Transcriptomic Analysis of the Chorioallantois of Mares with Nocardioform Placentitis

Nocardioform placentitis (NP) continues to result in episodic outbreaks of abortion and preterm birth in mares and remains a poorly understood disease. The objective of this study was to characterize the transcriptome of the chorioallantois (CA) of mares with NP. Term CA were collected from mares with gross lesions consistent with NP, and NP was subsequently confirmed in four CA based upon pathology and PCR for Amycolatopsis spp. CA samples were collected from the margin of the NP lesion (NPL, n=4) and grossly normal region (NPN, n=4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n=4). Transcriptome analysis identified 2,892 DEGs in CRL vs. NPL and 2,450 DEGs in NPN vs. NPL. Functional genomics analysis elucidated that inflammatory signaling, toll-like receptor signaling, inflammasome activation, chemotaxis, and apoptosis pathways dominate NP. The increased leukocytic infiltration in NPL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP3, and MMP8) and apoptosis-related genes, such as caspases (CASP3 and CASP7), which could explain placental separation associated with NP. Also, NP was associated with downregulation of several placenta-regulatory genes (ABCG2, GCM1, EPAS1, and NR3C1), angiogenesis-related genes (VEGFA, FLT1, KDR, and ANGPT2), and glucose transporter coding genes (GLUT1, GLUT10, and GLUT12), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could elucidate placental insufficiency accompanying NP. In conclusion, our findings revealed for the first time, the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during NP. Overall design: Chorioallantois (CA) samples were collected from the margin of the NP lesion (NPL, n=4) and grossly normal region (NPN, n=4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n=4). Total RNA was isolated from all CA samples using RNeasy Mini Kit (#74104; Qiagen), and DNA digestion was performed on-column using RNase-free DNase I (#79254: Qiagen), followed by cleanup procedures. Paired-end reads with 150 nucleotides in length were produced. A TruSeq Stranded mRNA library prep kit (Illumina, San Diego, CA) was used to prepare libraries for mRNA sequencing. Libraries were loaded onto an Agilent DNA 1000 chip and validated on an Agilent 2100 Bioanalyzer (Agilent). Quantitation was performed with the Illumina Library Quantification Kit, ABI Prism qPCR Mix from Kapa Biosystems. Libraries were run on a NextSeq 500 v2 (Illumina) 300cycles High Output kit in a 2x150 base pairs with paired-end reads. The Fastq files were evaluated for read quality using FastQC 0.11.4. Subsequently, Trim Galore 0.4.1 was used for the adapter and read quality trimming (Phred score threshold of 30). Reads were mapped to the Equus caballus reference genome (EquCab 3.0) using the software STAR 2.5.3a, then annotated with the equine reference annotation from NCBI using Cufflinks 2.2.1. Fragments per kilobase per million (FPKM) was used to determine the expression level of genes. Lastly, we used Cuffdiff 2.2.1 to calculate differentially expressed genes (DEGs) between groups. The significance level was set at the FDR-adjusted p-value of the test statistic < 0.05 using the Benjamini-Hochberg correction.