Effects of replacing inorganic salts of trace minerals with organic trace minerals in the diet of prepartum cows on quality of colostrum and immunity of newborn calves.

Our objectives were to evaluate the impact of supplementary trace mineral (TM) form-inorganic salts (STM; Co, Cu, Mn, Zn sulfates, and Na selenite) or organic (OTM; Co, Cu, Mn, Zn proteinates, and selenized yeast)-in the prepartum diet on quantity and quality of colostrum, passive immunity, antioxidant biomarkers, cytokine responses to lipopolysaccharide (LPS), health, and growth of newborn calves. Pregnant heifers (n = 100) and cows (n = 173) were enrolled at 45 d before calving, blocked by parity and body condition score, and allocated randomly to STM (50 heifers; 86 cows) or OTM (50 heifers; 87 cows) supplementation. Cows in both treatments were fed the same diet, except for the source of supplementary TM. Within 2 h of calving, dams and calves were separated, colostrum was harvested, the yield was measured, and a sample was saved for posterior analyses of colostrum quality. A subgroup of calves (n = 68) had a blood sample collected before colostrum feeding. After colostrum feeding, all samples and data collection were limited to 163 calves (STM = 82; OTM = 81) fed 3 L of good quality (Brix% >22) maternal colostrum via nipple bottle minutes after harvesting. Concentration of IgG in colostrum and serum was determined 24 h after colostrum feeding using radial immunodiffusion. Concentration of TM in colostrum and serum were performed by inductively coupled plasma mass spectrometry. Activity of glutathione peroxidase, ferric reducing ability of plasma, and concentration of superoxide dismutase were evaluated in plasma by colorimetric assays. Ex vivo whole blood stimulation with LPS was performed on d 7 of life to evaluate cytokine responses in a subgroup of 66 calves. Health events were recorded from birth to weaning, and body weight was recorded at birth (all calves) and on d 30 and 60 (heifers only). Continuous variables were analyzed by ANOVA and binary responses were analyzed by logistic regression. Complete replacement of STM by OTM in prepartum diet resulted in greater concentration of Se (461 vs. 543 ± 7 µg/g; ± SEM) but did not alter the concentration or total mass of other TM and IgG in colostrum. Female calves of the OTM group had greater concentration of Se in serum at birth (0.23 vs. 0.37 ± 0.05 µg/mL), were lighter in weight at birth (40.9 vs. 38.8 ± 0.6 kg) and weaning (93.2 vs. 89.7 ± 1.6 kg) than those of the STM group. Maternal treatments did not affect passive immunity or antioxidant biomarkers. On d 7, basal concentrations (log10 of concentration in pg/mL) of IFNγ (0.70 vs. 0.95 ± 0.083) and LPS-stimulated concentrations of CC chemokine ligand 2 (CCL2; 2.45 vs. 2.54 ± 0.026), CC chemokine ligand 3 (CCL3; 2.63 vs. 2.76 ± 0.038), IL-1α (2.32 vs. 2.49 ± 0.054), and IL-1ß (3.62 vs. 3.86 ± 0.067) were greater in OTM than in STM. Supplementation with OTM in pregnant heifers, but not in pregnant cows, reduced the incidence of preweaning health problems in their calves (36.4 vs. 11.5%). Complete replacement of STM by OTM in the prepartum diet did not cause major changes in colostrum quality, passive immunity, and antioxidant capacity, but increased cytokine and chemokine responses to LPS on d 7 of life and benefited preweaning health of calves born to primiparous cows.

Immunotolerance of dairy heifers in response to repeated exposure to bacterial lipopolysaccharide endotoxin.

