1. Introduction
Tick-borne diseases are amongst neglected tropical diseases leading to high prevalence and distribution morbidity [1]. Lack of current information on the prevalence and distribution of tick-borne diseases and their vectors in sub-Saharan Africa make it difficult to assess their true impact [1]. Tick-borne pathogens include Ehrlichia chaffeensis, Ehrlichia ewingii, E. canis, E. ruminantium, and Anaplasma phagocytophilum [2]. Kyasanur Forest disease virus and Crimean-Congo hemorrhagic fever virus are some of viruses transmitted by ticks [3] [4]. The most important tick-borne diseases affecting livestock in Kenya includes theileriosis, anaplasmosis, babesiosis, and heartwater [5]. Omondi et al. [6] reported canine ehrlichiosis (E. ruminantium, Ehrlichia canis, and Ehrlichia sp), anaplasmosis (Anaplasma ovis, Anaplasma platys and A. bovis), and rickettsiosis (Rickettsia aeschlimannii) in ticks collected around Lake Victoria and Lake Baringo.
Encroachment of human settlements into the game areas by the pastoral communities has led to the emergence and re-emergence of tick-borne zoonotic diseases [7]. Other activities such as commercial ranching, and tourism increases the risk of pathogen transmission [8]. This has exposed the humans to the bite of the vectors [9]. Several tick-borne zoonotic pathogens have been detected in wildlife protected areas in Kenya [10] [11]. In order to curb the spread of these tick-borne pathogens the government must impose strict measure to prevent human encroachment into wildlife areas.
There is limited knowledge of circulating tick-borne pathogens of medical importance in their biological vectors at human-wildlife-livestock interface in Kenya due to limited resources. This study aimed at addressing the gap in knowledge on the zoonotic tick-borne pathogens circulating at human-wildlife-livestock interfaces in the Tsavo National Reserve (TNR) and the Shimba Hills National Reserve (SHNR), Kenya, by surveying the tick diversity and associated tick-borne pathogens.
2. Materials and Methods
2.1. Study Area
Sampling approval was obtained from the Kenya’s Directorate of Veterinary Services and Ministry of Health. Informed consent was obtained from the livestock’s owner before sampling of the ticks at near Tsavo and Shimba Hills National Reserves, Kenya. The two selected human-wildlife-livestock interfaces were selected based on encroachment human settlement by the pastoral community and reported cases of tick-borne pathogens of economic and public health burden. The Tsavo National Park is a protected area in Kenya, and borders the Chyulu Hills National Park in Kenya and Mkomazi game reserve of Tanzania. The park is divided by the highway road that runs from Nairobi to Mombasa into Tsavo East national park (13,747 Km2) and Tsavo West national park (9065 Km2). The SHNR is a wildlife protected area in Kwale County. It is the largest forest area in east African and home to a variety of wildlife species.
This map is republished with data under CC BY licenses from the following sources: https://www.wri.org/resources/data-sets/kenya-gis-data from the World Institute Resources, 2007 [12]; https://africaopendata.org/dataset/kenya-counties-shapefile from openAfrica, 2015 [13]; and https://gadm.org/data.html from GADM, 2018 [14] (Figure 1).
2.2. Tick Collection and Identification
Sampling approval was obtained from the Kenya’s Directorate of Veterinary Services and Ministry of Health. Informed consent was obtained from the livestock’s owner before sampling of the ticks. Ticks were collected from restrained cattle hide using sterile forceps and placed into labeled falcon tubes. The tubes were then plugged with cotton swabs and transported in liquid nitrogen to the testing lab. They were stored at −80˚C until when analyzed [6]. The ticks were identified to genus and/or species level as per the morphological keys [15] using sterile forceps, petri-dishes and gloves. Ticks were pooled according to species, sex and sampling sites into groups of 1 - 11 for adults and 1 - 20 for nymphs as described by Oundo et al. [10]. Representative of these morphologically identified ticks were molecularly identified.
