Research Article Repellent Activities of Essential Oils of Some Plants Used Traditionally to Control the Brown Ear Tick, Rhipicephalus appendiculatus

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1 Journl of Prsitology Reserch, Article ID , 10 pges Reserch Article Repellent Activities of Essentil ils of Some Plnts Used Trditionlly to Control the Brown Er Tick, Rhipicephlus ppendicultus Wycliffe Wnzl, 1,2 Ahmed ssnli, 2,3 Wolfgng Richrd Mukbn, 2,4 nd Willem Tkken 5 1 Deprtment of Biologicl Sciences, School of Pure nd Applied Sciences, South Estern Keny University, P.. Box , Kitui, Keny 2 Behviourl nd Chemicl Ecology Deprtment, Interntionl Centre of Insect Physiology nd Ecology, Africn Insect Science for Food nd elth, P.. Box GP, Nirobi, Keny 3 Chemistry Deprtment, School of Pure nd Applied Sciences, Kenytt University, P.. Box GP, Nirobi, Keny 4 School of Biologicl Sciences, University of Nirobi, P.. Box GP, Nirobi, Keny 5 Lbortory of Entomology, Wgeningen University nd Reserch Centre, P.. Box 8031, 6700 E Wgeningen, The Netherlnds Correspondence should be ddressed to Wycliffe Wnzl; osundw1@yhoo.com Received 12 ctober 2013; Revised 16 December 2013; Accepted 17 December 2013; Published 19 Februry 2014 Acdemic Editor: C. Genchi Copyright 2014 Wycliffe Wnzl et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Essentil oils of eight plnts, selected fter n ethnobotnicl survey conducted in Bukusu community in Bungom County, western Keny (Tgetes minut, Tithoni diversifoli, Juniperus procer, Solnecio mnnii, Senn didymobotry, Lntn cmr, Securidc longepeduncult, nd oslundi opposit), were initilly screened (t two doses) for their repellence ginst brown er tick, Rhipicephlus ppendicultus, using dul-choice climbing ssy. The oils of T. minut nd T. diversifoli were then selected for more detiled study. Dose-response evlutions of these oils showed tht T. minut oil ws more repellent (RD 50 = mg) thn tht of T. diversifoli (RD 50 = mg). Gs chromtogrphy-linked mss spectrometric (GC-MS) nlyses showed different compositions of the two oils. T. minut oil is comprised minly of cis-ocimene (43.78%), dihydrotgetone (16.71%), piperitenone (10.15%),trns-tgetone (8.67%), 3,9-epoxy-p-menth-1,8(10)diene (6.47%), β-ocimene (3.25%), nd cis-tgetone (1.95%), wheres T. diversifoli oil is comprised minly of α-pinene (63.64%), β-pinene (15.00%), isocryophyllene (7.62%), nerolidol (3.70%), 1- tridecnol (1.75%), limonene (1.52%), nd sbinene (1.00%). The results provide scientific rtionle for trditionl use of rw products of these plnts in controlling livestock ticks by the Bukusu community nd ly down some groundwork for exploiting prtilly refined products such s essentil oils of these plnts in protecting cttle ginst infesttions with R. ppendicultus. 1. Introduction In sub-shrn Afric, Est Cost fever (ECF), cused by Theileri prv prv, Theiler, 1904, nd trnsmitted by the brown er tick, Rhipicephlus ppendicultus, Neumnn, 1901, is one of the mjor constrints to the development of the livestock industry [1, 2]. f the estimted 12.7 millionhed of cttle (both indigenous nd exotic), 76% re t risk to ECF [3]. Thediseseisssocitedwithupto10%mortlityinzebu clves in ECF endemic res nd cn cuse up to 100% mortlity in susceptible exotic nd indigenous breeds [3, 4]. Prevention, control, nd mngement of both vector nd pthogen hve continued to rely hevily on the ppliction of synthetic chemicl cricides on the host since their introduction in 1902 in sub-shrn Afric [5]. owever, this hsprovedtobecostlyndunsustinbleinnumberofwys [1]. The cricides cn eliminte ticks from the host, but they do not prevent continued reinfesttion from the source environment, where ticks spend 90% of their life. For effective mngement of hrmful ticks, n integrted combintion of tctics my need to be put in plce tht controls ticks on individul hosts s well s in the host environment in order

