Dietary Zinc Supplementation in Steers Modulates Labile Zinc Concentration and Zinc Transporter Gene Expression in Circulating Immune Cells.

Zinc (Zn) is critical for immune function, and marginal Zn deficiency in calves can lead to suboptimal growth and increased disease susceptibility. However, in contrast to other trace minerals such as copper, tissue concentrations of Zn do not change readily in conditions of supplementation or marginal deficiency. Therefore, the evaluation of Zn status remains challenging. Zinc transporters are essential for maintaining intracellular Zn homeostasis, and their expression may indicate changes in Zn status in the animal. Here, we investigated the effects of dietary Zn supplementation on labile Zn concentration and Zn transporter gene expression in circulating immune cells isolated from feedlot steers. Eighteen Angus crossbred steers (261 ± 14 kg) were blocked by body weight and randomly assigned to two dietary treatments: a control diet (58 mg Zn/kg DM, no supplemental Zn) or control plus 150 mg Zn/kg DM (HiZn; 207 mg Zn/kg DM total). After 33 days, Zn supplementation increased labile Zn concentrations (as FluoZin-3 fluorescence) in monocytes, granulocytes, and CD4 T cells (P < 0.05) but had the opposite effect on CD8 and γδ T cells (P < 0.05). Zn transporter gene expression was analyzed on purified immune cell populations collected on days 27 or 28. ZIP11 and ZnT1 gene expression was lower (P < 0.05) in CD4 T cells from HiZn compared to controls. Expression of ZIP6 in CD8 T cells (P = 0.02) and ZnT7 in B cells (P = 0.01) was upregulated in HiZn, while ZnT9 tended (P = 0.06) to increase in B cells from HiZn. These results suggest dietary Zn concentration affects both circulating immune cell Zn concentrations and Zn transporter gene expression in healthy steers.

Feeding Saccharomyces cerevisiae fermentation postbiotic products alters immune function and the lung transcriptome of preweaning calves with an experimental viral-bacterial coinfection.

Bovine respiratory disease causes morbidity and mortality in cattle of all ages. Supplementing with postbiotic products from Saccharomyces cerevisiae fermentation (SCFP) has been reported to improve growth and provide metabolic support required for immune activation in calves. The objective of this study was to determine effects of SCFP supplementation on the transcriptional response to coinfection with bovine respiratory syncytial virus (BRSV) and Pasteurella multocida in the lung using RNA sequencing. Twenty-three calves were enrolled and assigned to 2 treatment groups: control (n = 12) or SCFP-treated (n = 11, fed 1 g/d SmartCare in milk and 5 g/d NutriTek on starter grain; both from Diamond V Mills Inc.). Calves were infected with ∼104 median tissue culture infectious dose per milliliter of BRSV, followed 6 d later by intratracheal inoculation with ∼1010 cfu of Pasteurella multocida (strain P1062). Calves were euthanized on d 10 after viral infection. Blood cells were collected and assayed on d 0 and 10 after viral infection. Bronchoalveolar lavage (BAL) cells were collected and assayed on d 14 of the feeding period (preinfection) and d 10 after viral infection. Blood and BAL cells were assayed for proinflammatory cytokine production in response to stimulation with lipopolysaccharide (LPS) or a combination of polyinosinic:polycytidylic acid and imiquimod, and BAL cells were evaluated for phagocytic and reactive oxygen species production capacity. Antemortem and postmortem BAL and lesioned and nonlesioned lung tissue samples collected at necropsy were subjected to RNA extraction and sequencing. Sequencing reads were aligned to the bovine reference genome (UMD3.1) and edgeR version 3.32.1 used for differential gene expression analysis. Supplementation with SCFP did not affect the respiratory burst activity or phagocytic activity of either lung or blood immune cells. Immune cells from the peripheral blood of SCFP-supplemented calves produced increased quantities of IL-6 in response to toll-like receptor stimulation, whereas cells from the BAL of SCFP-treated calves secreted fewer proinflammatory cytokines and less tumor necrosis factor-α (TNF-α) and IL-6 in response to the same stimuli. Transcriptional responses in lung tissues and BAL samples from SCFP-fed calves differed from the control group. The top enriched pathways in SCFP-treated lungs were associated with decreased expression of inflammatory responses and increased expression of plasminogen and genes involved in glutathione metabolism, supporting effective lung repair. Our results indicate that supplementing with SCFP postbiotics modulates both systemic and mucosal immune responses, leading to increased resistance to bovine respiratory disease.

Modeling Human Respiratory Syncytial Virus (RSV) Infection: Recent Contributions and Future Directions Using the Calf Model of Bovine RSV Disease.

