Characterization of the Hepatic Transcriptome for Divergent Immune-Responding Sheep Following Natural Exposure to Gastrointestinal Nematodes.

Infections with gastrointestinal nematodes (GINs) reduce the economic efficiency of sheep operations and compromise animal welfare. Understanding the host's response to GIN infection can help producers identify animals that are naturally resistant to infection. The objective of this study was to characterize the hepatic transcriptome of sheep that had been naturally exposed to GIN parasites. The hepatic transcriptome was studied using RNA-Sequencing technology in animals characterized as high (n = 5) or medium (n = 6) based on their innate immune acute-phase (AP) response phenotype compared with uninfected controls (n = 4), and with biased antibody-mediated (AbMR, n = 5) or cell-mediated (CMR, n = 5) adaptive immune responsiveness compared to uninfected controls (n = 3). Following the assessment of sheep selected for innate responses, 0, 136, and 167 genes were differentially expressed (DE) between high- and medium-responding animals, high-responding and uninfected control animals, and medium-responding and uninfected control animals, respectively (false discovery rate (FDR) < 0.05, and fold change |FC| > 2). When adaptive immune responses were assessed, 0, 53, and 57 genes were DE between antibody- and cell-biased animals, antibody-biased and uninfected control animals, and cell-biased and uninfected control animals, respectively (FDR < 0.05, |FC| > 2). Functional analyses identified enriched gene ontology (GO) terms and metabolic pathways related to the innate immune response and energy metabolism. Six functional candidate genes were identified for further functional and validation studies to better understand the underlying biological mechanisms of host responses to GINs. These, in turn, can potentially help improve decision making and management practices to increase the overall host immune response to GIN infection.

Genomic Regions Associated with Resistance to Gastrointestinal Nematode Parasites in Sheep-A Review.

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.

Novel lncRNA regulatory elements in milk somatic cells of Holstein dairy cows associated with mastitis.

Despite regulatory elements such as long non - coding RNAs representing most of the transcriptome, the functional understanding of long non - coding RNAs in relation to major health conditions including bovine mastitis is limited. This study examined the milk somatic cell transcriptome from udder quarters of 6 Holstein dairy cows to identify differentially expressed long non - coding RNAs using RNA - Sequencing. Ninety - four differentially expressed long non - coding RNAs are identified, 5 of which are previously annotated for gene name and length, 11 are annotated for gene name and 78 are novel, having no gene name or length previously annotated. Significant inflammatory response and regulation of immune response pathways (false discovery rate < 0.05) are associated with the differentially expressed long non - coding RNAs. QTL annotation analysis revealed 31 QTL previously annotated in the genomic regions of the 94 differentially expressed long non - coding RNAs, and the majority are associated with milk traits. This research provides a better understanding of long non - coding RNAs regulatory elements in milk somatic cells, which may enhance current breeding strategies for more adaptable or high mastitis resistant cattle.

N-Acetylcysteine and Its Immunomodulatory Properties in Humans and Domesticated Animals.

N-acetylcysteine (NAC), an acetylated derivative of the amino acid L-cysteine, has been widely used as a mucolytic agent and antidote for acetaminophen overdose since the 1960s and the 1980s, respectively. NAC possesses antioxidant, cytoprotective, anti-inflammatory, antimicrobial, and mucolytic properties, making it a promising therapeutic agent for a wide range of diseases in both humans and domesticated animals. Oxidative stress and inflammation play a major role in the onset and progression of all these diseases. NAC's primary role is to replenish glutathione (GSH) stores, the master antioxidant in all tissues; however, it can also reduce levels of pro-inflammatory tumor necrosis factor-alpha (TNF-∝) and interleukins (IL-6 and IL-1ß), inhibit the formation of microbial biofilms and destroy biofilms, and break down disulfide bonds between mucin molecules. Many experimental studies have been conducted on the use of NAC to address a wide range of pathological conditions; however, its effectiveness in clinical trials remains limited and studies often have conflicting results. The purpose of this review is to provide a concise overview of promising NAC usages for the treatment of different human and domestic animal disorders.

The effects of timing of high immune response phenotyping in relation to weaning on immune responses of crossbred beef calves.