2 2 Journl of Prsitology Reserch to prevent host reinfesttion during grzing. ne possible strtegywouldbetousetickrepellentsonthehostndtickrepellent plnts in the psture (host environment), combined with plnts tht re ttrctive to ticks such s Aclyph fruticos Forssk. vr. villos utch (Fmily: Euphorbicee) surrounding the psture lnd so s to develop push-pull tick mnipultion system [6, 7]. Although the proposed strtegy ppers complex, it my be possible to chieve in zero/ semizero grzing, smll-scle free-rnge, nd tethering livestock frming systems. In others, such s pstorlism nd lrge-scle livestock frming systems, the deployment of wellformulted repellents dispensed from controlled-relese dispensers my be more prcticl. N,N-diethyl-3-methylbenzmide (DEET) is still considered the best vilble product, repelling wide vriety of insects, ticks, nd mites [8]. Though DEET is not expected to bioccumulte, the mounts present in the environment hve beenshowntobetoxictosomespeciesofzooplnktonnd fish [9, 10]. In humns, the repellent my cuse insomni, mood disturbnces, impired cognitive functions, seizures, toxic encephlopthy, nd llergic rections [11 13]. This hs led to serch for lterntive eco-friendly nd effective repellents. The potentil of some locl plnts nd plnt products to repel ticks from grzing res nd host nimls, respectively, hs been demonstrted previously [14 18]. Melinis minutiflor (molsses grss), tropicl grss lredy in use s livestock fodder [19, 20], covers crop nd mulch [21] ndfor thtching houses [22], it hs been shown to be toxic [15]nd repellent to ticks [14, 23, 24] swellsinsectsndsnkes [21]. ne study demonstrting potentil of molsses grss to control Amblyomm vriegtum nd R. ppendicultus, vectors of the livestock diseses hertwter (cowdriosis) nd ECF, respectively, hs been reported [24]. In ddition, severl other Kenyn locl shrubs, including Cleome hirt nd Gynndropsis gynndr, hve demonstrted potentil s tickrepellent psture plnts [25 27]. Anumberofstudieshveshownthtplnt-bsedrepellents cn be comprble to DEET or even better [28 32]. ne commercil repellent product is the Fle nd Tick Grnulr Repellent, which is mde from essentil oils of cedr, cinnmon, mint, nd lemon grss; it hs plesnt odour nd cn be sfely used outdoors for fle nd tick control [33]. Essentil oils of number of other plnts hve been shown to be repellent to ticks. These include Commiphor erythre nd C. myrrh [34], Cleome monophyll [35], cimum suve [36], Cleome hirt [27], nd G. gynndr [37]. Use of tick-repellent plnts in psture lnds or essentil oils on hosts nd their integrtion with other off-host or onhost tick control mesures could be prcticl nd provide economic wys of controlling not only livestock ticks but lso rthropod vectors [38 40]. In our previous survey of livestock tick control ethnoprctices mong Bukusu community in Bungom district, western Keny, we found widespred use of ethnobotnicls derived from locl/ntive plnts to control tick infesttions on cttle [41]. Blends of botnicls from one or more plnts re used either s on-host suspensions or burnt nd smoke used to fumigte cttle. ur follow up objective hs been to ssess the repellence of essentil oils of some of these plnts ginst R. ppendicultus dults in the lbortory, to chrcterize the chemicl constituent profiles of the more repellent ones, nd then to initite both off- nd on-host evlution of their efficcy in controlling the ticks in the field. In the present pper, we report the results obtined from repellence ssys of essentil oils of 8 plnts ginst R. ppendicultus dults nd results of more detiled study of two selected plnts, Tgetes minut L. nd Tithoni diversifoli (emsl.) A. Gry. 2. Mterils nd Methods 2.1. Selection of Eight Plnt Species. An ethnobotnicl survey ws previously conducted in the Bukusu community in Bungom County, western Keny, long the southern slopes nd foothills of Mount Elgon t ltitudes rnging from bout 1,300 m in the south to bout 3,500 m in the north [41]. The County is locted between ltitude 0 25 Snd0 53 Nnd longitude Wnd35 04 E. Specimens of 157 plnt