Abstract
Ticks harbor diverse Rickettsia spp., including several spotted fever group rickettsiae of veterinary and public health importance. However, information on tick-associated Rickettsia spp. in Zambia remains geographically limited. In this retrospective molecular survey, we examined 1,587 individual ticks collected from vegetation and domestic animals in eight provinces of Zambia during 2016–2018. Ticks were identified morphologically and screened for Rickettsia spp. by PCR targeting the citrate synthase gene (gltA). Representative gltA positive samples were further characterized by sequencing of ompA, ompB, htrA, sca4, and the 16S rRNA gene. In total, 204 ticks (12.9%) were tested positive for gltA and detected in all eight provinces examined. Sequencing of gltA revealed 26 sequence variants, of which 25 clustered within the spotted fever group and one belonged to transitional group rickettsiae. Multilocus phylogenetic analysis identified sequence variants related to recognized or putative tick-borne rickettsiae, including R. africae, R. conorii, R. massiliae, R. aeschlimannii, Candidatus R. jingxinensis, Candidatus R. rhabdomydis, and R. hoogstraalii. These findings indicate that ticks in Zambia harbor genetically diverse Rickettsia lineages and provide a geographically broad molecular baseline for future surveillance, isolation attempts, and genomic characterization of tick-borne rickettsiae in the region.
Keywords: Amblyomma variegatum, gltA, Rickettsia africae, spotted fever group rickettsiae, Zambia
INTRODUCTION
Spotted fever group (SFG) rickettsiae are obligate intracellular bacteria transmitted mainly by ticks and include several species of veterinary and public health importance. In Africa, Rickettsia africae, the agent of African tick-bite fever, is widely associated with Amblyomma ticks, whereas other SFG rickettsiae such as R. conorii, R. massiliae, and R. aeschlimannii have also been detected in ticks or implicated in human disease in different regions [17, 31, 35, 39, 42]. Because rickettsial infections often present as non-specific febrile illness, their occurrence is likely underestimated in areas where laboratory diagnosis is limited [4, 5, 40].
Zambia has diverse tick fauna associated with livestock, wildlife, domestic animals, and vegetation, providing ecological opportunities for the maintenance of tick-borne bacteria. Previous molecular studies in Zambia have detected several Rickettsia species or lineages in ticks, domestic animals, wildlife, rodents, and fleas, including lineages related to R. africae, R. conorii, R. massiliae, R. lusitaniae, R. hoogstraalii, and other undescribed or incompletely characterized rickettsiae [6,7,8, 19, 26, 28, 32,33,34]. These studies indicate that Rickettsia spp. are more diverse in Zambia than previously recognized. However, available data remain geographically uneven, and information from several provinces, particularly Copperbelt, Luapula, Muchinga, Northern, and North-Western Provinces, is still limited. In addition, serological evidence of exposure to SFG in residents, molecular detection of Rickettsia in human blood, and a travel-associated case of Mediterranean spotted fever after return from Zambia have been reported [16, 27, 30].
Molecular detection and sequence-based characterization of Rickettsia spp. in ticks are useful for defining local pathogen diversity and for identifying lineages that warrant further ecological, veterinary, and public health investigation. However, species-level identification of Rickettsia based on a single genetic marker can be unreliable, especially for closely related SFG lineages. Therefore, multilocus phylogenetic analysis provides a more informative approach for provisional assignment of detected sequence variants and for distinguishing recognized species from potentially novel or poorly characterized lineages [12, 41].
In this retrospective molecular survey, we examined ticks collected from vegetation and domestic animals in eight provinces of Zambia during 2016–2018. The objectives were to (i) describe the occurrence of Rickettsia spp. in major tick taxa, (ii) characterize the genetic diversity of detected Rickettsia lineages using citrate synthase gene (gltA) screening followed by multilocus phylogenetic analysis of representative samples, and (iii) generate a sequence-based reference dataset for future ecological, veterinary, and public health investigations of tick-borne Rickettsia spp. in Zambia.
MATERIALS AND METHODS
In this retrospective molecular survey, we analyzed ticks collected between 2016 and 2018 in eight provinces of Zambia: Central, Copperbelt, Eastern, Luapula, Muchinga, Northern, North-Western, and Western. Ticks were collected either from vegetation by flagging with flannel cloth or directly from domestic animals, as previously described [15, 18]. Permission for tick collection was obtained from the Department of Veterinary Services in accordance with the Animal Health Act No. 27 of 2010 of the Laws of Zambia. Tick sampling from cattle and pastures was conducted with the consent of the participating farmers. Field-collected ticks were placed in tubes and transported to the Hokudai Center for Zoonosis Control in Zambia for laboratory processing. During transport, the tubes were kept in insulated Styrofoam boxes with ice packs. The duration from collection to laboratory processing ranged from the same day to approximately five days, and the exact temperature during transport was not continuously monitored. Tick genera and, where possible, species were identified morphologically under a stereomicroscope using standard taxonomic keys [2, 43, 44].
Each tick was washed twice in 70% ethanol and then rinsed twice in sterile phosphate-buffered saline. Ticks were individually homogenized in 100 µL of high-glucose Dulbecco’s modified Eagle medium using a Micro Smash MS-100R bead beater (TOMY, Tokyo, Japan). The homogenate was divided into two 50 µL aliquots. One aliquot was used for RNA and DNA extraction with TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, and the other was stored at −80°C for future studies. Only DNA extracts were used in the present study.