Dairy cattle face a variety of stressful events on a daily basis. More specifically, climate change has resulted in more frequent heat stress events that increase the incidence of chronic bacterial infections by inducing conditions like leaky gut syndrome, whereby the integrity of the intestinal epithelium is compromised allowing for luminal bacterial lipopolysaccharide (LPS) endotoxin to infiltrate the host's bloodstream resulting in acute or chronic systemic stimulation of the innate immune system. Repeated exposure to LPS over a short period of time is reported to induce immunotolerance within the host. This LPS tolerance is an essential immunohomeostatic response that can protect against over activation of the inflammatory response during subsequent exposure to LPS. In the present study, Holstein calves (n = 20) were initially stress challenged with either saline, or 100, 200 or 400 ng/kg of LPS administered intramuscular, and again re-challenged with 200 ng/kg of LPS 2-weeks later. Serum was collected every 2 hr for 6 hr to profile changes in circulatory stress biomarkers after the repeated LPS exposures. Heifers that were initially challenged with 100, 200 and 400 ng/kg of LPS demonstrated significantly attenuated cortisol responses in the second challenge (p < 0.01, 0.01 and 0.05, respectively), whereas control animals who previously received saline demonstrated a strong cortisol response at 2 hr after receiving 200 ng/kg of LPS (p < 0.05). The cytokine/chemokine (IL-6, CCL2, CCL3 and CCL4) responses were also attenuated during the LPS rechallenge (p < 0.05). Finally, microRNA expression profiles were determined to assess the epigenetic response to repeated LPS exposure. Interestingly, miR-31 and miR-223 were downregulated in response to the second LPS challenge. The present study demonstrates the dynamic nature of the stress response in dairy cattle as it relates to the development of LPS tolerance. Understanding the roles of various stress biomarkers in the context of innate immune cell tolerance is essential for evaluating their impact on immune system homeostasis.

Neutrophil function and antibody production during the transition period: Effect of form of supplementary trace minerals and associations with postpartum clinical disease and blood metabolites.

Our objectives were to evaluate the effect of the form of supplementary trace minerals-inorganic salts (STM: Co, Cu, Mn, and Zn sulfates and Na selenite) or organic (OTM: Co, Cu, Mn, Zn proteinates, and selenized yeast)-fed at 100% of recommended levels in both pre- and postpartum diets on in vitro phagocytic activity of neutrophils, and in vivo IgG responses to an ovalbumin challenge during the transition period. In addition, we investigated the associations of these immunological responses with incidence of postpartum clinical diseases and the dynamic changes of metabolic markers during the transition period. Pregnant heifers and cows (n = 273) were enrolled at 45 ± 3 d before expected calving, blocked by parity and body condition score, and allocated randomly to STM or OTM supplementation. Cows in both treatments were fed the same diet, except for the form of supplementary trace minerals. Automatic feeding gates were used to assign treatments to individual cows. Blood was collected on d -7 ± 3 and 7 ± 3 relative to calving in a subgroup of cows (n = 131 and 133, respectively) to measure phagocytic activity of neutrophils in vitro using flow cytometry. Subcutaneous immunization with 0.5 mg of chicken egg ovalbumin was performed in a subgroup of cows (n = 181) on d -45, -21, and 3 relative to calving. Concentration of anti-ovalbumin IgG in serum was measured by ELISA on d -45, -21, 3, 7, and 21. Trace mineral concentrations in blood were measured by inductively coupled plasma mass spectrometry on d -45, -21, -7, 0, 7, and 21 relative to calving. Selected metabolites were measured on d -21, -10, -3, 0, 3, 7, 10, 14, and 21 relative to calving. Treatment did not affect the percentage of neutrophils performing phagocytosis on d -7 or 7 but the median fluorescence intensity of phagocytosis on d 7 was greater for OTM than STM. We found no differences between treatments in the level of anti-ovalbumin IgG in serum on any of the sampling days. Changes in neutrophil function from prepartum to postpartum were associated with incidence of postpartum clinical disease, postpartum feed intake and milk production, concentrations of Ca, K, Se, Mn, Co, and total protein in serum. Immunoglobulin G responses to ovalbumin injections were not associated with incidence of postpartum clinical disease but were associated with body weight, feed intake, energy balance, and concentrations of nonesterified fatty acids, aspartate aminotransferase, gamma-glutamyl transpeptidase, albumin, Na, P, and Cu in serum. In conclusion, replacement of STM by OTM improved one measure of phagocytic capacity of neutrophils in vitro, which was also greater in cows that did not develop postpartum clinical disease. The associations of innate and acquired immune responses with feed intake, energy balance, and circulating concentrations of key macro and micronutrients reinforce the importance of nutritional management for the health of dairy cows during the transition period.

