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Crimean-Congo hemorrhagic fever virus in hyalommid ticks, Northeastern Kenya.

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus, family Bunyaviridae, causes hemorrhagic disease in humans with a >30% casefatality rate. The virus was first described in 1944 in the Crimea in the former Soviet Union and later was found to be similar to a virus isolated in 1956 in the Belgian Congo (1).

Domestic ruminants are infected through tick bites and are able to infect more ticks to perpetuate the virus (2,3). The virus may be transmitted to humans by the bite of an infected tick or by contact with body fluids from an infected animal or person (4). The main vectors of CCHFV are ticks in the genus Hyalomma, family Ixodidae, with other ixodid ticks and ticks from the family Argasidae also contributing to transmission (2). The virus is transovarially transmitted among ticks (2,3); consequently, ticks are also reservoirs of CCHFV.

In Kenya, CCHFV has been detected on only 2 occasions: in Rhipicephalus pulchellus ticks collected in the 1970s from a dying sheep in a veterinary laboratory in the town of Kabete outside Nairobi (2) and from a person with Crimean-Congo hemorrhagic fever in western Kenya in October 2000 (5). Evidence of CCHFV activity in Kenya is limited, and although tick-borne arbovirus surveillance in Kenya has demonstrated circulation of a range of viruses, to our knowledge, detection of CCHFV has not been reported (6,7).

Crimean-Congo hemorrhagic fever is a substantial public health threat because of the associated high mortality rate (30%-60%), the potential for person-to-person transmission, the unavailability of a licensed vaccine, and the limited treatment options for infected persons (3,4). Entomologic surveillance is valuable for assessing the risk for human exposure and for identifying so-called hot spots for focused preventive action to minimize the effects of virus outbreaks. As part of ongoing entomologic arbovirus surveillance conducted by the United States Army Medical Research Unit in Kenya and the Kenya Medical Research Institute, ticks were collected from livestock in the semiarid areas of Kenya, where intense pastoralist farming is practiced, to assess the risk to the community for tick-borne arbovirus exposure.

The Study

Ticks were sampled in the villages of Diiso and ElHumow and at the livestock market and abattoirs in Garissa District, North Eastern Province of Kenya, during April-May 2008 (Figure). 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).

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 [micro]g/mL streptomycin, and 1 [micro]L/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 (1x medium 199 with Earle salts, 1% bovine albumin, 100 U/mL penicillin, 100 [micro]g/mL streptomycin, and 1 [micro]L/mL amphotericin B) under high containment. The homogenates were clarified by centrifugation at 1,500 rpm for 15 min at 4[degrees]C, and supernatants were stored at -80[degrees]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.

[FIGURE OMITTED]

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 3x more frequently in Diiso than in El-Humow (Table). 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 (Table) in which single DNA bands corresponding to the predicted 536-bp PCR product were detected.

Conclusions

The detection of CCHFV in pools of Hyalomma spp. ticks from Diiso village and the Garissa District slaughterhouse provides strong evidence of CCHFV presence in northeastern Kenya and indicates that CCHFV circulation in Kenya is underestimated. CCHFV was detected only in ticks collected from cattle and a camel. Livestock play a role in the amplification of the virus because the animals become viremic for 7 days (2,3), during which time they can infect more ticks. Our findings indicate that CCHFV circulates in northeastern Kenya with substantial involvement of camels and cattle. The detection of CCHFV in ticks from camels at the slaughterhouse also suggests the potential of exposure for abattoir workers. The presence of CCHFV among hyalommid ticks in northern Kenya highlights the risk to the resident population and requires the assessment of human exposure. Health care workers must therefore help create awareness among the population and take steps to prepare for and prevent outbreaks.

DOI: 10.320/eid1708.102064

Acknowledgment

We thank Dunstone Beti, Reuben Lugalia, John Gachoya, David Oulo, and Daniel Ngonga for technical support.

This work received financial support from the Global Disease Detection Program (Centers for Disease Control and Prevention) and the Armed Forces Health and Surveillance Center-Walter Reed Project, Kenya.

Dr Sang is the head of the Arbovirus and Viral Hemorrhagic Fever Laboratory at the Kenya Medical Research Institute. Her interests include the mechanisms of arbovirus maintenance and spread in ecosystems.

References

(1.) Watts DM, Ksiazek TG, Linthicum KJ, Hoogstraal H. CrimeanCongo hemorrhagic fever. In: Monath TP, editor. The arboviruses: epidemiology and ecology, vol. 2. Boca Raton (FL): CRC Press; 1989.

(2.) Hoogstraal H. The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. J Med Entomol. 1979;15:307-17.

(3.) Mardani M, Keshtkar-Jahromi M. Crimean-Congo hemorrhagic fever. Arch Iran Med. 2007;10:204-14.

(4.) Burney MI, Ghafoor A, Saleen M, Webb PA, Casals J. Nosocomial outbreak of viral hemorrhagic fever caused by Crimean hemorrhagic fever-Congo virus in Pakistan, January 1976. Am J Trop Med Hyg. 1980;29:941-7.