All DNA samples were screened for Rickettsia spp. by conventional PCR targeting a fragment of the gltA gene using primers gltA8211Fc and gltA8211Rc [14]. PCR reactions were performed in a final volume of 20 µL using Ex Taq HS (Takara Bio Inc., Kusatsu, Japan), with 2 µL of DNA template. The cycling conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min, with a final extension at 72°C for 5 min. Nuclease-free water was included as a negative control in each run. Amplicons were purified using ExoSAP-IT Express (Applied Biosystems, Foster City, CA, USA), and Sanger sequencing was performed using the same primers as those used for amplification. Chromatograms were assembled using ATGC software version 6.0.4 (GENETYX, Tokyo, Japan). For descriptive purposes, unique gltA sequences differing by at least one nucleotide over the analyzed fragment were treated as distinct gltA sequence variants and were tentatively labeled G1–G26, following a sequence-based classification approach used in a previous study [41]. These labels were used only as operational identifiers to compare sequence patterns and to select representative samples for multilocus characterization; they do not denote formal genotypes, strains, species, or taxonomic units.
For multilocus characterization, 1–5 specimens were selected from each gltA sequence variant based on sample availability and DNA quality, including singleton sequence variants whenever possible. Additional PCR assays were performed to amplify fragments of five loci: outer membrane protein A gene (ompA), outer membrane protein B gene (ompB), 17-kDa common antigen gene (htrA), surface cell antigen 4 gene (sca4), and the 16S rRNA gene, using previously published primer sets [1, 22, 36,37,38]. Each PCR reaction was performed in a final volume of 20 µL using Ex Taq HS (Takara Bio Inc.), with 2 µL of DNA template. The cycling conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec, locus-specific annealing for 30 sec, and 72°C for 1 min, except for the 16S rRNA gene, for which the extension step was performed at 72°C for 1 min 30 sec. A final extension was performed at 72°C for 5 min. Amplicons were purified, sequenced, and assembled as described above. Primer information and annealing temperatures are provided in Supplementary Table 1, and accession numbers of the sequences generated in this study are provided in Supplementary Table 2. The nucleotide sequences obtained were aligned with representative sequences of described Rickettsia species retrieved from GenBank using MUSCLE. Phylogenetic trees were inferred from each locus using the maximum likelihood method with 1,000 bootstrap replicates in MEGA X [21].
RESULTS
Among the 1,587 individual ticks examined, 204 were positive for Rickettsia DNA by gltA PCR, giving an overall PCR positivity of 12.9% (95% confidence interval [CI], 11.2–14.6%). Ticks that tested positive were detected in all eight provinces sampled. By province, PCR positivity ranged from 2.4% (8/333) in Copperbelt Province to 26.1% (46/176) in Central Province (Table 1). Among the tick taxa examined, four taxa (Rhipicephalus compositus, Rh. evertsi, Rh. simpsoni, and Rh. afranicus) were tested negative for gltA PCR. The observed positivity varied among tick taxa, ranging from 0% to 33.3%. The highest values were observed in Amblyomma pomposum (33.3%, 2/6) and A. variegatum (27.5%, 92/335) (Table 2).
Table 1. Province wise distribution of tested ticks, gltA PCR positive ticks, and detected sequence variants.
| Province | Tick taxon | gltA PCR | gltA sequence variants | |
|---|---|---|---|---|
| No. of tested (Male/Female/Nymph) | No. of positive (Male/Female/Nymph) | |||
| Central | Amblyomma variegatum | 38 (20/18/0) | 17 (6/11/0) | G1, G6, G12, G14, G15, G20 |
| Rhipicephalus decoloratus | 10 (0/10/0) | 1 (0/1/0) | G1 | |
| Haemaphysalis aciculifer | 26 (9/17/0) | 4 (2/2/0) | G9, G18 | |
| Haemaphysalis leachi complex | 1 (1/0/0) | 0 (0/0/0) | ||
| Hyalomma spp. | 1 (1/0/0) | 1 (1/0/0) | G19 | |
| Hyalomma truncatum | 1 (1/0/0) | 0 (0/0/0) | ||
| Rhipicephalus appendiculatus | 1 (1/0/0) | 0 (0/0/0) | ||
| Rhipicephalus lunulatus | 6 (3/3/0) | 1 (0/1/0) | G3 | |
| Rhipicephalus simus | 1 (1/0/0) | 1 (1/0/0) | G7 | |
