Transport stress induces paradoxical increases in airway inflammatory responses in beef stocker cattle
Data files
Jan 19, 2026 version files 147.05 GB
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allCells_clean_viral_S3.rds
1.90 GB
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BovLav101614_1.fq.gz
3.93 GB
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BovLav101614_2.fq.gz
3.94 GB
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BovLav105226_1.fq.gz
4.09 GB
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BovLav105226_2.fq.gz
4.11 GB
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BovLav106915_1.fq.gz
3.66 GB
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BovLav106915_2.fq.gz
3.68 GB
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BovLav107720_1.fq.gz
4.24 GB
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BovLav107720_2.fq.gz
4.34 GB
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BovLav12132_1.fq.gz
3.84 GB
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BovLav12132_2.fq.gz
3.87 GB
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BovLav12745_1.fq.gz
3.26 GB
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BovLav12745_2.fq.gz
3.31 GB
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BovLav13014_1.fq.gz
4.27 GB
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BovLav13014_2.fq.gz
4.27 GB
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BovLav253180_1.fq.gz
3.87 GB
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BovLav253180_2.fq.gz
3.86 GB
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BovLav255204_1.fq.gz
4.23 GB
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BovLav255204_2.fq.gz
4.22 GB
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BovLav420826_1.fq.gz
3.97 GB
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BovLav420826_2.fq.gz
4 GB
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BovLav429617_1.fq.gz
4.20 GB
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BovLav429617_2.fq.gz
4.22 GB
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BovLav442765_1.fq.gz
2.91 GB
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BovLav442765_2.fq.gz
2.96 GB
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BovLav461618_1.fq.gz
4.43 GB
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BovLav461618_2.fq.gz
4.44 GB
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BovLav463021_1.fq.gz
3.07 GB
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BovLav463021_2.fq.gz
3.10 GB
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BovLav465113_1.fq.gz
4.71 GB
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BovLav465113_2.fq.gz
4.75 GB
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BovLav466718_1.fq.gz
3.96 GB
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BovLav466718_2.fq.gz
3.98 GB
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BovLav58778_1.fq.gz
3.40 GB
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BovLav58778_2.fq.gz
3.40 GB
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BovLav587932_1.fq.gz
3.58 GB
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BovLav587932_2.fq.gz
3.58 GB
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BovLav742132_1.fq.gz
2.71 GB
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BovLav742132_2.fq.gz
2.74 GB
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Cibersortx_Analysis.R
14.73 KB
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counts.txt
18.46 MB
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filtered_counts.txt
3.72 MB
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final_analysis_code.R
20.48 KB
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README.md
14.02 KB
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Stress_Study_Dryad_Metadata_Submission.xlsx
21.18 KB
Abstract
The development of Bovine Respiratory Disease (BRD ) in beef cattle is associated with stressful events, including auction and transport. To characterize immunity at the primary site of pathogen colonization in BRD, we evaluated stress responses in lung cells from beef stocker cattle to understand mucosal immune changes. Stocker calves were sampled via bronchoalveolar lavage fluid collection within 24 hours of auction and transport to a new facility (Stressed, n=12), or allowed to acclimate for 2 months at the new facility before sampling (Acclimated, n=7). Lavage cellular RNA was extracted and sequenced for bulk RNA-seq gene expression. The resulting fastq files are included in this repository and named based on the calf's unique identifier at sampling. The metadata file provides the key to which experimental group each calf belongs, as well as pertinent sequencing metadata values and MD5 sums to ensure file quality. Raw data counts are included in the counts.txt file, and are organized with genes as the row names and calf unique identifiers as the column names. Code files for DESeq2 and CIBERSORTx analysis are included as final analysis code.R and Cibersortx analysis.R. Differential gene expression analysis of RNA sequencing data demonstrated a profound upregulation of inflammatory genes in Stressed calves compared to Acclimated calves, including expression of CXCL8, CSF3R, IL1B, and CCL22. The top pathway upregulation in Stressed calves involved neutrophil migration and chemotaxis, and cytokine signaling. To predict cellular population proportions from the data, CIBERSORTx was used to deconvolute bulk RNA-seq gene counts. This analysis showed that Stressed calves had significantly increased BALF neutrophils compared to Acclimated calves (p =0.003). Neutrophilic infiltration occurred in the absence of pathogen colonization of the lungs in most calves, as demonstrated by a multi-pathogen respiratory qPCR screen. As such, the stress induced an inflammatory response in the lungs not explained by pathogen exposure. This study provides strong evidence that shipping stress in beef stocker calves can paradoxically trigger increased rather than suppressed inflammatory pulmonary mucosal immune responses, which have important implications for the pathogenesis of BRD. All animals were sampled under institutional IACUC approval.
