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

2009; Elsevier BV; Volume: 15; Linguagem: Inglês

10.1111/j.1469-0691.2008.02228.x

1469-0691

Maja Gračner, Tatjana Avšič‐Županc, Volga Punda-Polić, Jan Dolinšek, D. Bouyer, David H. Walker, Jorge Zavala-Castro, Nikola Bradarić, Patricia A. Crocquet-Valdes, Darja Duh,

Mosquito-borne diseases and control

Rickettsia felis-like bacteria were previously identified in Haemaphysalis sulcata ticks from Croatia based on the molecular analysis of the complete gltA and partial ompB and 17 kDa genes. The ompA gene could not be amplified at the time [1Duh D Punda-Polic V Trilar T Petrovec M Bradaric N Avsic-Zupanc T Molecular identification of Rickettsia felis-like bacteria in Haemaphysalis sulcata ticks collected from domestic animals in southern Croatia.Ann NY Acad Sci. 2006; 1078: 347-351Crossref PubMed Scopus (26) Google Scholar]. A rickettsial endosymbiont was recently co-isolated with the embryotic cell line CCE3 obtained from a soft tick Carios capensis from Georgia, USA [2Mattila JT Burkhardt NY Hutcheson HJ Munderloh UG Kurtti TJ Isolation of cell lines and a rickettsial endosymbiont from the soft tick Carios capensis (Acari: Argasidae: Ornithodorinae).J Med Entomol. 2007; 44: 1091-1101Crossref PubMed Scopus (48) Google Scholar]. Interestingly, the gltA and 17 kDa sequences of the endosymbiont were 100% identical to the gene sequences of the above-mentioned Rickettsia detected in H. sulcata ticks. In order to confirm the identity of the two rickettsiae on a molecular level, we amplified the rickettsial ompA gene directly from H. sulcata ticks and from the same bacteria grown in the mosquito C6/36 cells. The obtained gene sequences were compared with each other and with the ompA gene of the C. capensis endosymbiont named Candidatus R. hoogstraalii. The DNA was extracted from three H. sulcata ticks and from the third passage of the isolate (Duh Darja, unpublished data) (Table 1). The complete ompA amplicons were obtained with two PCRs producing overlapping fragments A and B. The Platinum High Fidelity polymerase was used for the PCRs according to the manufacturer's instructions (Invitrogen Life TechnologiesTM; Carlsbad, CA, USA). The fragment A (FGW10/FGW7) and B (Rf190-1F/RGW5) primers were designed based on the alignment of known rickettsial ompA gene sequences and by using the genome walking technique. Fragments A and B were approximately 3000 and 4000 bp long, respectively, and they were successfully amplified from all DNA samples. One of the ompA amplicons was cloned with the TOPO XL PCR Cloning Kit (Invitrogen Life Technologies™) and the extracted plasmid DNA was used for the sequencing. In addition, the RNA was extracted with the Trizol reagent (Invitrogen Life Technologies™) from a stored half of a H. sulcata tick and from the third passage of the isolate. The RNA was transcribed and amplified with the SuperScript III One-Step RT-PCR with Platinum Taq High Fidelity (Invitrogen Life Technologies™). Five A and B amplicons of rickettsial and plasmid DNA were sequenced with the BigDye technology. Eighteen primers were needed to sequence the A fragment. Only part of the B fragment was sequenced using the other eight primers. Fragment B contains a tandem repeat region and completion of this region is in progress. The obtained sequences were assembled, aligned and examined for the sequence similarity. The phylogenetic tree was inferred with the Treecon software by the Kimura 80 model and neighbour-joining tree evaluation. Support for the tree nodes was calculated with 1000 bootstrap replicates. All primer sequences used in this study are available upon request. The unique ompA gene sequences were submitted to the GenBank and can be assessed under the Accession Numbers: 1A – EU816562, 2A – EU816563, 3A – EU816564, p1A – EU816566 and ISOA – EU816565 (Table 1). The ompA gene sequences (3317 and 3329 bp long) were compared between those obtained (i) directly from the tick's DNA and from the plasmid's DNA of the respective clone; (ii) directly from the tick's DNA and from the third passage of the isolate; (iii) directly from two ticks' DNA, considering that the ticks were collected from different sheep in the same location and (iv) directly from two