Genetic selection for immune response has the potential to increase the sustainability of the beef industry by breeding cattle that are productive yet with an increased capacity to resist disease. Determining the optimal time to immunophenotype beef cattle is crucial for the accurate prediction of an animal's immune response. The objective of this study was to determine the effect of time of immunophenotyping in relation to weaning on immune responses of beef calves. Antibody- (AMIR) and cell-mediated (CMIR) immune responses were measured on 97 calves on the day of weaning (WEANING, N = 56) or 2 mo post-weaning (POST-WEANING, N = 41). Within each period of immunophenotyping, on day 0, blood was collected, and calves received a 1.0 mL intramuscular injection of type 1 and 2 test antigens. On day 14, blood was collected, and baseline skinfold thickness (SFT) was measured. Calves received an intradermal injection of 0.1 mg of the type 1 antigen suspended in 0.1 mL phosphate buffered saline (PBS) in the right tail fold, and 0.1 mL of PBS in the left. Changes in SFT at 24 h was used to indicate CMIR. To assess AMIR, the titer of type 2 antigen-specific bovine immunoglobulin G in serum from blood collected on day 14 was determined by measuring optical density (OD) using an enzyme-linked immunosorbent assay (ELISA). Among heifers, AMIR was greater for the POST-WEANING group than for the WEANING group (P < 0.01). Among steers, AMIR was not different between the POST-WEANING group and the WEANING group (P = 1.0). Therefore, the AMIR of heifers may be more negatively affected by immunophenotyping at weaning than the AMIR of steers. For steers, CMIR was greater in the POST-WEANING group than the WEANING group (P < 0.001). For heifers, CMIR was not different between the POST-WEANING group and the WEANING group (P = 0.22). The CMIR of steers may be more negatively affected by immunophenotyping at weaning than the CMIR of heifers. Calf age was not associated with AMIR or CMIR for calves phenotyped at weaning or post-weaning. The effect of sire nested within dam age was significant for CMIR for calves in the POST-WEANING group (P < 0.01), but not for calves in the WEANING group (P = 0.67). The results suggest that measuring immunocompetence at weaning may not be representative of a calf's genetic ability to mount an effective immune response, and immunophenotyping should be performed outside the weaning period.

Understanding the optimal time to immunophenotype beef calves is important for the accurate estimation of their genetic ability to resist disease. The compound stressors experienced by a calf during weaning may have a similar impact on the immune system as chronic stress. Therefore, the immune response phenotype of a calf immunophenotyped during the weaning period may not truly reflect the animal's genuine capacity for immune response. To accurately identify cattle with a superior capacity for immune response, with the goal of genetically selecting cattle for immunocompetence, immunophenotypes must be measured accurately. In this study, the effect of time of immunophenotyping in relation to weaning on immune responses of beef calves was determined. Calves immunophenotyped at weaning had lesser antibody-mediated and cell-mediated immune responses than calves immunophenotyped 2 mo post-weaning, this effect was influenced by sex. Sire affected immune responses when calves were immunophenotyped 2 mo post-weaning, but not when calves were immunophenotyped at weaning, indicating that when immunophenotyped post-weaning, the genetic component of a calf's immune response is quantified without being obscured by other environmental factors.

Impact of heat stress on dairy cattle and selection strategies for thermotolerance: a review.

Climate change is a problem that causes many environmental issues that impact the productivity of livestock species. One of the major issues associated with climate change is an increase of the frequency of hot days and heat waves, which increases the risk of heat stress for livestock species. Dairy cattle have been identified as being susceptible to heat stress due to their high metabolic heat load. Studies have shown heat stress impacts several biological processes that can result in large economic consequences. When heat stress occurs, dairy cattle employ several physiological and cellular mechanisms in order to dissipate heat and protect cells from damage. These mechanisms require an increase and diversion in energy toward protection and away from other biological processes. Therefore, in turn heat stress in dairy cattle can lead numerous issues including reductions in milk production and reproduction as well as increased risk for disease and mortality. This indicates a need to select dairy cattle that would be thermotolerant. Various selection strategies to confer thermotolerance have been discussed in the literature, including selecting for reduced milk production, crossbreeding with thermotolerant breeds, selecting based on physiological traits and most recently selecting for enhanced immune response. This review discusses the various issues associated with heat stress in dairy cattle and the pros and cons to the various selection strategies that have been proposed to select for thermotolerance in dairy cattle.

