antibody-mediated immune responses (AMIR) to determine associations between immune response (IR) and economically important diseases of dairy cattle. In total, 699 Holsteins were classified as high, average, or low for CMIR, AMIR, and overall IR (combined CMIR and AMIR), and associations with mastitis, metritis, ketosis, displaced abomasums, and retained fetal membranes were determined. The incidence of mastitis was higher among average cows as compared with cows classified as high AMIR [odds ratio (OR) = 2.5], high CMIR (OR = 1.8), or high IR (OR = 1.8). Low-CMIR cows had a higher incidence of metritis (OR = 11.3) and low-IR cows had a higher incidence of displaced abomasum (OR = 4.1) and retained fetal membrane (OR = 2.8) than did average responders. Results of this study show that cows classified as high immune responders have lower occurrence of disease, suggesting that breeding cattle for enhanced IR may be a feasible approach to decrease the incidence of infectious and metabolic diseases in the dairy industry. [ABSTRACT FROM AUTHOR]

The objectives of this study were to estimate genetic parameters of cell-mediated (CMIR) and antibody-mediated (AMIR) immune response (IR) traits of Holstein cattle on a national scale and to associate estimated breeding values of CMIR, AMIR, and overall IR with routinely evaluated traits in Canada. In collaboration with the Canadian Bovine Mastitis Research Network, 445 Holstein cows from 42 herds across Canada were immunized to measure delayed-type hypersensitivity as an indicator of CMIR and serum antibody for AMIR to putative type 1 and type 2 test antigens, respectively. Primary (d 14) and secondary (d 21) AMIR were measured for both IgG1 and IgG2. A series of uni- and bivariate linear animal models were used to estimate genetic parameters and breeding values for CMIR and the 4 AMIR traits. The models included the fixed effects of parity and stage of lactation and the random effects of herd-technician, animal, and residual. Heritability of CMIR was 0.19 (SE = 0.10) and for AMIR traits ranged from 0.16 to 0.41 (SE = 0.09--0.11) depending on time and antibody isotype. The genetic correlations between CMIR and AMIR were negative and ranged from -0.13 to -0.45 (SE = 0.32--0.46). The results indicate adaptive immune response traits are moderately heritable and provide a potential for genetic selection. The negative genetic correlations between CMIR and AMIR indicate the importance of considering both traits in breeding for overall disease resistance. Significant beneficial associations between the reproductive traits number of services and first service to conception were found, which may indicate these reproductive traits would improve with genetic selection for enhanced immune response. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate potential differences in udder health and immune response traits among Holstein-Friesian (HF), Norwegian Red (NR), and NR x HF (NRX) cows on 30 commercial Irish dairy farms. A total of 648 second-lactation cows (HF n = 274, NR n = 207, and NRX n = 167) were immunized with hen egg white lysozyme (HEWL) to induce antibody-mediated immune response (AMIR). Candida albicans was used to induce a cell-mediated immune response, with in vivo delayed-type hypersensitivity used as the indicator. Antibody response to HEWL was measured by ELISA. Udder health defined as mean somatic cell score (SCS) over the lactation, mean SCS within 30 d of the beginning of the immunization, peak SCS for each individual cow during lactation, and incidence of mastitis were statistically superior for the NR. The NR had a greater primary AMIR, producing greater concentrations of anti-HEWL immunoglobulin G on d 14 compared with HF and NRX. No difference was observed among the breed groups in the magnitude of secondary AMIR (d 21 post-immunization) or cell-mediated immune response. The proportion of high and low responders was similar across breed groups. Cows with high AMIR and high cell-mediated immune response had significantly lower mean SCS within 30 d of the start of the immunization, but greater occurrence of clinical mastitis, recorded as a binary trait over the course of the lactation. Otherwise, no significant difference in udder health was evident between cows designated as high and low responders. Although differences in mean breed group SCS values were in line with group mean AMIR values, no association was found among the traits when correlated on an individual cow basis. Results highlight the superiority of the NR with regard to udder health and suggest that improvements to udder health may result from crossbreeding with the NR. However, the immune response traits investigated proved to be inconsistent indicators of udder health. [ABSTRACT FROM AUTHOR]