Dynamic changes in Holstein heifer circulatory stress biomarkers in response to lipopolysaccharide immune challenge.

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

Endotoxin-induced cytokine, chemokine and white blood cell profiles of variable stress-responding sheep.

Individual variation of the hypothalamic-pituitary-adrenal (HPA) axis response to stress could contribute to variable stress resiliency of livestock. During stress events, the innate immune system can also become activated and work in concert with the neuroendocrine system to restore homeostasis, while minimizing tissue damage. The purpose of this study was to assess immune function in variable stress-responding sheep in response to bacterial lipopolysaccharide (LPS) endotoxin immune-challenge. High (HSR, n = 12), middle (MSR, n = 12), and low-stress responders (LSR, n = 12) were selected from a population of 112 female lambs and classified based on serum cortisol concentration after receiving an intravenous bolus of LPS (400 ng/kg). Blood was collected from the jugular vein at 0 and 4 hrs post-LPS challenge to monitor changes in serum pro- and anti-inflammatory cytokines and chemokines, and white blood cell populations. Rectal temperature was recorded hourly to monitor fever. HSR had the greatest increase in rectal temperature and strongest pro-inflammatory IL-6 and IFN-γ cytokine responses compared to MSR and LSR. HSR and MSR had stronger anti-inflammatory IL-10 cytokine and CCL2 chemokine responses than LSR. White blood cell counts changed between 0 and 4 h; however, no differences were detected among the variable stress response groups. The distinct inflammatory response in variable stress responding sheep could contribute to individual differences in stress resiliency and this warrants investigation in the context of other types of stress.

LAY SUMMARYAcute inflammation was studied in sheep stress-phenotyped using bacterial lipopolysaccharide. Sheep selected based on stress responsiveness (i.e. serum cortisol concentration) have different immune responses to bacterial stress. Specifically, high-stress responders have a more pronounced inflammatory response than low-stress responders.

CRISPR-Cas9-mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate-induced inflammation in bovine mammary epithelial cells.

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in the recognition of microbial pathogens and host alarmins. Ligation to TLR4 initiates a signaling cascade that leads to inflammation. Polymorphisms in bovine TLR4 have been associated with Mycobacterium avium ssp. paratuberculosis (MAP) susceptibility and resistance, the cause of Johne's disease, and milk somatic cell score, a biomarker of mastitis. Although the contribution of TLR4 to recognition of bacterial lipopolysaccharide (LPS) has been well characterized, its role in MAP recognition is less certain. Clustered regularly interspaced short palindromic repeats-Cas9 mediated gene editing was performed to generate TLR4 knockout (KO) mammary epithelial cells to determine if TLR4 expression is involved in the initiation of the host inflammatory response to MAP cell lysate (5 and 10 µg/mL) and Escherichia coli LPS (5 µg/mL). The absence of TLR4 in KO cells resulted in enhanced expression of key inflammatory genes (TNFA and IL6), anti-inflammatory genes (IL10 and SOCS3), and supernatant cytokine and chemokine levels (TNF-α, IL-6, IL-10, CCL3) in response to the MAP cell lysate (10 µg/mL). However, in response to LPS, the KO cells showed reduced expression of key inflammatory genes (TNFA, IL1A, IL1B, and IL6) and supernatant cytokine levels (TNF-α, IL-6, CCL2, IL-8) as compared with unedited cells. Overall, these results confirm that TLR4 is essential for eliciting inflammation in response to LPS; however, exacerbated gene and protein expression in TLR4 KO cells in response to MAP cell lysate suggests a different mechanism of infection and host response for MAP, at least in terms of how it interacts with TLR4. These novel findings show potential divergent roles for TLR4 in mycobacterial infections, and this may have important consequences for the therapeutic control of inflammation in cattle.

Impact of feeding n-3 fatty acids to layer breeders and their offspring on concentration of antibody titres against infectious bronchitis, and Newcastle diseases and plasma fatty acids in the offspring.