Identifying hepatic genes regulating the ovine response to gastrointestinal nematodes using RNA-Sequencing.

Gastrointestinal nematode (GIN) infections are considered the most important disease of grazing sheep and due to increasing anthelmintic resistance, chemical control alone is inadequate. Resistance to Gastrointestinal nematode infection is a heritable trait, and through natural selection many sheep breeds have higher resistance. Studying the transcriptome from GIN-exposed and GIN-unexposed sheep using RNA-Sequencing technology can provide measurements of transcript levels associated with the host response to Gastrointestinal nematode infection, and these transcripts may harbor genetic markers that can be used in selective breeding programs to enhance disease resistance. The objective of this study was to compare liver transcriptomes of sheep naturally exposed to Gastrointestinal nematode s, with either high or low parasite burdens, to GIN-unexposed control sheep in order to identify key regulator genes and biological processes associated with Gastrointestinal nematode infection. Differential gene expression analysis revealed no significant differentially expressed genes (DEG) between sheep with a high or low parasite burden (p-value ≤0.01; False Discovery Rate (FDR) ≤ 0.05; and Fold-Change (FC) of > ±2). However, when compared to the control group, low parasite burden sheep showed 146 differentially expressed genes (64 upregulated and 82 downregulated in the low parasite burden group relative to the control), and high parasite burden sheep showed 159 differentially expressed genes (57 upregulated and 102 downregulated in the low parasite burden group relative to the control) (p-value ≤0.01; FDR ≤0.05; and FC of > ±2). Among these two lists of significant differentially expressed genes, 86 differentially expressed genes (34 upregulated, 52 downregulated in the parasited group relative to the control) were found in common between the two parasite burden groups compared to the control (GIN-unexposed sheep). Functional analysis of these significant 86 differentially expressed genes found upregulated genes involved in immune response and downregulated genes involved in lipid metabolism. Results of this study offer insight into the liver transcriptome during natural Gastrointestinal nematode exposure that helps provide a better understanding of the key regulator genes involved in Gastrointestinal nematode infection in sheep.

Expression Profile of miRNA from High, Middle, and Low Stress-Responding Sheep during Bacterial Endotoxin Challenge.

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.

Correction: Karrow et al. Maternal COVID-19 Vaccination and Its Potential Impact on Fetal and Neonatal Development. Vaccines 2021, 9, 1351.

In the original publication [...].

Immunoceuticals: Harnessing Their Immunomodulatory Potential to Promote Health and Wellness.

Knowledge that certain nutraceuticals can modulate the immune system is not new. These naturally occurring compounds are known as immunoceuticals, which is a novel term that refers to products and systems that naturally improve an individual's immuno-competence. Examples of immunoceuticals include vitamin D3, mushroom glycans, flavonols, quercetin, omega-3 fatty acids, carotenoids, and micronutrients (e.g., zinc and selenium), to name a few. The immune system is a complex and highly intricate system comprising molecules, cells, tissues, and organs that are regulated by many different genetic and environmental factors. There are instances, such as pathological conditions, in which a normal immune response is suboptimal or inappropriate and thus augmentation or tuning of the immune response by immunoceuticals may be desired. With infectious diseases, cancers, autoimmune disorders, inflammatory conditions, and allergies on the rise in both humans and animals, the importance of the use of immunoceuticals to prevent, treat, or augment the treatment of these conditions is becoming more evident as a natural and often economical approach to support wellness. The global nutraceuticals market, which includes immunoceuticals, is a multi-billion-dollar industry, with a market size value of USD 454.55 billion in 2021, which is expected to reach USD 991.09 billion by 2030. This review will provide an overview of the immune system, the importance of immunomodulation, and defining and testing for immunocompetence, followed by a discussion of several key immunoceuticals with clinically proven and evidence-based immunomodulatory properties.

