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. 2023 Apr 3;12(4):552.
doi: 10.3390/pathogens12040552.

Diversity and Phylogeny of Cattle Ixodid Ticks and Associated Spotted Fever Group Rickettsia spp. in Tunisia

Myriam Kratou  1 , Hanene Belkahia  1 , Rachid Selmi  1   2 , Rihab Andolsi  1 , Mokhtar Dhibi  3 , Moez Mhadhbi  3 , Lilia Messadi  1 , Mourad Ben Said  1   3   4
Affiliations

Diversity and Phylogeny of Cattle Ixodid Ticks and Associated Spotted Fever Group Rickettsia spp. in Tunisia

Myriam Kratou et al. Pathogens. .

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.

Keywords: Rickettsia species; Tunisia; cattle ticks; genotyping; molecular species identification; phylogenetic analysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Map of Tunisia showing investigated governorates. Legend: The districts of the governorate of Bizerte are written in white, those of the governorate of Manouba in yellow and those of the governorate of Ariana in blue. Abbreviations: SJ: Sejnane; ME: Metline; MB: Menzel Bourguiba; MT: Mateur; JM: Joumine; EH: El Mabtouh; KA: Kalâat El Andalous; BH: Bach Hamba; SO: Sidi Othmen; DH: Dhniba; TB: Tebourba; BT: El Battan; MG: Mornaguia; BJ: Bjaoua; SH: Sanhaja; DJ: Djedeida.
Figure 2
Figure 2
Phylogenetic tree representing partial sequences (320 bp) of the mitochondrial 16S rRNA gene isolated from analyzed tick specimens belonging to Hy. marginatum, Hy. excavatum and Hy. rufipes with those of the Hyalomma species published in GenBank using the neighbor-joining method. Legend: Branche-related numbers represent the bootstrap percentages over 1000 iterations supporting the nodes (only percentages greater than 50% are shown). The host, genotype, strain, isolate or clone, country of origin and GenBank accession number are indicated. The sequences of Rickettsia spp. newly obtained in this study are represented in bold and marked with an asterisk. A partial sequence of the mitochondrial 16S rRNA gene isolated from a Rh. sanguineus s.l. tick was added as an out-group sequence. Note: Our GenBank accession numbers related to each genotype present in the tree are shown in Table 3 and Table 5.
Figure 3
Figure 3
A phylogenetic analysis of partial sequences (320 bp) of the mitochondrial 16S rRNA gene isolated from the revealed tick specimens of the Rhipicephalus sanguineus sensu lato complex with those of other Rhipicephalus species published in GenBank using the neighbor-joining method. Legend: Branche-related numbers represent the bootstraps rate over 1000 iterations supporting the nodes (only percentages greater than 50% are shown). The host, strain, isolate or clone, country of origin and GenBank accession number are indicated. The sequences of Rh. sanguineus s.l. newly obtained in this study are represented in bold and marked with an asterisk. A partial sequence of the mitochondrial 16S rRNA gene isolated from the Hy. marginatum tick was added as an out-group sequence. Note: Our GenBank accession numbers related to each genotype are shown in Table 6.
Figure 4
Figure 4
Phylogenetic tree of Rickettsia species inferred with partial ompB sequences (382 bp) of Rickettsia spp. obtained in this study, with selected sequences representative of the Rickettsia genus. Legend: Numbers over the branches indicate the percentage of replicated trees in which the associated taxa clustered together in the bootstrap test (1000 replicates, only percentages greater than 50% are represented). The partial ompB sequences representative of different Rickettsia spp. genotypes obtained in this study are indicated in bold and marked with an asterisk. The host or vector, genotype, strain or isolate name, country of origin and GenBank accession number are indicated. One R. prowazekii ompB partial sequence was added as an out-group. Note: Our GenBank accession numbers related to each genotype are shown in Table 3, Table 5 and Table 6.
Figure 5
Figure 5
Neighbor-joining tree based on the alignment of partial ompA sequences (490 bp) using the neighbor-joining method showing the novel obtained sequences from Tunisian cattle ruminant ticks. Legend: Bootstrap values (1000 replicates) are indicated in each node (only percentages greater than 50% are shown). The genotypes of Rickettsia spp. obtained in the present study are indicated in bold and marked with an asterisk. The host or vector, genotype, strain or isolate name, country of origin and GenBank accession number are represented. One R. felis ompA partial sequence was added as an out-group. Note: Our GenBank accession numbers related to each genotype are shown in Table 3, Table 5 and Table 6.
Figure 6
Figure 6
Phylogenetical relationships based on nucleotide multiple alignments of partial Rickettsia spp. gltA sequences (341 bp). Legend: Numbers over the branches indicate the percentages of replicated trees in which the associated taxa clustered together in the bootstrap test (1000 replicates, only percentages greater than 50% are represented). The only R. massiliae gltA genotype revealed in this study from positive samples is represented in bold and marked with an asterisk. The host or vector, genotype, sequence type, strain or isolate name, country of origin and GenBank accession number are indicated. One R. prowazekii gltA partial sequence was added as an out-group. Note: Our GenBank accession numbers related to each genotype are shown in Table 3, Table 5 and Table 6.
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