Grad Student Competition
Effects of a parenteral innate immune stimulant on calf vaccine responses and bronchoalveolar lavage fluid cell gene expression
| Date/Time: |
8/28/2026
11:00
|
| Author: |
Grace M Jakes |
| Clinic: |
Colorado State University |
| City, State, ZIP: |
Fort Collins, CO 80521 |
G.M. Jakes, BS
1
;
S. Else, BS
2
;
K. Park, BS, MS
3
;
S. Dow, DVM, PhD, DACVIM (SAIM)
3
;
S.M. Raabis, DVM, PhD DACVIM (LAIM)
3
;
1Department of Microbiology, Immunology, and Pathology, Colorado State University, 80521
2Department of Animal Sciences, Colorado State University, 80521
3Department of Clinical Sciences, Colorado State University 80521
Introduction:
Reducing and refining antimicrobial use in cattle is essential to advance animal health and antimicrobial stewardship. Non-specific innate immune stimulation can mitigate respiratory disease and may reduce the need for antimicrobials, however the mechanism of protection requires further research. Innate immune stimulants may improve health by augmenting vaccine responses. The interaction of innate immune stimulants with vaccination and associated health outcomes has not been fully described, especially at the mucosal level or in the lung. Optimizing vaccine responses through innate immune stimulation may provide protective benefit. Therefore, the objective of this study was to evaluate the capacity of an innate immune stimulant to alter immune parameters following vaccination in pre-weaned dairy calves.
Materials and methods:
Pre-weaned 4-week-old calves were intramuscularly administered 1 mL of liposomal Toll-like receptor (TLR)3/TLR9 complex (LTC, n=7), or an equal amount of a buffered sucrose diluent (CON, n=6). Calves were vaccinated 10 days later with a multivalent vaccine against BRSV, BHV-1, BVD-1, BVD-2, and PI-3 (Bovi-Shield GOLD®, Zoetis). Bronchoalveolar lavage fluid (BALF) was collected from calves before administration of LTC, 1 day after vaccination, and 28 days after vaccination. BALF cells were stimulated with 300 ng/mL LPS for 48 hours and supernatants were collected for evaluation of cytokine production via ELISA. An aliquot of BALF cells were extracted for bulk RNA sequencing (RNA-seq), completed on an Illumina NovaSeq instrument. Sequences were aligned to the bovine genome (ARS-UCD1.3) using a STAR alignment pipeline. Differential gene expression analysis was completed in R using DESeq2, with clusterProfiler used for gene set enrichment analysis. Serum was collected from all calves before LTC administration, and at 4 and 5 weeks post vaccination to evaluate titers against the major viral pathogens included in the multivalent vaccine. Statistical tests were performed in Prism8 (GraphPad). For the cytokine analysis, normality of data was confirmed using Shapiro-Wilk test and a 2-way repeated measures ANOVA was completed, assessing treatment, time, and treatment x time interaction. Significance was set to p<0.05.
Results:
No calf developed clinical respiratory disease signs through 8 weeks of age in this study. Calves had high titers against viral pathogens before vaccination with the multivalent vaccine, and these titers remained high throughout the study. LPS stimulated IL-6 production was greatest in calves at the 1-day post vaccination timepoint. LTC calves tended to have reduced IL-6 cytokine production in response to LPS stimulation as compared to CON (p=0.078), and there was a treatment x time interaction where LTC calves had reduced change in IL-6 production following vaccination (p=0.035). RNA-seq gene expression profiles demonstrated that LTC calves had increased B cell associated signaling in their BALF immune cells at 1-day post vaccination, including genes BLK, CD19, and CD79B. As compared to pre-vaccine and LTC administration, BALF immune cells at 28 days post vaccination in all calves regardless of treatment had profound upregulation of both T cell activation and inflammatory gene pathways, including genes CD8A, LCK, and IL33.
Significance:
In this study, administration of a liposomal TLR3-/TLR9 agonist to calves 10 days prior to vaccination altered alveolar macrophage ex vivo responsiveness to LPS over time and significantly altered BALF RNA-seq gene expression profiles. All calves had adequate passive transfer as demonstrated by serum total protein, which may explain high titers prior to vaccination. While no calf was treated for BRD during this study, inflammatory signaling in BALF at 28 days post vaccination merits screening for BRD pathogens via qPCR, which is pending. Overall, non-specific immune stimulation altered both innate and adaptive arms of the immune response, which may have implications for future immune functionality.