(5.) Dunster L, Dunster M, Ofula V, Beti D, Kazooba-Voskamp F, Burt F, et al. First documentation of human Crimean-Congo hemorrhagic fever, Kenya. Emerg Infect Dis. 2002;8:1005-6.

(6.) Johnson BK, Chanas AC, Squires EJ, Shockley P, Simpson DI, Parsons J, et al. Arbovirus isolations from ixodid tick infesting livestock, Kano Plain, Kenya. Trans R Soc Trop Med Hyg. 1980;74:732-7. doi:10.1016/0035-9203(80)90188-1

(7.) Sang R, Onyango C, Gachoya J, Mabinda E, Konongoi S, Ofula V, et al. Tickborne arbovirus surveillance in market livestock, Nairobi, Kenya. Emerg Infect Dis. 2006;12:1074-80.

(8.) Statoids. Administrative districts of countries. Districts of Kenya. http://www.statoids.com/yke.html

(9.) Matthysse JG, Colbo MH. The ixodid ticks of Uganda together with species pertinent to Uganda because of their present known distribution. New York: Oxford University Press; 1987.

(10.) Okello-Onen J, Hassan SM, Essuman S. Taxonomy of African ticks. Nairobi (Kenya): International Centre of Insect Physiology and Ecology Science Press; 1999.

(11.) O'Guinn ML, Lee JS, Kondig JP, Fernandez Rand, Carbajal F. Field detection of eastern equine encephalitis virus in the Amazon Basin region of Peru using reverse transcription-polymerase chain reaction adapted for field identification of arthropod-borne pathogens. Am J Trop Med Hyg. 2004;70:164-71.

(12.) Burt FJ, Leman PA, Smith JF, Swanepoel R. The use of reverse transcription-polymerase chain reaction for the detection of viral nucleic acid in the diagnosis of Crimean-Congo hemorrhagic fever. J Virol Methods. 1998;70:129-37. doi:10.1016/S0166-0934(97)00182-1

Address for correspondence: Rosemary Sang, Kenya Medical Research Institute, Centre for Virus Research, PO Box 54626, Nairobi 00200, Kenya; email: rsang@wrp-nbo.org

The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.

Author affiliations: Kenya Medical Research Institute, Nairobi, Kenya (R. Sang, J. Lutomiah, E. Chepkorir); US Army Medical Research Unit Nairobi (H. Koka, A. Makio, C. Ochieng, S. Yalwala, J. Mutisya, L. Musila, J.H. Richardson, D. Schnabel); and Centers for Disease Control and Prevention, Fort Collins, Colorado, USA (B.R. Miller)
Table. 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 *

                                                   No. pools
Site and animal      Tick species      No. ticks    tested

El-Humow village
  Goat                Amblyomma             64         16
                      variegatum
  Goat                 Am. gemma            47          6
  Cattle              Am. lepidum           48          6
  Camel             Rhipicephalus           94         12
                    appendiculatus
  Camel            Hyalomma rufipes         17          3
  Cattle              Hy. rufipes          124         16
  Sheep               Hy. rufipes           50          7
  Camel              Hy. truncatum          22          3
  Cattle             Hy. truncatum         623         81
  Sheep              Hy. truncatum          24          3
  Cattle              Hyaloma sp.           73         10
  Cattle            Rh. pulchellus         748         94
  Sheep             Rh. pulchellus         162         21

Diiso village
  Cattle              Hy. rufipes          160         20
  Camel              Hy. truncatum       1,034        132
  Cattle             Hy. truncatum       1,513        191
  Camel              Hyalomma sp.          191         24
  Cattle             Hyalomma sp.          192         59
  Camel             Rh. pulchellus         168         21
  Cattle            Rh. pulchellus       1,297        163
  Goat              Rh. pulchellus         420         53

Livestock market
  Cattle             Hy. truncatum         266         34
  Cattle             Hyalomma sp.            7          1

Slaughterhouse
  Camel               Hy. rufipes           90         12
  Camel              Hy. truncatum         642         81
  Camel              Hyalomma sp.           76         19
  Camel             Rh. pulchellus         448         56

Totals                                   8,600      1,144

                     No. CCHFV-
Site and animal    positive pools

El-Humow village
  Goat                   0

  Goat                   0
  Cattle                 0
  Camel                  0

  Camel                  0
  Cattle                 0
  Sheep                  0
  Camel                  0
  Cattle                 0
  Sheep                  0
  Cattle                 0
  Cattle                 0
  Sheep                  0

Diiso village
  Cattle                 3
  Camel                  4
  Cattle                14
  Camel                  0
  Cattle                 1
  Camel                  0
  Cattle                 0
  Goat                   0

Livestock market
  Cattle                 0
  Cattle                 0

Slaughterhouse
  Camel                  1
  Camel                  0
  Camel                  0
  Camel                  0
Totals                  23

* CCHFV, Congo-Crimean hemorrhagic fever virus.
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Article Details
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Title Annotation:DISPATCHES
Author:Sang, Rosemary; Lutomiah, Joel; Koka, Hellen; Makio, Albina; Chepkorir, Edith; Ochieng, Caroline; Ya
Publication:Emerging Infectious Diseases
Article Type:Report
Geographic Code:6KENY
Date:Aug 1, 2011
Words:1900
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