species, which were documented to hve vried effects on livestock ticks [41], were collected for txonomic exmintion t the herbrium of the School of Biologicl Sciences, University of Nirobi, Keny. The potentil efficcy of ech plnt species in protecting cttle ginst tick infesttions ws ssessed following four-level protocol proposed by einrich nd coworkers [42], nd eight plnt species were selected for initil lbortory screening [41, 43]. Voucher specimens of these plnts were deposited t the University of Nirobi erbrium, nd comprised of Tgetes minut L. (029-BGM- Mwi/2002), Tithoni diversifoli (emsl.) A. Gry (015-BGM- Muf/2002), Juniperus procer Endl. (134-BGM-Elg/2002), Solnecio mnii (ook. f.) C. Jeffrey. (106-BGM-Mwi/2002), Senn didymobotry (Fresen.). S. Irwin nd Brneby (132- BGM-Web/2002), Lntn cmr L. (043-BGM-Mwi/2002), Securidc longepeduncult Fres. (018-BGM-Mec/2002), nd oslundi opposit Vhl. (133-BGM-Bul/2002) Experimentl Ticks. Theticksused(thebrownertick, Rhipicephlus ppendicultus Neumnn, 1901) were obtined from the colonies t the Interntionl Livestock Reserch Institute(ILRI)ndbredintheinsectrytICIPE,Nirobi, Keny. Rering conditions nd mngement of ticks were s described previously [44, 45]. All the experiments were conducted using the newly emerged dult ticks of mixed sexes Isoltion of Essentil ils. The eril prts of ech of the eight plnts were collected from the southern slopes nd foothills of Mount Elgon in western Keny during the month ofaugustndllowedtodryinwell-ventiltedroomfor1-2 weeks.echplntmterilwscutintosmllpiecesnd bout 1 kg ws hydrodistilled using Clevenger-type pprtus for 8 h [46]. Essentil oil of ech plnt ws collected in 2mLvilsndstoredt 20 C in freezer until required for biossys or nlyses Dul-Choice Repellence Assys. A dul-choice tick repellence climbing ssy [47] tht exploits the behviour of R. ppendicultus to climb up grss stems to wit potentil

3 Journl of Prsitology Reserch 3 hosts pssing by [48, 49]wsused.Therepellenceofessentil oils of the eight plnts ginst R. ppendicultus ws first compredt0.1mgnd50mgdoses.themostrepellentoils(tht of T. minut nd tht of T. diversifoli)werethenselected for more detiled study. These plnts re lso highly rnked by livestock holders of Bukusu community in livestock tick prevention nd control [41]. The oils of the plnts were diluted serilly with dichloromethne (GC grde) to provide 0.5 mg to mg/10 μlofsolutions.anliquotof20μl of ech dose ws pplied to filter pper strip on the glss tubes, with n equivlent volume of dichloromethne dded to the control filter pper strip. The-set up ws llowed to equilibrte for 30 min before five dult R. ppendicultus of mixed ge nd sex were relesed on the bse of the climbing set-up ssy [47]. bservtions were mde over 1-hour period, nd the number of ticks bove the filter pper strip on the control glsstube(nc)ndontheglsstubewithtestmterils(nt) ws recorded t 15, 30, 45, nd 60 min. Twenty replictes for ech dose were crried out, ech time with fresh, nïve dult ticks. Initil comprison of the responses of ticks in the set-up with nd without residul dichloromethne on both sides showed no bis for either side nd no effects of the residul solvent on the dult ticks. The repellency of ech dose ws clculted using the formul: (number of ticks in control rm number of ticks in treted rm/totl responding ticks) 100. Dose-response dt were subjected to probit nlysis using the % repellencies from the replicted experiments [47]. 