Dataset DOI: 10.5061/dryad.02v6wwqh6
Description of the data and file structure
This study was conducted to evaluated differences in gene expression from bronchoalveolar lavage fluid cells between Stressed calves (collected within 24 hours of arrival at a new facility, n=12) and Acclimated calves that experienced the same stress, but were allowed to acclimated for 2 months to the facility before sampling (n=7). All cattle were Bos Taurus, Angus cross cattle and were sampled between 6-8 months of age. After sampling cattle via aseptic technique, samples were transported on ice to the processing lab, where they were washed with 1X phosphate-buffered saline (PBS), filtered through a sterile 70 micron filter, and pelleted via centrifugation at 500g for 10 minutes. Supernatant was removed and cells were lysed using RLT lysis buffer from Qiagen (Mini Kit). Next, the Qiagen Mini Kit for RNA extraction was used to extract all samples for bulk RNA.
RNA libraries were sequenced by Novogene Corp. using an Illumina Novaseq 6000 platform (Novogene Co., Sacramento, CA). RNA quality was evaluated using an Agilent 2100 Bioanalyzer system to verify minimum RNA integrity levels.
Libraries were sequenced on an Illumina PE150 (Novaseq) platform for 40M raw reads per sample. Raw data were filtered using Fastp (version 0.23.2) by removing reads containing adapters and for Phred scores >30. The filtered reads were then aligned with STAR (version 2.7.10b), ARS-UCD1.3 genome assembly. Aligned reads were annotated and counted using FeatureCounts (version 2.0.1), and differentially expressed genes were identified using DEseq2. Further biological interpretations including gene ontology enrichment and gene set enrichment analysis were then performed using clusterProfiler. CIBERSORTx analysis completed using best practices as outlined in CIBERSORTx documentation. Briefly, no batch correction and b batch correction methods evaluated for best correlation fit with prior single-cell RNA-seq dataset (included in the allCells_clean_viral_S3.rds file), and b batch selected for downstream analysis.
Data files in this submission include FastQ files for each sample (Reads 1 and 2), as well as a raw counts file (counts.txt). The prior single-cell dataset is included as a Seurat object .rds file, and can be visualized using the Seurat package of R. The filtered_counts.txt file includes the same information as the raw counts.txt file, but is appropriately formatted for the CIBERSORTx analysis pipeline. For the raw FastQ data, file names do not include designation of "Stressed" or "Acclimated" grouping, but this information is included in the description for each data file, the description of the counts.txt file, and in the metadata file.
Files and variables
The raw data are presented in compressed FastQ files that have not been trimmed or aligned to the bovine genome. Read 1 is designated in any file that ends in _1.fq.gz, and read 2 is designated in any file that ends in _2.fq.gz. Each sample is named with a "BovLav" prefix followed by the calf's ear tag number. Metadata relating to which experimental group each sample corresponds to is indicated in the counts.txt file's variables, as well as in each file's description, and in the Stress_Study_Dryad_Metadata_Submission.xlsx metadata file. In addition to the raw FastQ files, a raw counts file (from STAR alignment to Bos Taurus genome ARS-UCD1.3, and FeatureCounts output) is included where gene IDs and associated gene data are in rows, and individual samples/respective sample gene counts are in columns. A filtered counts file that is appropriately configured for use in the CIBERSORTx pipeline is included as well. The single-cell data is included as a .rds file, and can be visualized using the Seurat package of R. R code files are also included in this dataset submission for convenience of viewers. R code files are split into DEseq analysis and all traditional RNA-seq analysis, and then a separate R code file for the CIBERSORTx analysis.
File: counts.txt
Description: Raw counts file output from STAR alignment and FeatureCounts output.