| Rhipicephalus spp. | 91 (50/41/0) | 21 (14/7/0) | G1, G3, G4, G5, G13 | |
| Sub-total | 176 (87/89/0) | 46 (24/22/0) | ||
| Copperbelt | Amblyomma variegatum | 17 (12/5/0) | 8 (7/1/0) | G1, G8 |
| Rhipicephalus decoloratus | 73 (6/67/0) | 0 (0/0/0) | ||
| Rhipicephalus microplus | 45 (4/41/0) | 0 (0/0/0) | ||
| Haemaphysalis leachi complex | 2 (0/2/0) | 0 (0/0/0) | ||
| Rhipicephalus appendiculatus | 7 (6/1/0) | 0 (0/0/0) | ||
| Rhipicephalus lunulatus | 1 (0/1/0) | 0 (0/0/0) | ||
| Rhipicephalus linnaei | 19 (8/11/0) | 0 (0/0/0) | ||
| Rhipicephalus afranicus | 49 (25/24/0) | 0 (0/0/0) | ||
| Rhipicephalus zambeziensis | 8 (3/5/0) | 0 (0/0/0) | ||
| Rhipicephalus spp. | 112 (6/106/0) | 0 (0/0/0) | ||
| Sub-total | 333 (70/263/0) | 8 (7/1/0) | ||
| Eastern | Amblyomma pomposum | 1 (1/0/0) | 0 (0/0/0) | |
| Amblyomma variegatum | 15 (14/1/0) | 4 (3/1/0) | G1 | |
| Amblyomma spp. | 8 (0/0/8) | 5 (0/0/5) | G1, G2 | |
| Rhipicephalus microplus | 22 (3/19/0) | 1 (0/1/0) | G2 | |
| Hyalomma truncatum | 56 (34/22/0) | 4 (2/2/0) | G2 | |
| Rhipicephalus appendiculatus | 5 (3/2/0) | 2 (1/1/0) | G7 | |
| Rhipicephalus zambeziensis | 1 (0/1/0) | 0 (0/0/0) | ||
| Rhipicephalus spp. | 48 (0/18/30) | 3 (0/2/1) | G1, G2 | |
| Sub-total | 156 (55/63/38) | 19 (6/7/6) | ||
| Luapula | Amblyomma variegatum | 36 (13/23/0) | 11 (2/9/0) | G1, G11, G17, G26 |
| Amblyomma spp. | 1 (0/0/1) | 0 (0/0/0) | ||
| Rhipicephalus microplus | 34 (4/30/0) | 0 (0/0/0) | ||
| Rhipicephalus spp. | 1 (0/0/1) | 0 (0/0/0) | ||
| Rhipicephalus appendiculatus | 1 (0/1/0) | 0 (0/0/0) | ||
| Sub-total | 73 (17/54/2) | 11 (2/9/0) | ||
| Muchinga | Amblyomma variegatum | 60 (35/25/0) | 26 (15/11/0) | G1, G6, G8, G14, G17, G21, G22, G23, G24 |
| Argas walkerae | 20 (NA) | 2 (NA) | G16 | |
| Rhipicephalus microplus | 46 (5/41/0) | 5 (1/4/0) | G3 | |
| Haemaphysalis leachi complex | 1 (0/1/0) | 0 (0/0/0) | ||
| Rhipicephalus lunulatus | 136 (40/96/0) | 18 (3/15/0) | G1, G3, G15, G25 | |
| Rhipicephalus linnaei | 12 (6/6/0) | 2 (0/2/0) | G7 | |
| Rhipicephalus zambeziensis | 46 (25/21/0) | 11 (7/4/0) | G4, G5 | |
| Rhipicephalus spp. | 3 (0/2/1) | 0 (0/0/0) | ||
| Sub-total | 324 (111/192/1, NA:20) | 64 (26/36/0, NA:2) | ||
| Northern | Amblyomma pomposum | 5 (3/2/0) | 2 (0/2/0) | G1 |
| Amblyomma variegatum | 42 (23/19/0) | 13 (4/9/0) | G1, G6, G8, G11 | |
| Rhipicephalus microplus | 5 (0/5/0) | 0 (0/0/0) | ||
| Haemaphysalis leachi complex | 9 (6/3/0) | 1 (1/0/0) | G13 | |
| Rhipicephalus appendiculatus | 12 (9/3/0) | 0 (0/0/0) | ||
| Rhipicephalus compositus | 1 (0/1/0) | 0 (0/0/0) | ||
| Rhipicephalus evertsi | 1 (0/1/0) | 0 (0/0/0) | ||
| Rhipicephalus lunulatus | 10 (2/8/0) | 1 (0/1/0) | G3 | |
| Rhipicephalus linnaei | 36 (26/10/0) | 3 (2/1/0) | G4, G7 | |
| Rhipicephalus simpsoni | 1 (1/0/0) | 0 (0/0/0) | ||
| Rhipicephalus sulcatus | 6 (4/2/0) | 1 (1/0/0) | G7 | |
| Rhipicephalus zambeziensis | 7 (5/2/0) | 1 (0/1/0) | G7 | |
| Rhipicephalus spp. | 49 (19/30/0) | 5 (2/3/0) | G4, G5 | |
| Sub-total | 184 (98/86/0) | 27 (10/17/0) | ||
| North-Western | Amblyomma variegatum | 56 (12/44/0) | 6 (0/6/0) | G1 |
| Amblyomma spp. | 12 (1/0/11) | 1 (0/0/1) | G1 | |
| Rhipicephalus appendiculatus | 1 (1/0/0) | 0 (0/0/0) | ||
| Rhipicephalus lunulatus | 19 (10/9/0) | 6 (1/5/0) | G1, G6 | |
| Rhipicephalus simus | 8 (5/3/0) | 0 (0/0/0) | ||
| Rhipicephalus zambeziensis | 6 (3/3/0) | 0 (0/0/0) | ||
| Rhipicephalus spp. | 44 (5/38/1) | 5 (0/5/0) | G3, G8, G12 | |
| Sub-total | 146 (37/97/12) | 18 (1/16/1) | ||
| Western | Amblyomma variegatum | 71 (50/21/0) | 7 (5/2/0) | G1 |
| Amblyomma spp. | 2 (0/0/2) | 0 (0/0/0) | ||
| Rhipicephalus decoloratus | 16 (0/16/0) | 0 (0/0/0) | ||
| Hyalomma spp. | 5 (5/0/0) | 0 (0/0/0) | ||
| Hyalomma truncatum | 1 (1/0/0) | 0 (0/0/0) | ||
| Rhipicephalus appendiculatus | 3 (0/3/0) | 0 (0/0/0) | ||
| Rhipicephalus evertsi | 23 (18/5/0) | 0 (0/0/0) | ||
| Rhipicephalus lunulatus | 33 (20/13/0) | 0 (0/0/0) | ||
| Rhipicephalus linnaei | 25 (14/11/0) | 4 (3/1/0) | G2, G10 | |
| Rhipicephalus spp. | 16 (4/12/0) | 0 (0/0/0) | ||
| Sub-total | 195 (112/81/2) | 11 (8/3/0) | ||
| Total | 1,587 (587/925/55, NA:20) | 204 (84/111/7, NA:2) | ||
NA: not applicable.
