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Published on: December 23, 2022
In vitro propagation and genome sequencing of three 'atypical' Ehrlichia ruminantium isolates
Junita Liebenberg1, Helena C Steyn, Antoinette I Josemans
1Vaccines and Diagnostics Development Programme, ARC-Onderstepoort Veterinary Research, Pretoria. liebenbergj@arc.agric.za.
Abstract:
Three isolates of Ehrlichia ruminantium (Kümm 2, Omatjenne and Riverside), the causative agent of heartwater in domestic ruminants, were isolated in Ixodes scapularis (IDE8) tick cell cultures using the leukocyte fraction of infected sheep blood. All stocks were successfully propagated in IDE8 cells, whereas initiation attempts using endothelial cell cultures were unsuccessful. Therefore, the new technique should be included in any attempt to isolate field strains of E. ruminantium to enhance the probability of getting E. ruminantium isolates which might not be initiated in endothelial cells. Draft genome sequences of all three isolates were generated and compared with published genomes. The data confirmed previous phylogenetic studies that these three isolates are genetically very close to each other, but distinct from previously characterised E. ruminantium isolates. Genome comparisons indicated that the gene content and genomic synteny were highly conserved, with the exception of the membrane protein families. These findings expand our understanding of the genetic diversity of E. ruminantium and confirm the distinct phenotypic and genetic characteristics shared by these three isolates.
Insights
New tick cell culture methods successfully isolated Ehrlichia ruminantium, the heartwater pathogen, from sheep blood. Genomic analysis revealed these isolates are distinct, highlighting Ehrlichia ruminantium genetic diversity.
Area of Science:
- Veterinary Microbiology
- Tick-borne Diseases
- Genomics
Background:
- Heartwater, caused by Ehrlichia ruminantium, significantly impacts domestic ruminants.
- Previous isolation methods for E. ruminantium have limitations.
- Understanding the genetic diversity of E. ruminantium is crucial for disease control.
Purpose of the Study:
- To optimize the isolation of Ehrlichia ruminantium using tick cell cultures.
- To characterize the draft genomes of three distinct E. ruminantium isolates.
- To compare these isolates with previously identified strains.
Main Methods:
- Isolation of E. ruminantium from infected sheep blood leukocytes using Ixodes scapularis (IDE8) tick cell cultures.
- Propagation of isolates in IDE8 cells and attempts with endothelial cells.
- Generation and comparative analysis of draft genome sequences.
Main Results:
- Successful isolation and propagation of three E. ruminantium isolates (Kümm 2, Omatjenne, Riverside) in IDE8 cells.
- Isolation attempts using endothelial cells were unsuccessful, indicating the utility of the IDE8 method.
- Genomic analysis confirmed the isolates are closely related to each other but distinct from other known E. ruminantium strains.
- High conservation of gene content and genomic synteny, with variations noted in membrane protein families.
Conclusions:
- The leukocyte fraction in IDE8 tick cell culture is an effective method for isolating E. ruminantium, especially for strains difficult to initiate in endothelial cells.
- These three isolates represent a distinct genetic group within E. ruminantium.
- The findings contribute to a deeper understanding of E. ruminantium genetic diversity and its implications for heartwater disease management.