Variables
- Geneid: The gene ID as defined in ARS-UCD1.3 genome build
- Chr: Chromosome of gene
- Start: Start of gene
- End: End of gene
- Strand: Strand of gene
- Length: Length of gene
- BovLav101614: Stressed calf sample 9, collected within 24hr of arrival at novel backgrounding facility
- BovLav105226: Stressed calf sample 4, collected within 24hr of arrival at novel backgrounding facility
- BovLav106915: Stressed calf sample 5, collected within 24hr of arrival at novel backgrounding facility
- BovLav107720: Stressed calf sample 6, collected within 24hr of arrival at novel backgrounding facility
- BovLav12132: Stressed calf sample 1, collected within 24hr of arrival at novel backgrounding facility
- BovLav12745: Stressed calf sample 2, collected within 24hr of arrival at novel backgrounding facility
- BovLav13014: Stressed calf sample 3, collected within 24hr of arrival at novel backgrounding facility
- BovLav253180: Stressed calf sample 10, collected within 24hr of arrival at novel backgrounding facility
- BovLav255204: Stressed calf sample 11, collected within 24hr of arrival at novel backgrounding facility
- BovLav420826: Acclimated calf sample 1, collected after about 2 months at backgrounding facility
- BovLav429617: Acclimated calf sample 2, collected after about 2 months at backgrounding facility
- BovLav442765: Stressed calf sample 7, collected within 24hr of arrival at novel backgrounding facility
- BovLav461618: Acclimated calf sample 3, collected after about 2 months at backgrounding facility
- BovLav463021: Acclimated calf sample 4, collected after about 2 months at backgrounding facility
- BovLav465113: Acclimated calf sample 5, collected after about 2 months at backgrounding facility
- BovLav466718: Acclimated calf sample 6, collected after about 2 months at backgrounding facility
- BovLav58778: Stressed calf sample 8, collected within 24hr of arrival at novel backgrounding facility
- BovLav587932: Stressed calf sample 12, collected within 24hr of arrival at novel backgrounding facility
- BovLav742132: Acclimated calf sample 7, collected after about 2 months at backgrounding facility
File: BovLav12132_1.fq.gz
Description: Read 1 of Stressed calf sample 1, collected within 24hr of arrival at novel backgrounding facility
File: BovLav12132_2.fq.gz
Description: Read 2 of Stressed calf sample 1, collected within 24hr of arrival at novel backgrounding facility
File: BovLav12745_1.fq.gz
Description: Read 1 of Stressed calf sample 2, collected within 24hr of arrival at novel backgrounding facility
File: BovLav12745_2.fq.gz
Description: Read 2 of Stressed calf sample 2, collected within 24hr of arrival at novel backgrounding facility
File: BovLav58778_1.fq.gz
Description: Read 1 of Stressed calf sample 8, collected within 24hr of arrival at novel backgrounding facility
File: BovLav58778_2.fq.gz
Description: Read 2 of Stressed calf sample 8, collected within 24hr of arrival at novel backgrounding facility
File: BovLav13014_1.fq.gz
Description: Read 1 of Stressed calf sample 3, collected within 24hr of arrival at novel backgrounding facility
File: BovLav101614_1.fq.gz
Description: Read 1 of Stressed calf sample 9, collected within 24hr of arrival at novel backgrounding facility
File: BovLav101614_2.fq.gz
Description: Read 2 of Stressed calf sample 9, collected within 24hr of arrival at novel backgrounding facility
File: BovLav13014_2.fq.gz
Description: Read 2 of Stressed calf sample 3, collected within 24hr of arrival at novel backgrounding facility
File: BovLav105226_1.fq.gz
Description: Read 1 of Stressed calf sample 4, collected within 24hr of arrival at novel backgrounding facility
File: BovLav105226_2.fq.gz
Description: Read 2 of Stressed calf sample 4, collected within 24hr of arrival at novel backgrounding facility
File: BovLav106915_1.fq.gz
Description: Read 1 of Stressed calf sample 5, collected within 24hr of arrival at novel backgrounding facility
File: BovLav106915_2.fq.gz
Description: Read 2 of Stressed calf sample 5, collected within 24hr of arrival at novel backgrounding facility
File: BovLav253180_1.fq.gz
Description: Read 1 of Stressed calf sample 10, collected within 24hr of arrival at novel backgrounding facility
File: BovLav253180_2.fq.gz
Description: Read 2 of Stressed calf sample 10, collected within 24hr of arrival at novel backgrounding facility
File: BovLav107720_1.fq.gz
Description: Read 1 of Stressed calf sample 6, collected within 24hr of arrival at novel backgrounding facility
File: BovLav107720_2.fq.gz
Description: Read 2 of Stressed calf sample 6, collected within 24hr of arrival at novel backgrounding facility