Table 2. PCR positivity for Rickettsia spp. by tick taxon.
| Tick taxon | No. positive / No. tested | PCR positivity (%) | 95% CI (exact, %) |
|---|---|---|---|
| Amblyomma pomposum | 2/6 | 33.3 | 4.3–77.7 |
| Amblyomma variegatum | 92/335 | 27.5 | 22.8–32.6 |
| Amblyomma spp. | 6/23 | 26.1 | 10.2–48.4 |
| Argas walkerae | 2/20 | 10 | 1.2–31.7 |
| Haemaphysalis aciculifer | 4/26 | 15.4 | 4.4–34.9 |
| Haemaphysalis leachi complex | 1/13 | 7.7 | 0.2–36.0 |
| Hyalomma truncatum | 4/58 | 6.9 | 1.9–16.7 |
| Hyalomma spp. | 1/6 | 16.7 | 0.4–64.1 |
| Rhipicephalus afranicus | 0/49 | 0 | 0.0–7.3 |
| Rhipicephalus appendiculatus | 2/30 | 6.7 | 0.8–22.1 |
| Rhipicephalus compositus | 0/1 | 0 | 0.0–97.5 |
| Rhipicephalus decoloratus | 1/99 | 1 | 0.0–5.5 |
| Rhipicephalus evertsi | 0/24 | 0 | 0.0–14.2 |
| Rhipicephalus linnaei | 9/92 | 9.8 | 4.6–17.8 |
| Rhipicephalus lunulatus | 26/205 | 12.7 | 8.5–18.0 |
| Rhipicephalus microplus | 6/152 | 3.9 | 1.5–8.4 |
| Rhipicephalus simpsoni | 0/1 | 0 | 0.0–97.5 |
| Rhipicephalus simus | 1/9 | 11.1 | 0.3–48.2 |
| Rhipicephalus sulcatus | 1/6 | 16.7 | 0.4–64.1 |
| Rhipicephalus zambeziensis | 12/68 | 17.6 | 9.5–28.8 |
| Rhipicephalus spp. | 34/364 | 9.3 | 6.6–12.8 |
| Total | 204/1,587 | 12.9 | 11.2–14.6 |
All 204 samples that tested positive for gltA PCR were successfully sequenced. Sequence analysis identified 26 distinct gltA sequence variants, designated G1–G26. In the gltA phylogenetic tree, 25 sequence variants clustered within the SFG rickettsiae, whereas sequence variant G16 clustered with transitional group (TRG) rickettsiae related to R. hoogstraalii (Fig. 1). Several sequence variants were detected only in a single tick taxon in the present dataset: G16 in Argas walkerae, G9 and G18 in Haemaphysalis aciculifer, G11, G14, G17, G20–G24, and G26 in A. variegatum, and G25 in Rh. lunulatus (Table 1).
Fig. 1.
Maximum-likelihood phylogeny of rickettsiae inferred from gltA sequences (537 bp). Bootstrap support values ≥70% (1,000 replicates) are shown at the internal nodes. Sequences obtained in this study are highlighted in red. Sample counts for each sequence variant are indicated as n=x. SFG, spotted fever group; TG, typhus group; TRG, transitional group.
To further characterize the detected Rickettsia lineages, 56 representative samples were selected for multilocus sequencing. Successful sequences were obtained from 49, 37, 49, 41, and 33 samples for ompA, ompB, htrA, sca4, and the 16S rRNA gene, respectively. BLASTn top hits were broadly consistent with the phylogenetic placements, although the closest named species differed among loci for several sequence variants (Supplementary Table 3). Phylogenetic analyses based on ompA and ompB provided additional support for the lineage placement of representative sequence variants (Fig. 2A and 2B). Analyses based on htrA, sca4, and the 16S rRNA gene showed broadly concordant placements where sequences were available and are presented in Supplementary Fig. 1.
Fig. 2.
Phylogenetic trees were constructed using sequences of the ompA (491 bp) (A) and ompB (770 bp) (B) genes. Maximum-likelihood analyses were conducted. Bootstrap support values ≥70% (1,000 replicates) are indicated at the internal nodes. Sequences obtained in this study are highlighted in red, and gltA sequence variants are shown in parentheses.
Based on concordant phylogenetic clustering in at least three loci, 12 sequence variants (G1, G6, G8, G11, G12, G14, G15, G17, G20, G22, G23, and G24) were provisionally regarded as R. africae-like lineages. Four sequence variants (G3, G4, G7, and G25) clustered with R. massiliae, two sequence variants (G10 and G13) with R. conorii, and one sequence variant (G19) with R. aeschlimannii. Two sequence variants (G9 and G18) clustered with Candidatus R. jingxinensis, and one sequence variant (G5) showed affinity to Candidatus R. rhabdomydis in the ompA and ompB phylogenies. Two sequence variants (G2 and G26) were represented only by gltA sequences, and one sequence variant (G21) by gltA and ompA sequences; therefore, these sequence variants were not assigned to species level.
DISCUSSION
This study expands the molecular evidence for tick-associated Rickettsia spp. in Zambia by demonstrating not only widespread geographic detection but also substantial sequence variant level diversity across tick taxa and provinces. Among 1,587 individually examined ticks, Rickettsia DNA was detected in 204 specimens, and sequence analysis identified 26 gltA sequence variants. Most sequence variants clustered within the SFG, whereas one sequence variant belonged to TRG rickettsiae. Consistent with and extending previous reports from Zambia, the detected lineages included not only Amblyomma-associated R. africae-like sequence variants but also sequence variants related to R. conorii, R. massiliae, R. aeschlimannii, Candidatus R. jingxinensis, Candidatus R. rhabdomydis, and R. hoogstraalii. Thus, the main contribution of this study is the demonstration that tick-associated rickettsial diversity in Zambia is geographically broad and can be resolved more clearly by sequence variant level and multilocus analyses than by overall PCR positivity alone. These findings also suggest that rickettsial surveillance in Zambia should be designed to capture both dominant, widely distributed lineages and less frequent, tick taxon-associated lineages. A surveillance strategy focused only on overall PCR positivity or on A. variegatum-associated R. africae-like lineages may underestimate the ecological and potential public health diversity of tick-associated rickettsiae in the region.