2.5.DetermintionoftheCompositionofT.minutndT. diversifoli Essentil ils. GC-MS nlyses of the two oils were performed with VG Msslb qudruple gs chromtogrphy-mss spectrometer. Chromtogrphic seprtions were chieved using fused silic cpillry column (ewlett Pckrd, 50 m 0.32 mm ID) coted with Crbowx 20 M (0.3 μm film thickness) with helium s the crrier gs. All the GC-MS nlyses were mde in the splitless mode with helium s the crrier gs. The oven temperture ws progrmmed from 60 Cfor7min,to120 Ct5 C per min, then to 180 Ct10 C per min, nd finlly to 220 Ct20 Cper min, where it ws mintined for l0 min. Constituents of the essentil oils were identified by nlysis of their mss spectr, direct comprison of these with those in the Wiley NBS nd NIST dtbses, nd coinjections with uthentic stndrds (from Sigm Chemicl Compny, Poole, UK nd Aldrich Chemicl Compny, Gillinghm, UK) on ewlett Pckrd P 5890A Gs Chromtogrph equipped with flme ioniztion detector (t 230 C). A fused silic cpillry column (ewlett Pckrd, 50 m 0.22 mm 0.33 mm CD) coted with methyl silicon (0.3μm film thickness) ws used with nitrogen s the crrier gs. All GC nlyses were performed in the splitless mode with the injector temperture t 270 Cnd oven temperture progrmme similr to tht in GC-MS nlyses Dt Anlysis. Dose-response dt were subjected to simple regression nd probit nlysis using the percent Men (%) repellency b Plnts 0.1 mg dose 50 mg dose b bc Figure 1: The repellent effect of essentil oils of eight plnts t doses of 0.1 mg nd 50 mg (net oil) ginst newly emerged Rhipicephlus ppendicultus dults. Plnt species 1 is Tgetes minut, 2is Tithoni diversifoli, 3isoslundi opposit, 4isSolnecio mnnii, 5is Lntn cmr,6isjuniperus procer,7issenn didymobotry nd 8isSecuridc longepeduncult. For given repellent dose, mens cpped by the sme lphbeticl letters re not significntly different t P < (Student-Newmn-Keuls test). repellency vlues obtined from replicted experiments nd regression model developed bsed on bc b Probit [Π (dose1)] =β 0 +β 1 x+, (1) where β 0 is the coefficient of the model representing yintercept, β 1 is the coefficient of the model representing dose1, x is the vrious concentrtions of essentil oils, dose1 is the Log 10 (dose), is the error term (residul term) representing the difference between the ctul observed vlue nd tht predicted by the model (the predictor (regressor) vrible, x is the dose of the essentil oil), nd Π is the repellency probbility. Student-Newmn-Keuls test ws used to compre the men vlues of repellency obtined for vrious doses of the repellent effects [50]. Percent repellency vlues were trnsformed into probbilities, while essentil oil doses were trnsformed into logrithms to bse 10 nd lines for regression models fitted using R softwre for Microsoft windows. These models were used to estimte repellent effects of the two essentil oils t RD 50 nd RD 75 [8, 51]. 3. Results 3.1.ScreeningoftheEssentililsIsoltedfromtheSelected Eight Plnt Species. The results of repellency tests following the screening of the essentil oils isolted from the eight plnts (T. minut, T. diversifoli, J. procer, S. mnnii, S. didymobotry, L. cmr, S. longepeduncult, nd. opposit)t 0.1 mg nd 50 mg doses re shown in Figure 1.Somevrition ws found in the repellent effect of the essentil oils t the two doses with tht of S. longepeduncult showing the lest repellent effect t both doses nd tht of T. minut showing the b bc c b

4 4 Journl of Prsitology Reserch highest repellent effect t the lower dose (80.1 ± 4.7%). The essentil oil of T. minut ndoneoftheothersixplnts(t. diversifoli) were therefore selected for more detiled biossy Dose-Response Repellency of the Essentil ils of T. minut nd T. diversifoli. The repellence of the two essentil oils t different doses is shown in Figures 2() nd 2(b).The essentil oil of T. minut ws found to be significntly more repellent thn tht of T. diversifoli t ll corresponding doses (P < 0.05). In boththe essentiloilsoft. minut nd T. diversifoli, there ws significnt correltion between repellence nd dose (Person Correltion, α = 0.01). Model development of the biossy dt of the two essentil oils llowed estimtion of RD 50 nd RD 75 (Tble 2). Previous work t ICIPE, Nirobi, Keny, tested vrious DEET doses under the sme lbortory conditions s described previously [27, 35, 37] nd determined their percent repellence ginst R. ppendicultus (Tble 3),withwhichwecompredthecurrentdose-response repellencies cused by the essentil oils of T. minut nd T. diversifoli Mjor Chemicl