
==== Front
Sci One Health
Sci One Health
Science in One Health
2949-7043
Elsevier

S2949-7043(24)00013-1
10.1016/j.soh.2024.100074
100074
Full Length Article
Molecular detection and phylogenetic characterization of Rickettsia in ticks collected from leopard tortoise (Geochelone pardalis) in rural Zambia
Khumalo Cynthia Sipho a
Mulavu Malala b
Changula Katendi c
Mubemba Benjamin d
Bubala Nchimunya e
Martin Anne C. f
Ng'ombwa Innocent Billy g
Nalubamba King Shimumbo h
Chitanga Simbarashe bij
Muleya Walter a
Simulundu Edgar edgar.simulundu@macharesearch.org
ek⁎
a Department of Biomedical Sciences, School of Veterinary Medicine, University of Zambia, Lusaka, Zambia
b Department of Biomedical Sciences, School of Health Sciences, University of Zambia, Lusaka, Zambia
c Paraclinical Sciences Department, School of Veterinary Medicine, University of Zambia, Lusaka, Zambia
d Department of Wildlife Sciences, School of Natural Resources, Copperbelt University, Kitwe, Zambia
e Macha Research Trust, Choma, Zambia
f Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
g Department of National Parks and Wildlife, Wildlife Veterinary Unit, Chilanga, Zambia
h Clinical Studies Department, School of Veterinary Medicine, University of Zambia, Lusaka, Zambia
i Department of Preclinical Studies, School of Veterinary Medicine, University of Namibia, Windhoek, Namibia
j School of Life Sciences, College of Agriculture, Engineering and Sciences, University of KwaZulu-Natal, Durban, South Africa
k Department of Disease Control, School of Veterinary Medicine, University of Zambia, Zambia
⁎ Corresponding author. edgar.simulundu@macharesearch.org
08 8 2024
2024
08 8 2024
3 10007428 5 2024
3 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
In sub-Saharan Africa, limited studies have investigated zoonotic pathogens that may be harboured by ticks infesting reptiles such as tortoises. Here, we report the presence of pathogenic Rickettsia in ticks (Amblyomma marmoreum) collected from the leopard tortoise (Geochelone pardalis) in rural Zambia. Using polymerase chain reaction, 56% (49/87) of ticks were positive for the Rickettsia outer membrane protein (ompB) gene. Multi-locus sequence and phylogenetic analysis based on the ompB, ompA, and citrate synthase (gltA) genes showed that the ticks carried R. africae, and other Rickettsia spp. closely related to R.raoultii, R.massiliae, R.tamurae and R.monacensis. Given the proximity between humans, livestock, and wildlife in these habitats, there exists a considerable risk of transmission of zoonotic Rickettsia to human populations in this rural setting. These results call for heightened awareness and further research into the dynamics of tick-borne diseases in regions where humans and animals coexist, particularly in the context of tortoise-associated ticks as vectors. Understanding and addressing these potential disease vectors is crucial for effective public health measures and the prevention of Rickettsia zoonoses.

Highlights

• Tortoise-associated Amblyomma marmoreum harbours zoonotic Rickettsia africae and diverse unidentified Rickettsia spp. of unknown zoonotic potential.

• Surveillance efforts are needed to reduce the impact of tick-borne rickettsial infections in humans living in proximity to livestock and wildlife.

• The study stresses "One Health" approaches to combat vector-borne diseases.

Keywords

Rickettsia
Leopard tortoise
Phylogenetic analysis
Amblyomma marmoreum
Zambia
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pmc1 Introduction

Ticks serve as vectors of numerous emerging zoonotic pathogens of humans and animals [1,2]. Wildlife acts not only as a source, but also as a maintenance host for most tick-borne pathogens (TBPs) [3]. This is especially true for long-living reptiles such as tortoises infested with ticks, although they are rarely recognized as important reservoirs of pathogens [4]. In the recent past, many tick species infesting amphibians and reptiles have been reported, with some species such as Amblyomma rotundatum, A. marmoreum and Bothriocroton hydrosauri, showing a marked preference for infesting these animals [5,6]. In Zambia, A. marmoreum has been reported to infest reptiles such as the puff adder (Bitis arietans) [7] and the leopard tortoise (Geochelone pardalis) [8]. A. marmoreum is believed to be a vector of Ehrlichia ruminantium [9], the causative agent of heart water, a deadly disease of domestic and wild ruminants. Further, Rickettsia and Ehrlichia present in Amblyomma and Hyalomma ticks infesting G. pardalis tortoises exported from Zambia to Japan indicate possible circulation of these pathogens in tortoises in the country [10].