The objective of this study was to compare the immune response of Holstein and Norwegian Red x Holstein calves on 13 commercial Canadian dairy farms. Data were collected on 135 calves, 68 Holstein and 67 Norwegian Red x Holstein calves aged between 2 and 6 mo. The calves were immunized with hen egg white lysozyme to induce antibody-mediated immune response. Candida albicans was used as an in vivo indicator of cell-mediated immune response, with delayed-type hypersensitivity used as the indicator. Antibody response to hen egg white lysozyme (IgG, IgG1, and IgG2) was measured by ELISA. Calves of both breed groups produced a significant primary and secondary antibody-mediated immune response, as well as a delayed-type hypersensitivity reaction. The Norwegian Red x Holstein produced a greater primary IgG antibody-mediated immune response (d 14, and d 14 minus d 0) when compared with the Holstein. No differences were observed between the breeds for secondary response or anti-hen egg white lysozyme isotype (IgG1 or IgG2) production or the ratio of IgG1:IgG2. There was no effect of breed on delayed-type hypersensitivity. Nonetheless, high and low immune responders could be identified in both breed groups, but with no difference in the proportion of high and low responders observed for either antibody-mediated immune response or cell-mediated immune response between breed groups. [ABSTRACT FROM AUTHOR]

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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Gastrointestinal nematodes (GINs) can be a major constraint and global challenge to the sheep industry. These nematodes infect the small intestine and abomasum of grazing sheep, causing symptoms such as weight loss, diarrhea, hypoproteinemia, and anemia, which can lead to death. The use of anthelmintics to treat infected animals has led to GIN resistance, and excessive use of these drugs has resulted in residue traced in food and the environment. Resistance to GINs can be measured using multiple traits, including fecal egg count (FEC), Faffa Malan Chart scores, hematocrit, packed cell volume, eosinophilia, immunoglobulin (Ig), and dagginess scores. Genetic variation among animals exists, and understanding these differences can help identify genomic regions associated with resistance to GINs in sheep. Genes playing important roles in the immune system were identified in several studies in this review, such as the CFI and MUC15 genes. Results from several studies showed overlapping quantitative trait loci (QTLs) associated with multiple traits measuring resistance to GINs, mainly FEC. The discovery of genomic regions, positional candidate genes, and QTLs associated with resistance to GINs can help increase and accelerate genetic gains in sheep breeding programs and reveal the genetic basis and biological mechanisms underlying this trait.

In cattle, maternal immunoglobulins are transferred through colostrum to provide passive immunity to the neonatal calf once they are absorbed into circulation. Cows can be assessed for antibody- and cell-mediated immune responses (AMIR and CMIR, respectively), and through estimated breeding values (EBV) and genomic parent averages (GPA), cows can be classified as having high, average, or low immune response (IR). The objective of this study was to identify associations of colostral IgG concentrations with IR in dairy cows. High IR dairy cows identified by GPA or EBV were hypothesized to produce higher colostral IgG concentrations than cows with average or low IR. Colostrum was collected from Holstein dairy cows from 3 large commercial herds (n = 590) in the United States and 1 research herd at the Ontario Dairy Research Centre (n = 275) in Canada. For the US herds, IR GPA were available through genotyping. For the Canadian herd, IR EBV were available through phenotyping and pedigree information. Colostral IgG concentrations were measured by radial immunodiffusion and analyzed using general linear