Rickettsia positive ticks were detected in all eight provinces examined. Province level PCR positivity ranged from 2.4% in Copperbelt Province to 26.1% in Central Province, although these values should not be interpreted as directly comparable prevalence estimates because sampling effort, tick species composition, host sources, and collection methods differed among provinces. Nevertheless, the detection of positive ticks in Copperbelt, Luapula, Muchinga, Northern, and North-Western Provinces expands molecular information from areas where Rickettsia data have been relatively limited. Muchinga Province was particularly sequence variant rich, with 16 gltA sequence variants detected, whereas sequence variant G1 was found in all eight provinces. These patterns suggest that both widely distributed and geographically restricted rickettsial lineages circulate in Zambia.
Amblyomma variegatum was the dominant contributor to Rickettsia positive ticks in this dataset from both quantitative and phylogenetic perspectives. This species accounted for 92 (45.1%) of the 204 ticks that tested positive for gltA PCR, and gltA PCR positive individuals were detected in all eight provinces where this species was collected. It also harbored the largest number of gltA sequence variants, including multiple sequence variants provisionally assigned to the R. africae clade. In particular, sequence variant G1 was detected in all eight provinces and was recovered from A. variegatum in each province, although it was also detected in other tick taxa. These findings suggest that G1 represents a broadly distributed rickettsial lineage in Zambia, while the detection of multiple additional R. africae-like sequence variants in A. variegatum indicates localized diversity among rickettsiae associated with this tick species. This pattern is consistent with previous studies in Zambia and other parts of sub-Saharan Africa that have linked Amblyomma ticks with SFG rickettsiae, particularly R. africae [7, 8, 17, 19, 20, 23, 26, 29, 32,33,34]. Because R. africae is the causative agent of African tick-bite fever [17, 23], the broad detection of R. africae-like sequence variants in A. variegatum supports the role of this tick species as a key target for veterinary and public health surveillance in Zambia.
The present data also show that Rickettsia diversity in Zambia cannot be understood by focusing only on A. variegatum or R. africae-like sequence variants. Several sequence variants related to other SFG rickettsiae were detected in Rhipicephalus, Hyalomma, and Haemaphysalis ticks. Sequence variants related to R. massiliae, R. conorii, and R. aeschlimannii are notable because these species, or closely related lineages, have been reported from ticks in Africa and other regions and include recognized or suspected agents of human rickettsioses [31, 35, 39, 42]. In Zambia, serological evidence of exposure to SFG rickettsiae, molecular detection of rickettsiae in human blood, and a travel-associated Mediterranean spotted fever case have also been reported [16, 27, 30]. Therefore, surveillance and diagnostic consideration in Zambia should not be restricted to African tick-bite fever alone.
The sequence variant distribution suggested a degree of structure by tick taxon, although this should be interpreted cautiously. Some sequence variants were detected only from particular tick taxa in the present dataset, including G9 and G18 from Haemaphysalis aciculifer, G16 from Argas walkerae, and G25 from Rhipicephalus lunulatus. The detection of Candidatus R. jingxinensis-like sequence variants in H. aciculifer is noteworthy because this lineage was originally reported from East Asia and related organisms have recently been associated with human infection [3, 13, 24, 25]. Similarly, sequence variant G5 showed affinity to Candidatus R. rhabdomydis in the ompA and ompB phylogenies, suggesting that incompletely characterized rickettsiae related to wildlife- or small-mammal-associated lineages may also occur in Zambian ticks [10]. In addition, the detection of an R. hoogstraalii-related sequence variant in A. walkerae indicates that soft ticks may contribute to the overall rickettsial diversity in Zambia [33].
The multilocus approach was essential for interpreting the detected sequence variants. Concordant phylogenetic placement across multiple loci allowed provisional assignment of several sequence variants to recognized or putative rickettsial clades, whereas sequence variants represented by only gltA or by limited loci were not assigned to species level. This conservative approach is appropriate because species-level identification of closely related Rickettsia spp. based on a single marker can be unreliable [12, 41]. For example, G2 and G26 were represented only by gltA sequences, and G21 by gltA and ompA sequences, and therefore could not be confidently assigned to species level. Similarly, G5 showed different closest named relatives depending on the locus, illustrating the difficulty of interpreting incompletely characterized lineages using partial gene fragments. Isolation, whole-genome sequencing, and genome-based taxonomic comparison will be required to clarify the biological and taxonomic significance of these sequence variants [9, 12].
This study has several limitations. First, the ticks were collected during 2016–2018, and the results should be interpreted as a retrospective molecular baseline rather than a direct estimate of current Rickettsia prevalence in Zambia. Second, tick sampling was not standardized across provinces, hosts, habitats, seasons, or tick taxa, limiting statistical comparisons among geographic areas or species. Third, individual-level information on whether each tick was collected from vegetation or domestic animals was not consistently available. PCR detection also does not confirm bacterial viability or vector competence, and positive results in host-derived ticks may include DNA derived from blood meals rather than from established infection in the tick [11]. Despite these limitations, the geographic breadth of sampling, individual tick testing, and multilocus characterization provide useful baseline information for future ecological, veterinary, and public health investigations.