File: BovLav255204_1.fq.gz
Description: Read 1 of Stressed calf sample 11, collected within 24hr of arrival at novel backgrounding facility
File: BovLav255204_2.fq.gz
Description: Read 2 of Stressed calf sample 11, collected within 24hr of arrival at novel backgrounding facility
File: BovLav442765_1.fq.gz
Description: Read 1 of Stressed calf sample 7, collected within 24hr of arrival at novel backgrounding facility
File: BovLav442765_2.fq.gz
Description: Read 2 of Stressed calf sample 7, collected within 24hr of arrival at novel backgrounding facility
File: BovLav420826_1.fq.gz
Description: Read 1 of Acclimated calf sample 1, collected after about 2 months at backgrounding facility
File: BovLav420826_2.fq.gz
Description: Read 2 of Acclimated calf sample 1, collected after about 2 months at backgrounding facility
File: BovLav463021_1.fq.gz
Description: Read 1 of Acclimated calf sample 4, collected after about 2 months at backgrounding facility
File: BovLav463021_2.fq.gz
Description: Read 2 of Acclimated calf sample 4, collected after about 2 months at backgrounding facility
File: BovLav742132_1.fq.gz
Description: Read 1 of Acclimated calf sample 7, collected after about 2 months at backgrounding facility
File: BovLav742132_2.fq.gz
Description: Read 2 of Acclimated calf sample 7, collected after about 2 months at backgrounding facility
File: BovLav429617_1.fq.gz
Description: Read 1 of Acclimated calf sample 2, collected after about 2 months at backgrounding facility
File: BovLav429617_2.fq.gz
Description: Read 2 of Acclimated calf sample 2, collected after about 2 months at backgrounding facility
File: BovLav587932_1.fq.gz
Description: Read 1 of Stressed calf sample 12, collected within 24hr of arrival at novel backgrounding facility
File: BovLav587932_2.fq.gz
Description: Read 2 of Stressed calf sample 12, collected within 24hr of arrival at novel backgrounding facility
File: BovLav461618_1.fq.gz
Description: Read 1 of Acclimated calf sample 3, collected after about 2 months at backgrounding facility
File: BovLav466718_1.fq.gz
Description: Read 1 of Acclimated calf sample 6, collected after about 2 months at backgrounding facility
File: BovLav461618_2.fq.gz
Description: Read 2 of Acclimated calf sample 3, collected after about 2 months at backgrounding facility
File: BovLav466718_2.fq.gz
Description: Read 2 of Acclimated calf sample 6, collected after about 2 months at backgrounding facility
File: BovLav465113_1.fq.gz
Description: Read 1 of Acclimated calf sample 5, collected after about 2 months at backgrounding facility
File: BovLav465113_2.fq.gz
Description: Read 2 of Acclimated calf sample 5, collected after about 2 months at backgrounding facility
File: Stress_Study_Dryad_Metadata_Submission.xlsx
Description: This is the metadata file for each of the samples. It describes the study and provides additional sequencing metadata, the MD5 Sums of each FastQ file, as well as which experimental group each calf is assigned to.
File: allCells_clean_viral_S3.rds
Description: This file is the Seurat object output for the single-cell dataset. 10 calves are included in this dataset, comprising over 50,000 cells. This dataset was used to create the deconvolution matrix for the CIBERSORTx analysis pipeline. To inspect and use this dataset, please load in the Seurat package of R.
File: filtered_counts.txt
Description: This file is a counts file, similar to the raw data counts.txt file, but configured for the CIBERSORTx pipeline.
File: Cibersortx_Analysis.R
Description: This is the code used for CIBERSORTx analysis.
File: final_analysis_code.R
Description: This is the code used for DESeq2 analysis and all associated figures.
Code/software
Raw data were filtered using Fastp (version 0.23.2) by removing reads containing adapters and for Phred scores >30. The filtered reads were then aligned with STAR (version 2.7.10b), ARS-UCD1.3 genome assembly. Aligned reads were annotated and counted using FeatureCounts (version 2.0.1), and differentially expressed genes were identified using DEseq2. Further biological interpretations including gene ontology enrichment and gene set enrichment analysis were then performed using clusterProfiler. CIBERSORTx analysis completed using best practices as outlined in CIBERSORTx documentation. Briefly, no batch correction and b batch correction methods evaluated for best correlation fit with prior single-cell RNA-seq dataset, and b batch selected for downstream analysis.
Other publicly accessible locations of the data:
- NCBI GEO Database, Accession number: GSE299548