Despite the potential for tortoises, due in part to their longevity, to serve as a source of zoonotic pathogens to various vectors including ticks, there is limited evidence of the presence of Rickettsia species in tortoise-associated ticks. From the few available studies, Rickettsia species from all groups, including Spotted fever group, Typhus group and the ancestral group R. belli have been detected from tortoise-associated ticks [[10], [11], [12]]. Considering the proximity of reptiles to humans and the evidence of zoonotic pathogens in arthropods associated with these animals, it is important to screen reptiles and/or arthropod vectors for infectious diseases as part of public health risk mitigation. This study aimed to detect and phylogenetically characterize Rickettsia species in ticks collected from tortoises in rural Zambia, where humans, livestock, and wildlife like tortoises live in close proximity.

2 Materials and methods

2.1 Study site and sample collection

This study was conducted in Macha, a rural area located in Choma District, Southern Province, Zambia. The major economic activity of people living in this area is subsistence agriculture, particularly livestock production. During the two rainy seasons of 2020 and 2021 (from around November to April), 87 ticks (68 adults and 19 nymphs) were collected from leopard tortoises (n = 48) in residential areas around the Macha Hospital catchment area. Morphological identification of the ticks was conducted as previously described [13].

2.2 DNA extraction and molecular detection

Ticks were washed with phosphate-buffered saline and 70% alcohol and DNA extraction was done using the InviMag® Universal Kit (Invitek Molecular, Berlin, Germany) according to the manufacturer's instructions. For molecular detection of Rickettsia species, polymerase chain reaction (PCR) was conducted using Taq® 2X Master Mix (New England BioLabs, Massachusetts, USA) reaction mix (Suppl Table 1), using primers targeting the ompB [14], gltA [15] and ompA [16] genes (Suppl Table 2). Samples were first screened using the ompB gene. Samples that tested positive were analyzed using primers targeting the gltA [15] and ompA [16] genes to further confirm the presence of Rickettsia spp. and to genetically characterize the species through sequencing of the obtained amplicons. Amplified DNA fragments were visualized on 1.5% agarose gel stained with ethidium bromide. Positive samples were purified using Wizard® SV Gel and Clean-Up System (Promega, Madison, WI, USA). Purified DNA was then subjected to sequencing using the Big dye™ Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, Waltham, Massachusetts, USA) followed by ethanol precipitation and capillary electrophoresis on the SeqStudio™ Genetic Analyzer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

2.3 Phylogenetic analysis

Obtained sequences were assembled and edited using GENETYX ATGC software, version 12.0 (GENETYX Co., Tokyo, Japan). Thereafter, alignments of the resulting sequences for the three target genes were conducted using the multiple sequence alignment program (MAFFT) [17]. Final alignments were then uploaded to the online IQ-tree tool (IQTREE Web Server: Fast and accurate phylogenetic trees under maximum likelihood (univie.ac.at) for the construction of the maximum likelihood (ML) phylogeny using auto-selected substitution model with Bayesian Information Criterion (BIC) for tree improvement otherwise using default settings [18]. Branch support was estimated using 1,000 bootstrapping replicates. TempEst (version 1.5.1) was then used to estimate the best-fitting root of the resulting ML tree using the heuristic residual mean squared function to ensure that the variance of the root-to-tip distances is minimised [19]. The resultant tree file was then edited using iTOL (Interactive Tree of Life) (https://itol.embl.de/) [20].