models in SAS. Based on a prediction equation, cows in US herds with a CMIR GPA of 1 would have colostral IgG concentrations 6.3 g/L higher on average than cows with a CMIR GPA of 0. High CMIR cows produced statistically greater colostral IgG concentrations (least squares mean ± standard error of the mean, 107.5 ± 7.7 g/L) than low CMIR cows (91.4 ± 7.1 g/L), with intermediate values for average CMIR cows (105.1 ± 5.6 g/L). No differences were found among AMIR categories in US cows. The Canadian herd showed a trend for cows with high CMIR EBV (continuous variable) to produce greater colostral IgG. No differences were observed among high, average, and low AMIR EBV classifications in Canadian cows. The findings suggest that selective breeding of Holstein cows to enhance CMIR could contribute to higher-quality colostrum in succeeding generations., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Animals respond to stress by activating a wide array of physiological and behavioral responses that are collectively referred to as the stress response. MicroRNAs (miRNAs) are small, noncoding RNAs that play key roles in the regulation of homeostasis. There are many reports demonstrating examples of stress-induced miRNA expression profiles. The aim of this study was to determine the circulatory miRNA profile of variable stress-responding lambs ( n = 112) categorized based on their cortisol levels as high (HSR, 336.2 ± 27.9 nmol/L), middle (MSR, 147.3 ±9.5 nmol/L), and low (LSR, 32.1 ± 10.4 nmol/L) stress responders post-LPS challenge (400 ng/kg iv ). Blood was collected from the jugular vein at 0 (T0) and 4 h (T4) post-LPS challenge, and miRNAs were isolated from four animals from each group. An array of 84 miRNAs and 6 individual miRNAs were evaluated using qPCR. Among 90 miRNAs, there were 48 differentially expressed (DE) miRNAs (log fold change (FC) > 2 < log FC) in the HSR group, 46 in the MSR group, and 49 in the LSR group compared with T0 (control) samples. In the HSR group, three miRNAs, miR-485-5p, miR-1193-5p, and miR-3957-5p were significantly ( p < 0.05) upregulated, while seven miRNAs, miR-376b-3p, miR-376c-3p, miR-411b-5p, miR-376a-3p, miR-376b-3p, miR-376c-3p, and miR-381-3p, were downregulated ( p < 0.05) as compared to the LSR and MSR groups. Functional analysis of DE miRNAs revealed their roles in Ras and MAPK signaling, cytokine signaling, the adaptive immune system, and transcription pathways in the HSR phenotype, implicating a hyper-induced acute-phase response. In contrast, in the LSR group, enriched pathways included glucagon signaling metabolic regulation, the transportation of amino acids and ions, and the integration of energy metabolism. Taken together, these results indicate variation in the acute-phase response to an immune stress challenge, and these miRNAs are implicated in regulating responses within cortisol-based phenotypes.

The variety of drugs available to treat neurodegenerative diseases is limited. Most of these drug's efficacy is restricted by individual genetics and disease stages and usually do not prevent neurodegeneration acting long after irreversible damage has already occurred. Thus, drugs targeting the molecular mechanisms underlying subsequent neurodegeneration have the potential to negate symptom manifestation and subsequent neurodegeneration. Neuroinflammation is a common feature of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis, and is associated with the activation of the NLRP3 inflammasome, which in turn leads to neurodegeneration. Inflammasome activation and oligomerisation is suggested to be a major driver of disease progression occurring in microglia. With several natural products and natural product derivatives currently in clinical trials, mushrooms have been highlighted as a rich and largely untapped source of biologically active compounds in both in vitro and in vivo neurodegenerative disease models, partially supported by successful clinical trial evaluations. Additionally, novel high-throughput methods for the screening of natural product compound libraries are being developed to help accelerate the neurodegenerative disease drug discovery process, targeting neuroinflammation. However, the breadth of research relating to mushroom natural product high-throughput screening is limited, providing an exciting opportunity for further detailed investigations.