1. The impact of feeding sources of n-3 fatty acids (FA) to ISA Brown and Shaver White breeders and their offspring on antibody titres and plasma FA profile was examined.2. Breeders were fed either a control diet (CON); CON + 1% microalgae (DMA: Aurantiochytrium limacinum) as a source of docosahexaenoic acid; or CON + 2.6% of a co-extruded mixture of full-fat flaxseed (FFF) as a source of α-linolenic acid. Day-old female offspring were assigned to diets (breeder-offspring): 1) CON-CON, 2) CON-DMA, 3) CON - FFF, 4) DMA - CON, 5) DMA - DMA, 6) FFF - CON or 7) FFF - FFF, followed by a standard layer diet through 18 weeks of age (WOA) to 42 WOA.3. Antibody titres against infectious bronchitis (IBV) and Newcastle disease (NDV) were measured at six days and six WOA, and plasma FA profile was measured at 18 and 42 WOA.4. Pullets from FFF-fed breeders had higher antibody titres against IBV and NDV than pullets fed DMA (P < 0.05). Feeding FFF to offspring increased plasma ∑n-3 FA at 18 and 42 WOA, whereas feeding DMA to offspring reduced ∑n-6 FA at 18 WOA.5. In conclusion, independent of breeder strain, alpha linoleic acid (ALA) and DHA sources showed varied responses. Feeding breeders FFF increased plasma concentration of antibody titres and n-3 FA whereas DMA reduced plasma concentration of ∑n-6 FA.

Short communication: Characterizing ovine serum stress biomarkers during endotoxemia.

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

Impact of feeding microalgae (Aurantiochytrium limacinum) and co-extruded mixture of full-fat flaxseed as sources of n-3 fatty acids to ISA brown and Shaver white breeders and progeny on pullet skeletal attributes at hatch through to 18 weeks of age.

Impact of feeding n-3 fatty acids (FA) to ISA brown and Shaver white breeders and their progeny on bone development in pullets was investigated. Breeders were fed Control (CON); CON + 1% microalgae (DMA: Aurantiochytrium limacinum) as the source of docosahexaenoic acid; and CON + 2.6% of a co-extruded mixture of full-fat flaxseed (FFF) and pulses mixture as source of α-linolenic acid. Test diets (DMA and FFF) were balanced for total n-3 FA and n-6: n-3 FA ratio. Samples of day-old progeny were euthanized for bone mineral content (BMC) and tibia collagen type II. The remaining pullets were fed posthatch treatments as follows: from breeder CON: CON (CON-CON), DMA (CON-DMA), and FFF (CON-FFF), from breeder DMA: CON (DMA-CON) and DMA (DMA-DMA) and from breeder FFF: CON (FFF-CON) and FFF (FFF-FFF). A total of 60 pullets per posthatch diets were reared in cages (12 pullets/cage, n = 5) with free access to feed and water, bled at 6, 12, and 18 wk of age (WOA) for bone turnover markers and necropsied at 18 WOA for tibia and femur samples. Day-old pullets from breeder fed CON had greater BMC (P < 0.001) relative to those from breeders fed other diets. There was strain and diet interaction (P ≤ 0.024) on tibia breaking strength (TBS) and tibia cortical ash concentration at 18 WOA such that diet responses were only observed in Shaver white pullets. In this context, TBS of DMA-DMA and FFF-FFF was greater than for pullets originating from CON breeder, and the cortical ash weight of DMA-DMA and FFF-FFF pullets was 23.8 and 20.2%, respectively, higher than for CON-CON pullets. In conclusions, the strain effects were strong on tibia attributes on 18-week-old pullets. Breeder feeding of n-3 FA was more effective when concomitant with posthatch feeding of n-3 FA in supporting the skeletal strength and cortical bone development in Shaver white pullets. Further investigations are warranted to establish the impact these strategies on skeletal health during laying cycle.

Effect of algae or fish oil supplementation and porcine maternal stress on the adrenal transcriptome of male offspring fed a low-quality protein diet.

