DOI: 10.5061/dryad.02v6wwqh6
title: Transport stress induces paradoxical increases in airway inflammatory responses in beef stocker cattle

description: 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.
description: # Transport stress induces paradoxical increases in airway inflammatory
 responses in beef stocker cattle Dataset DOI:
 [10.5061/dryad.02v6wwqh6](https://doi.org/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 &gt;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 &gt;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