Background: In mammalian species, hypoxia is a prominent feature of inflammation. The role of hypoxia in regulating macrophage responses via alteration in metabolic pathways is well established. Recently, oxidative burst-induced hypoxia has been shown in murine macrophages after phagocytosis. Despite the available detailed information on the regulation of macrophage function at transcriptomic and epigenomic levels, the association of genetic polymorphism and macrophage function has been less explored. Previously, we have shown that host genetics controls approximately 80% of the variation in an oxidative burst as measured by nitric oxide (NO - ). Further studies revealed two clusters of transcription factors (hypoxia-related and inflammatory-related) are under the genetic control that shapes macrophages' pro-inflammatory characteristics., Material and Methods: In the current study, the association between 43,066 autosomal Single Nucleic Polymorphism (SNPs) and the ability of MDMs in production of NO - in response to E. coli was evaluated in 58 Holstein cows. The positional candidate genes near significant SNPs were selected to perform functional analysis. In addition, the interaction between the positional candidate genes and differentially expressed genes from our previous study was investigated., Results: Sixty SNPs on 22 chromosomes of the bovine genome were found to be significantly associated with NO - production of macrophages. The functional genomic analysis showed a significant interaction between positional candidate genes and mitochondria-related differentially expressed genes from the previous study. Further examination showed 7 SNPs located in the vicinity of genes with roles in response to hypoxia, shaping approximately 73% of the observed individual variation in NO - production by MDM. Regarding the normoxic condition of macrophage culture in this study, it was hypothesized that oxidative burst is responsible for causing hypoxia at the cellular level., Conclusion: The results suggest that the genetic polymorphism via regulation of response to hypoxia is a candidate step that perhaps shapes macrophage functional characteristics in the pathway of phagocytosis leading to oxidative burst, hypoxia, cellular response to hypoxia and finally the pro-inflammatory responses. Since all cells in one individual carry the same alleles, the effect of genetic predisposition of sensitivity to hypoxia will likely be notable on the clinical outcome to a broad range of host-pathogen interactions., Competing Interests: MS was employed by Select Sires Inc. The commercial use of the phenotyping method of Monocyte-Derived Macrophages that was described in the manuscript is protected by the Patent Cooperation Treaty (PCT/CA2020/050997) and pending US patents (App. No. 62/878,377 and 62/941,927). ME is the founder of Biomics Analytica Inc. and BM is the CEO of Immunogenetic Inc. These companies are commercializing the SNPs that were described in the manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Emam, Tabatabaei, Sargolzaei and Mallard.)

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-1β, 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., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Reactive Nitrogen Species (RNS) are a group of bactericidal molecules produced by macrophages in response to pathogens in a process called oxidative burst. Nitric oxide (NO - ) is a member of RNS produced from arginine by inducible Nitric Oxide Synthase (iNOS) enzyme. The activity of iNOS and production of NO - by macrophages following stimulation is one of the indicators of macrophage polarization towards M1/proinflammatory. Production of NO - by bovine monocyte-derived macrophage (MDM) and mouse peritoneal macrophages has been shown to be strongly associated with host genetic with the heritability of 0.776 in bovine MDM and 0.8 in mouse peritoneal macrophages. However, the mechanism of genetic regulation of macrophage response has remained less explored. In the current study, the transcriptome of bovine MDMs was compared between two extreme phenotypes that had been classified as high and low responder based on NO - production. The results showed that 179 and 392 genes were differentially expressed (DE) between high and low responder groups at 3 and 18 hours after exposure to Escherichia coli, respectively. A set of 11 Transcription Factors (TFs) (STAT1, IRF7, SPI1, STAT4, IRF1, HIF1A, FOXO3, REL, NFAT5, HIC1, and IRF4) at 3 hours and a set of 13 TFs (STAT1, IRF1, HIF1A, STAT4, ATF4, TP63, EGR1, CDKN2A, RBL1, E2F1, PRDM1, GATA3, and IRF4) at 18 hours after exposure to E. coli were identified to be differentially regulated between the high and low responder phenotypes. These TFs were found to be divided into two clusters of inflammatory- and hypoxia-related TFs. Functional analysis revealed that some key canonical pathways such as phagocytosis, chemotaxis, antigen presentation, and cell-to-cell signalling are enriched among the over-expressed genes by high responder phenotype. Based on the results of this study, it was inferred that the functional characteristics of bovine MDMs are associated with NO-based classification. Since NO - production is strongly associated with host genetics, this study for the first time shows the distinct proinflammatory profiles of macrophages are controlled by the natural genetic polymorphism in an outbred population. In addition, the results suggest that genetics can be considered as a new dimension in the current model of macrophage polarization which is currently described by the combination of stimulants, only.

