Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato and is infrequently isolated in Europe. In contrast, this species is by far the most predominant in North Africa and in Portugal. In this study, we analyzed the genetic diversity, at several loci, of a large population of isolates from free-living Ixodes ricinus ticks collected in Tunisia and Morocco. We found a moderate diversity of the whole genome by using pulsed-field gel electrophoresis as well as in the ospA gene sequences, compared to a high level of strain homogeneity in the small noncoding ribosomal spacer. In contrast, a high diversity of this locus has been previously reported for Portuguese isolates. We hypothesize that B. lusitaniae strains isolated in North Africa constitute a clone of Portuguese origin.Within the complex Borrelia burgdorferi sensu lato, three species, B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, are known to cause a broad spectrum of human manifestations that are described as Lyme borreliosis. The genetic diversity of pathogenic species has been largely investigated both at the species level and at the strain level within each species (41). Otherwise, although its pathogenicity for humans is only suspected (31, 33), Borrelia valaisiana represents a widely distributed species in ticks throughout Europe and Asia and therefore has been extensively studied (12,22,32,38,40).In contrast to these species, little data are available concerning Borrelia lusitaniae. Only sporadic isolates have been characterized from ticks collected in the Czech Republic, Moldavia, Ukraine, and Belarus (20), in Slovakia (11), in Spain (3), in Poland (25), in France (30), in Switzerland (15-17), or in Turkey (13), and these isolates did not show any significant diversity. However, a study conducted in Portugal revealed a large diversity among B. lusitaniae strains isolated from a local tick population (6). Moreover, it is noteworthy that, among the three first Portuguese isolates identified as B. lusitaniae, two different types were demonstrated (20,26). Hence, these studies suggested a higher diversity among B. lusitaniae around the Mediterranean basin. However, all these studies only focused on the small ribosomal rrf-rrl spacer and, therefore, the global diversity of this species remained unknown.Recently, we reported that B. lusitaniae was, by far, the most prevalent species, ranging from 96.6 to 100% of the B. burgdorferi sensu lato species identified in Tunisia and Morocco (35,44,46). The report of some Lyme borreliosis cases in North Africa (1, 27) and the recent isolation in Portugal of B.lusitaniae from the skin of a human patient with chronic lesions (5) prompted us to characterize this species as a potential human pathogen. Therefore, we investigated the molecular characterization and the genetic diversity of B. lusitaniae isolated from free-living Ixodes ricinus ticks in Tunisia and Morocco. For these purposes we studied two single loci (ospC and ospA) and the noncoding intergenic spacer (r...
To investigate whether ticks of the genus Ixodes are infected by Borrelia burgdorferi complex, 490 unfed Ixodes ricinus ticks were collected by flagging in three different areas of Tunisia in 1998. DNAs extracted from 81 adults, 60 nymphs and 38 larvae were analysed after genic amplification of the non-coding spacer between the two copies of the rrl-rrf genes of B. burgdorferi sl. The prevalence of B. burgdorferi sl. in adults, nymphs and larvae was found to be 34, 33.3 and 2.6%, respectively. All DNAs (n = 61) but one were identified as belonging to different genotypes of B. lusitaniae by analysis of the restriction fragment length polymorphism of amplification products. In addition, 290 adults, 14 nymphs and 7 larvae were used to inoculate BSK-H medium to isolate spirochetes. Fifteen strains were isolated from adult ticks in the humid areas of Tunisia, whereas only one was obtained from larvae. Isolates were identified as B. lusitaniae (15/16) and B. garinii (1/16). These results provide new evidence for the existence of Lyme borreliosis in North Africa.
In the present study, we report the sympatric occurrence of Ixodes ricinus (Linnaeus, 1758) and Ixodes inopinatus (Estrada-Peña, Nava, and Petney, 2014) in Tunisia. In total, 173 adult Ixodes ticks were collected from four sites (El Jouza, Tamra, Aïn Soltan, and Jbel Zaghouan) between February and April 2017, a period corresponding to the peak of activity of I. ricinus in North Africa. The morphological characters corresponded to both species; thus, we generated a total of 28 16S rRNA sequences and compared them with previously published data in GenBank. The two species were sympatric in Tamra, Aïn Soltan, and El Jouza, whereas collections in Jbel Zaghouan only yielded I. inopinatus. These results indicate that the two taxa are widespread in the humid area of northern Tunisia. The one tick collected in Jbel Zaghouan suggests that the distribution of at least I. inopinatus might extend to the sub-humid area. More studies are needed to fully comprehend the systematic status of the two taxonomic entities using multiple molecular markers and morphological characters; integrating these two identification methods are a necessary step toward a better understanding of the ecology and epidemiology of tick-borne diseases in Tunisia.
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