Temporal impact of metritis and systemic antibiotics on immune cell populations inferred from the blood leukocyte transcriptome

Description

Supplemental material for manuscript entitled "Temporal impact of metritis and systemic antibiotics on immune cell populations inferred from the blood leukocyte transcriptome" submitted to JDS Communications.

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Twenty-three first-lactation Holstein cows were enrolled in the study. Cases of metritis were identified by the herdsman based on clinical signs at 7 to 10 d postpartum (fetid red-brown watery vaginal discharge with a flaccid uterus) and confirmed by our research team. Cows diagnosed with metritis (n=11) were matched with healthy control (n=12) cows that calved within the same wk. Cows were either antibiotic-treated [metritis-treated (n=5) and healthy-treated (n=6)] with ceftiofur hydrochloride (i.m. 1.25 g/d for 3 d; Excenel RTU, Zoetis, Parsippany-Troy Hills, NJ, USA) or left untreated [metritis-untreated (n=6) and healthy-untreated (n=6)]. Blood samples were collected weekly beginning at disease diagnosis and before treatment (wk 1; 7.3 ± 1.5 d postpartum), 1 wk after treatment (wk 2; 13.8 ± 1.4 d postpartum) and 3 wk after treatment (wk 4; 26.8 ± 1.6 d postpartum). RNA was extracted from blood. The MU Genomics Technology Core performed the RNA library preparation and sequencing. Stranded RNA-seq libraries were constructed using the poly-A enrichment method with the Illumina Stranded mRNA Prep (Illumina, San Diego, CA, USA). Each purified library was sequenced on a NovaSeq 6000 sequencer (Illumina) using a single NovaSeq S2-PE100 flow cell with an average read count of approximately 60 million paired reads per sample (range: 46–78 million). The program fqtrim (https://ccb.jhu.edu/software/fqtrim/) was used to remove the sequence adaptors of raw sequence reads (fastq) and perform quality trimming. The trimmed paired-end reads were aligned to the Ensembl Bos taurus reference genome ARS-UCD1.2 using the Hisat2 mapper [https://daehwankimlab.github.io/hisat2/; (Kim et al., 2015)]. Subread's featureCounts was used to quantify the number of reads mapped to exon sequences of annotated genes using the ARS-UCD1.2 Bos taurus gene annotation file (release 102) from Ensembl (Liao et al., 2014). Differentially expressed genes (DEG) between groups were determined by fitting the read counts to a generalized linear model on edgeR robust (Zhou et al., 2014). A transcript was defined as differentially expressed for each group comparison if the FDR-adjusted P-value was < 0.05. The DEG lists were uploaded to the Database for Annotation, Visualization, and Integrated Discovery (DAVID v2024q4; https://davidbioinformatics.nih.gov/home.jsp/) to detect significantly enriched Gene Ontology (GO) Biological Process Direct terms (GOTERM_BP_DIRECT) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Enrichment of GO terms and KEGG pathways were significant if the FDR-adjusted P-value was < 0.05. Lists with DEG were then uploaded to ShinyGO 0.85 (http://bioinformatics.sdstate.edu/go/) (Ge et al., 2020) to detect enriched cell types using the CellMarker.2024 database (Hu et al., 2023) and the Enrichr program (https://maayanlab.cloud/Enrichr) (Chen et al., 2013; Kuleshov et al., 2016; Xie et al., 2021).

Institutions

• University of Missouri

  Missouri, Columbia

Categories

Funders

• Eunice Kennedy Shriver National Institute of Child Health and Human Development

  National Institutes of Health

  Bethesda

  Grant ID: R01HD092254

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