Grad Student Competition
Stress alters respiratory innate immune profiles in beef stocker cattle
| Date/Time: |
9/12/2025
11:30
|
| Author: |
Grace Jakes |
| Clinic: |
Colorado State University |
| City, State, ZIP: |
Fort Collins, CO 80521 |
G.M. Jakes, BS
1
;
D.T. Ammons, DVM, PhD
1
;
E. Silva, PhD
4
;
R. Hunter, DVM
3
;
S. Dow, DVM, PhD, DACVIM-SAIM
1
;
S.M. Raabis, DVM, PhD, DACVIM-LAIM
2
;
1Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, 80521
2Department of Clinical Sciences, Colorado State University, Fort Collins, CO, 80521
3Hunter Cattle, Wheatland, WY, 82201
4U.S. Department of Agriculture, Agricultural Research Service, National Bio and Agro-Defense Facility, Manhattan, KS, 66502
Introduction:
Bovine respiratory disease (BRD) is a leading cause of morbidity and mortality in beef cattle globally. Stress due to transport and weaning is associated with BRD risk, yet the mechanism of immune susceptibility following stress is less clear. The mucosal immune response to BRD is integral in mitigating pathology, yet the understanding of functional immune states and interactions between immune cell populations at the respiratory mucosal surface can be limited by a relative lack of species-specific reagents. Single-cell RNA sequencing (scRNA-seq) enables the evaluation of transcriptional profiles of individual cells, supporting the identification of unique cell populations and functional states of diverse immune cell subsets. To better characterize stress-induced changes to respiratory mucosal immunity, we sought to evaluate lung immune cells following stress using scRNA-seq. Our hypothesis was that stress would be associated with a reduced immune response due to the suppressive effects of stress on lymphocyte numbers.
Materials and methods:
Stocker calves were purchased at auction and transported to a commercial backgrounding facility. Bronchoalveolar lavage fluid (BALF) was collected from a subset of calves within 24 hours (Stressed; n=5) and from calves allowed to acclimate to the facility for 2-3 months prior to sampling (Acclimated; n=5). Calves were randomly selected and screened for sampling based on an absence of lung consolidation on lung ultrasound and a rectal temperature <103.5 ℉. BALF sampling was performed at the backgrounding facility and samples were processed using a 10X Genomics Chromium iX platform, with 5,000 cells targeted per sample. Raw sequencing data were aligned to a customized reference including the bovine genome (Ensembl release 113, Bos_taurus.ARS-UCD1.3.113), and viral genomes including Bovine Respiratory Syncytial Virus (BRSV), Bovine Herpesvirus (BHV-1), Bovine Coronavirus, and Bovine Viral Diarrhea Virus (BVDV) using Cell Ranger (version 7.1.0). Downstream processing and analysis were completed in R (version 4.5.0) using the Seurat package (version 5.0.1) with differential expression analysis completed using DESeq2. T-tests were conducted to compare cellular population proportions between Stressed and Acclimated cattle with significance set to 0.05. An aliquot of BALF from each calf was screened for BRD pathogens, including Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis, Pasteurella multocida, BRSV, BHV-1, BVDV, and Bovine Coronavirus via qPCR.
Results:
In total, we profiled 59,322 cells, which included 5 major cell types: macrophages, neutrophils, T cells, actively proliferating macrophages, and B cells. Neutrophils were the major cell type significantly overrepresented in Stressed calves (17.5-fold increase in cellular proportion, P = 0.0079), while T cells and B cell proportions did not significantly differ between groups (P = 0.15, P = 0.84 respectively). Monocyte proportions were also increased in Stressed calves (2-fold mean increase, P = 0.0079), while M2 macrophage (high expressing CCL22, CD24) proportions were reduced in Stressed animals (1.7-fold, P = 0.016). Despite mean neutrophil proportions of 35%, all Stressed calves were negative for respiratory pathogens by qPCR (Ct cutoff > 35).
Significance:
These findings indicate that in contrast to our hypothesis, stress may immunomodulate some calves to experience inflammatory immune responses in the first days after transport and comingling. While stress has classically been considered immunosuppressive, Stressed calves in this study did not experience a significant reduction in adaptive immune cell numbers and instead saw a shift from immunomodulatory to proinflammatory innate immune cell populations in their BALF. These findings are important to the immunological management of calves in the first days after stress and indicate that the host response to environmental stressors may result in respiratory mucosal inflammation in the first days after arrival.