A key characteristic of mushroom polysaccharides that elicit an immunomodulatory response is that they are rich in β-glucans and low in α-glucans. In this study we analysed nine commercially available preparations from three mushroom species, Reishi (Ganoderma lucidum), Shiitake (Lentinula edodes) and Maitake (Grifola frondosa), for β- and α-glucan content. Based on β- and α-glucan content we selected three extracts to combine into a formula and evaluated the ability of the individual extracts and formula to impact on the expression of cytokines IL-1α, IL-6, IL-10 and TNF-α in human macrophages with and without LPS stimulation. The majority of mushroom extracts and the formula were found to be highly potent immuno-stimulators possessing EC50 values lower than 100 μg/mL. Interestingly the mushroom formula had lower EC50 values in TNF-α expression from LPS stimulated macrophages compared to the individual extracts, suggesting a potential synergistic effect of the mushroom formula. A response additivity graph and curve-shift analysis illustrated that indeed the mushroom formula exhibited an immuno-stimulatory synergistic effect on the expression of the majority of cytokines evaluated in both LPS stimulated and non-stimulated human macrophages, with IL-10 having an antagonistic response. This study represents the first report of a synergistic immuno-modulatory response in human macrophages elicited from a mushroom formula rationally derived from β- and α-glucan content., Competing Interests: This research was sponsored by Integria Healthcare (Australia) Pty Ltd., and was conducted collaboratively with DNL and HW who are employees of Integria Healthcare (Australia) Pty Ltd. DNL and HW quantified the glucan levels in the mushroom extracts, were involved in the preliminary cytokine expression study design, preliminary data analysis, editing and revising of the manuscript, and decision to publish. BM and JT are employees of Griffith University and designed and conducted the cytokine expression analysis as well as the synergy analysis. They acquired the cytokine expression data, analysed and interrupted the data, wrote, edited and revised the manuscript, and initiated the decision to publish. A patent application (International PCT Application No. PCT/AU2019/050295) based in part on these results has been filed by Integria Healthcare (Australia) Pty Ltd naming BM, DNL, HW and JT as inventors. DNL and HW are employees of Integria Healthcare (Australia) Pty Ltd, which is the sponsor of MediHerb® Mushroom Forte. We confirm that this does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

The dynamic interaction between the host and pathogens, along with environmental factors, influences the regulation of mammalian immune responses. Therefore, comprehensive in vivo immune-phenotyping during an active response to a pathogen can be complex and prone to confounding effects. Evaluating critical fundamental aspects of the immune system at a cellular level is an alternative approach to reduce this complexity. Therefore, the objective of the current study was to examine an in vitro model for functional phenotyping of bovine monocyte-derived macrophages (MDM), cells which play a crucial role at all phases of inflammation, as well influence downstream immune responses. As indicators of MDM function, phagocytosis and nitric oxide (NO - ) production were tested in MDM of 16 cows in response to 2 common bacterial pathogens of dairy cows, Escherichia coli and Staphylococcus aureus. Notable functional variations were observed among the individuals (coefficient of variation: 33% for phagocytosis and 70% in the production of NO - ). The rank correlation analysis revealed a significant, positive, and strong correlation (rho = 0.92) between NO - production in response to E. coli and S. aureus, and a positive but moderate correlation (rho = 0.58) between phagocytosis of E. coli and S. aureus. To gain further insight into this trait, another 58 cows were evaluated solely for NO - response against E. coli. The pedigree of the tested animals was added to the statistical model and the heritability was estimated to be 0.776. Overall, the finding of this study showed a strong effect of host genetics on the in vitro activities of MDM and the possibility of ranking Holstein cows based on the in vitro functional variation of MDM., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Background: Technical feasibility of RNA quantification by real time RT-PCR has led to enormous utilization of this method. However, real time PCR results need to be normalized due to the high sensitivity of the method and also to eliminate technical variation. Normalization against a reference gene that is constitutively transcribed and has minimum variation among samples is the ideal method. Nevertheless, many studies have shown that there is no general reference gene(s) with ideal characteristics and candidate reference genes should be tested before being used as a "normalizer" in each study., Methods: The current study investigated the effects of previous exposure of the host to experimental test antigens and culturing time on the expression of 11 candidate genes when blood mononuclear cells (BMCs) were cultured and treated in-vitro by hen egg white lysozyme, Candida albicans extract and a mitogen. Mononuclear cells were isolated and cultured from 12 bovine blood samples representing 3 different immunological