In conclusion, ticks from multiple provinces of Zambia harbored diverse Rickettsia lineages, including R. africae-like sequence variants frequently detected in A. variegatum and additional lineages related to R. conorii, R. massiliae, R. aeschlimannii, Candidatus R. jingxinensis, Candidatus R. rhabdomydis, and R. hoogstraalii. These findings indicate that Rickettsia surveillance in Zambia should not rely on overall PCR positivity alone, but should incorporate sequence variant level analysis by tick taxon and province. In particular, A. variegatum remains a central target for monitoring R. africae-like lineages, whereas Rhipicephalus, Hyalomma, Haemaphysalis, and argasid ticks may provide additional information on other SFG rickettsial lineages and TRG rickettsiae. Future studies combining standardized tick sampling, bacterial isolation, genome sequencing, and clinical or serological surveillance will be needed to clarify the ecology, pathogenic potential, and veterinary public health significance of these lineages.
CONFLICT OF INTEREST
The authors declare no conflicts of interest associated with this manuscript.
Supplementary Material
Acknowledgments
We thank Mr. Sakae Kashihara and Mr. Kenji Yokoi of the Japan International Cooperation Agency (JICA) for coordinating field activities. We also thank the staff of the Department of National Parks and Wildlife, the Ministry of Fisheries and Livestock, and the Hokudai Center for Zoonosis Control in Zambia (HUCZCZ) for their support during tick collection and laboratory work. This study was supported by the Japan Agency for Medical Research and Development (AMED; grant numbers JP25wm0225034, JP223fa627005, and JP25wm0125008), the Science and Technology Research Partnership for Sustainable Development (SATREPS) program of JICA and AMED (grant number JP23jm0110019), and JSPS KAKENHI (grant numbers JP22K06018, JP24KK0133, and JP25K02166).
Funding Statement
This study was supported by the Japan Agency for Medical Research and Development (AMED; grant numbers JP25wm0225034, JP223fa627005, and JP25wm0125008), the Science and Technology Research Partnership for Sustainable Development (SATREPS) program of JICA and AMED (grant number JP23jm0110019), and JSPS KAKENHI (grant numbers JP22K06018, JP24KK0133, and JP25K02166).
REFERENCES
- 1.Anstead CA, Chilton NB. 2013. A novel Rickettsia species detected in vole ticks (Ixodes angustus) from western Canada. Appl Environ Microbiol 79: 7583–7589. doi: 10.1128/AEM.02286-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Apanaskevich DA, Horak IG. 2008. The genus Hyalomma Koch, 1844. V. Re-evaluation of the taxonomic rank of taxa comprising the H. (Euhyalomma) marginatum Koch complex of species (Acari: Ixodidae) with redescription of all parasitic stages and notes on biology. Int J Acarol 34: 13–42. doi: 10.1080/01647950808683704 [DOI] [Google Scholar]
- 3.Bang MS, Kim CM, Pyun SH, Kim DM, Yun NR. 2021. Molecular investigation of tick-borne pathogens in ticks removed from tick-bitten humans in the southwestern region of the Republic of Korea. PLoS One 16: e0252992. doi: 10.1371/journal.pone.0252992 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Biggs HM, Behravesh CB, Bradley KK, Dahlgren FS, Drexler NA, Dumler JS, Folk SM, Kato CY, Lash RR, Levin ML, Massung RF, Nadelman RB, Nicholson WL, Paddock CD, Pritt BS, Traeger MS. 2016. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever and other spotted fever group rickettsioses, ehrlichioses, and anaplasmosis −United States. MMWR Recomm Rep 65: 1–44. doi: 10.15585/mmwr.rr6502a1 [DOI] [PubMed] [Google Scholar]
- 5.Blair PW, Alharthi S, Londoño AF, Wailagala A, Manabe YC, Dumler JS. Acute Febrile Illness and Sepsis in Uganda Study Teams. 2025. Rickettsioses as underrecognized cause of hospitalization for febrile illness, Uganda. Emerg Infect Dis 31: 1708–1717. doi: 10.3201/eid3109.250479 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Chitanga S, Chambaro HM, Moonga LC, Hayashida K, Yamagishi J, Muleya W, Changula K, Mubemba B, Simbotwe M, Squarre D, Fandamu P, Nalubamba KS, Qiu Y, Hirofumi S, Simulundu E. 2021. Rickettsia lusitaniae in Ornithodoros porcinus ticks, Zambia. Pathogens 10: 1306. doi: 10.3390/pathogens10101306 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Chitanga S, Chibesa K, Sichibalo K, Mubemba B, Nalubamba KS, Muleya W, Changula K, Simulundu E. 2021. Molecular detection and characterization of Rickettsia species in ixodid ticks collected from cattle in southern Zambia. Front Vet Sci 8: 684487. doi: 10.3389/fvets.2021.684487 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Chitimia-Dobler L, Dobler G, Schaper S, Küpper T, Kattner S, Wölfel S. 2017. First detection of Rickettsia conorii ssp. caspia in Rhipicephalus sanguineus in Zambia. Parasitol Res 116: 3249–3251. doi: 10.1007/s00436-017-5639-z [DOI] [PubMed] [Google Scholar]
- 9.Diop A, El Karkouri K, Raoult D, Fournier PE. 2020. Genome sequence-based criteria for demarcation and definition of species in the genus Rickettsia. Int J Syst Evol Microbiol 70: 1738–1750. doi: 10.1099/ijsem.0.003963 [DOI] [PubMed] [Google Scholar]