3 Results

3.1 PCR and molecular detection

This study investigated the presence of Rickettsia species in ticks collected from tortoises in Macha, Choma District, Zambia. All collected ticks (n = 87) were identified as A. marmoreum (Fig. 1). On PCR screening, 56.3% (n = 49), 36.8% (n = 32), and 31% (n = 27) ticks were positive for the Rickettsia ompB, ompA, and gltA genes, respectively (Suppl Table 3). Sequencing was attempted on all positive samples. From the positive samples, 26/49, 30/32, and 23/27 nucleotide sequences of the respective genes were obtained. Of the sequences obtained, only 23 ompB, 16 ompA, and 22 gltA sequences were successfully assembled. These sequences were used for further analysis.Fig. 1 Amblyomma marmoreum tick (red arrow) on the back of the leopard tortoise (Geochelone pardalis) during tick collection.

Fig. 1

3.2 Blast analysis

Using the Basic Local Alignment Search Tool (BLASTn) (https://blast.ncbi.nlm.nih.gov/Blast.cgi), sequences obtained were compared to other sequences in the GenBank and percentage similarities are as shown in Suppl Table 4. On ompB, 10 sequences showed high sequence similarity (98%–100%) to R. africae. The highest sequence similarity by BLAST analysis of the remaining ompB sequences (n = 13) was to R. monacensis (96% similarity) which was detected in 1981 in South Korea (GenBank accession EU883092.1). The majority (n = 13) of gltA sequences (n = 13) were like various R. africae sequences in Africa including Zambia and from France. The remaining gltA nucleotide sequences (n = 7) from this study showed similarity to R. raoultii (n = 3) and R. monacensis (n = 4). Similarly, the majority of ompA nucleotide sequences (n = 11) were like various R. africae sequences in Africa including Zambia, Antigua, and France. The remaining ompA nucleotide sequences (n = 5) identified with R. tamurae (n = 1) and R. monacensis (n = 4). The ranges for sequence similarity on both gltA and ompA were 90%–100%. All sequences generated in this study were deposited in GenBank under accession numbers LC775369 to LC775391 for ompB, LC775856 to LC775877 for gltA, and LC775586 to LC775601 for ompA (Suppl Table 4).

3.3 Phylogenetic analysis

Some sequences on all three genes were removed from the analysis because they were shorter than other sequences on the same gene and hence comparison could not be reliable. Phylogenetic analysis of ompB gene sequences showed most of the samples (n = 14) from this study clustered closely with R. africae reference sequences from within Africa, but there was no signal for monophyly based on the country of origin with those previously detected in Zambia (Fig. 2). While phylogenetic assignment placed the majority of the ompB sequences (n = 14) in the R. africae clade, some sequences (n = 6) could not be definitively assigned this species as they were negative for either of the other two screening genes (ompB and gltA) and hence were designated as Rickettsia sp. in the tree. The second group of six sequences formed a distinct reciprocally monophyletic group to a clade mainly consisting of R. monacensis sequences from Asia, Europe, and the USA with a very strong branch support. Though these ompB sequences showed close relationships with R. monacensis on BLAST, they could not be assigned this species as the ompA and gltA sequences were negative for the same. It is a requirement that a test sample should be positive on all the three genes for Rickettsia species assignment when using short fragments of genes [21]. Three sequences of the ompB gene from this study formed a distinct cluster and were not closely related to any of the known Rickettsia species.Fig. 2 Phylogenetic analysis of a 496 bp region within RickettsiaompB gene detected in ticks of tortoises from Macha. Tip labels of all sequences generated in this study are shown in red text while reference sequences used are shown by their GenBank accession numbers and country of origin/or by Rickettsia sp. Branches supported by bootstrap values ≥ 70 are indicated in blue. The tree scale shows nucleotide substitution per variable site.

Fig. 2

Phylogenetic analysis of the gltA gene (Fig. 3) demonstrated similar trends as that of the ompB gene tree, in that 13 sequences determined in this study clustered with those of R. africae detected mainly in African countries including Zambia, South Africa, Madagascar, and Senegal. However, there was no signal for monophyly based on country of origin with those previously detected in Zambia. Three samples, namely MA38-AFA, MA317-AMAb and MA336-AMAa, clustered independently but showed close relation to R. raoultii detected in China and Crimea. The gltA sequences of samples MA27-AFAb, MA27-AMAb, MA331-AMAb and MA341-RFN clustered together with R. tamurae and R. monacensis from Japan, China, and Crimea forming distinct reciprocally monophyletic groups with these species within this bigger cluster (Fig. 3). Further, as observed with ompB analysis, some gltA sequences were designated as Rickettsia sp. despite them having support for species assignment phylogenetically owing to these gltA sequences being negative for the respective Rickettsia species on either ompB or ompA, or both (Suppl Table 4) [21].Fig. 3 Phylogenetic analysis of a 536 bp region within Rickettsia gltA gene detected in ticks of tortoises from Macha. Tip labels of all sequences generated in this study are shown in red text while reference sequences used are shown by their GenBank accession numbers and country of origin/or type of Rickettsia sp. Branches supported by bootstrap values ≥ 70 are indicated in blue. The tree scale shows nucleotide substitution per variable site.