The human orthopneumovirus (human respiratory syncytial virus [RSV]) is a leading cause of respiratory disease in children worldwide and a significant cause of infant mortality in low- and middle-income countries. The natural immune response to the virus has a preponderant role in disease progression, with a rapid neutrophil infiltration and dysbalanced T cell response in the lungs associated with severe disease in infants. The development of preventive interventions against human RSV has been difficult partly due to the need to use animal models that only partially recapitulate the immune response as well as the disease progression seen in human infants. In this brief review, we discuss the contributions of the calf model of RSV infection to understanding immunity to RSV and in developing vaccine and drug candidates, focusing on recent research areas. We propose that the bovine model of RSV infection is a valuable alternative for assessing the translational potential of interventions aimed at the human population.

Subcutaneous Bacillus Calmette-Guérin Administration Induces Innate Training in Monocytes in Preweaned Holstein Calves.

The bacillus Calmette-Guérin (BCG) vaccine, administered to prevent tuberculosis, is a well-studied inducer of trained immunity in human and mouse monocytes. We have previously demonstrated that aerosol BCG administration induces innate training in calves. The current study aimed to determine whether s.c. BCG administration could induce innate training, identify the cell type involved, and determine whether innate training promoted resistance to bovine respiratory syncytial virus (BRSV) infection, a major cause of bovine respiratory disease in preweaned calves. A total of 24 calves were enrolled at 1-3 d of age and blocked by age into two treatment groups (BCG, n = 12; control, n = 12). BCG was given s.c. to preweaned calves. The control calves received PBS. We observed a trained phenotype, demonstrated by enhanced cytokine production in response to in vitro stimulation with LPS (TLR-4 agonist) in PBMCs and CD14+ monocytes from the BCG group 2 wk (IL-1ß, p = 0.002) and 4 wk (IL-1ß, p = 0.005; IL-6, p = 0.013) after BCG administration, respectively. Calves were experimentally infected via aerosol inoculation with BRSV strain 375 at 5 wk after BCG administration and necropsied on day 8 postinfection. There were no differences in disease manifestation between the treatment groups. Restimulation of bronchoalveolar lavage fluid cells isolated on day 8 after BRSV infection revealed enhanced IL-1ß (p = 0.014) and IL-6 (p = 0.010) production by the BCG group compared with controls. In conclusion, results from our study show that s.c. administration of the BCG vaccine can induce trained immunity in bovine monocytes and influence cytokine production in the lung environment after BRSV infection.

Immunization with a mucosal, post-fusion F/G protein-based polyanhydride nanovaccine protects neonatal calves against BRSV infection.

Human respiratory syncytial virus (HRSV) is a leading cause of death in young children and there are no FDA approved vaccines. Bovine RSV (BRSV) is antigenically similar to HRSV, and the neonatal calf model is useful for evaluation of HRSV vaccines. Here, we determined the efficacy of a polyanhydride-based nanovaccine encapsulating the BRSV post-fusion F and G glycoproteins and CpG, delivered prime-boost via heterologous (intranasal/subcutaneous) or homologous (intranasal/intranasal) immunization in the calf model. We compared the performance of the nanovaccine regimens to a modified-live BRSV vaccine, and to non-vaccinated calves. Calves receiving nanovaccine via either prime-boost regimen exhibited clinical and virological protection compared to non-vaccinated calves. The heterologous nanovaccine regimen induced both virus-specific cellular immunity and mucosal IgA, and induced similar clinical, virological and pathological protection as the commercial modified-live vaccine. Principal component analysis identified BRSV-specific humoral and cellular responses as important correlates of protection. The BRSV-F/G CpG nanovaccine is a promising candidate vaccine to reduce RSV disease burden in humans and animals.

Identification of a DRB3*011:01-restricted CD4+ T cell response against bovine respiratory syncytial virus fusion protein.

Although Human Respiratory Syncytial Virus (HRSV) is a significant cause of severe respiratory disease with high morbidity and mortality in pediatric and elderly populations worldwide there is no licensed vaccine. Bovine Respiratory Syncytial Virus (BRSV) is a closely related orthopneumovirus with similar genome structure and high homology between structural and nonstructural proteins. Like HRSV in children, BRSV is highly prevalent in dairy and beef calves and known to be involved in the etiology of bovine respiratory disease, in addition to being considered an excellent model for HRSV. Commercial vaccines are currently available for BRSV, though improvements in efficacy are needed. The aims of this study were to identify CD4+ T cell epitopes present in the fusion glycoprotein of BRSV, an immunogenic surface glycoprotein that mediates membrane fusion and a major target of neutralizing antibodies. Overlapping peptides representing three regions of the BRSV F protein were used to stimulate autologous CD4+ T cells in ELISpot assays. T cell activation was observed only in cells from cattle with the DRB3*011:01 allele by peptides from AA249-296 of the BRSV F protein. Antigen presentation studies with C-terminal truncated peptides further defined the minimum peptide recognized by the DRB3*011:01 allele. Computationally predicted peptides presented by artificial antigen presenting cells further confirmed the amino acid sequence of a DRB3*011:01 restricted class II epitope on the BRSV F protein. These studies are the first to identify the minimum peptide length of a BoLA-DRB3 class II-restricted epitope in BRSV F protein.

