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Comparative OmpA gene analysis of Rickettsia felis-like bacteria detected in Haemaphysalis sulcata ticks and isolated in mosquito C6/36 cell line

Objectives: The immunodominant OmpA protein of SFG Rickettsia plays a role in cell invasion and contains a domain of different number of tandem repeats, which dictates the genetic diversity of SFG Rickettsia. This protein is therefore used for the diagnosis of rickettsioses, species characterization and is considered as a potential vaccine candidate. The focus of our present study is the molecular characterization of the ompA gene amplified from a Rickettsia felis-like bacterium previously detected in Haemaphysalis sulcata ticks and from the same bacterium grown in mosquito C6/36 cells. Methods: DNA was extracted from 4 H. sulcata ticks and from the third passage of the isolate. Amplicons of complete ompA were obtained with two PCRs producing overlapping fragments named A and B. Primer sets were designed based on the alignment of rickettsial ompA sequences and using genome walking. Distinctive ompA amplicons were also cloned. In addition, RNA was extracted from stored halves of the ticks. It was transcribed and amplified by one step RT-PCR. Seven A and B amplicons were sequenced. Eighteen primers were needed to sequence fragment A. Sequencing of fragment B, which contains tandem repeats, is in process. Sequences were assembled, aligned and examined for sequence similarity. The phylogenetic trees were drawn with Treecon. Results: The ompA gene was successfully amplified from both H. sulcata ticks and from the bacteria isolated and cultured in C6/36 cells. Amplified fragments A and B were approximately 3000 and 4000 bp long, respectively. Fragment A sequences were compared between those obtained (i) directly from tick DNA and from the respective clone ; (ii) directly from tick DNA and from the isolate ; (iii) directly from tick DNA, but ticks were collected in different locations. Two, 16 and 45 nucleotide changes were noted in first, second and third sources of DNA, respectively. Changes in all samples were point mutations with the exception of insertions seen only in the second DNA source. RT-PCR of complete ompA resulted in an approximately 6500 bp long product. Phylogenetic analysis of Rickettsia felis-like bacteria based on partial ompA showed a maximum of 90.9 % identity with other SFG Rickettsia. Conclusions: Results of analysis of ompA sequences from fragment A demonstrated the presence of a unique species of Rickettsia. Given the substantial genetic divergence of the new Rickettsia, establishment of the isolate in C6/36 cells and TEM evidence of Rickettsia in internal organs of infected ticks, we propose the name of a new SFG species Rickettsia croatica, strain Kastelanii.

Rickettsiae; OmpA gene; Haemaphsalis sulcata

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