Fig. 3

Phylogenetic analysis of the ompA gene revealed that most of the sequences under study were closely related to those of R. africae obtained mostly from various African countries including Zambia, but there was no signal detected for monophyly with those previously detected in Zambia (Fig. 4). The ompA gene sequence of sample MA 317 AMAb clustered with uncultured Rickettsia sp. detected in Malaysia. The rest of the sequences (n = 4) clustered together and were closely related to Rickettsia sp. previously detected in Zambia (Fig. 4).Fig. 4 Phylogenetic analysis of a 520 bp region within Rickettsia ompA gene detected in ticks of tortoises from Macha. Tip labels of all sequences generated in this study are shown in red text while reference sequences used are shown by their GenBank accession numbers and country of origin/or by Rickettsia sp. Branches supported by bootstrap values ≥ 70 are indicated in blue. The tree scale shows nucleotide substitution per variable site.

Fig. 4

4 Discussion

This study detected Rickettsia in A. marmoreum ticks infesting tortoises in rural Zambia. Detection of Rickettsia in tortoises-associated ticks has been recorded in Qatar, Israel, Algeria, and Madagascar [4,[22], [23], [24]]. Compared to the Rickettsia proportion of 56.3% observed in this study (based on the ompB gene), elsewhere, they found a prevalence of 53% [23] while others, found a lower prevalence of 25% [24]. This shows the potential of tortoises-associated ticks to carry and transmit Rickettsia. Other Rickettsia species detected were closely related to R. raoultii, R. massiliae, R. tamurae, and R. monacensis based on ompA, ompB and gltA genes and all can cause infections in humans. The presence of R. africae was not unexpected as recent studies have reported its presence in ticks infesting cattle in Zambia [25] with a prevalence of 18.6% as well as in tortoise-associated ticks in Madagascar and Qatar with prevalence rates of 100% and 14.3%, respectively [4,22]. R. africae is the causative agent of African tick-bite fever (ATBF) and is most prevalent in sub-Saharan Africa, commonly transmitted by Amblyomma tick species [26].

These zoonotic Rickettsia were detected in ticks associated with tortoises' frequent residential areas. This increases opportunities for transmission of Rickettsia to humans. Considering the lifespan of Geochelone species (over 50 years), these pathogens are likely to be circulating for a prolonged period, thus increasing opportunities for exposure of rickettsial pathogens to other hosts if no intervention is made [27]. Specifically, these findings hold significant implications for public health and eco-epidemiology in the region. By expanding the scope of host species and vectors known to harbour Rickettsia, this study provides invaluable insights into the potential transmission dynamics of the detected rickettsial pathogens within the local ecosystem. Furthermore, it underscores the interconnectedness of wildlife, domestic animals, and humans in the epidemiology of tick-borne diseases.

Rickettsia in Amblyomma ticks hosted by tortoises is not uncommon [4], and this is attributed to the ability of Amblyomma ticks to absolutely transmit Rickettsia both transovarially and trans-stadialy [28]. Whilst previous studies have shown the presence of Rickettsia species in different tick species associated with tortoises across Africa and the world, Rickettsia species have been detected in Amblyomma sparsum on tortoises imported into Japan from Zambia. Additionally, Rickettsia species have been detected in humans, livestock, and pet animals in Zambia [10,25,29,30]. This is the second report of Rickettsia in ticks collected from tortoises in Zambia. Our findings contribute to the growing body of knowledge on the spatial eco-epidemiology of Rickettsia in Zambia, which is critical in informing prevention and control strategies.