Effects of feeding a Saccharomyces cerevisiae fermentation product and ractopamine hydrochloride to finishing beef steers on growth performance, immune system, and muscle gene expression.

The objective of this study was to determine impacts on immune parameters, anti-oxidant capacity, and growth of finishing steers fed a Saccharomyces cerevisiae fermentation product (SCFP; NaturSafe; Diamond V, Cedar Rapids, IA) and ractopamine hydrochloride (RAC; Optaflexx; Elanco Animal Health, Greenfield, IN). Angus-crossbred steers (N = 288) from two sources were utilized in this 90-d study. Steers were blocked by source, stratified by initial body weight to pens of six steers, and pens randomly assigned to treatments (16 pens per treatment). Three treatments compared feeding no supplemental SCFP (control; CON) and supplemental SCFP for 57 d (SCFP57), and 29 d (SCFP29) before harvest. Supplementation of SCFP was 12 g per steer per d, and all steers were fed RAC at 300 mg per steer per d for 29 d before harvest. Blood samples were collected from3 steers per pen, and muscle samples were collected from 1 steer per pen at 57, 29 (start of RAC), and 13 (midRAC) days before harvest. Blood was analyzed from 2 steers per pen for ferric reducing anti-oxidant power (FRAP). Muscle gene expression of myokines, markers of anti-oxidant and growth signaling were assessed. Individual animal BW were also collected on 57, 29, 13, and 1 d before being harvested at a commercial facility (National Beef, Tama, IA). Data were analyzed using the Mixed procedure of SAS 9.4 (Cary, NC) with pen as the experimental unit. The model included fixed effects of treatment and group. Increased BW compared to CON was observed days -29, -13, and -1 in SCFP57 steers (P ≤ 0.05), with SCFP29 being intermediate days -13 and -1. Overall G:F was improved in SCFP29 and SCFP57 (P = 0.01). On day -29, FRAP was greater in SCFP57 than CON (P = 0.02). The percent of gamma delta T cells and natural killer cells in both SCFP29 and SCFP57 was greater than CON on day -13 (P = 0.02). There were no treatment × day effects for muscle gene expression measured (P ≥ 0.25). Interleukin 6 tended to decrease in SCFP29 and SCFP57 on day -13 (P = 0.10). No other treatment effects were observed for muscle gene expression. Muscle gene expression of interleukin 15 was increased (P = 0.01), and expression of interleukin 8 was decreased (P = 0.03) due to RAC feeding. Increased growth in SCFP-fed cattle may be related to changes in anti-oxidant capacity and the immune system.

Saccharomyces cerevisiae fermentation products (SCFP) can provide additional support for improved growth performance. This study investigated the effects of supplementing a SCFP (NaturSafe; Diamond V, Cedar Rapids, IA; 12 g per steer per d) for 29 (SCFP29) or 57 (SCFP57) d before harvest when also feeding ractopamine hydrochloride (RAC; 300 mg per steer per d; Optaflexx, Elanco Animal Health, Greenfield, IN) for 29 d before harvest. Compared to steers not fed SCFP (CON), SCFP29 and SCFP57 had improved gain:feed for the entire feeding period. Steers supplemented with SCFP had increased percentages of gamma delta T cells and natural killer cells 13 d before harvest compared to CON. Gene expression of cytokine and anti-oxidant signaling in muscle were changed in all treatments during RAC compared to before RAC. Improvements in growth during RAC with SCFP supplementation may be due to the changes in anti-oxidant and cytokine signaling in muscle.

Effects of supplemental Zn concentration and trace mineral source on immune function and associated biomarkers of immune status in weaned beef calves received into a feedlot.