Components of the Essentil ils of Tgetes minut nd Tithoni diversifoli. Gs chromtogrphy (GC) in combintion with gs chromtogrphy/mss spectrometry (GC-MS) seprted the chemicl components in the mixtures of the essentil oils of T. minut nd T. diversifoli plnts, nd the mjor representtive GC/GC- MSprofilesreshowninTbles1() nd 1(b), respectively. The mjor chemicl components of T. minut essentil oil were cis-ocimene (43.78%), dihydrotgetone (16.71%), piperitenone (10.15%), trns-tgetone (8.67%), 3.9-epoxyp-menth-1.8(10)diene (6.47%), β-ocimene (3.25%), cistgetone (1.95%), nd β-cryophyllene (0.84%). Those chemicl components of the essentil oil of T. diversifoli were minly α-pinene (63.64%), β-pinene (15.00%), isocryophyllene (7.62%), nerolidol (3.70%), 1-tridecnol (1.75%), limonene (1.52%), nd sbinene (1.00%). 4. Discussion In previous study, we undertook survey of indigenous knowledgeofthebukusucommunityofwesternkenyon livestock ticks, the risks they pose nd ethnoprctices ssocited with their mngement [41]. The study showed tht the Bukusu community hs ccumulted rich ethnoveterinry knowledge nd prctices nd tht on-host use of ethnobotnicl suspensions nd fumigtion of host nimls with voltiles from burning plnt products (prepred from one or more of 157 plnts) constitute importnt methods of controlling the ticks. In the present study, repellence of essentil oils ssocited with some of the plnts ws used to ssess one possible mode of ction of the plnt products on ticks. Eight of these plnts were selected for screening ginst the dults of the brown er tick following four-level ssessment protocol (bsed on dditionl ethnobotnicl informtion on similr use of the plnt elsewhere, reported phytochemicl profile of the plnt or relted species, nd ny documented bioctivity % Men (%) repellency e de de dc d d d dc Dose (mg) () cd Dose (mg) (b) Figure 2: Men repellency percentge of different doses of Tgetes minut () nd Tithoni diversifoli (b) essentil oils ginst newly emerged dults, Rhipicephlus ppendicultus, in dul-choice ssy. Mens with the sme lphbeticl letters re not significntly different t P < (Student-Newmn-Keuls test). dt of the plnt extrcts or their phytochemicls) proposed by einrich nd coworkers [42]. The essentil oils of these plnts showed some vrition in repellence ginst newly emerged R. ppendicultus dult ticks. This ws prticulrly pprent t the lower dose (0.1 mg), with the essentil oil of T. minut showing the highest repellence nd tht of S. longepeduncult showing the lest repellent effect. Comprehensive repellence studies with T. minut nd T. diversifoli oils t eight doses confirmed the higher repellence of the former ginst R. ppendicultus. Interestingly, its repellent effect is comprble to tht of commonly used repellent DEET t 0.1 mg dose (with essentil oil of T. minut producing repellent effect of 80.1 ± 4.9% compred with tht of DEED, 84.0 ± 3.9%). Additionlly, the results obtined by this study re compred fvourbly with the results reported by Nchu nd coworkers [52] for the essentil oil of T. minut plnts growing in Pretori, South Afric, ginst ylomm rufipes Koch.. rufipes is lso widely distributed in Afric nd cn trnsmit disese-cusing virl nd protozon (e.g., Crimen-Congo hemorrhgic fever nd Bbesi, resp.) pthogens to livestock nd humns like [53, 54]. Although the T. minut oil obtined in the present study shres series of mjor constituents (e.g., cis-ocimene, dihydrotgetone, piperitenone, tgetone, nd β-ocimene) with tht isolted in Pretori, South Afric [52], there re lso some chemotypic differences between the two, reflected in different proportions of these compounds nd the presence of some constituents in one chemotype (e.g., 3-methyl-2-(2-methyl-2-butenyl)-furn c c bc b b