Offspring adrenal function may be negatively affected in utero by maternal stressors such as microbial infection. Maternal supplementation with immunomodulatory compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) may help minimize the adverse effects of maternal stress on fetal hypothalamic-pituitary-adrenal development and improve offspring health. Presently, n-3 PUFA sources are primarily fish-based, but n-3 PUFA microalgae (AL) may be an alternative. Previously, it was determined that maternal AL or fish oil (FO) supplementation to sows, in addition to maternal stress induced by Escherichia coli lipopolysaccharide (LPS) challenge appeared to have a greater influence on the stress response of male offspring compared to females. To further elaborate on these findings, this study assessed the effects of maternal AL or FO supplementation combined with a maternal LPS challenge on adrenal gene expression in male offspring fed a nursery diet containing low-quality protein sources. Forty-eight sows were fed gestation diets starting on gestation day (gd) 75 containing either 3.12% AL, 3.1% FO, or a control diet containing 1.89% corn oil. On gd 112, half the sows in each treatment were administered 10 â€‹µg/kg LPS i.m. Piglets were weaned at 21 days of age onto a common low-quality plant-based protein diet, and one week after weaning, four piglets per sow were administered 40 â€‹µg/kg LPS i.m. Two hours later, the piglets were euthanized to obtain adrenal tissue, and total RNA was extracted to carry out transcriptome analysis using the Affymetrix GeneChip WT Plus assay and subsequent validation by real-time PCR. Analysis revealed that adrenal steroidogenesis, fatty acid metabolism and immune function were significantly influenced by maternal diet and stress. Increased expression of immune-related genes including lymphocyte antigen 96, TLR-2 and NF-κB suggests that maternal AL supplementation may increase offspring sensitivity to inflammation after weaning. Decreased expression of lymphocyte antigen 96 in male offspring from sows receiving maternal LPS challenge also suggests a possible role of maternal stress in diminishing the offspring immune response to immune stress challenge. Increased expression of the genes encoding the 11BHSD2 enzyme in offspring from sows fed FO may also reduce the magnitude of the stress response. These data provide insight to the immune and metabolic mechanisms that may be influenced by maternal diet and stress.

Microalgae supplementation to late gestation sows and its effects on the health status of weaned piglets fed diets containing high- or low-quality protein sources.

Maternal stress, such as a bacterial infection occurring in late gestation, may predispose offspring to a variety of diseases later in life. It may also alter programming of developing systems within the fetus, such as the hypothalamic-pituitary-adrenal (HPA) axis and immune system. Dietary supplementation during the last trimester of pregnancy with immune-modulating compounds may be a means of reducing potential adverse effects of maternal stress on the developing fetus. Essential omega-3 polyunsaturated fatty acids (n-3 PUFA) such as docosahexanoic acid (DHA) and eicosapentanoic acid are well-known for their immune-modulating and anti-inflammatory properties. Sources of these n-3 PUFA include fish products such as fish oil and microalgae, which may be a suitable alternative to fish-based products. The aim of this study was to determine the effect of supplementing gestating sow diets with n-3 PUFA and inducing an immune stress challenge in late gestation on piglet growth and immune responsiveness when placed on either a high- or low-quality protein diet after weaning. Forty-eight sows were fed gestation diets containing either 3.12% microalgae, 3.1% fish oil or a corn oil control diet containing 1.89% corn oil starting on gestation day (gd) 75. On gd112, half the sows in each treatment were immune stress challenged with bacterial lipopolysaccharide (LPS) endotoxin (10 µg/kg administered i.m). After farrowing, piglet BW gain was monitored weekly during lactation and pigs were weaned at 21 days of age. One week after weaning, four piglets per sow were immune stress challenged with LPS (40 µg/kg administered i.m.). At the same time, four piglets per sow were vaccinated with the novel antigens chicken ovalbumin (OVA) and Candida cellular antigen (CAA) and received booster vaccinations two weeks later. Four weeks after the initial vaccination, a transdermal hypersensitivity immune challenge was performed using the same antigens. Blood samples were also collected to quantify IgG antibody responses to both antigens. PUFA enrichment in sow blood and piglet brain was detected after sows were on feed for 40 days. Piglet growth was increased in pigs fed a high-quality diet in nursery phase 1. Concentrations of the cytokines IL-1ra, IL-6 and IL-10 were elevated in pigs fed a high-quality protein diet following LPS immune challenge. Overall, it appears that in the current study piglet nursery diet quality was more important for determining piglet health and growth than maternal diet and immune stress.

