MOLECULAR CHARACTERIZATION OF TICKS, TICKBORNE VIRUSES AND RICKETTSIAL PATHOGENS IN TICKS COLLECTED FROM SELECTED COUNTIES IN KENYA

Abstract

Ticks are excellent vectors of pathogens that cause morbidity and mortality in man, livestock and wildlife. At least 900 species of ticks are known globally. With the expansion of ticks into new habitats as a result of climate change and their heterogeneous distribution, accurate identification is important in understanding the epidemiology of tick-borne diseases (viral, parasitic or bacterial) so as to establish the most effective and targeted control strategies. In Kenya, there has been an increased focus on ticks due to their ability to transmit diverse tick-borne diseases. This study sought to characterize ticks, tick-borne viruses and rickettsial pathogens in ticks collected from selected counties of Turkana, West Pokot, Isiolo, Baringo, Marsabit and Kwale in Kenya. To determine distribution and diversity, archived ticks collected from livestock in the study sites were identified to the species level using morphological identification keys and pooled then homogenized. A subset of representative individual (unpooled) ticks (n=30) of different species at each of the four sites (Turkana, Isiolo, Baringo and West Pokot counties) were also subjected to molecular identification and characterization to determine genetic diversity. DNA was extracted from 120 tick homogenates and PCR was performed using the mitochondrion cytochrome c oxidase (cox1) gene. The PCR products were cleaned using Exo-sap, sequenced and sequences compared with those on GenBank by Blast analysis. Sequences with identities scores of 96–100% were used in phylogenetic analysis. Thirdly, the supernatant from the pooled tick samples were also inoculated in Vero cells and those that showed virus growth by exhibiting cytopathic effect (CPE) were harvested. The virus isolates obtained were sequenced and phylogenetic analysis was performed. Lastly, DNA was also extracted from the 379 pooled tick homogenates that were randomly selected from the 2259 pooled ticks to 13 represent each of the six counties. PCR was carried out using three rickettsial primer sets gltA, OmpA and OmpB. A subset of the PCR amplicons that were not identified adequately were further sequenced using the 16S rRNA primer. A total of 15,773 ticks were identified from six sites. The highest number of ticks had been collected from Turkana (n = 806, 35.7%), followed by Isiolo (n = 409, 18.1%), Baringo (n = 403, 17.8%), Kwale (n=232, 10.3%), Marsabit (n=253, 11.2%) while West Pokot had the least collection (n = 156, 6.9%) (Chi-square test=863.7, df=5, p <0.001). Most of the ticks were collected from camels (33%), cattle (26.6%), sheep (22.3%) and goats (18.1%) respectively. Thirteen tick species were identified across the six sites and pooled (n = 2259). Some of the tick species that were morphologically identified across the sites included: R. appendiculatus H. marginatum, H. truncatum, A. gemma, R. pulchellus, H. dromedarii, A. variegatum, A. lepidum and R. evertsi. Following molecular characterization of the ticks, 84 sequences from the mitochondrial cox1 gene marker were obtained and phylogenetic analysis done. There were no differences in molecular characterization of Amblyomma species in comparison to morphological identification. However, there were some differences in the Hyalomma and Rhipicephalus species identification by molecular analysis in comparison to morphological identification. In addition, fourteen virus isolates were obtained from the n = 2259 tick pools inoculated. Ten (10) isolates were from Isiolo County, two (2) each from Baringo and Kwale Counties. Two novel viruses, a Thogoto-like virus and another in the genus Bandavirus, tentatively named Kinna virus which was closely related to Guertu virus, were identified by Whole Genome Sequencing. Kinna is the site in Isiolo County where the positive ticks were collected. The other 12 isolates obtained in this study are to be sequenced. Moreover, the prevalence of rickettsial pathogens by the gltA, OmpA, rompB primers was 21.9%, 35.6% and 41.7% respectively. 123 (32.5%, 95% CI: 27.9–37.3%) of the 379 samples were positive by at least two primers. This proportion varied significantly by region (p=0.003) from a low of 15.5% in Turkana to a high of 45.3% in Isiolo. Tick species of veterinary and medical importance were found across the sites using morphological identification techniques. Tick species identification using morphological and molecular tools for Amblyomma species were comparable. However, there was disparity in the data reported for the morphological identification and molecular classification of some of the Hyalomma and Rhipicephalus tick species. The inconsistency in data comparing the Hyalomma species was attributed to introgressive hybridization in this species while for R. appendiculatus species, the inconsistency was due to misidentification. This study reports the isolation of a novel virus closely related to Guertu virus and Severe Fever with thrombocytopenia syndrome virus (genus Bandavirus) from a tick collected in Isiolo County, tentatively named Kinna virus. This novel-tick-borne virus like other emerging life threatening Bunyaviruses may pose a risk to public health in Isiolo County. In addition, a novel Thogoto-like virus, was also isolated and will be characterized. Therefore, this study provides more data on the viruses’ endemic in pastoral communities. This study also reports a high prevalence of rickettsial pathogens in all the sites and highlights the need for surveillance and detection of the disease in human populations at the sites. This is a preventable disease as treatment is available. Overall, the study has highlighted the public health importance of ticks and tick-borne pathogens. The data obtained from this study will inform evidence-based policies geared towards tick control, population sensitization and disease control. The sequences obtained will be useful in the development of diagnostics or therapeutic interventions.