Multispacer typing of Rickettsia isolates from humans and ticks in Tunisia revealing new genotypes

Parasites & Vectors Multispacer typing of Rickettsia isolates from humans and ticks in Tunisia revealing new genotypes Abir Znazen 0 Fatma Khrouf 2 Nihel Elleuch 0 Dorra Lahiani 1 Chakib Marrekchi 1 Youmna M'Ghirbi 2 Mounir Ben Jemaa 1 Ali Bouattour 2 Adnene Hammami 0 0 Laboratory of Microbiology, Research Laboratory MPH, Habib Bourguiba University Hospital of Sfax , Sfax , Tunisia 1 Infectious diseases department, Hedi Chaker University Hospital of Sfax , Sfax , Tunisia 2 Laboratory of entomology, Pasteur Institute , Tunis , Tunisia Background: Rickettsioses are important remerging vector born infections. In Tunisia, many species have been described in humans and vectors. Genotyping is important for tracking pathogen movement between hosts and vectors. In this study, we characterized Rickettsia species detected in patients and vectors using multispacer typing (MST), proposed by Founier et al. and based on three intergenic spacers (dksA-xerC, rmpE- tRNAfMet, mppA-pruC) sequencing. Methods: Our study included 25 patients hospitalized during 2009. Ticks and fleas were collected in the vicinity of confirmed cases. Serology was performed on serum samples by microimmunofluorescence using Rickettsia conorii and Rickettsia typhi antigens. To detect and identify Rickettsia species, PCR targeting ompA, ompB and gltA genes followed by sequencing was performed on 18 obtained skin biopsies and on all collected vectors. Rickettsia positive samples were further characterized using primers targeting three intergenic spacers (dksA-xerC, rmpE- tRNAfMet and mppA-purC). Results: A rickettsial infection was confirmed in 15 cases (60%). Serology was positive in 13 cases (52%). PCR detected Rickettsia DNA in four biopsies (16%) allowing the identification of R. conorii subsp israelensis in three cases and R. conorii subsp conorii in one case. Among 380 collected ticks, nine presented positive PCR (2.4%) allowing the identification of six R. conorii subsp israelensis, two R. massiliae and one R. conorii subsp conorii. Among 322 collected fleas, only one was positive for R. felis. R. conorii subsp israelensis strains detected in humans and vectors clustered together and showed a new MST genotype. Similarly, R. conorii subsp conorii strains detected in a skin biopsy and a tick were genetically related and presented a new MST genotype. Conclusions: New Rickettsia spotted fever strain genotypes were found in Tunisia. Isolates detected in humans and vectors were genetically homogenous despite location differences in their original isolation suggesting epidemiologic circulation of these strains. Rickettsia; Multispacer typing; Intergenic spacers; Vectors; Spotted fever rickettsioses; Tunisia - Background Rickettsiae are Gram negative obligate intracellular rods belonging to the subgroup of alpha Proteobacteriae. These bacteria are closely related to arthropods that act as their vectors and reservoirs [1]. After transmission through a tick or flea bite, some pathogenic species cause polymorphic clinical features, essentially eruptive fever associated or not to inoculation eschar (tache noire) or isolated fever. A total of 28 species are validated into the genus Rickettsia, among which approximately 20 are recognized as human pathogens. Formerly, the classification of Rickettisae was based on serology and divided the genus into two sero-groups: typhus group and spotted fever group (SFG). Molecular and phylogenetic analyses classified the genus Rickettsia into at least three groups: SFG, typhus group, the R. bellii group (ancestral) [2,3]. Rickettsioses are considered to be important emerging vector born infections of humans worldwide. In Tunisia, R. conorii subsp conorii, the agent of Mediterranean spotted fever (MSF), was previously thought to be the unique species causing spotted fever rickettsiosis [4]. In recent years, many studies based on both serological and molecular techniques described a variety of species causing rickettsioses. Thus, R. conorii, R. typhi, R. aeschlimannii and R. felis were characterized by serology [5,6]. Using molecular methods, R. conorii subsp conorii and R. conorii subsp israelensis were detected in humans [7,8], R. monacensis and R. helvetica in Ixodes ricinus [9] and recently R. massiliae in Rhipicephalus sanguineus [10]. Molecular typing of infectious agents is important since it provides a better understanding of ecological niches and the spread of microorganisms. In rickettsiology, the bacterial dynamic between the bacterium, vectors and hosts is not completely studied. The genotyping of strains detected in human samples and in arthropods could help further our understanding of the circulating strains and identifying hypervirulent strains. For Rickettsia genus, Fournier et al. proposed a multispacer typing (MST) combining three spacers to distinguish rickettsial genotypes [11]. Indeed, intergenic spacers were shown to be better conserved and less submitted to selection pressure in intracellular bacteria. Herein, we aim to characterize rickettsial species detected both in humans and in vectors, using the multispacer typing method. Methods Patients Our study was conducted during 2009 at the Infectious Diseases Department of Hedi Chaker University of Sfax Tunisia. Included patients were suspected to have rickettsial infection on the basis of clinical presentation (fever associated to cutaneous rash) and epidemiologic features (exposition to ticks and or fleas with or without a history of bite). All the subjects provided informed consent. The study was approved by the Habib Bourguiba University hospital ethics committee. Skin biopsies performed on the cutaneous rash using a punch (of 4 mm diameter) and serum samples were collected. The skin biopsies were stored at 80C until their use. Tick and flea collection Ticks and fleas, feeding on domestic animals, were collected in the vicinity of households with serologically or clinically confirmed human rickettsiosis cases from July to October 2009 in Sfax. They were manually collected from dogs, sheep and goats. All specimens were identified to species level using appropriate taxonomic keys [12,13]. All ticks and fleas were stored in 70% ethanol at room temperature until DNA extraction. Serology Serology was performed by microimmunofluorescence assay using R. conorii and R. typhi antigens provided by the Unit des Rickettsies in Marseille France as described previously [14]. Titers higher than 1:32 for IgM and 1:128 for IgG were considered positive. DNA extraction For skin biopsies and collected arthropods, DNA extraction was performed using QIAamp DNA tissue extraction kit (Qiagen, Hilden, Germany) according to manufacturers instructions. DNA extracts were stored at 20C until their use. PCR amplification and sequencing To identify species detected in skin biopsies and ectoparasite vectors, ompA, ompB and gltA genes were amplified and sequenced using primers previously reported [15,16]. Molecular typing was pe (...truncated)