Genetic mechanisms regulating the host response during mastitis.

Mastitis is a very costly and common disease in the dairy industry. The study of the transcriptome from healthy and mastitic milk somatic cell samples using RNA-Sequencing technology can provide measurements of transcript levels associated with the immune response to the infection. The objective of this study was to characterize the Holstein milk somatic cell transcriptome from 6 cows to determine host response to intramammary infections. RNA-Sequencing was performed on 2 samples from each cow from 2 separate quarters, one classified as healthy (n = 6) and one as mastitic (n = 6). In total, 449 genes were differentially expressed between the healthy and mastitic quarters (false discovery rate <0.05, fold change >±2). Among the differentially expressed genes, the most expressed genes based on reads per kilobase per million mapped reads (RPKM) in the healthy group were associated with milk components (CSN2 and CSN3), and in the mastitic group they were associated with immunity (B2M and CD74). In silico functional analysis was performed using the list of 449 differentially expressed genes, which identified 36 significantly enriched metabolic pathways (false discovery rate <0.01), some of which were associated with the immune system, such as cytokine-cytokine interaction and cell adhesion molecules. Seven functional candidate genes were selected, based on the criteria of being highly differentially expressed between healthy and mastitic groups and significantly enriched in metabolic pathways that are relevant to the inflammatory process (GLYCAM1, B2M, CD74, BoLA-DRA, FCER1G, SDS, and NFKBIA). Last, we identified the differentially expressed genes that are located in quantitative trait locus regions previously known to be associated with mastitis, specifically clinical mastitis, somatic cell count, and somatic cell score. It was concluded that multiple genes within quantitative trait locus regions could potentially affect host response to mastitis-causing agents, making some cows more susceptible to intramammary infections. The identification of potential candidate genes with functional, statistical, biological, and positional relevance associated with host defense to infection will contribute to a better understanding of the underlying genetic architecture associated with mastitis. This in turn will improve the sustainability of agricultural practices by facilitating the selection of cows with improved host defense leading to increased resistance to mastitis.

Nucleotide-rich yeast extract fed to broiler chickens challenged with Eimeria: impact on growth performance, jejunal histomorphology, immune system, and apparent retention of dietary components and caloric efficiency1.

Nucleotide-rich yeast extract (YN) was investigated for its effects on growth performance, jejunal histomorphology and mucosal immunoglobulin A (IgA), immune organs weight and apparent retention (AR) of components in broiler chickens challenged with Eimeria. A total of 336 day-old male chicks (Ross x Ross 708) were placed in floor pens and provided a corn-soybean meal-based diet without or with YN (500 g/mt) (n = 14). On day 10, 7 replicates per diet were orally administered with 1 mL of sporulated E. acervulina and E. maxima oocysts and the rest (non-challenged control) administered equivalent distilled water creating a 2 × 2 factorial arrangement for the post-challenge period (day 11 to 35). Feed intake (FI), BWG, and FCR responses were measured in pre- and post-challenge periods. Excreta samples were collected on day 14 to 17 and 31 to 34 for oocyst count and AR of components, respectively. On day 15 and 35, 5 birds/pen were necropsied for intestinal samples. Spleen, bursa, and thymus weights were also recorded at both time points and breast yield on day 35. Diet had no effect (P > 0.05) on pre-challenge growth performance. Interaction (P = 0.046) between Eimeria and YN on FI was such that Eimeria challenge increased FI (day 11 to 35) in non-YN birds. There was no interaction (P > 0.05) between Eimeria and YN on other post-challenge responses. Eimeria reduced (P < 0.05) BWG, FCR, caloric efficiency, day 15 jejunal villi height and IgA concentration, and increased (P < 0.01) day 15 spleen weight. On day 35, YN increased bursa weight (1.57 vs. 1.78 mg/g BW, P = 0.04). There was a tendency for an interaction effect (P = 0.09) on day 35 thymus weight, such that in challenged birds, YN fed birds tended to have a lighter thymus relative to non-YN fed birds. In conclusions, independent of Eimeria challenge, supplemental YN had no effect on growth performance, caloric efficiency, and intestinal function but increased immune organ weights.