3. Laboratory Procedure
3.1. DNA Extraction
Ticks were homogenized in a 1.5-ml sterile microcentrifuge tubes containing 750 mg of 2-mm yttria-stabilized zirconium using a handheld battery-operated
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Figure 1. Map of ticks sampling locations near the TNR in Taita Taveta County, Kenya, and near the SHNR in Kwale County, Kenya. The map was prepared using common-license shapefiles in QGIS software. TNR, Tsavo National Reserve; SHNR, Shimba Hills National Reserve.
homogenizer oxide bead (Glen Mills, Clifton, NJ). DNA was extracted from the homogenate using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) as per the manufacture’s protocol. The concentration and purity of the DNA were evaluated using a Nanodrop spectrophotometer (Thermo Fisher Scientific Wilmington, USA) and extracts stored at −20˚C.
3.2. Molecular Detection and Identification of Tick-Borne Pathogens
Tick-borne pathogens were screened using HRM and characterized by sequencing of specific RT-PCR products of Anaplasma, Ehrlichia, and Rickettsia using genus-specific primers (Table 1). For HRM, a 10 µl reaction volume was prepared containing a final concentration of 1× HOT FIREPol Eva-Green HRM mix (Solis BioDyne, Tartu, Estonia), 500 nM of the respective forward and reverse primers (Table 1), 100 ng of template DNA and 5 μl nuclease free water. DNA extracts from Anaplasma phagocytophilum, Ehrlichia ruminantium, and Rickettsia africae were used as positive controls while nuclease-free water was used as a negative control. Minimum infection rate was calculated using the following formula: [number of pathogen positive tick pools/total number of ticks of that species tested] × 1000. The MIR assumes that only one tick is positive in a pool.
3.3. Data Analysis
Geneious software version 8.1.9 was used to edit and align all nucleotide sequences using the MAFFT plugin [16]. GenBank database using the Basic Local Alignment Search Tool (https://www.ncbi.nih.gov/BLAST/) was used in sequence identities. The map was prepared using QGIS software version 3.12.1 (QGIS) to show the sampling sites.
4. Results
4.1. Tick Diversity and Abundance
At near Tsavo National Reserve, a total of 317 (281 adult ticks and 36 nymphs) were collected, representing seven species (Table 2). Amblyomma ticks dominated the collections with Amblyomma gemma the most sampled species (n = 135, 42.6%). Other Amblyomma species sampled was Amblyomma variegatum (n = 40, 12.62%). Four species of Rhipicephalus ticks were collected including; R. appendiculatus (n = 44, 13.9%), R. evertsi (n = 1, 0.31%), R. decoloratus (n = 5, 1.6%), and Rhipicephalus pulchellus (n = 91, 28.7%). A single Hyalomma specimen was also collected (Table 2).
Near Shimba Hills game reserve 240 adult ticks were collected representing eight species (Table 2) and again Amblyomma gemma dominated the collection (n = 156, 65%). Other Amblyomma species sampled included; A. lepidum (n = 5, 2.1%), A. variegatum (n = 15, 6.3%). Rhipicephalus ticks included R. appendiculatus (n = 18, 7.5%), R. evertsi (n = 6, 2.5%), R. decoloratus (n = 4, 1.7%), R. pulchellus (n = 34, 14.2%). Two representatives of Hyalomma scupense were also collected (n = 2, 0.83%) (Table 2).
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Table 1. PCR primers pairs used in the study.
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Table 2. Abundance and diversity of tick species collected in near Tsavo and Shimba Hills National Reserves, Kenya.
4.2. Tick-Borne Pathogens Identified
At near Tsavo National Reserve (TNR) in Taita Taveta County, a total of three pools of Rhipicephaline appendiculatus were positive for Theileria parva (GenBank accession Number OL451869-OL451871), two pools of Rhipicephaline evertsi for Anaplasma platys (GenBank accession Number OL451873-OL451874) and one pool of Amblyomma variegatum nymphs for Rickettsia africae (GenBank accession Number OL466919) as shown in Table 3.
