Zoonotic Rickettsia Species in Small Ruminant Ticks From Tunisia

Abstract

Tick-borne rickettsioses present a significant public health threat among emerging tick-borne diseases. In Tunisia, little is known about tick-borne Rickettsia pathogens. Therefore, the aim of this study was to investigate the presence of Rickettsia species in small ruminant ticks from Tunisia. Adult ticks (n = 694) were collected from goats and sheep in northern Tunisia. Obtained ticks were identified as Rhipicephalus turanicus (n = 434) and Rhipicephalus sanguineus sensu lato (n = 260). Selected ticks (n = 666) were screened for the presence of Rickettsia spp. by PCR targeting a partial sequence of the ompB gene followed by sequence analysis. Rickettsial DNA was detected in 122 (18.3%) tested tick samples. The infection rates in Rh. turanicus and Rh. sanguineus s.l. ticks were 23.4 and 9.5%, respectively. The overall prevalence of rickettsial DNA was markedly higher in ticks collected from goats (23.2%) compared to those infesting sheep (7.9%). The detection of rickettsial DNA was significantly higher in ticks from the governorate of Beja (39.0%) than those from the governorate of Bizerte (13.9%). Two additional genes, the outer membrane protein A gene (ompA) and the citrate synthase gene (gltA), were also targeted for further characterization of the detected Rickettsia species. Genotyping and phylogenetic analysis based on partial sequences (n = 106) of the three different genes revealed that positive ticks are infected with different isolates of two Spotted Fever Group (SFG) Rickettsia, namely, Rickettsia massiliae and Rickettsia monacensis, closely related to those infecting camels and associated ticks from Tunisia, and humans and small ruminant ticks from neighboring countries like Italy, France, and Spain.

Keywords: Rhipicephalus ticks; Rickettsia species; Tunisia; genotyping; molecular survey; phylogenetic analysis.

Figure 1

Map of the Tunisian studied regions. (A) Geographical position of Tunisia in the African continent. (B) Map of Tunisia showing investigated governorates. (C) Position of studied localities according to bioclimatic areas.

Figure 2

Neighbor-joining tree based on the alignment of partial ompB sequences (382 bp) of Rickettsia spp. obtained in this study with selected sequences representative of the Rickettsia genus. Numbers over the branches indicate the percentage of replicated trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates, only percentages >50% were represented). The six partial ompB sequences representative of different Rickettsia spp. genotypes obtained in this study are indicated in bold. The host or vector, the genotype, strain or isolate name, the country of origin, and the GenBank accession number are indicated. One R. prowazekii ompB partial sequence was added as an outgroup.

Figure 3

Phylogenetic tree of Rickettsia species inferred with partial sequences (490 bp) of the ompA gene using the neighbor-joining method showing the novel obtained sequences (n = 11) from Tunisian small ruminant ticks. Bootstrap values (1,000 replicates) are indicated in each node (only percentages >50% are shown). The 11 genotypes of Rickettsia spp. obtained in the present study are indicated in bold. The host or vector, the genotype, strain or isolate name, the country of origin, and the GenBank accession number are represented. One R. felis ompA partial sequence was added as an outgroup.

Figure 4

Phylogenetical relationships based on nucleotide multiple alignments of partial Rickettsia spp. gltA sequences (341 bp). Numbers over the branches indicate the percentage of replicated trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates, only percentages >50% were represented). The only R. massiliae gltA genotype revealed in this study from 25 positive samples is represented in bold. The host or vector, the genotype, sequence type, strain or isolate name, the country of origin, and the GenBank accession number are indicated. One R. prowazekii gltA partial sequence was added as an outgroup.