Rickettsioses are endemic worldwide. Many epidemiological features of these infections need to be clarified. Genotyping of human and vector strains could help to identify circulating strains. Actually, many techniques are used for identification and genetic characterization of bacterial isolates. Using multispacer typing we characterized different Rickettsia species in specimens from ticks and humans from Tunisia.
Tracking the movement of causative organisms between animals and humans allows the evaluation of the risk of human infectious diseases [21, 22]. In the public health setting, molecular typing of infectious agents is important for the study of bacterial population dynamics, the understanding of the ecological niches occupied by specific pathogen types in the environment, the spread of pathogens in outbreaks, the detection of disseminated antibiotic resistant strains in managed care facilities, the identification of hypervirulent strains, and the monitoring failures in live vaccination programs [23, 24]. Molecular typing schemes based on the sequencing of intergenic regions have been developed. MST is a nucleotide sequencing based on genotyping method that uses highly variable intergenic spacers as typing markers. It is the most suitable genotyping procedure for evaluating the population structure of intracellular bacteria. Thus, it was developed for Bartonella and Coxiella[25, 26]. This method was shown to be discriminatory not only for Rickettsia conorii but also for other Rickettsia species. When first described by Fournier et al., MST identified 27 genotypes among 39 R. conorii studied strains . The method was then applied to 22 R. conorii strains detected in Algeria and 7 genotypes could be differentiated . Using this MST, 15 rickettsial isolates obtained from humans or ticks over a 26-year period in various areas of China were demonstrated to belong to R. sibirica subsp. sibirica. In Russia, also, tick born rickettsioses was confirmed to be caused by a R. heilongjangensis strain different from the prototype strain HLJ054 .
We were able to genotype Rickettsia species detected in our specimens directly without being cultured. In fact, the MST genotyping is PCR based. Of note, none of our samples showed nonspecific amplification and all amplicons provided a single band on the electrophoresis profile. In the study of Wenjun et al. the MST genotyping was also applied directly to specimens .
Among 20 analyzed skin biopsies, R. conorii subsp conorii was detected in only one patient while R. conorii subsp israelensis was detected in three patients. For two patients with Israeli spotted fever (ISF), epidemiologic and clinical characteristics were previously reported . In ticks also, R. conorii subsp israelensis was more frequently detected than R. conorii subsp conorii. Contrary to the previously adopted hypothesis, that MSF caused by R. conorii subsp conorii is the most common rickettsioses in Tunisia, in our study R. conorii subsp israelensis was the most frequent agent detected in both humans and ticks. This could suggest that cases of ISF occurring during 2009 are epidemic. In fact, the phylogenetic tree showed that human and tick isolates were genetically homogenous despite the differences in location of their original isolation (Table 1). Moreover, ISF isolates were grouped in a separate cluster and presented a new MST genotype. The obtained genotype showed a genetic diversity with the single ISF strain included in the study of Fournier et al.. The two genotypes differed in two spacers. As previously reported , one of our patients (P75) was suspected to be infected in Libya, suggesting the occurrence of the new genotype in other North African countries. Nevertheless, no ISF strain has been reported until now from neighboring countries. Further investigations are needed to define the distribution of this species and its genotypes in North Africa. Previously, we speculated that strains circulating in Libya could be more invasive since the patient (P75) presented a more severe disease. Boillat et al. also reported a case of a severe form of ISF with a fatal outcome of a patient suspected to be infected in Libya . However, the strain detected in patient P75 was genetically homogenous to the other strains. Furthermore, in Portugal, Vitorino et al. found no correlation between virulence of ISF strains and the dksA-xerC profile .
R. conorii subsp conorii strains detected in a skin biopsy and a tick clustered together suggesting the circulation of this strain between humans and vectors. This strain presented a new rpmE-tRNAfMet genotype and thus a new MST genotype, different from that previously reported in Tunisia . In fact, Wenjun et al. studied the phylogenetic position of 61 R. conorii subsp conorii isolates, and reported MST genotype 3 in many Mediterranean regions (Algeria, Tunisia, Spain and France). In this study, seven genotypes among 22 skin biopsies were identified, revealing heterogeneity in circulating species in Algeria . Our findings emphasize the distribution of varying genotypes of R. conorii in North Africa.
Infections caused by a variety of Rickettsia species have been previously described in Tunisia. R. felis, was diagnosed in many human cases by serology from our region and from the centre of Tunisia [5, 6]. In our work, the DNA of the bacteria was detected in a flea. The strain found was similar to wild type (URRWXCa12) for two spacers. However, amplification of the spacer rpmE-tRNAfMet failed. This could be explained by the direct amplification from specimens without culturing strains. Similarly to other north African countries (Morocco and Algeria) [31, 32], R. massiliae was detected by PCR in Rhipicephalus sanguineus in Tunisia. This species was recently found in ticks analyzed by reverse line blot Assay . MST genotypes of our strains were genetically homogenous with the R. massiliae AZT80 strain. This strain was first described in Spain as Rickettsia massiliae strain Bar29  then in Arizona , which suggests its ubiquity.