- 10.Essbauer S, Hofmann M, Kleinemeier C, Wölfel S, Matthee S. 2018. Rickettsia diversity in southern Africa: A small mammal perspective. Ticks Tick Borne Dis 9: 288–301. doi: 10.1016/j.ttbdis.2017.11.002 [DOI] [PubMed] [Google Scholar]
- 11.Estrada-Peña A, Gray JS, Kahl O, Lane RS, Nijhof AM. 2013. Research on the ecology of ticks and tick-borne pathogens—methodological principles and caveats. Front Cell Infect Microbiol 3: 29. doi: 10.3389/fcimb.2013.00029 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Fournier PE, Dumler JS, Greub G, Zhang J, Wu Y, Raoult D. 2003. Gene sequence-based criteria for identification of new rickettsia isolates and description of Rickettsia heilongjiangensis sp. nov. J Clin Microbiol 41: 5456–5465. doi: 10.1128/JCM.41.12.5456-5465.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Gao Y, Wang Y, Du L, Liu X, Sun Y, Zhang Y, Wu H, Zhang S, Wang Z, Mao L, Deng B. 2025. Human infection with Candidatus Rickettsia jingxinensis: First identification and clinical characteristics. PLoS Negl Trop Dis 19: e0013685. doi: 10.1371/journal.pntd.0013685 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Gaowa, Ohashi N, Aochi M, Wuritu, Wu D, Yoshikawa Y, Kawamori F, Honda T, Fujita H, Takada N, Oikawa Y, Kawabata H, Ando S, Kishimoto T. 2013. Rickettsiae in ticks, Japan, 2007–2011. Emerg Infect Dis 19: 338–340. doi: 10.3201/eid1902.120856 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Harima H, Orba Y, Torii S, Qiu Y, Kajihara M, Eto Y, Matsuta N, Hang’ombe BM, Eshita Y, Uemura K, Matsuno K, Sasaki M, Yoshii K, Nakao R, Hall WW, Takada A, Abe T, Wolfinger MT, Simuunza M, Sawa H. 2021. An African tick flavivirus forming an independent clade exhibits unique exoribonuclease-resistant RNA structures in the genomic 3′-untranslated region. Sci Rep 11: 4883. doi: 10.1038/s41598-021-84365-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Ichikawa T, Qiu Y, Ando S, Takeuchi Y, Nagasaka A. 2024. The case of Mediterranean spotted fever of the traveler returned from Zambia. Ticks Tick Borne Dis 15: 102347. doi: 10.1016/j.ttbdis.2024.102347 [DOI] [PubMed] [Google Scholar]
- 17.Jensenius M, Fournier PE, Kelly P, Myrvang B, Raoult D. 2003. African tick bite fever. Lancet Infect Dis 3: 557–564. doi: 10.1016/S1473-3099(03)00739-4 [DOI] [PubMed] [Google Scholar]
- 18.Kajihara M, Simuunza M, Saasa N, Dautu G, Mori-Kajihara A, Qiu Y, Nakao R, Eto Y, Furumoto H, Hang’ombe BM, Orba Y, Sawa H, Simulundu E, Fukushi S, Morikawa S, Saijo M, Arikawa J, Kabilika S, Monze M, Mukonka V, Mweene A, Takada A, Yoshimatsu K. 2021. Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia. PLoS Negl Trop Dis 15: e0009452. doi: 10.1371/journal.pntd.0009452 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Khumalo CS, Mulavu M, Changula K, Mubemba B, Bubala N, Martin AC, Ng’ombwa IB, Nalubamba KS, Chitanga S, Muleya W, Simulundu E. 2024. Molecular detection and phylogenetic characterization of Rickettsia in ticks collected from leopard tortoise (Geochelone pardalis) in rural Zambia. Sci One Health 3: 100074. doi: 10.1016/j.soh.2024.100074 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Kimita G, Mutai B, Nyanjom SG, Wamunyokoli F, Waitumbi J. 2016. Phylogenetic variants of Rickettsia africae, and incidental identification of “Candidatus Rickettsia moyalensis” in Kenya. PLoS Negl Trop Dis 10: e0004788. doi: 10.1371/journal.pntd.0004788 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35: 1547–1549. doi: 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Labruna MB, Whitworth T, Bouyer DH, McBride J, Camargo LMA, Camargo EP, Popov V, Walker DH. 2004. Rickettsia bellii and Rickettsia amblyommii in Amblyomma ticks from the state of Rondônia, western Amazon, Brazil. J Med Entomol 41: 1073–1081. doi: 10.1603/0022-2585-41.6.1073 [DOI] [PubMed] [Google Scholar]
- 23.Ledger KJ, Innocent H, Lukhele SM, Dorleans R, Wisely SM. 2022. Entomological risk of African tick-bite fever (Rickettsia africae infection) in Eswatini. PLoS Negl Trop Dis 16: e0010437. doi: 10.1371/journal.pntd.0010437 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Liu H, Li Q, Zhang X, Li Z, Wang Z, Song M, Wei F, Wang S, Liu Q. 2016. Characterization of rickettsiae in ticks in northeastern China. Parasit Vectors 9: 498. doi: 10.1186/s13071-016-1764-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Lu M, Ji Y, Zhao H, Wang W, Tian J, Duan C, Qin X, Guo Y, Chen G, Lei F, Meng C, Li K. 2023. Circulation of multiple Rickettsiales bacteria in ticks from Sichuan province, Southwest China. Microb Pathog 183: 106313. doi: 10.1016/j.micpath.2023.106313 [DOI] [PubMed] [Google Scholar]