ATBF is the second most common cause of systemic febrile illness among travellers in Africa, after malaria [25]. These findings indicate that tick-borne disease may contribute to cases of non-malaria febrile illnesses in the area as recent studies show a dramatic reduction and sustained low transmission of malaria cases in the study area [28]. In addition, the population of humans in the study area has frequent exposure to livestock and wildlife including tortoises which are frequently seen in residential areas, especially during the rainy season (from around November to April). A. marmoreum is also known to parasitize cattle and hence humans in close contact with livestock such as herders are at increased risk of infestation and possible infection with the pathogens they may carry, such as zoonotic Rickettsia [29]. Considering this, we echo our previous call for routine screening of causative agents of ATBF in humans presenting with non-malarial febrile illness in this area and the country at large [30]. This recommendation is especially critical given that human rickettsiosis is a newly emerging tick-borne zoonotic disease worldwide, and diagnosis remains challenging due to healthcare providers’ lack of awareness and limited diagnostic capabilities in most Zambian healthcare facilities [30]. Just recently, a case of Mediterranean spotted fever (MSF), an infectious disease caused by R. conorii was reported in a Japanese traveller who had returned from Lusaka, Zambia [31]. This is the first case of MSF in Zambia highlighting the potential threat of MSF in urban areas especially that dog brown tick (Rhipicephalus sanguineus sensu lato), the vector of the R. conorii, has been found in various areas of Zambia.

In conclusion, this study has shown that tortoise-associated A. marmoreum harbours zoonotic R. africae and diverse unidentified Rickettsia of unknown zoonotic potential. This represents a significant advancement in our knowledge of tick-borne diseases in Zambia. Further, these findings underscore the need for continued research and surveillance efforts to mitigate the impact of rickettsial infections on both human and animal populations. This is critical for humans living in proximity with these animals, particularly among patients with non-malarial febrile illnesses. This also implies that physicians should also consider rickettsioses for unexplained febrile illnesses among individuals seeking healthcare in this region. Considering that tick control would be a challenging undertaking in these reptiles, community sensitization on the risk posed by tortoise-associated ticks in transmitting zoonotic diseases could be a viable intervention strategy. Additionally, more studies to assess the burden and risk that ticks infesting tortoises and other wildlife pose to public health are warranted not only in this rural setting but in other regions as well, especially in countries importing tortoises for breeding or as a source of exotic protein. This necessitates the use of interdisciplinary approaches by combining knowledge from epidemiology, veterinary medicine, and ecology to provide a more thorough understanding of disease dynamics to aid in the creation of all-encompassing intervention strategies.

Funding statement

This work was supported by a grant from the National Institute of Allergy and Infectious Diseases of the 10.13039/100000002 National Institutes of Health ‘Spatial eco-epidemiology of tick-borne rickettsial pathogens’ under award number R01AI136035 [PI – Gaff H], through a sub-award to the University of Zambia [PI – Chitanga S]. The content is solely the responsibility of the authors and does not necessarily represent the views of the National Institutes of Health.

Ethical statement

Permission to conduct this study was obtained from the Ministry of Tourism and Arts and was issued as a Research Capture and Release Permit in accordance with section 43 (1) of the Zambia Wildlife Act No. 14 of 2015.

Data availability

All data generated in this study are included within the article along with their supplementary files.

CRediT authorship contribution statement

Cynthia Sipho Khumalo: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Malala Mulavu: Writing – review & editing, Methodology, Data curation. Katendi Changula: Writing – review & editing, Supervision, Methodology, Investigation. Benjamin Mubemba: Writing – review & editing, Supervision, Software, Formal analysis, Data curation, Conceptualization. Nchimunya Bubala: Writing – review & editing. Anne C. Martin: Writing – review & editing, Methodology. Innocent Billy Ng'ombwa: Writing – review & editing. King Shimumbo Nalubamba: Writing – review & editing, Methodology. Simbarashe Chitanga: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. Walter Muleya: Writing – review & editing, Writing – original draft, Validation, Supervision, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Edgar Simulundu: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgments

The authors would like to thank the Department of National Parks and Wildlife for supporting this study.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.soh.2024.100074.
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