Low-risk, weaned Angus-crossbred steers (n = 72; 284 ± 25 kg) were used in a 42-d receiving study. Steers were housed in pens (n = 6 steers per pen) equipped with GrowSafe bunks for determination of individual animal feed disappearance. Dietary treatments (n = 24 steers per treatment) included: 1) trace minerals (TM) from an organic source (Availa4; Zinpro Corp., Eden Prairie, MN) at 7 g·steer-1·d-1; for 42 d (ORG); 2) ORG for entire 42-d plus AvailaZn (Zn amino acid complex, Zinpro Corp., Eden Prairie, MN) to provide 1,000 mg Zn·steer-1·d-1 for first 14 d (ORG+Z); 3) inorganic TM sources to supplemented at equivalent concentration as in ORG for 42-d (ING). Cattle were weighed on day -1, 0, 14, 41, and 42. Whole blood was collected (n = 72 steers) on day 0, 14, and 42. Liver biopsies were conducted (n = 36 steers; 3 steers per pen) on day 0, 14, and 42. Flow cytometry measures were conducted using whole blood on day 1, 14, and 42 for determination of circulating frequencies of immune cell populations. There was a tendency for improved overall average daily gain (P = 0.07) where both ORG and ORG+Z were greater than ING. Final body weight did not differ (P = 0.21) and overall dry matter intake was unaffected by dietary treatment (P ≥ 0.18). However, overall gain-to-feed ratio was improved (P = 0.01) in steers supplemented organic TM (ORG and ORG+Z) compared to ING. Plasma Zn concentration did not differ at any time point during the study (P ≥ 0.20). Liver Zn concentration did not differ between treatments on day 0 or 42; however, on day 14 ING tended (P = 0.09) to be greater than ORG+Z with ORG being intermediate. Plasma Cu was unaffected by dietary treatment (P ≥ 0.34) on day 0, 14, and 42. Plasma Fe did not differ on day 0 or 42 but tended to be greater in ORG and ORG+Z compared to ING (P = 0.08) on day 14. Dietary treatment did not alter (P ≥ 0.22) liver Fe or Mn concentration at any time point. Frequency of total circulating natural killer (NK) and CD8 T cells measured on day 0, 14, and 42 did not differ (P ≥ 0.07). However, cell surface markers of activation (CD16, CD44, and CD8) on NK cells measured on day 14 did differ because of treatment (P ≤ 0.05). Results presented herein indicate TM from an organic source supplemented to steers during receiving can positively influence growth rate and feed efficiency. Regardless of source, TM supplementation affected markers of immune function but did not influence the prevalence of circulating NK and CD8 T-cell populations.

The receiving phase of the beef cattle production cycle occurs when calves are initially placed into the feedlot. During this time cattle are often exposed to stressors such as new environments, unfamiliar feedstuffs, and new pathogens. Together these stressors can result in lesser feed consumption. Along with lower total feed consumption, it is during this time that cattle likely require greater amounts of specific trace minerals (TM) to mount an effective immune response and maintain adequate growth. Therefore, this study aimed to evaluate the effects of supplemental Zn concentration and TM source on the immune function and associated biomarkers of immune status in weaned beef calves received into a feedlot. In this study, the more bioavailable, organic TM source supplemented to steers during receiving positively influenced growth rate and feed efficiency. Plasma TM concentration of steers in this study was adequate and was minimally influenced by TM source or concentration. These results also show TM supplementation, regardless of source, can alter markers of activation within immune cell populations.

Future of biomedical, agricultural, and biological systems research using domesticated animals.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

Use of Thoracic Ultrasonography to Improve Disease Detection in Experimental BRD Infection.

Bovine respiratory disease (BRD) is caused by complex interactions between viral and bacterial pathogens, host immune status, and environmental stressors. In both clinical and research settings, current methods for detecting BRD in calves commonly focus on visual indicators such as attitude, nasal discharge, and cough, in addition to vital signs such as rectal temperature and respiration rate. Recently, thoracic ultrasonography (TUS) has become more commonly used in clinical settings, in addition to physical examination to diagnose BRD. To assess the value of performing TUS during experimental BRD infection, 32 calves were challenged with bovine respiratory syncytial virus, to mimic a viral infection, and 30 calves were infected with Mannheimia haemolytica, to mimic a bacterial infection. TUS was performed at regular intervals using a standardized method and scoring system in addition to daily clinical scoring. Although overall correlations between clinical scores and TUS scores were generally weak (maximum R2 = 0.3212), TUS identified calves with abnormal lung pathology that would have otherwise been misclassified on the basis of clinical scoring alone, both on arrival and throughout the studies. In addition, TUS had an increased correlation with gross lung pathology on necropsy (maximum R2 = 0.5903), as compared to clinical scoring (maximum R2 = 0.3352). Our results suggest that TUS can provide additional information on calf health at enrollment and throughout a study and may provide an alternative to terminal studies, due to the high correlation with lung pathology at necropsy.

The Effects of Ursolic Acid Treatment on Immunopathogenesis Following Mannheimia haemolytica Infections.