ticks' DNA, considering that the ticks were collected in different locations. The results of the comparison are presented in Table 1. One, 14, 0 and 40 nucleotide changes were observed in option (i), (ii), (iii) and (iv), respectively. The nucleotide changes in all the options were point mutations with the exception of the insertions seen only in the second option. The RT-PCR of the complete ompA resulted in approximately 6500 bp long product for both tested RNAs. The phylogenetic analysis of the Rickettsia species from H. sulcata and C. capensis ticks and of other SFG rickettsiae was based on 1333 bp of the ompA because this is the maximum length of the Candidatus R. hoogstraalii gene sequence deposited in the GenBank. The Rickettsia species from the H. sulcata ticks showed 99.3% similarity to the Candidatus R. hoogstraalii, whereas up to 94.3% similarity was noted when this part of the ompA gene was compared with the other SFG rickettsiae. In particular, we determined that the novel Rickettsia species from H. sulcata were 94.3% similiar to R. monacensis, 94.0% to R. australis, 93.9% to R. felis and 88.8% to R. conorii.TABLE 1Comparative ompA gene sequence analysis of Rickettsia felis-like bacteria detected in Haemaphysalis sulcata ticks and isolated in the mosquito C6/36 cell lineHost ID/ locationTick IDTick DNA (direct ompA PCR)Plasmid DNA (cloned ompA PCR)RNA, RT-PCR(Option) sequence comparison/similaritySheep #57 Pribude-Muc#5951A, 1B – posp1A, p1B – posND(i) 1A and p1A: 99.9%Sheep #244/ Lecevica-Dugobabe#1362A, 2B – posNDpos(ii) 3A and ISOA: 99.8%Sheep #238/ Lecevica-Dugobabe#893A, 3B – posNDND(iii) 2A and 3A: 100%Third passage Rickettsia isolateISOISOA, ISOB – posNDpos(iv) 1A and 2A: 98.7%pos, positive; ND, not done; p, plasmid. Open table in a new tab pos, positive; ND, not done; p, plasmid. The immunodominant OmpA protein of SFG rickettsiae plays an important role in cell invasion and contains a domain of different numbers of tandem repeats, which dictates a genetic diversity of SFG rickettsiae [3Stenos J Walker DH The rickettsial outer-membrane protein A and B genes of Rickettsia australis, the most divergent rickettsia of the spotted fever group.Int J System Evol Microbiol. 2000; 50: 1775-1779PubMed Google Scholar]. The protein is therefore used for the molecular diagnosis of rickettsioses, species characterisation and it is considered as a potential vaccine candidate [4Zavala-Castro JE Small M Keng C Bouyer DH Zavala-Velazquez J Walker DH Transcription of the Rickettsia felis ompA gene in naturally infected fleas.Am J Trop Med Hyg. 2005; 73: 662-666PubMed Google Scholar]. The successful amplification of the ompA gene sequence described in this study confirmed the presence of rickettsiae in H. sulcata ticks collected from sheep in southern Croatia. In addition, the comparison of ompA sequences obtained directly from ticks and of those obtained from cloned amplicon and from bacteria isolated in a mosquito cell line indicates the existence of one particular species of Rickettsia in H. sulcata ticks. The Candidatus R. hoogstraalii was desribed recently in a soft bodied C. capensis tick collected from the nest of a brown pelican in coastal Georgia, USA [2Mattila JT Burkhardt NY Hutcheson HJ Munderloh UG Kurtti TJ Isolation of cell lines and a rickettsial endosymbiont from the soft tick Carios capensis (Acari: Argasidae: Ornithodorinae).J Med Entomol. 2007; 44: 1091-1101Crossref PubMed Scopus (48) Google Scholar]. The phylogenetic analysis of published and newly acquired gene sequences demonstrated that both rickettsiae are most likely to be the same species given the gene sequence-based criteria for the identification of new Rickettsia species [5Fournier PE Dumler JS Greub G Zhang J Wu Y Raoult D Gene sequence-based criteria for identification of new rickettsia isolates and description of Rickettsia heilongjiangensis sp. nov.J Clin Microbiol. 2003; 41: 5456-5465Crossref PubMed Scopus (269) Google Scholar]. Due to the wide geographic distribution of novel rickettsiae, their presence in different species of ticks collected from different hosts, and molecular similarity to human pathogens such as R. felis and R. australis, it is important to ascertain the pathogenetic potential of the Candidatus R. hoogstraalii.