statuses. The expression of candidate housekeeping genes were measured by real-time RT-PCR at 4 and 24 hours post culture. The expression of candidate genes were first compared between the two time points in untreated samples. Constitutively expressed genes were further tested in linear mixed effects models to examine the effect of previous host exposure and in-vitro treatments., Results: Our findings showed that the expression of the most common reference genes, β-actin, and Glyceraldehydes-3-phosphate dehydrogenase (GAPDH), are significantly decreased at 24 hours after culturing BMCs, even without any treatment. The effect of culturing time was also significantly influenced the expression of 18s ribosomal RNA, β2-microglobulin, Tyrosine 3-monooxygenase/tryptophan 5-monoxygenase activation protein, zeta polypeptide (YWHAZ) in BMCs. Only the expression of C-terminal binding protein 1 (CTBP1) and RAD50 among all tested genes were consistent after treatment of cultured BMCs with C. albicans whole yeast extract and Hen Egg White Lysozyme (HEWL), respectively. In addition, expressions of CTBP1, and RAD50 were independent from previous exposure of the host to the antigen., Conclusions: The results of this study demonstrated inconsistent expression of commonly used reference genes in untreated cultured BMCs over time. As this condition applies to negative controls in real time RT-PCR study designs, normalization against these genes can largely deceive the outcome, especially in kinetic studies. Moreover, the potential effects of immunological memory on the expression of reference genes should be considered if BMCs are collected from different individuals under different environmental conditions and if these cells are treated in-vitro by an antigen.

Immunocompetence of livestock plays an important role in farm profitability because it directly affects health maintenance. Genetics significantly influences the immune system, and the genotypic structure of modern fast-growing chickens has been changed, particularly after decades of breeding for higher production. Therefore, this study was designed to help determine if intensive breeding programs have adversely affected immunocompetence or whether the immune response profiles are controlled to greater extent by genetic background. Thus, 3 indigenous chicken populations from different genetic backgrounds and 2 globally available modern broiler strains, Ross 308 and Cobb 500, were evaluated for various aspects of immune response. These included antibody responses against sheep red blood cells and Brucella abortus antigen, as well as some aspects of cell-mediated immunocompetence by toe web swelling test and in vitro blood mononuclear cell proliferation. Significant differences (P < 0.05) in antibody responses to both antigens and cellular proliferation were observed among populations but not consistently between modern commercial strains versus the indigenous populations. In fact, the immune response profiles of Cobb 500 were similar to the indigenous populations, but varied compared with the other commercial strain. In addition, considerable variation was recorded between indigenous populations for all responses measured in this study. The results of this study suggest that the variation observed in immune responses between these strains of chickens is most likely due to differences in the genetic background between each strain of chicken rather than by commercial selection programs for high production.

Background: Staphylococcus aureus is one of the most prevalent pathogens to cause mastitis in dairy cattle. Intramammary infection of dairy cows with S. aureus is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. A sub-population of S. aureus, known as small colony variant (SCV), displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. Therefore, it was hypothesized that the host immune response will be different for SCV than its parental or typical strains of S. aureus. In this study, the local and systemic immune protein responses to intramammary infection with three strains of S. aureus, including a naturally occurring bovine SCV strain (SCV Heba3231), were characterized. Serum and casein-depleted milk cytokine levels (interleukin-8, interferon-γ, and transforming growth factor-β1), as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three S. aureus strains. Furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of S. aureus intramammary infection., Results: Serum IL-8, IFN-γ, and TGF-β1 responses differed in dairy cows challenged with different strains of S. aureus. Changes in overall serum haptoglobin concentrations were observed for each S. aureus challenge group, but there were no significant differences observed between groups. In casein-depleted milk, strain-specific differences in the host IFN-γ response were observed, but inducible IL-8 and TGF-β1 concentrations were not different between groups. Proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection. Notably, the protein, component-3 of the proteose peptone (CPP3), was differentially expressed between the S. aureus treatment groups, implicating it as a potential antimicrobial peptide involved in host defense against S. aureus intramammary infection., Conclusions: Intramammary infection of dairy cattle with S. aureus causes an up-regulation of serum and milk immune-related proteins, and these responses vary depending on the infecting strain.