Bovine respiratory disease complex (BRDC) is a costly economic and health burden for the dairy and feedlot cattle industries. BRDC is a multifactorial disease, often involving viral and bacterial pathogens, which makes it difficult to effectively treat or vaccinate against. Mannheimia haemolytica (MH) are common commensal bacteria found in the nasopharynx of healthy cattle; however, following environmental and immunological stressors, these bacteria can rapidly proliferate and spread to the lower respiratory tract, giving rise to pneumonic disease. Severe MH infections are often characterized by leukocyte infiltration and dysregulated inflammatory responses in the lungs. IL-17A is thought to play a key role in this inflammatory response by inducing neutrophilia, activating innate and adaptive immune cells, and further exacerbating lung congestion. Herein, we used a small molecule inhibitor, ursolic acid (UA), to suppress IL-17A production and to determine the downstream impact on the immune response and disease severity following MH infection in calves. We hypothesized that altering IL-17A signaling during MH infections may have therapeutic effects by reducing immune-mediated lung inflammation and improving disease outcome. Two independent studies were performed (Study 1 = 32 animals and Study 2 = 16 animals) using 4-week-old male Holstein calves, which were divided into 4 treatment group including: (1) non-treated and non-challenged, (2) non-treated and MH-challenged, (3) UA-treated and non-challenged, and (4) UA-treated and MH-challenged. Based on the combined studies, we observed a tendency (p = 0.0605) toward reduced bacterial burdens in the lungs of UA-treated animals, but did not note a significant difference in gross (p = 0.3343) or microscopic (p = 0.1917) pathology scores in the lungs. UA treatment altered the inflammatory environment in the lung tissues following MH infection, reducing the expression of IL-17A (p = 0.0870), inflammatory IL-6 (p = 0.0209), and STAT3 (p = 0.0205) compared to controls. This reduction in IL-17A signaling also appeared to alter the downstream expression of genes associated with innate defenses (BAC5, DEFB1, and MUC5AC) and lung remodeling (MMP9 and TIMP-1). Taken together, these results support our hypothesis that IL-17A signaling may contribute to lung immunopathology following MH infections, and further understanding of this inflammatory pathway could expand therapeutic intervention strategies for managing BRDC.

Development of surface engineered antigenic exosomes as vaccines for respiratory syncytial virus.

Respiratory syncytial virus (RSV) is one of the main pathogens associated with lower respiratory tract infections in infants and young children worldwide. Exosomes secreted by antigen presenting cells (APCs) can elicit immune responses by carrying major histocompatibility complex (MHC) class I molecules complexed with antigenic peptides and other co-stimulating factors. Therefore, we developed novel immunomagnetic nanographene particles to sequentially isolate, surface engineer, and release intact dendritic cell (DC) exosomes for use as a potential vaccine platform against RSV. The H-2Db-restricted, immunodominant peptides from RSV (M187-195 and NS161-75) were introduced to MHC-I on DC-derived exosomes to express peptide/MHC-I (pMHC-I) complexes. A mouse model of RSV infection was used to define the immunogenicity of surface engineered exosomes for activating virus-specific immune responses. Ex vivo assays demonstrated that engineered exosomes carrying RSV-specific peptides can elicit interferon-gamma (IFN-γ) production by virus-specific CD8+ T cells isolated from RSV-infected C57BL/6 mice. In vivo assays demonstrated that subcutaneous administration of both M187-195 and NS161-75 engineered exosomes to mice, with or without additional adjuvant, appeared safe and well tolerated, however, did not prime antigen-specific CD8+ T cell responses. Surface engineered exosomes are immunogenic and promising for further development as a vaccine platform.

Feeding Saccharomyces cerevisiae fermentation products lessens the severity of a viral-bacterial coinfection in preweaned calves.