5 Journl of Prsitology Reserch 5 Tble 1: The mjor chemicl components in the essentil oils of Tgetes minut () nd Tithoni diversifoli (b) identified using cpillry column gs chromtogrphy in combintion with gs chromtogrphy/mss spectrometry. RT (min) Chemicl nme IUPAC nme Moleculr formul () M + (g/mol) Density (g/cm 3 ) CAS number Structure Reltive bundnce (%) cis-cimene 3,7-Dimethyl-1,3,7-octtriene C Dihydrotgetone 2,6-Dimethyloct-7-en-4-one C Piperitenone 2-Methyl-6-propn-2- ylidenecyclohex-2-en-1-one C trns-tgetone (5E)-2,6-dimethyloct-5,7-dien-4-one C ,9 Epoxy-p-menth-1,8 (10) diene C β-cimene (3E)-3,7-Dimethyloct-1,3,6-triene C cis-tgetone (Z)-2,6-Dimethyloct-5,7-dien-4-one C β-cryophyllene (trns) 3,6-Dimethylidene-4,5,7,7tetrhydro-3-1-benzofurn 4,11,11-Trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene C Bicyclogermcrene (1R, 4E,8E, 10S)-4,8,11,11- Tetrmethylbicyclo[8.1.0]undec-4,8- diene C

6 6 Journl of Prsitology Reserch RT (min) Chemicl nme IUPAC nme Moleculr formul () Continued. M + (g/mol) Density (g/cm 3 ) CAS number Structure Reltive bundnce (%) AR-turmerone (6S)-2-Methyl-6-(4- methylphenyl)hept-2-en-4-one RT (min) Compound IUPAC nme C Moleculr formul (b) M + (g/mol) Density (g/cm 3 ) CAS registry number Structure 0.50 Reltive bundnce (%) α-pinene 2,6,6-Trimethylbicyclo[3.1.1]hept-2- ene C β-pinene 6,6-Dimethyl-2- methylenebicyclo[3.1.1]heptne C Isocryophyllene (1R,4Z,9S)-4,11,11-trimethyl-8- methylidenebicyclo[7.2.0]undec-4-ene C Nerolidol (6E)-3,7,11-Trimethyl-1,6,10- dodectrien-3-ol C Tridecnol 1-Tridecnol C Limonene 1-Methyl-4-(1-methylethenyl)- cyclohexene C

7 Journl of Prsitology Reserch 7 RT (min) Compound IUPAC nme Moleculr formul (b) Continued. M + (g/mol) Density (g/cm 3 ) CAS registry number Structure Reltive bundnce (%) Sbinene 4-Methylene-1-(1- methylethyl)bicyclo[3.1.0]hexne C α-copene (1R,2S,6S,7S,8S)-8-Isopropyl-1,3- dimethyltricyclo[ ,7 ]dec-3-ene C α-gurjunene C Cyclodecene (4R,4R)-1,1,4,7-Tetrmethyl- 1,2,3,4,4,5,6,7bocthydrocycloprop[e]zulene 6,10-Dimethyl-3-(1-methylethyl)-6- cyclodecene-1,4-dione C Key: M + : moleculr weight. RT: retention time in minutes (min.). IUPAC: The IUPAC nomenclture system of orgnic chemistry is systemtic method of nming orgnic chemicl compounds s recommended by the Interntionl Union of Pure nd Applied Chemistry (IUPAC). CAS registry numbers: Are unique numericl identifier (vlues) numbers ssigned by the chemicl bstrcts service (CAS) to every chemicl described in the open scientific literture (currently including those chemicls described from t lest 1957 through the present) nd including elements, isotopes, orgnic nd inorgnic compounds, ions, orgnometllics, metls, nonstructurble mterils, nd so forth.

8 8 Journl of Prsitology Reserch Tble 2: Probit nlysis of dose-response reltionship of Tgetes minut nd Tithoni diversifoli essentil oils t RD 50 nd RD 75 generted by regression model: Probit [Π(dose1)] = dose1 for the essentil oil of T. minut nd the regression model: Probit [Π(dose1)] = dose1 for the essentil oil of T. diversifoli. Plnt species Repellence probbility Repellent dose (mg) Upper confidence limit t 95% Lower confidence limit t 95% Tgetes minut Tithoni diversifoli Tble 3: Men percent repellence (±SE) of N,N-diethyl-3-methylbenzmide (DEET) evluted in dul-choice ssy ginst newly emerged dult ticks, Rhipicephlus ppendicultus, t the Interntionl Centre of Insect Physiology nd Ecology, Nirobi, Keny, under the sme lbortory conditions s the current studies. Repellent dose (mg) Repellency (%) ± ± ± ± 3.6 Sources: Ndung u et l. [35], 1995; Lwndeet l. [37], 1998; Ndung u et l.[27], in T. minut oil collected in Pretori, South Afric) tht were not detected in the other. Which constituents contribute to the repellence of the essentil oil of T. minut ginst the two tick species, respectively, must wit detiled subtrctive ssys [55] with synthetic blends of the mjor constituents of the two essentil oils with ech component missing t time. Nchu nd coworkers [52]lso found tht T. minut oil significntly delyed moulting of. rufipes engorged nymphs. In nother study reported by Krishn nd coworkers [56], most eggs of the coleoptern beetle, Tribolium cstneum, exposed to the vpours