Investigation of HCAR2 antagonists as a potential strategy to modulate bovine leukocytes.

BACKGROUND: Dairy cows experiencing ketosis after calving suffer greater disease incidence and are at greater risk of leaving the herd. In vitro administration of beta-hydroxybutyric acid (BHBA; the primary blood ketone) has inhibitory effects on the function of bovine leukocytes. BHBA is a ligand of HCAR2 and the activation of these receptors promotes an anti-inflammatory response which may be related with immunosuppression observed in transition dairy cattle. The objective of this study was to identify and test antagonists for HCAR2 in bovine immune cells cultured with BHBA. RESULTS: We observed expression of HCAR2 at the protein level within lymphocytes, monocytes, and granulocytes. The proportion of cells expressing HCAR2 tended to be greater in mid-lactation compared to early lactation cows; the increase was a result of increased proportion of T and B cells expressing HCAR2. Stimulation of HCAR2 with niacin or BHBA promoted Ca2+ mobilization in neutrophils and mononuclear cells. Mononuclear cells treated with BHBA had diminished intracellular Ca2+ responses when HCAR2 was knocked down by siRNA silencing, indicating Ca2+ mobilization was mediated by HCAR2 signaling. Two candidate antagonists for HCAR2, synthesized from niacin (NA-1 and NA-5), were tested; monocytes and neutrophils pre-treated with NA-1 and NA-5 had reduced Ca2+ mobilization after incubation with BHBA. Furthermore, NA-5 but not NA-1 prevented BHBA-associated reductions in cyclic AMP. CONCLUSIONS: We demonstrated that HCAR2 is present on bovine leukocytes and has greater expression later in lactation. We confirmed that BHBA and niacin derived HCAR2 antagonists alter bovine leukocyte activity. Our results demonstrate that both BHBA and niacin affect bovine leukocyte Ca2+ mobilization in a HCAR2-dependent manner.

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.

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.

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.

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.