Responses of broiler chickens to Eimeria challenge when fed a nucleotide-rich yeast extract.

Nucleotide-rich yeast extract (YN) was investigated for effects on growth performance, jejunal physiology, and cecal microbial activity in Eimeria-challenged broiler chickens. A total of 360-day-old male chicks (Ross × Ross 708) were placed on floor pens and provided a corn-soybean meal-based diet without or with YN (500 g/MT; n = 12). On d 10, 6 replicates per diet were orally administered with 1 mL of E. acervulina and E. maxima sporulated oocysts and the rest (non-challenged control) were administered with 1 mL of distilled water. On d 15, 5 birds/pen were then necropsied for intestinal lesion scores, histomorphology and cecal digesta pH, short chain fatty acids (SCFA), and microbial community using Illumina Miseq platform. Supplemental YN improved (P = 0.01) Feed conversion ratio (FCR) during the prechallenge phase (d 0 to 10). In the postchallenge period (d 11 to 15), Eimeria depressed (P < 0.05) Body weight gain (BWG) relative to non-challenged birds, whereas YN-fed birds had a higher (P = 0.05) BWG compared to that of non-YN-fed birds. There was an interaction between YN and Eimeria on jejunal villi height (VH) (P = 0.001) and expression of cationic amino acid transporter 1(CAT1) (P = 0.04). Specifically, in the absence of Eimeria, YN-fed birds had a shorter VH (892 vs. 1,020 µm) relative to that of control but longer VH (533 vs. 447 µm) in the presence of Eimeria. With respect to CAT1, YN-fed birds had a higher (1.65 vs. 0.78) expression when subjected to Eimeria than when not challenged. Independently, Eimeria decreased (P < 0.01) the jejunal expression of maltase, Na glucose transporter 1 and occludin genes, ceca digesta abundance of genus Clostridium cluster XlVa and Oscillibacter but increased (P < 0.01) jejunal proliferating cell nuclear antigen and interleukin 10. Interaction between YN and Eimeria was observed for ceca digesta pH (P = 0.04) and total SCFA (P = 0.01) such that YN increased SCFA in the absence of Eimeria but reduced SCFA and increased pH in the presence of Eimeria. In summary, Eimeria impaired performance and gut function and shifted gut microbiome; YN improved performance independently, attenuated Eimeria damage on indices of gut function, and modulated cecal microbiome.

The genetic architecture of milk ELISA scores as an indicator of Johne's disease (paratuberculosis) in dairy cattle.

Johne's disease (or paratuberculosis), caused by Mycobacterium avium ssp. paratuberculosis (MAP) infection, is a globally prevalent disease with severe economic and welfare implications. With no effective treatment available, understanding the role of genetics influencing host infection status is essential to develop selection strategies to breed for increased resistance to MAP infection. The main objectives of this study were to estimate genetic parameters for the MAP-specific antibody response using milk ELISA scores in Canadian Holstein cattle as an indicator of resistance to Johne's disease, and to unravel genomic regions and candidate genes significantly associated with MAP infection. After data editing, 168,987 milk ELISA records from 2,306 herds, obtained from CanWest Dairy Herd Improvement, were used for further analyses. Variance and heritability estimates for MAP infection status were determined using univariate linear animal models under 3 scenarios: (a) SCEN1: the complete data set (all herds); (b) SCEN2: herds with at least one suspect or test-positive animal (ELISA optical density ≥0.07); and (c) SCEN3: herds with at least one test-positive animal (ELISA optical density ≥0.11). Heritability estimates were calculated as 0.066, 0.064, and 0.063 for SCEN1, SCEN2, and SCEN3, respectively. The correlations between estimated breeding values for resistance to MAP infection and other economically important traits, when significant, were favorable and of low magnitude. Genome-wide association analyses identified important genomic regions on Bos taurus autosome (BTA)1, BTA7, BTA9, BTA14, BTA15, BTA17, BTA19, and BTA25 showing significant association with MAP infection status. These regions included 2 single nucleotide polymorphisms located 2 kb upstream of positional candidate genes CD86 and WNT9B, which play key roles in host immune response and tissue homeostasis. This study revealed the genetic architecture of MAP infection in Canadian Holstein cattle as measured by milk ELISA scores by estimating genetic parameters along with the identification of genomic regions potentially influencing MAP infection status. These findings will be of significant value toward implementing genetic and genomic evaluations for resistance to MAP infection in Holstein cattle.

