Ticks were sampled in the villages of Diiso and El-Humow and at the livestock market and abattoirs in Garissa District, North Eastern Province of Kenya, during April–May 2008 (). Garissa District is in a semi-arid to arid ecologic zone that receives sporadic rainfall from March to May; vegetation consists primarily of Acacia-Commiphora
bushes. Its population is largely composed of nomadic herders who travel between districts in northern Kenya in search of water and pasture (8
Location of Garissa District (A, box) in North Eastern Province, Kenya, and tick collection sites (B).
Ticks were picked by hand from infested livestock, stored in labeled sterile vials, and transported in liquid nitrogen to the Kenya Medical Research Institute laboratory. In the laboratory, ticks were washed in sterile water, rinsed first with 70% ethanol, and then rinsed with minimum essential medium containing antimicrobial agents (100 U/mL penicillin, 100 μg/mL streptomycin, and 1 μL/mL amphotericin B). They were identified to species by using taxonomic keys (9,10
) and pooled in groups of 2 to 10 by species, sex, collection date and site, and host. The tick pools were homogenized by using 90-mesh alundum sand in a prechilled, sterile mortar and pestle with 1.6 mL–2 mL ice-cold bovine albumin 1 medium (1× medium 199 with Earle salts, 1% bovine albumin, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 μL/mL amphotericin B) under high containment. The homogenates were clarified by centrifugation at 1,500 rpm for 15 min at 4°C, and supernatants were stored at –80°C.
Viral RNA was extracted from tick homogenates by using Trizol-LS (Invitrogen, Carlsbad, CA, USA) reagent, according to the manufacturer’s instructions. RNA was screened by reverse transcription PCR (11
) to amplify a 536-bp fragment of the gene encoding for the nucleocapsid protein in the small (S) segment of the CCHFV genome by using the following primers (12
): CCHF F2 (5′-TGGACACCTTCACAAACTC-3′) and R3 (5′-GACAAATTCCCTGCACCA-3′), positions 135–153 and 653- 670, respectively, on the reference strain CCHFV 10200.
Electrophoresis of the PCR products was performed by using 1% agarose gels in Tris-acetate-EDTA buffer containing ethidium bromide; product bands were visualized and documented with the Canon UVP PhotoDoc-It gel imaging system (UVP, LLC, Upland, CA, USA) mounted with a digital camera. The PCR products of a subset of 4 of the CCHFV-positive homogenates were purified by using the QIAquick PCR Purification Kit (QIAGEN Sciences, Germantown, MD, USA), according to the manufacturer’s instructions, and sequenced by using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the ABI 3730 and automated 3130xl Genetic Analyzer (Applied Biosystems). The sequences were analyzed by using the Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi
) and the GenBank database to confirm the identity of the virus. Data (including tick species, collection site, animal host, and virologic test results) were entered into an Excel database (Microsoft Corp., Redmond, WA, USA) and analyzed by using pivot tables. A total of 8,600 ticks, of 3 genera and 8 species, were sampled primarily from camels, cattle, goats, and sheep, principally Hyalomma rufipes
and Hy. truncatum
. Ticks of the genus Hyalomma
were sampled 3× more frequently in Diiso than in El-Humow (). CCHFV was detected in 23 Hyalomma
spp. tick pools from Diiso and the Garissa slaughterhouse, including 4 pools of Hy. rufipes
(3 from cattle and 1 from a camel), 18 pools of Hy. truncatum
(14 from cattle and 4 from camels), and 1 unidentified Hyalomma
species () in which single DNA bands corresponding to the predicted 536-bp PCR product were detected.
Ticks species sampled from different livestock animals from 4 sites within Garissa District, northeastern Kenya, and CCHFV infection detected from tick pools, April–May 2008*