Zoonotic Tick Borne Pathogens in Ticks at Human-Wildlife-Livestock interfaces in Kwale County, Kenya

Background Ticks are vectors that can harbor pathogens like viruses, protozoa and bacteria that can cause zoonotic diseases in human. Human gets infected through tick bites where the pathogens are passed to the human blood. One tick bite can lead to transmission of more than one disease. Ticks are obligate hematophagous, infest most vertebrates globally. Lack of surveillance information about the tick-borne pathogens and diseases has made it impossible to assess its impact to the community. Materials and Methods We morphologically identified ticks collected from two game reserves in Kenya, locations near the Shimba Hills National Reserve (SHNR) and Tsavo National Reserve (TNR). We identified tick-borne pathogens by HRM analysis and sequencing of specific RT-PCR products of Anaplasma, Ehrlichia, and Rickettsia. Results A total of 317 (281 adult ticks and 36 nymphs) was collected at near Tsavo National Reserve in Taita Taveta County, which includes seven species. Amblyomma was the most sampled genus with Amblyomma gemma being the most sampled species (n=135, 42.6%). Other Amblyomma species sampled was Amblyomma Variegatum (n= 40, 12.62%). Greatest species diversity was identified in Rhipicephalus genus with four species identified that includes; Rhipicephalus appendiculatus (n=44, 13.9%), Rhipicephalus Averts (n=1, 0.31%), Rhipicephalus Decoloratus (n=5, 1.6%), Rhipicephalus Pulchellus (n= 91, 28.7%). A single species of Hyalomma sp. was sampled. From near Shimba Hill game reserve (SHNR), a total of 240 adult’s ticks were sampled that comprises of eight species. Amblyomma was the most sampled genus and again Amblyomma gemma being the most sampled species (n=156, 65 %). Other Amblyomma species sampled includes; Amblyomma Lepidum (n= 5, 2.1 %), Amblyomma Variegatum (n= 15, 6.3 %). Greatest species diversity was also identified in Rhipicephalus genus with four species identified that includes; Rhipicephalus appendiculatus (n=18, 7.5 %), Rhipicephalus Averts (n=6, 2.5 %), Rhipicephalus Decoloratus (n=4, 1.7 %), Rhipicephalus Pulchellus (n= 34, 14.2 %). The least sampled species was a single species of Hyalomma Scupense (n=2, 0.83 %). At near Tsavo National Reserve (TNR) in Taita Taveta County, a total of three pools of Rhipicephaline appendiculatus were positive for Theileria parva, two pools of Rhipicephaline evertsi for Anaplasma platys and one pool of Amblyomma variegatum nymphs for Rickettsia africae. From near Shimba Hill game reserve (SHNR), Kwale County, Rickettsia africae pathogen was detected in two pools of Am. variegatum and one pool of Am. Gemma. Rickettsia sp. and Anaplasma sp. were detected in Am. Gemma and Rh. evertsi respective. R. aeschlimannii was detected in a pool of Am. Gemma. These findings highlight the risk of transmission of zoonotic R. africae and unclassified Rickettsia spp. to humans causing African tick-bite fever and other spotted-fever group rickettsioses.


Introduction
Arthropod vectors like ticks can harbor viruses, bacteria, and protozoa which can cause diseases in wildlife and livestock (1,2). They are obligate hematophagous that parasitize most vertebrates in almost every region in the world (3). Ticks are second to mosquitoes as vectors with 800 species which depends on the blood of most vertebrates and cause disease in human (3). Ticks belong to class Arachnida, only two families, the Ixodidae (hard tick) and Argasidae (soft ticks) have medical importance. These families can be differentiated by the presence of the scutum, hard ticks possess the scutum and soft ticks lack the scutum (4). Humans get the infection through a tick bite, direct contact from viremia tissues of humans or animals, and the disease is characterized by hemorrhagic (5) and a single tick bite can transmit more than one pathogen (6).
The vector-borne infections have been a problem for both animals and humans causing black death plagues in Europe and yellow fever in America in 1th centaury (7). Tick-borne diseases include: Ehrlichiachaffeensis, Ehrlichiaewingii, E. canis, E. ruminantium, and Anaplasma phagocytophilum, and transmission is by ticks (8). Tick-borne diseases are amongst neglected tropical diseases leading to high prevalence and distribution morbidity (9). Lack of the tick-borne zoonotic information in sub-Saharan Africa made it impossible to assess their impact (9).
Hard ticks harbor intracellular bacteria that show mutualistic and pathogenic associations to the host, the bacteria can be transmitted to mammals including humans (10). The most important tickborne disease in Kenya includes Theileriosis, anaplasmosis, babesiosis, and heartwater (11). A study was done along with Lake Victoria and Lake Baringo about the ticks molecular and pathogenic diversity by Omondi et al (12) reported pathogens like canine ehrlichiosis (E. ruminantium, Ehrlichia canis, and Ehrlichia sp). anaplasmosis (Anaplasma ovis, Anaplasm a platys and A. bovis) rickettsiosis (Rickettsia aeschlimannii) in ticks.
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 ticks' diversity and determining tick-borne pathogens.

