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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 Ixodesticks 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. ricinusin 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. inopinatusmight 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.

ABSTRACTBorrelia lusitaniaeis a species within the complex Borrelia burgdorferisensu 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 ricinusticks 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 ospAgene 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. lusitaniaestrains isolated in North Africa constitute a clone of Portuguese origin.

To investigate whether ticks of the genus Ixodesare infected by Borrelia burgdorfericomplex, 490 unfed Ixodes ricinusticks 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 noncoding spacer between the two copies of the rrl–rrfgenes of B. burgdorferisl. The prevalence of B. burgdorferisl. 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. lusitaniaeby 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.

Tick-borne encephalitis virus (TBEV) is the etiologic agent of tick-borne encephalitis (TBE) and transmitted by Ixodes ricinus(Linnaeus, 1758) in Europe. The geographical distribution of I. ricinusin the Palearctic region covers also northern Africa, including northwestern Tunisia. While the eco-epidemiology of TBE in Europe is well documented, no data concerning TBEV from northern Africa are available. We investigated whether TBEV is circulating in Tunisia. A total of 877 adult I. ricinuscollected from northwestern Tunisia were examined in pools for the presence of TBEV by nRT-PCR. Viral RNA was detected in one pool of three engorged ticks, yielding a minimum infection rate of 0.11 % (1/877). Phylogenetic analysis showed that the Tunisian TBEV strain belongs to the European lineage. We report for the first time the presence of TBEV in I. ricinusfrom northern Africa. Therefore, more studies are needed to assess the public health importance of TBEV in northern Africa.