As an extension of a former study, the objectives of this study were to evaluate purebred Holstein (HO; n=140) and crossbred Norwegian Red × Holstein (NRFX; n=142) calves for antibody (AMIR) and cell-mediated immune responses (CMIR) as well as survival. Blood was collected on d 0, 14, and 21, and calves were immunized on d 0 and 14 with type 1 (Candida albicans) and type 2 (hen egg white lysozyme) antigens, which have been shown to induce CMIR and AMIR, respectively. Day 21 background skin-fold measurements of either side of the tail-fold were taken and intradermal injections of test (type 1 antigen) and control (phosphate saline buffer) were administered. Day 23 final skin-fold measurements were taken to assess delayed type hypersensitivity as an indicator of CMIR. Survival data were obtained from CanWest Dairy Herd Improvement. Statistical Analysis System general linear models were used to analyze all immune response and survival data and to determine statistical significance between breeds. Results showed that NRFX had greater primary IgM, IgG, IgG1, and secondary IgG1 antibody response, as well as greater primary IgG1:IgG2 ratio to the type 2 antigen compared with HO. The NRFX also had greater primary IgG1 and IgG2, and secondary IgG2 antibody response as well as greater primary IgG1:IgG2 ratio to the type 1 antigen. The NRFX calves had a tendency toward greater survival from age at immune response testing to calving. No difference was observed between breeds for other secondary antibody response traits or delayed type hypersensitivity. Results indicate NRFX have greater AMIR and therefore may have enhanced defense against extracellular pathogens. This may contribute to increased survival compared with HO. Both breeds, however, likely have similar defense against intracellular pathogens, because no differences in CMIR were observed. In general, these results may suggest that crossbreeding could improve resistance to certain diseases in dairy calves, resulting in decreased input costs to producers for crossbred calves compared with purebred calves. However, more research with larger sample sizes and different breeds should be conducted to confirm these results and obtain a complete picture of the benefits of crossbreeding on immune response traits in calves., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Selection for production traits with little or no emphasis on health traits has led to an increase in the incidence of disease in Canadian dairy herds. We describe here a patented protocol for estimating the breeding value for immune responsiveness in heifers that combines measures of both cell-mediated (CM) and antibody-mediated (AM) immune responses (IR). The ability of putative type 1 and type 2 antigens used to induce CMIR and AMIR, respectively, was assessed in replacement Holstein heifers, and the effects of age and pregnancy on type 1 and type 2 IR bias were estimated. Results demonstrated that the type 1 and type 2 antigens induced polarized type 1 and type 2 responses in heifers regardless of age and pregnancy status, and can therefore be used to identify animals with superior overall immune responsiveness. However, age and pregnancy status had significant effects on adaptive IR profiles, highlighting the need for appropriate statistical modeling of such effects when ranking animals on their ability to mount CM and AMIR. Responses became increasingly type 1 biased as heifers approached 12 mo of age, from which point, responses then became increasingly type 2 biased with age and length of gestation. Knowledge of how age and pregnancy influence the dynamics of type 1 and type 2 IR bias is expected to improve our ability to select animals with enhanced immune responsiveness and aid in the development of effective vaccines through strategic targeting of vaccine components to recipients., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Persistence of bovine Staphylococcus aureus mastitis may be associated with the small colony variant (SCV) form that is adapted to intracellular life and resists elimination by the immune system. This study evaluated antibody-mediated (AMIR) and cell-mediated immune responses (CMIR) to two bovine SCV forms and their parent strains isolated from cows with mastitis. Four groups of healthy cows, five cows/treatment group, were challenged by the intramammary route with naturally occurring bovine SCV Heba3231, its parent strain 3231, a hemB mutant displaying the SCV phenotype or its parent strain, Newbould 305. Blood and milk samples were collected at day 0 before challenge and at days 1, 14, 21 and 36 post-challenge to