- 26.Makayi C, Simainga S, Mertens-Scholz K, Chitimia-Dobler L, Fischer S, Labruna MB, Mans BJ, Dautu G, Brangsch H, Simuunza M, Groschup MH, Schulz A. 2026. Pathogen screening of Zambian ticks: new insights on the occurrence of tick-borne pathogens in the country. Parasit Vectors 19: 156. doi: 10.1186/s13071-026-07353-w [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Moonga LC, Hayashida K, Mulunda NR, Nakamura Y, Chipeta J, Moonga HB, Namangala B, Sugimoto C, Mtonga Z, Mutengo M, Yamagishi J. 2021. Molecular detection and characterization of Rickettsia asembonensis in human blood, Zambia. Emerg Infect Dis 27: 2237–2239. doi: 10.3201/eid2708.203467 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Moonga LC, Hayashida K, Nakao R, Lisulo M, Kaneko C, Nakamura I, Eshita Y, Mweene AS, Namangala B, Sugimoto C, Yamagishi J. 2019. Molecular detection of Rickettsia felis in dogs, rodents and cat fleas in Zambia. Parasit Vectors 12: 168. doi: 10.1186/s13071-019-3435-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Nakao R, Qiu Y, Igarashi M, Magona JW, Zhou L, Ito K, Sugimoto C. 2013. High prevalence of spotted fever group rickettsiae in Amblyomma variegatum from Uganda and their identification using sizes of intergenic spacers. Ticks Tick Borne Dis 4: 506–512. doi: 10.1016/j.ttbdis.2013.07.001 [DOI] [PubMed] [Google Scholar]
- 30.Okabayashi T, Hasebe F, Samui KL, Mweene AS, Pandey SG, Yanase T, Muramatsu Y, Ueno H, Morita C. 1999. Short report: prevalence of antibodies against spotted fever, murine typhus, and Q fever rickettsiae in humans living in Zambia. Am J Trop Med Hyg 61: 70–72. doi: 10.4269/ajtmh.1999.61.70 [DOI] [PubMed] [Google Scholar]
- 31.Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, Abdad MY, Stenos J, Bitam I, Fournier PE, Raoult D. 2013. Update on tick-borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev 26: 657–702. doi: 10.1128/CMR.00032-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Phiri BSJ, Kattner S, Chitimia-Dobler L, Woelfel S, Albanus C, Dobler G, Küpper T. 2023. Rickettsia spp. in Ticks of South Luangwa Valley, Eastern Province, Zambia. Microorganisms 11: 167. doi: 10.3390/microorganisms11010167 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Qiu Y, Simuunza M, Kajihara M, Chambaro H, Harima H, Eto Y, Simulundu E, Squarre D, Torii S, Takada A, Hang’ombe BM, Sawa H, Sugimoto C, Nakao R. 2021. Screening of tick-borne pathogens in argasid ticks in Zambia: Expansion of the geographic distribution of Rickettsia lusitaniae and Rickettsia hoogstraalii and detection of putative novel Anaplasma species. Ticks Tick Borne Dis 12: 101720. doi: 10.1016/j.ttbdis.2021.101720 [DOI] [PubMed] [Google Scholar]
- 34.Qiu Y, Simuunza M, Kajihara M, Ndebe J, Saasa N, Kapila P, Furumoto H, Lau ACC, Nakao R, Takada A, Sawa H. 2022. Detection of tick-borne bacterial and protozoan pathogens in ticks from the Zambia-Angola border. Pathogens 11: 566. doi: 10.3390/pathogens11050566 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Raoult D, Fournier PE, Abboud P, Caron F. 2002. First documented human Rickettsia aeschlimannii infection. Emerg Infect Dis 8: 748–749. doi: 10.3201/eid0807.010480 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Regnery RL, Spruill CL, Plikaytis BD. 1991. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol 173: 1576–1589. doi: 10.1128/jb.173.5.1576-1589.1991 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Roux V, Raoult D. 2000. Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB). Int J Syst Evol Microbiol 50: 1449–1455. doi: 10.1099/00207713-50-4-1449 [DOI] [PubMed] [Google Scholar]
- 38.Sekeyova Z, Roux V, Raoult D. 2001. Phylogeny of Rickettsia spp. inferred by comparing sequences of ‘gene D’, which encodes an intracytoplasmic protein. Int J Syst Evol Microbiol 51: 1353–1360. doi: 10.1099/00207713-51-4-1353 [DOI] [PubMed] [Google Scholar]
- 39.Spernovasilis N, Markaki I, Papadakis M, Mazonakis N, Ierodiakonou D. 2021. Mediterranean spotted fever: current knowledge and recent advances. Trop Med Infect Dis 6: 172. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Stewart AG, Stewart AGA. 2021. An update on the laboratory diagnosis of Rickettsia spp. infection. Pathogens 10: 1319. doi: 10.3390/pathogens10101319 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Thu MJ, Qiu Y, Matsuno K, Kajihara M, Mori-Kajihara A, Omori R, Monma N, Chiba K, Seto J, Gokuden M, Andoh M, Oosako H, Katakura K, Takada A, Sugimoto C, Isoda N, Nakao R. 2019. Diversity of spotted fever group rickettsiae and their association with host ticks in Japan. Sci Rep 9: 1500. doi: 10.1038/s41598-018-37836-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Vitale G, Mansuelo S, Rolain JM, Raoult D. 2006. Rickettsia massiliae human isolation. Emerg Infect Dis 12: 174–175. doi: 10.3201/eid1201.050850 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Walker JB, Keirans JE, Horak IG. 2000. The Genus Rhipicephalus (Acari, Ixodidae): A Guide to the Brown Ticks of the World. Cambridge University Press, Cambridge. [Google Scholar]
- 44.Walker AR, Bouattour A, Camicas JL, Estrada-Peña A, Horak IG, Latif AA, Pegram RG, Preston PM. 2003. Ticks of Domestic Animals in Africa: A Guide to Species Identification. Bioscience Reports, Edinburgh. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.