At Shimba Hills, Rickettsia africae was detected in two pools (GenBank accession Number OL466921-OL466922) of A. variegatum and one pool of A. gemma. Rickettsia sp. (GenBank accession Number OL466920) and Anaplasma sp. (GenBank accession Number OL451872) were detected in pools of A. gemma
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Table 3. Tick-borne pathogens identified.
and R. evertsi respectively. Rickettsia aeschlimannii (GenBank accession Number OL466924) was detected in a pool of A. gemma.
5. Discussion
Detection of tick-borne zoonotic diseases at the human-wildlife-livestock interfaces of Shimba Hills National Reserve and the Tsavo National Reserve, gives an insight into the possibility of tick-borne zoonotic diseases spilling over from the wildlife to livestock and humans. Most of the tick species identified in this study have been incriminated as the vectors of tick-borne diseases of public health and veterinary concern [17].
Rickettsia africae is a zoonotic tick-borne pathogen which causes African tick bite fever in humans and manifests with headache, fever, rush, myalgia and skin lesion at the site of tick bite [11]. It is often misdiagnosed at the clinical setting because it shares symptoms with common febrile illnesses including malaria. This calls for robust surveillance and inclusions of R. africae in hospitals and other clinical settings. In this study, R. africae was detected in A. gemma and A. variegatum ticks. These findings are consistent with those that confirm the circulation of R. africae among the Amblyomma species [10] [11] [18]. Further, R. africae was detected in nymphs of A. variegatum ticks collected from cattle.
Rickettsia aeschlimannii is also considered to be a zoonotic tick-borne pathogen which has been reported in patients travelling from Africa [19] and it is understood that migratory birds play an important role in the epidemiology of R. aeschlimannii by passive transportation of R. aeschlimannii-infected ticks from one region to another [20]. This pathogen has been detected in detected in Hyalomma ticks in Kenya [6], which are thought to serves as reservoirs for R. aeschlimannii [21]. However, in the present study, R. aeschlimannii was detected in A. gemma. According to our knowledge this is the first time R. aeschlimannii has been detected in A. gemma.
Uncharacterized Rickettsia sp. was detected in Am. variegatum ticks. Although its pathogenicity was unknown, it can be a public health threat in future [19]. Many known Rickettsia species of public health concern today, were not initially considered to be harmful to humans. This species needs further characterization with additional markers such as ompA, sca4, 17kDa [10].
Anaplasma platys is a TBP infecting dogs which is transmitted by brown dog ticks (Rhipicephalus sanguineus s.l.) [22]. The pathogen has been reported also to infect humans causing mild headache, lethargy and myalgia [23] [24]. In the current study, Anaplasma platys was detected in R. evertsi.
Theileria parva is a TBP of veterinary importance which causes East Coast Fever in cattle [15]. In this current study, Theileria parva was detected in R. appendiculatus ticks. These findings were consistent with those of Oundo et al. [10]. The cattle get infected by coming into close proximity to buffaloes which are natural reservoir for T. parva [25].
6. Conclusion
The detection of Rickettsia africae, Rickettsia aeschlimannii, and Anaplasma platys gives an insight into the possibility of transmission of zoonotic tick-borne pathogens from wildlife to humans at human-wildlife-livestock interfaces. Since persons infected with these pathogens present with fever, it is paramount to include tests that can diagnose these infections in clinical settings.
Acknowledgements
We acknowledge the staff of the Msambweni Department of vector borne diseases for their technical assistance.
Data Availability Statement
Sequences obtained in this study have been deposited in the GenBank database under the following accession numbers: OL466919-OL466924 (Rickettsia 16S rRNA), OL451872-OL451874 (Anaplasma 16S rRNA), and OL451869-OL451871 (Theileria 18S rRNA).
Author’s Contributions
Conceptualization: MNC, MWM
Data curation: All authors.
Formal analysis: All authors.
Investigation: SKG
Methodology: All authors
Project administration: SKG
Resources: SKG
Supervision: MNC, MWM
Validation: MNC, MWM
Visualization: SKG, MNC, MWM
Writing-original draft: All authors.
Writing-review & editing: All authors.