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 o C until when analysed (12). The ticks were identified to the genus and/or species level as per the morphological keys (13) using sterile forceps and sterile petridish while on sterile 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. (14).

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 homogenizer oxide beads (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. Briefly, 20 µl of proteinase K was added into the 1.5-ml eppendorf tube with ticks' homogenates. Buffer ATL was mixed into the mixture and vortexed. Two hundred microliter of absolute ethanol was added and vortexed.
The content was transferred into the spin column before being centrifuged at 8000 rpm for 1 minute. The flow-through together with the collection tube was discarded. 500 µl of buffer AW1 was added into the spin-column that was placed into a new collection tube and centrifuged for 1 minute. Again the flow-through together with the collection tube was discarded. 500 µl of buffer AW2 was added into the spin-column that was placed in another new collection tube and centrifuged at 13000 rpm for 3 minutes. Only the flow-through was discarded and the collection tube re-used. Centrifugation of the spin column was done for 3 minutes at 13000 rpm. 200 µl of buffer AE was added into the spin column that was placed into a new 1.5-ml eppendorf tube and incubated at room temperatures for 1 minute. The spin column was centrifuged for 1 minute at 6 8000 rpm to extract the elute. The concentration and purity of the DNA were evaluated using a Nanodrop spectrophotometer (Thermo Fisher Scientific Wilmington, USA) and stored at -20°C.

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 containing final concentration of 1x 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 Ricketia 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] x 1000. The MIR assumes that only one tick is positive in a pool.

Tick diversity and abundance
At near Tsavo National Reserve in Taita Taveta County, a total of 317 (281 adult ticks and 36 nymphs) was collected which includes seven species (  Table 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.  Rickettsia aeschlimannii is also considered to be a zoonotic tick borne pathogen which has been reported on patients travelling from Africa (22). This pathogen has been detected in detected in Hyalomma ticks in Kenya (12). It is believed ticks of the genus Hyalomma serves as reservoirs for R. aeschlimannii (23). However, in the present study, R. aeschlimannii was detected in Am.
gemma. According to our knowledge this is the first time R. aeschlimannii has been detected in Am. gemma. Migratory birds may play an important role in the epidemiology of R. aeschlimannii by carrying R. aeschlimannii infetcted ticks from one region to another (24).
Uncharacterized Rickettsia sp. was detected in Am. variegatum ticks. Although its pathogenicity was unknown, it can be a public health threat in future (25). Many known Rickettsia spps. of public health concern today, were initially considered not to be harmful to humans. This species needs further characterization with better makers such as ompA, sca4, 17kDa (14).
Anaplasma platy is a TBP infecting dogs which is transmitted by brown dog ticks (Rhipicephalus sanguineus) (26). The pathogen has been reported also to infect humans causing mild headache, lethargy and myalgia (27). Humans get infected by coming into contact with dogs infested with ticks infected with Anaplasms platy (28). In the current study, Anaplasma platy was detected in

Rh. Evertsi.
Theileria parva is a TBP of veterinary importance which causes East Coast Fever in cattle (13). In this current study, Theileria parva was detected in Rh.appendiculatus ticks. These findings were consistent with those of Oundo et al. (14). The cattle get infected by coming into close proximity to buffaloes which are natural reservior for T.parva (29).

Conclusion
The detection of Rickettsia africae, Rickettsia aeschlimannii, and Anaplasma platy 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 like, any febrile illness, it is paramount to include tests that can diagnose these infections at clinical settings.