Short communication: Uncovering quantitative trait loci associated with resistance to Mycobacterium avium ssp. paratuberculosis infection in Holstein cattle using a high-density single nucleotide polymorphism panel.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of Johne's disease in cattle. Johne's disease is a disease of significant economic, animal welfare, and public health concern around the globe. Therefore, understanding the genetic architecture of resistance to MAP infection has great relevance to advance genetic selection methods to breed more resistant animals. The objectives of this study were to perform a genome-wide association study of previously analyzed 50K genotypes now imputed to a high-density single nucleotide polymorphism panel (777K), aiming to validate previously reported associations and potentially identify additional single nucleotide polymorphisms associated with antibody response to MAP infection. A principal component regression-based genome-wide association study revealed 15 putative quantitative trait loci (QTL) associated with the MAP infection phenotype (serum or milk ELISA tests) on 9 different chromosomes (Bos taurus autosomes 5, 6, 7, 10, 14, 15, 16, 20, and 21). These results validated previous findings and identified new QTL on Bos taurus autosomes 15, 16, 20, and 21. The positional candidate genes NLRP3, IFi47, TRIM41, TNFRSF18, and TNFRSF4 lying within these QTL were identified. Further functional validation of these genes is now warranted to investigate their roles in regulating the immune response and, consequently, cattle resistance to MAP infection.

Type I and type II cytokine production of CD4+ T-cells in immune response biased dairy cattle around calving.

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

In vitro bioassessment of the immunomodulatory activity of Saccharomyces cerevisiae components using bovine macrophages and Mycobacterium avium ssp. paratuberculosis.

The yeast Saccharomyces cerevisiae and its components are used for the prevention and treatment of enteric disease in different species; therefore, they may also be useful for preventing Johne's disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). The objective of this study was to identify potential immunomodulatory S. cerevisiae components using a bovine macrophage cell line (BOMAC). The BOMAC phagocytic activity, reactive oxygen species production, and immune-related gene (IL6, IL10, IL12p40, IL13, IL23), transforming growth factor ß, ARG1, CASP1, and inducible nitric oxide synthase expression were investigated when BOMAC were cocultured with cell wall components from 4 different strains (A, B, C, and D) and 2 forms of dead yeast from strain A. The BOMAC phagocytosis of mCherry-labeled MAP was concentration-dependently attenuated when BOMAC were cocultured with yeast components for 6 h. Each yeast derivative also induced a concentration-dependent increase in BOMAC reactive oxygen species production after a 6-h exposure. In addition, BOMAC mRNA expression of the immune-related genes was investigated after 6 and 24 h of exposure to yeast components. All yeast components were found to regulate the immunomodulatory genes of BOMAC; however, the response varied among components and over time. The in vitro bioassessment studies reported here suggest that dead yeast and its cell wall components may be useful for modulating macrophage function before or during MAP infection.

Short communication: Ovine leukocyte telomere length is associated with variation in the cortisol response to systemic bacterial endotoxin challenge.

Stress has been associated with biological aging and numerous age-related diseases. This may be due, in part, to accelerated shortening of telomeres, which are critical genomic structures that cap and protect chromosomal ends. Dysfunction of the hypothalamic-pituitary-adrenal axis may indirectly contribute to telomere shortening if an animal reacts too strongly or weakly to a stressor, leading to accelerated biological aging. In this study, outbred Rideau-Arcott sheep were stress challenged with Escherichia coli endotoxin and classified as high, middle, or low cortisol responders to investigate a potential relationship between cortisol response and age, and telomere length. In the present study, no association was found between age and telomere length. The study, however, revealed shorter telomeres in high and low cortisol responders compared with the middle cortisol responders, which suggests that health and longevity may be compromised in extreme high- and low-stress-responding sheep.