determine antigen-specific immunoglobulin (Ig) IgG(1) and IgG(2) antibody responses as indicators of type 2 and type 1 responses, respectively. At day 24 post-challenge cows in each group were inoculated with the UV-killed homologous strain intradermally in the neck to induce delayed-type hypersensitivity (DTH) as an indicator of CMIR. The SCV Heba3231 and 3231 strains induced significant IgG(1) and IgG(2) antibody responses in sera and in sera and milk whey, respectively. The hemB SCV mutant and Newbould 305 strains induced significant IgG(1) antibody in milk whey, and in sera and milk whey, respectively. The SCV Heba3231 and 3231 strains induced DTH, the hemB mutant induced intermediate hypersensitivity, and Newbould 305 failed to induce DTH. These results indicate marked differences in immune responses induced by parent and SCV forms of the same strain of S. aureus and by the two wild-type strains. This is the first study to evaluate both AMIR and CMIR in the context of persistent bovine mastitis to different and genetically characterized strains of S. aureus including two SCVs. The findings expand our understanding of immune responses to persistent S. aureus mastitis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Suboptimal innate and immune mechanisms of host resistance during the peripartum period may contribute to increased incidence of mastitis. To evaluate associations between antibody response to ovalbumin and milk production during the peripartum period, 136 Holstein cows and heifers from three herds with known antibody response profiles, were evaluated for projected 305-d milk, protein, and fat yield. Using a previously described index (Wagter et al., 2000), cows were quantitatively classified based on their profile of antibody response to ovalbumin into high, average, or low antibody response groups. The single-effect antibody response group contributed significantly to variation in fat and protein yield, but not milk yield. The interaction between antibody response and parity significantly contributed to the variation in milk, fat, and protein yields; therefore the effects of group were reported on a within-parity basis. Among first-parity cows, low responders had a higher fat and protein yield than high or average antibody responder animals. Among older cows (parity 3 or greater) milk yield was significantly higher for those in the high antibody response group compared with average and low response groups. However, no significant differences in fat or protein yields were observed between high and low antibody response groups. These results suggest the possibility to select cows for enhanced immune response with no adverse effects on yield. That first-parity cows with low antibody response produce more fat and protein may be offset by the fact that mastitis occurrence was highest in this group in two out of three herds investigated. Selection for high immune response may prove beneficial to herd life by maintaining optimal yield, yet minimizing occurrence of disease.

A quantitative approach was developed to classify Holstein cows and heifers based on phenotypic variation of serum antibody response and to determine associations with peripartum mastitis. Using an index, 136 cows and heifers were classified into high (Group 1), average (Group 2), or low (Group 3) antibody groups following immunization with ovalbumin at wk -8, -3, and 0 relative to parturition. The ranking of groups based on the quantitative index of serum antibody response to ovalbumin were similar for sera and whey antibody such that Group 1 > Group 2 > Group 3. Animals were also vaccinated with Escherichia coli J5 (Rhône Mérieux, Lenexa, KS) at wk -8 and -3 relative to parturition. The ranking of groups for E. coli J5 was similar to that observed for serum and whey antibody to ovalbumin. Serum and whey IgG1 and IgG2 concentrations were measured at wk 0, 3, and 6 but differences between groups were not significant. There was no occurrence of mastitis for Group 1 animals in two of the herds. In contrast, Group 1 animals from the third herd had the highest occurrence of mastitis; however, these cases all occurred in first-parity heifers. According to pooled data across all herds, Group 3 animals had the highest occurrence of mastitis. Heritability estimates of serum antibody response to ovalbumin varied between 0.32 to 0.64 depending on week relative to parturition. Heritability estimates of serum antibody response to E. coli J5 also varied between 0.13 to 0.88 depending upon week relative to parturition. These results indicate that high peripartum antibody may be beneficial in some herds.