Detection of Neorickettsia risticii in antemortem fecal and postmortem fetal samples, with genomic insights from complete genome sequencing of a strain recovered from an aborted equine fetus.

Research Overview

• This study investigates the presence of Neorickettsia risticii, the bacterium causing Potomac horse fever, in samples from live horses and aborted equine fetuses.
• It also presents the first complete genome sequencing of N. risticii obtained directly from an aborted equine fetus, providing genomic insights into the pathogen.

Background

• Neorickettsia risticii is an intracellular bacterium responsible for Potomac horse fever (PHF), a disease in horses marked by symptoms such as diarrhea, fever (pyrexia), and laminitis.
• While N. risticii infection has occasionally been linked to mares’ abortions, no extensive retrospective studies or genomic analyses of the pathogen in aborted fetal tissues had been conducted prior to this study.

Objective of the Study

• To detect N. risticii in fecal samples from live, clinically ill horses and in colon samples from aborted equine fetuses.
• To perform a complete genome sequencing of N. risticii directly from a fetal colon sample to gain genomic insights.

Sample Collection and Detection

• Fecal samples: 546 from clinically ill horses collected from January 1, 2017, to December 31, 2024.
• Colon samples: 833 from aborted equine fetuses collected from September 20, 2018, to December 31, 2024.
• Detection method: Real-time PCR used to identify presence of N. risticii DNA.
• Results:
  • 11.5% (about 63 samples) of fecal samples were positive for N. risticii.
  • 1.08% (about 9 samples) of fetal colon samples tested positive.

Seasonal and Gestational Findings

• Positive cases in live horses mostly occurred from May to September.
• Fetal cases were detected between September and December.
• Detected aborted fetuses ranged from 5 to 8 months of gestational age.
• Microscopic examination of fetal samples revealed evidence of colitis, an inflammation of the colon, suggesting pathology linked to the infection.

Genome Sequencing and Analysis

• Methodology:
  • A shotgun metagenomic sequencing approach was applied directly to archived fetal colon tissue.
  • Sequencing was carried out using the MiSeq platform.
• Results:
  • Obtained a complete genome sequence of the N. risticii strain named KY18-EqFetus.
  • Genome size was 879,923 base pairs with a GC content of 41.3%.
  • Compared to the reference strain Illinois, KY18-EqFetus showed 99.72% nucleotide identity.
  • Identified 2024 genetic variants, including insertions, deletions, and nucleotide polymorphisms.
  • Notably, three genes coding for small hypothetical proteins present in the reference genome were missing in this strain, which could imply functional differences or host adaptation.

Significance and Contributions

• This is the first comprehensive retrospective study assessing N. risticii detection in both live (antemortem fecal) and postmortem (fetal colon) equine samples.
• The study provides the first complete genome assembly of N. risticii directly from an aborted equine fetus, opening avenues to understanding its genomic variations potentially linked to pathogenesis or abortion.
• Seasonal patterns of infection in live horses versus fetal cases were documented, suggesting temporal infection dynamics.
• The findings highlight the role of N. risticii in equine abortions and provide a genomic resource for future comparative and functional studies of the pathogen.