We have previously reported that supplementation with Saccharomyces cerevisiae fermentation products (SCFP) ameliorates clinical signs and lung pathology following experimental bovine respiratory syncytial virus (BRSV) infection in preweaned dairy calves. The objectives of this study were to determine the effect of SCFP supplementation on the metabolic and endocrine responses, and disease outcome of a viral-bacterial coinfection in preweaned calves. Twenty-seven, 1- to 2-d-old Holstein-Angus cross calves were enrolled in the study; one SCFP calf was removed from the trial during the pre-challenge phase due to complications from nephritis. Calves were assigned to two treatment groups: control or SCFP-treated, base milk replacer with 1 g/d SCFP (Smartcare, soluble formula) and calf starter top dressed with 5 g/d SCFP (NutriTek, insoluble formula). Calves were infected with BRSV on day 21, followed 6 d later by intratracheal inoculation with Pasteurella multocida (PM). Calves were euthanized on day 10 post-viral infection. Calves receiving SCFP had reduced thoracic ultrasonography scores on day 7 post-viral infection (P = 0.03) and a tendency toward reduced scores on day 10 post-viral infection (P = 0.09). Calves receiving SCFP also had less severe lung pathology scores at necropsy (P = 0.06). No differences between treatments were observed in lung viral loads (P = 0.48) or bacterial lung recovery (P = 0.34); however, there was a distinction in the lung location for PM recovery, with PM isolated more frequently from the cranial lobes in SCFP-treated calves, but more frequently from the caudal lobes of control calves. Calves treated with SCFP tended (P = 0.07) to have higher serum IL-6 concentrations following the coinfection. Calves treated with SCFP had lower concentrations of serum nonesterified fatty acids and beta-hydroxybutyric acid compared with controls following experimental challenge (P = 0.03 and P = 0.08, respectively), suggesting metabolic changes favoring growth and development. There were no differences between groups in gene expression of insulin receptor, insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), growth hormone receptor, or haptoglobin in the liver. Results from this study suggest that supplementing with SCFP may moderate the impact of a respiratory viral-bacterial coinfection on preweaned calves through metabolic and immune modifications.

Effect of bis-glycinate bound zinc or zinc sulfate on zinc metabolism in growing lambs.

To assess the efficacy of bis-glycinate bound Zn, 36 crossbred wethers (34 ± 2 kg) were sorted by body weight into three groups and stagger started on a Zn-deficient diet (18 mg Zn/kg dry matter [DM]; 22.5% neutral detergent fiber [NDF]) for 45 d prior to a 15-d metabolism period (10 d adaptation and 5 d collection). On day 46, lambs were randomly assigned to dietary treatments (four lambs treatment-1group-1): no supplemental Zn (CON) or 15 mg supplemental Zn/kg DM (ZINC) as Zn sulfate (ZS) or bis-glycinate (GLY; Plexomin Zn, Phytobiotics). Blood was collected from all lambs on days 1, 44, 56, and 61. Liver, jejunum, and longissimus dorsi samples were collected after euthanasia on day 61. Gene expression was determined via quantitative real-time polymerase chain reaction. Data were analyzed using ProcMixed of SAS (experimental unit = lamb; fixed effects = treatment, group, and breed) and contrast statements assessed the effects of supplemental Zn concentration (ZINC vs. CON) and source (GLY vs. ZS). After 15 d of Zn supplementation, plasma Zn concentrations were greater for ZINC vs. CON and GLY vs. ZS (P ≤ 0.01); tissue Zn concentrations were unaffected (P ≥ 0.27). Liver Cu concentrations were lesser for ZINC vs. CON (P = 0.03). Longissimus dorsi Mn concentrations were greater for ZINC vs. CON (P = 0.05) and tended to be lesser for GLY vs. ZS (P = 0.09). Digestibility of DM, organic matter (OM), and NDF was lesser for ZINC vs. CON (P ≤ 0.05); acid detergent fiber digestibility tended to be greater for GLY vs. ZS (P = 0.06). Nitrogen retention (g/d) tended to be greater for GLY vs. ZS (P = 0.10), and N apparent absorption was lesser for ZINC vs. CON (P = 0.02). Zinc intake, fecal output, retention, and apparent absorption were greater for ZINC vs. CON (P ≤ 0.01). Apparent absorption of Zn was -5.1%, 12.8%, and 15.0% for CON, ZS, and GLY, respectively. Nitrogen and Zn retention and apparent absorption were not correlated for CON (P ≥ 0.14) but were positively correlated for ZINC (retention: P = 0.02, r = 0.52; apparent absorption: P < 0.01, r = 0.73). Intestinal expression of Zn transporter ZIP4 was lesser for ZINC vs. CON (P = 0.02). Liver expression of metallothionein-1 (MT1) tended to be greater for GLY vs. ZS (P = 0.07). Although Zn apparent absorption did not differ between sources (P = 0.71), differences in post-absorptive metabolism may be responsible for greater plasma Zn concentrations and liver MT1 expression for GLY-supplemented lambs, suggesting improved bioavailability of GLY relative to ZS.

A Recombinant BCG Vaccine Is Safe and Immunogenic in Neonatal Calves and Reduces the Clinical Disease Caused by the Respiratory Syncytial Virus.

