ABSTRACT
Background: Until now, there has been limited information on the prevalence and the phylogeny of Borrelia burgdorferi sensu lato in Ixodes ticks in Tunisia, particularly in Ixodes inopinatus. Methods: The present study aimed to determine the prevalence and the phylogeny of B. burgdorferi s.l., in coexisted I. ricinus and I. inopinatus ticks collected from Northern Tunisia. One hundred questig ticks were collected during winter 2020 by tick-dragging method in Beja gouvernorate located in the north of Tunisia. Real-time PCR panel targeting B. burgdorferi s.l. 23S rRNA gene were performed. Positive DNA samples were subjected to conventional PCRs targeting 457 bp fragment of the Borrelia sp. flagellin (fla) gene using primers FlaF/FlaR. The identified Borrelia sp. isolate underwent partial sequence analysis to determine genospecies and evaluate their phylogenetic position. Results: The study revealed a prevalence rate of 28% (28/100) for B. burgdorferi sensu lato in the Ixodes ticks. The prevalence rates across tick species and genders did not show significant variations (p > 0.05). Interestingly, the study underlines the coexistence of I. inopinatus and I. ricinus sharing the same geographic areas in Northern Tunisia. Furthermore, DNA of B. lusitaniae was detected in I. inopinatus ticks for the first time in Tunisia. Revealed B. lusitaniae bacterium is similar to previously identified strains in Mediterranean region, but distinct from those isolated exclusively from countries of Eastern and Central Europe, such as Serbia, Romania, and Poland. This study highlights the prevalence of B. burgdorferi s.l. in I. ricinus/I. inopinatus ticks, and reveals B. lusitaniae in I. inopinatus ticks for the first time in Tunisia. Conclusion: These findings suggest the involvement of I. inopinatus as a potential vector of this pathogenic genospeciess in Tunisia. This may help understanding the ecology of Ixodes ticks, the natural infection and the transmission dynamics of Borrelia species in this country.
ABSTRACT
Tick-borne rickettsioses are mainly caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the Rickettsia genus. So far, the causative agents of SFG rickettsioses have not been detected in cattle ticks from Tunisia. Therefore, the aim of this study was to investigate the diversity and phylogeny of ticks associated with cattle from northern Tunisia and their associated Rickettsia species. Adult ticks (n = 338) were collected from cattle in northern Tunisia. The obtained ticks were identified as Hyalomma excavatum (n = 129), Rhipicephalus sanguineus sensu lato (n = 111), Hyalomma marginatum (n = 84), Hyalomma scupense (n = 12) and Hyalomma rufipes (n = 2). After DNA extraction from the ticks, 83 PCR products based on the mitochondrial 16S rRNA gene were sequenced and a total of four genotypes for Rh. sanguineus s.l., two for Hy. marginatum and Hy. excavatum and only one for Hy. scupense and Hy. rufipes were recorded, with the occurrence of one, two and three novel genotypes, respectively, for Hy. marginatum, Hy. excavatum and Rh. sanguineus s.l. mitochondrial 16S rRNA partial sequences. The tick DNA was tested for the presence of Rickettsia spp. by using PCR measurements and sequencing targeting three different genes (ompB, ompA and gltA). Of the 338 analyzed ticks, 90 (26.6%), including 38 (34.2%) Rh. sanguineus s.l., 26 (20.1%) Hy. excavatum, 25 (29.8%) Hy. marginatum and one (50%) Hy. rufipes tick, were positive for Rickettsia spp. Based on 104 partial sequences of the three analyzed genes, the BLAST analysis and phylogenetic study showed the infection of Hy. excavatum, Hy. marginatum and Rh. sanguineus s.l. tick specimens with R. massiliae, R. aeschlimannii and R. sibirica subsp. mongolitimonae and one Hy. rufipes tick specimen with R. aeschlimannii. In addition, coinfection with R. massiliae and R. aeschlimannii was reported in one Hy. marginatum and one Rh. sanguineus s.l. tick specimen, while a coinfection with R. massiliae and R. sibirica subsp. mongolitimonae was recorded in one Rh. sanguineus s.l. tick specimen. In conclusion, our study reports, for the first time in Tunisia, the infection of cattle ticks belonging to Hyalomma and Rhipicephalus genera with zoonotic Rickettsia species belonging to the SFG group.
ABSTRACT
Bovine anaplasmosis caused by Anaplasma marginale is a disease responsible for serious animal health problems and great economic losses all over the world. Thereby, the identification of A. marginale isolates from various bioclimatic areas in each country, the phylogeographic analysis of these isolates based on the most informative markers, and the evaluation of the most promising candidate antigens are crucial steps in developing effective vaccines against a wide range of A. marginale strains. In order to contribute to this challenge, a total of 791 bovine samples from various bioclimatic areas of Tunisia were tested for the occurrence of A. marginale DNA through msp4 gene fragment amplification. Phylogeographic analysis was performed by using lipA and sucB gene analyses, and the genetic relationship with previously characterized A. marginale isolates and strains was analyzed by applying similarity comparison and phylogenetic analysis. To evaluate the conservation of OmpA protein vaccine candidate, almost complete ompA nucleotide sequences were also obtained from Tunisian isolates, and various bioinformatics software were used in order to analyze the physicochemical properties and the secondary and tertiary structures of their deduced proteins and to predict their immunodominant epitopes of B and T cells. A. marginale DNA was detected in 19 bovine samples (2.4%). Risk factor analysis shows that cattle derived from subhumid bioclimatic area were more infected than those that originated from other areas. The analysis of lipA phylogeographic marker indicated a higher diversity of Tunisian A. marginale isolates compared with other available worldwide isolates and strains. Molecular, phylogenetic, and immuno-informatics analyses of the vaccine candidate OmpA protein demonstrated that this antigen and its predicted immunodominant epitopes of B and T cells appear to be highly conserved between Tunisian isolates and compared with isolates from other countries, suggesting that the minimal intraspecific modifications will not affect the potential cross-protective capacity of humoral and cell-mediated immune responses against multiple A. marginale worldwide strains.