Immuno-phenotyping of Canadian beef cattle: adaptation of the high immune response methodology for utilization in beef cattle.

The high immune response (HIR) methodology measures the genetic performance of the adaptive immune system to identify and breed animals with balanced and robust immunity. The HIR methodology has previously been used in dairy and swine to reduce disease but has not been fully investigated in beef cattle. The first objective of the current study was to examine whether the HIR methodology as standardized for use in dairy cattle was appropriate for use in beef cattle. The second objective was to determine the earliest age for immune response phenotyping of beef calves. In this study, beef calves (n = 295) of various ages, as well as mature beef cows (n = 170) of mixed breeds, were immunized using test antigens to assess their antibody- (AMIR) and cell-mediated immune responses (CMIR). Heritability for AMIR and CMIR was estimated at 0.43 and 0.18, respectively. The HIR methodology was appropriate for use in beef cattle; beef calves as young as 2-3 wk of age were capable of mounting AMIR responses comparable with those seen historically in mature Holstein dairy cows. Three-week-old beef calves mounted CMIR responses comparable with those of Holstein cows, but 9-mo-old calves and mature beef cows had significantly higher CMIR responses than Holsteins. The HIR methodology can be used to measure both AMIR and CMIR in beef calves as young as 3 wk of age.

Concentration and heritability of immunoglobulin G and natural antibody immunoglobulin M in dairy and beef colostrum along with serum total protein in their calves.

Immunoglobulin (Ig) G and natural antibody (NAb) IgM are passively transferred to the neonatal calf through bovine colostrum. Maternal IgG provides pathogen- or vaccine-specific protection and comprises about 85% of colostral Ig. NAb-IgM is less abundant but provides broad and nonspecific reactivity, potentially contributing to protection against the dissemination of pathogens in the blood (septicemia) in a calf's first days of life. In the dairy and beef industries, failure of passive transfer (FPT) of colostral Ig (serum total protein [STP] <5.2 g/dL) is still a common concern. The objectives of this study were to: (1) compare colostral IgG concentrations and NAb-IgM titers between dairy and beef cows; (2) assess the effect of beef breed on colostral IgG; (3) compare passive transfer of colostral Ig in dairy and beef calves; and (4) estimate the heritability of colostral IgG and NAb-IgM. Colostrum was collected from Holstein dairy (n = 282) and crossbred beef (n = 168) cows at the University of Guelph dairy and beef research centers. Colostral IgG was quantified by radial immunodiffusion and NAb-IgM was quantified by an enzyme-linked immunosorbent assay. In dairy (n = 308) and beef (n = 169) calves, STP was estimated by digital refractometry. Beef cows had significantly greater colostral IgG (146.5 ± 9.5 standard error of the mean [SEM] g/L) than dairy cows (92.4 ± 5.2 g/L, P <0.01). Beef cows with a higher proportion of Angus ancestry had significantly lower colostral IgG (125.5 ± 5.8 g/L) than cows grouped as "Other" (142.5 ± 4.9 g/L, P = 0.02). Using the FPT cutoff, 13% of dairy and 16% of beef calves had FPT; still, beef calves had a significantly larger proportion with excellent passive transfer (STP ≥6.2 g/dL, P <0.01). The heritability of colostral IgG was 0.04 (±0.14) in dairy and 0.14 (±0.32) in beef. Colostral NAb-IgM titers in dairy (12.12 ± 0.22, log2 [reciprocal of titer]) and beef cows (12.03 ± 0.19) did not differ significantly (P = 0.71). The range of NAb-IgM titers was 9.18-14.60, equivalent to a 42-fold range in antibody concentration. The heritability of colostral NAb was 0.24 (±0.16) in dairy and 0.11 (±0.19) in beef cows. This study is the first to compare colostral NAb-IgM between dairy and beef cows. Based on the range in NAb-IgM titers and the heritability, selective breeding may improve colostrum quality and protection for neonatal calves in the early days of life.