The human respiratory syncytial virus (hRSV) constitutes a major health burden, causing millions of hospitalizations in children under five years old worldwide due to acute lower respiratory tract infections. Despite decades of research, licensed vaccines to prevent hRSV are not available. Development of vaccines against hRSV targeting young infants requires ruling out potential vaccine-enhanced disease presentations. To achieve this goal, vaccine testing in proper animal models is essential. A recombinant BCG vaccine that expresses the Nucleoprotein of hRSV (rBCG-N-hRSV) protects mice against hRSV infection, eliciting humoral and cellular immune protection. Further, this vaccine was shown to be safe and immunogenic in human adult volunteers. Here, we evaluated the safety, immunogenicity, and protective efficacy of the rBCG-N-hRSV vaccine in a neonatal bovine RSV calf infection model. Newborn, colostrum-replete Holstein calves were either vaccinated with rBCG-N-hRSV, WT-BCG, or left unvaccinated, and then inoculated via aerosol challenge with bRSV strain 375. Vaccination with rBCG-N-hRSV was safe and well-tolerated, with no systemic adverse effects. There was no evidence of vaccine-enhanced disease following bRSV challenge of rBCG-N-hRSV vaccinated animals, suggesting that the vaccine is safe for use in neonates. Vaccination increased virus-specific IgA and virus-neutralization activity in nasal fluid and increased the proliferation of virus- and BCG-specific CD4+ and CD8+ T cells in PBMCs and lymph nodes at 7dpi. Furthermore, rBCG-N-hRSV vaccinated calves developed reduced clinical disease as compared to unvaccinated control calves, although neither pathology nor viral burden were significantly reduced in the lungs. These results suggest that the rBCG-N-hRSV vaccine is safe in neonatal calves and induces protective humoral and cellular immunity against this respiratory virus. These data from a newborn animal model provide further support to the notion that this vaccine approach could be considered as a candidate for infant immunization against RSV.

Applications of Nanovaccines for Disease Prevention in Cattle.

Vaccines are one of the most important tools available to prevent and reduce the incidence of infectious diseases in cattle. Despite their availability and widespread use to combat many important pathogens impacting cattle, several of these products demonstrate variable efficacy and safety in the field, require multiple doses, or are unstable under field conditions. Recently, nanoparticle-based vaccine platforms (nanovaccines) have emerged as promising alternatives to more traditional vaccine platforms. In particular, polymer-based nanovaccines provide sustained release of antigen payloads, stabilize such payloads, and induce enhanced antibod- and cell-mediated immune responses, both systemically and locally. To improve vaccine administrative strategies and efficacy, they can be formulated to contain multiple antigenic payloads and have the ability to protect fragile proteins from degradation. Nanovaccines are also stable at room temperature, minimizing the need for cold chain storage. Nanoparticle platforms can be synthesized for targeted delivery through intranasal, aerosol, or oral administration to induce desired mucosal immunity. In recent years, several nanovaccine platforms have emerged, based on biodegradable and biocompatible polymers, liposomes, and virus-like particles. While most nanovaccine candidates have not yet advanced beyond testing in rodent models, a growing number have shown promise for use against cattle infectious diseases. This review will highlight recent advancements in polymeric nanovaccine development and the mechanisms by which nanovaccines may interact with the bovine immune system. We will also discuss the positive implications of nanovaccines use for combating several important viral and bacterial disease syndromes and consider important future directions for nanovaccine development in beef and dairy cattle.

Supplementing a Saccharomyces cerevisiae fermentation product modulates innate immune function and ameliorates bovine respiratory syncytial virus infection in neonatal calves.

The objectives of this study were to determine the effects of oral supplementation with Saccharomyces cerevisiae fermentation products (SCFP; SmartCare and NutriTek; Diamond V, Cedar Rapids, IA) on immune function and bovine respiratory syncytial virus (BRSV) infection in preweaned dairy calves. Twenty-four Holstein × Angus, 1- to 2-d-old calves (38.46 ± 0.91 kg initial body weight [BW]) were assigned two treatment groups: control or SCFP treated, milk replacer with 1 g/d SCFP (SmartCare) and calf starter top-dressed with 5 g/d SCFP (NutriTek). The study consisted of one 31-d period. On days 19 to 21 of the supplementation period, calves were challenged via aerosol inoculation with BRSV strain 375. Calves were monitored twice daily for clinical signs, including rectal temperature, cough, nasal and ocular discharge, respiration effort, and lung auscultation. Calves were euthanized on day 10 postinfection (days 29 to 31 of the supplementation period) to evaluate gross lung pathology and pathogen load. Supplementation with SCFP did not affect BW (P = 0.762) or average daily gain (P = 0.750), percentages of circulating white blood cells (P < 0.05), phagocytic (P = 0.427 for neutrophils and P = 0.460 for monocytes) or respiratory burst (P = 0.119 for neutrophils and P = 0.414 for monocytes) activity by circulating leukocytes either before or following BRSV infection, or serum cortisol concentrations (P = 0.321) after BRSV infection. Calves receiving SCFP had reduced clinical disease scores compared with control calves (P = 0.030), reduced airway neutrophil recruitment (P < 0.002), reduced lung pathology (P = 0.031), and a reduced incidence of secondary bacterial infection. Calves receiving SCFP shed reduced virus compared with control calves (P = 0.049) and tended toward lower viral loads in the lungs (P = 0.051). Immune cells from the peripheral blood of SCFP-treated calves produced increased (P < 0.05) quantities of interleukin (IL)-6 and tumor necrosis factor-alpha in response to toll-like receptor stimulation, while cells from the bronchoalveolar lavage (BAL) of SCFP-treated calves secreted less (P < 0.05) proinflammatory cytokines in response to the same stimuli. Treatment with SCFP had no effect on virus-specific T cell responses in the blood but resulted in reduced (P = 0.045) virus-specific IL-17 secretion by T cells in the BAL. Supplementing with SCFP modulates both systemic and mucosal immune responses and may improve the outcome of an acute respiratory viral infection in preweaned dairy calves.

