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.

Heartwater is a tick-borne disease caused by the intracellular rickettsial parasite Ehrlichia ruminantium and transmitted by Amblyomma hebraeum ticks. Heartwater is problematic in endemic areas because it causes high mortality in ruminants and leads to economic losses that threaten productivity and food security. This may indicate that there is augmented genetic diversity in the field, which may result in isolates that are more virulent than the Ball3 and Welgevonden isolates. The genetic diversity of E. ruminantium was investigated in this study, focussing on the pCS20 gene region and four polymorphic open reading frames (ORFs) identified by subtractive hybridisation. The 16S ribosomal ribonucleic acid gene confirmed E. ruminantium in brain, blood and tick genomic deoxyribonucleic acid samples (n = 3792) collected from 122 farms that were randomly selected from seven provinces of South Africa where heartwater is endemic. The conserved E. ruminantium pCS20 quantitative polymerase chain reaction (qPCR) assay was used to scan all collected field samples. A total of 433 samples tested positive with the qPCR using the pCS20 gene region, of which 167 were sequenced. The known stocks and field samples were analysed, and phylogenetic trees were generated from consensus sequences. A total of 25 new clades were identified; of these, nine isolates from infected blood could be propagated in cell cultures. These clades were not geographically confined to a certain area but were distributed amongst heartwater-endemic areas in South Africa. Thus, the knowledge of strain diversity of E. ruminantium is essential for control of heartwater and provides a basis for further vaccine development.