of essentil oil of specific genotype of T. minut filed to htch. Both these effects hve been ttributed to tgetone, one of the mjor constituents of T. minut previously shown to hve growth disrupting bioctivities on rthropods [57]. Since trns-tgetone is prominent constituent of T. minut essentil oil of Bungom chemotype (8.7%, compred with 1.6% in the Pretori chemotype), it will be interesting to see if similr repellent effects re lso observed with nymphl R. ppendicultus. In this study, essentil oil of T. diversifoli growing in Bumgom ws found to be less repellent thn tht of T. minut. This is reflected in its very different terpenoid profile. owever, the plnt is highly vlued for its tick control property by the Bukusu community in Bungom [41]. Interestingly,itislsousedbytheKikuyucommunityincentrl Keny to control livestock ticks [58]ndbytheLuyhndLuo communitiesinwesternkenytocontrolinsectpestsin rble frming systems [59, 60]. This suggests other possible modes of ction of T. diversifoli phytochemicl profile. Follow-up studies on other potentil modes of ction of the essentil oil nd nonvoltile constituents of the plnt my help to shed light on this question. 5. Conclusion In conclusion, the present study provides some scientific rtionle for the incorportion of some botnicls in Bukusu ethnoprctices in western Keny to control tick infesttions on cttle. The study lso lys down some groundwork for follow-up studies on other possible effects of the phytochemicls of the plnts studied nd for exploiting prtilly refined products such s essentil oils in protecting cttle ginst infesttionsby R. ppendicultus nd other tick species. Conflict of Interests The uthors declre tht they hve no competing interests of ny kind tht could inppropritely influence (bis) the publiction of this pper. Funding This reserch work ws jointly supported by the Interntionl Foundtion for Sciences, Stockholm, Sweden, nd the rgniztion for the Prohibition of Chemicl wepons, The gue, The Netherlnds, through Grnt AB/ The first uthor wishes to cknowledge the finncil, logistic, nd mteril support received from the Interntionl Centre of Insect Physiology nd Ecology (ICIPE) under the Africn Regionl Postgrdute Progrmme in Insect Science (ARP- PIS) nd Wgeningen University nd Reserch Centre, Lbortory of Entomology under sndwich Ph.D. Fellowship. Acknowledgments The uthors wish to thnk Mr. Simon M. Mthenge from the University of Nirobi erbrium for identifiction of plnt species; ICIPE s stff for offering their technicl nd dministrtive support, nd Bukusu community ethnoprctitioners nd livestock frmers for their coopertion, prticiption nd support throughout the study period. They lso wish to thnk the locl dministrtion of the Government of Keny for supporting, prticipting, nd giving permission to smple plnts for isoltion of essentil oil. References [1] R.A.I.Norvl,B.D.Perry,ndA.S.Young,The Epidemiology of Theileriosis in Afric, Acdemic Press, London, UK, 1992.

9 Journl of Prsitology Reserch 9 [2] J.M.lwoch,B.Reyers,F.A.Engelbrecht,ndB.F.N.Ersmus, Climte chnge nd the tick-borne disese, Theileriosis (Est Cost fever) in sub-shrn Afric, Journl of Arid Environments,vol.72,no.2,pp ,2008. [3] A. J. Lwrence, J. A. de Vos, nd A. D. Irvin, Est cost fever, in Infectious Diseses of LivesTock With Specil Reference to Southern Afric, G.R.ThomsonndR.C.Tustin,Eds.,Cpe Town xford University Press, Cpe Town, South Afric, [4] K.S.Mbogo,P.D.Kriuki,N.Mcrdy,ndR.Pyne,Trining Mnul For Veterinry Stff on Immuniztion Aginst Est Cost Fever Using the ECFiM System, KenyAgriculturlReserch Institute nd verses Development Administrtion of UK, Nirobi, Keny, [5] Future of livestock industries in Est nd southern Afric, in Proceedings of the Workshop eld t Kdom Rnch otel, Zimbbwe Interntionl Livestock Centre for Afric (ILCA 92),J. A. Ktegile nd S. Mubi, Eds., pp , Addis Abb, Ethiopi, July [6]A.ssnli,.erren,Z.R.Khn,J.A.Pickett,ndC.M. 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