Understanding how breed influences immunoglobulin (Ig) G and natural antibody (NAb) IgM concentrations in colostrum can improve bovine colostrum quality and calf health. Maternal colostral IgG is abundant, persistent, and pathogen specific. Natural antibody-IgM is less abundant but mediates broad, short-lived, nonspecific pathogen protection, and potentially important against septicemia. Colostral IgG and NAb-IgM concentrations were compared between dairy and beef cows and among cross-bred beef cows. Heritabilities were calculated to assess the practicality of selective breeding. Serum total protein (STP) in neonatal dairy and beef calves was estimated using refractometry. Colostrum from beef cows had higher concentrations of IgG than dairy cows. Beef cows with higher Angus ancestry produced colostrum with lower IgG concentrations than other mixed breeds. Heritability of colostral IgG was low (0.04­0.14). Failure of passive transfer was similar in dairy and beef calves, but a significantly larger proportion of beef calves had excellent STP (≥6.2 g/dL). There were no differences in NAb-IgM titers between dairy and beef cows or among beef breeds. Colostral NAb-IgM varied widely among individuals (42-fold) and was moderately heritable (0.11­0.24). These results suggest that selective breeding to improve colostrum quality is feasible and practical to improve calf health.

Maternal COVID-19 Vaccination and Its Potential Impact on Fetal and Neonatal Development.

Vaccines have been developed at "warp speed" to combat the COVID-19 pandemic caused by the SARS-CoV-2 coronavirus. Although they are considered the best approach for preventing mortality, when assessing the safety of these vaccines, pregnant women have not been included in clinical trials. Thus, vaccine safety for this demographic, as well as for the developing fetus and neonate, remains to be determined. A global effort has been underway to encourage pregnant women to get vaccinated despite the uncertain risk posed to them and their offspring. Given this, post-hoc data collection, potentially for years, will be required to determine the outcomes of COVID-19 and vaccination on the next generation. Most COVID-19 vaccine reactions include injection site erythema, pain, swelling, fatigue, headache, fever and lymphadenopathy, which may be sufficient to affect fetal/neonatal development. In this review, we have explored components of the first-generation viral vector and mRNA COVID-19 vaccines that are believed to contribute to adverse reactions and which may negatively impact fetal and neonatal development. We have followed this with a discussion of the potential for using an ovine model to explore the long-term outcomes of COVID-19 vaccination during the prenatal and neonatal periods.

Response to Oxidative Burst-Induced Hypoxia Is Associated With Macrophage Inflammatory Profiles as Revealed by Cellular Genome-Wide Association.

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.

Effect of in-vitro heat stress challenge on the function of blood mononuclear cells from dairy cattle ranked as high, average and low immune responders.

BACKGROUND: The warming climate is causing livestock to experience heat stress at an increasing frequency. Holstein cows are particularly susceptible to heat stress because of their high metabolic rate. Heat stress negatively affects immune function, particularly with respect to the cell-mediated immune response, which leads to increased susceptibility to disease. Cattle identified as having enhanced immune response have lower incidence of disease. Therefore, the objective of this study was to evaluate the impact of in vitro heat challenge on blood mononuclear cells from dairy cattle, that had previously been ranked for immune response, in terms of heat shock protein 70 concentration, nitric oxide production, and cell proliferation. RESULTS: Blood mononuclear cells from dairy cattle classified as high immune responders, based on their estimated breeding values for antibody and cell-mediated responses, produced a significantly greater concentration of heat shock protein 70 under most heat stress treatments compared to average and low responders, and greater cell-proliferation across all treatments. Similarly, a trend was observed where high responders displayed greater nitric oxide production compared to average and low responders across heat treatments. CONCLUSION: Overall, these results suggest that blood mononuclear cells from high immune responder dairy cows are more thermotolerant compared to average and low immune responders.

The bioactivity of colostrum and milk exosomes of high, average, and low immune responder cows on human intestinal epithelial cells.