Innate Immunomodulation in Food Animals: Evidence for Trained Immunity?

Antimicrobial resistance (AMR) is a significant problem in health care, animal health, and food safety. To limit AMR, there is a need for alternatives to antibiotics to enhance disease resistance and support judicious antibiotic usage in animals and humans. Immunomodulation is a promising strategy to enhance disease resistance without antibiotics in food animals. One rapidly evolving field of immunomodulation is innate memory in which innate immune cells undergo epigenetic changes of chromatin remodeling and metabolic reprogramming upon a priming event that results in either enhanced or suppressed responsiveness to secondary stimuli (training or tolerance, respectively). Exposure to live agents such as bacille Calmette-Guerin (BCG) or microbe-derived products such as LPS or yeast cell wall ß-glucans can reprogram or "train" the innate immune system. Over the last decade, significant advancements increased our understanding of innate training in humans and rodent models, and strategies are being developed to specifically target or regulate innate memory. In veterinary species, the concept of enhancing the innate immune system is not new; however, there are few available studies which have purposefully investigated innate training as it has been defined in human literature. The development of targeted approaches to engage innate training in food animals, with the practical goal of enhancing the capacity to limit disease without the use of antibiotics, is an area which deserves attention. In this review, we provide an overview of innate immunomodulation and memory, and the mechanisms which regulate this long-term functional reprogramming in other animals (e.g., humans, rodents). We focus on studies describing innate training, or similar phenomenon (often referred to as heterologous or non-specific protection), in cattle, sheep, goats, swine, poultry, and fish species; and discuss the potential benefits and shortcomings of engaging innate training for enhancing disease resistance.

The Immunology of Bovine Respiratory Disease: Recent Advancements.

Bovine respiratory disease (BRD) remains a leading cause of morbidity, mortality, and economic loss to the cattle industry. The continued high prevalence of the disease underlines a gap in understanding of the host immune response to respiratory infection. The host immune response is beneficial and detrimental, required for clearing the disease but often leading to tissue damage and long-term defects in lung function. This article highlights advancements made in understanding innate and adaptive immunity in BRD, factors that predispose animals to BRD, and novel intervention strategies that may lead to changes in the approach to treating and controlling BRD.

Characterization of local and circulating bovine γδ T cell responses to respiratory BCG vaccination.

The Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine is administered parenterally to infants and young children to prevent tuberculosis (TB) infection. However, the protection induced by BCG is highly variable and the vaccine does not prevent pulmonary TB, the most common form of the illness. Until improved TB vaccines are available, it is crucial to use BCG in a manner which ensures optimal vaccine performance. Immunization directly to the respiratory mucosa has been shown to promote greater protection from TB in animal models. γδ T cells play a major role in host defense at mucosal sites and are known to respond robustly to mycobacterial infection. Their positioning in the respiratory mucosa ensures their engagement in the response to aerosolized TB vaccination. However, our understanding of the effect of respiratory BCG vaccination on γδ T cell responses in the lung is unknown. In this study, we used a calf model to investigate the immunogenicity of aerosol BCG vaccination, and the phenotypic profile of peripheral and mucosal γδ T cells responding to vaccination. We observed robust local and systemic M. bovis-specific IFN-γ and IL-17 production by both γδ and CD4 T cells. Importantly, BCG vaccination induced effector and memory cell differentiation of γδ T cells in both the lower airways and peripheral blood, with accumulation of a large proportion of effector memory γδ T cells in both compartments. Our results demonstrate the potential of the neonatal calf model to evaluate TB vaccine candidates that are to be administered via the respiratory tract, and suggest that aerosol immunization is a promising strategy for engaging γδ T cells in vaccine-induced immunity against TB.