Bovine milk contains bioactive components that are nutritionally and immunologically important to calves and humans. Dairy cows classified as high (H) immune responders using the patented high immune response technology have higher concentrations of immunoglobulin and specific antibodies in sera and milk compared with average (A) and low (L) responders. MicroRNA post-transcriptionally regulate expression of milk bioactive components and are enriched in extracellular vesicles known as exosomes, which protect them from degradation. The bioactivity of colostrum and milk exosomes at the human intestinal epithelial barrier remains to be explored, particularly in the context of the high immune response technology. Therefore, the purpose of this study was to evaluate the functional role of bovine milk exosomes compared with colostrum exosomes from H, A, and L responders at the intestinal interface using human colorectal adenocarcinoma epithelial (Caco-2) cells. Exosomes were isolated by successive ultracentrifugation and confirmed by western blot analysis for the presence of common exosomal proteins (CD9, CD63, and heat shock protein 70). Fluorescent labeling of exosomes using PKH67 dye confirmed their uptake by Caco-2 cells, demonstrating their potential bioavailability. The MTT assays showed that colostrum and milk exosomes maintain Caco-2 metabolic activity and are not cytotoxic to these cells. Specifically, metabolic activity after co-incubation with colostrum and milk exosomes from H responder cows was significantly greater than after co-incubation with exosomes from L responders. Caspase 3 activity, an indicator of apoptosis, was significantly lower after co-incubation of Caco-2 cells with milk exosomes compared with colostrum exosomes, suggesting that unlike colostrum exosomes, particularly those from L responders, milk exosomes do not activate the caspase 3 pathway in Caco-2 cells. This study helps us better understand the functional importance of colostrum and milk exosomes from dairy cows and emphasizes differences in functionality among exosomes from H, A, and L immune responders.

Identification of Ovine Serum miRNAs Following Bacterial Lipopolysaccharide Challenge.

Host-pathogen interactions are complex and influenced by host genetic and epigenetic modifications. Recently, the significance of microRNAs (miRNAs) in pathogenic infection and the regulation of immune response has been highlighted. However, information on miRNAs' role in the course of inflammation is still very limited in small ruminants. The present study was intended to identify changes in the expression of circulatory miRNAs post-lipopolysaccharide (LPS)-challenge. In this study, young ewes (n = 18) were challenged with Escherichia coli LPS (400 ng/kg i.v.) and blood samples were collected for serum miRNA isolation at two-time points; prior to challenge (T0), and 4 h (T4) post-challenge, reflecting the peak cortisol response. A total of 91 miRNAs were profiled, including 84 miRNAs on a commercial ovine miRNA-PCR array, and seven individual miRNAs. Forty five miRNAs were differentially expressed (DE) with 35 being up-regulated (Fold regulation, FR > 2) and 10 being down-regulated (FR < 1, p < 0.05) at T4. Among the up-regulated miRNAs, 14 were significantly (p < 0.05) induced, including oar-miRs: 369-3p, 495-3p, 376a-3p, 543-3p, 668-3p, 329a-3p, 655-3p, 411a-5p, and 154a-3p, which were located on ovine chromosome 18 forming four miRNA clusters within 10 kb. The elevated miRNAs belonged to different functional classes, playing roles in activating the hypothalamic-pituitary-adrenal axis; increasing cell survival and differentiation; and inducing inflammatory responses and targeted PI3K-Akt and MAPK signaling and chemokine signaling pathways. In summary, these results reveal the dynamic nature of ovine serum miRNAs during LPS-induced stress and highlight the potential role of identified miRNA-clusters on chromosome 18 to understand the regulation of the acute-phase response. Some of these identified circulating miRNAs may also serve as stress biomarkers for livestock in the future.

Transcriptomic Profiles of Monocyte-Derived Macrophages in Response to Escherichia coli is Associated with the Host Genetics.

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.

Genetic and Epigenetic Regulation of Immune Response and Resistance to Infectious Diseases in Domestic Ruminants.

Infectious diseases are the outcome of complex interactions between the host, pathogen, and environment. After exposure to a pathogen, the host immune system uses various mechanisms to remove the pathogen. However, environmental factors and characteristics of pathogens can compromise the host immune responses and subsequently alter the outcome of infection. In this article, genetic and epigenetic factors that shape the individual variation in mounting protective responses are reviewed. Different approaches that have been used by researchers to investigate the genetic regulation of immunity in ruminants and various sources of genetic information are discussed.

The effect of host genetics on in vitro performance of bovine monocyte-derived macrophages.

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.