Ethnobotanicals for management of the brown ear tick Rhipicephalus appendiculatus in western Kenya. Wycliffe Wanzala. 4^1 w

Transcription

1 Ethnbtanicals fr management f the brwn ear tick Rhipicephalus appendiculatus in western Kenya Wycliffe Wanzala 4^ w

2 Prmtren: Prf.dr. J.C. van Lenteren C-prmtren: Prf. dr. ir. W. Takken Prf. A.Hassanali Prmtiecmmissie: Prf. dr.ir. M.W. Sabelis Prf. dr. H.H.T. Prins Dr. W.J. de Kgel Hgleraar in de Entmlgie, Wageningen Universiteit Persnlijk Hgleraar bij de leerstelgrep Medische en Veterinaire Entmlgie, Wageningen Universiteit Internatinal Centre f Insect Physilgy and Eclgy, Kenya Universiteit van Amsterdam Wageningen Universiteit Plant Research Internatinal, Wageningen Universiteit and Research Centrum Prf. dr.ir. A.J. van der Zijpp Wageningen Universiteit Dit nderzek is uitgeverd binnen de CT de Wit nderzeksschl Prductin Eclgy and Resurce Cnservatin

3 Wycliffe Wanzala Ethnbtanicals fr management f the brwn ear tick Rhipicephalus appendiculatus in western Kenya Prefschrift ter verkrijging van de graad van dctr p gezag van de rectr magnificus van Wageningen Universiteit Prf. dr. M.J. Krpff inhet penbaar te verdedigen p wensdag 3maart, 2009 des namiddags te vier uur in de Aula

4 Wanzala, W. (2009) Ethnbtanicals fr management f the brwn ear tick Rhipicephalus appendiculatus in western Kenya PhD thesis Wageningen University - with references - with summaries in Dutch and English ISBN

5 Summary This thesis describes the results f a study t assess the effect f ethnbtanical prducts n the behaviur f the brwn ear tick Rhipicephalus appendiculatus, the main vectr f East Cast fever in sub-saharan Africa. Ethnknwledge f the Bukusu peple in western Kenya n tick cntrl and management was evaluated t identify plants that affect livestck ticks, using participatry actin research appraches. Mre than 50 plant species spread ver 0 genera and 5 families were identified and dcumented. Frm these, eight plants were selected and their essential ils extracted and used fr screening in the labratry n their behaviural effects n ticks. Frm these, the plants Tagetes minuta and Tithnia diversiflia were chsen fr further studies. The essential ils f these tw plants were further extracted and used in labratry and field biassays. Frm the labratry assay, using a dual-chice apparatus, it was fund that essential ils f bth T. minuta and T. diversiflia affect tick climbing behaviur, representing a repellent respnse. Dse respnse effects were bserved. On steers, differential effects t the essential ils were bserved with R. appendiculatus, which prefer t feed mainly inside the ears f the hst animal. It was fund that treatment f the ear regin with the essential ils f bth T. minuta and T. diversiflia significantly deterred ticks frm reaching the ear. The essential ils f T. minuta and T. diversiflia were evaluated in the field and significantly shwn t affect R. appendiculatus and ther ticks naturally attached t the hst animals. The essential il f T. minuta affects R. appendiculatus and ther ticks mre than the essential il f T. diversiflia. The results suggest the ptential fr essential ils t be incrprated in the n-hst "push" and "push-pull" strategy fr the cntrl and management f R. appendiculatus, ther affected livestck ticks and assciated tick-brne diseases amng the resurcelimited livestck farming cmmunity in trpical Africa.

6 Tablef Cntents General intrductin Part I Use f ethnbtanicals fr tick cntrl 2 Traditinal knwledge n tick cntrl within the Bukusu cmmunity in Bungma district, western Kenya 4 Part II Labratry evaluatin f essential ils 3 Repellent activities f essential ils f indigenus plants against the brwn ear tick Rhipicephalus appendiculatus 59 4 Attractive and repellent hst durs guide tickst their respective feeding sites 75 5 Dual and n-chice assays n theeffect f the essential il f Tagetes minuta n Rhipicephalus appendiculatus 83 Part III Field evaluatin f essential ils 6 The effect f essential ils f Tagetes minuta and Tithnia diversiflia n n-hst behaviur f the brwn ear tick Rhipicephalus appendiculatus 97 7 Essential ils f Tagetes minuta and Tithnia diversiflia affect rientatin t cattle and attachment sitepreference f the brwn ear tick, Rhipicephalus appendiculatus 09 8 Essential ils f indigenus plants prtect livestck frm infestatins with Rhipicephalus appendiculatus and ther tick species in herds grazing in natural pastures in Kenya 2 9 Summarizing discussin 33 Appendix 43 Appendix 2 63 References 7 Samenvatting 223 Acknwledgements 225 Curriculum vitae 227 Publicatins 229

7 General intrductin General Intrductin Research cncept Glbally, knwledge, skills, innvatins, techniques and practices f indigenus and lcal cmmunities have evlved s well ver human generatins that they are inherent in peple's cultural life. These centuries' ld practical ethnknwledge and cumulative ethnexperiences have been acquired by trial and errr, which has caused many a fatality in the prcess (Le Strange, 977). The evlutin f ethnknwledge invlved the inventin and develpment f ethnremedies fr almst every existing prblem affecting humans and their livestck (Marina et al., 200; Gueye, 2002). Succeeding generatins and civilizatins imprved upn these ethnremedies t suit their lcal needs and aspiratins and t ensure that they passed them n t the succeeding civilizatin (Marina et al., 200). Tday, indigenus knwledge is a resurce t facilitate sustainable develpment and imprve the standards f living f the pr peple (IK and DM, 2004; Akall, 2003). Indigenus knwledge is cnsidered an underutilized surce f infrmatin that may be used t fster agricultural and ecnmic develpment in less develped cuntries (Singh, 2002; Akall, 2003). In trpical Africa, livestck farming is seriusly affected by many indigenus infectius diseases. These diseases are a majr cnstraint t sustainable rural livelihds (Wanyangu et al., 996). Many f these diseases are transmitted by ticks, which serve as disease vectrs (Nrval et al., 992). In the curse f many centuries, African livestck farmers have develped ethnknwledge n varius methds f tick-brne disease cntrl and management, fr instance by selecting plants that serve as pharmaceuticals r as antitick substances (Marina et al., 200). Hwever, few studies have been dne t cnfirm the underlying science fthese cmmunity-specific ethnbtanical prducts. In the current thesis, the ethnknwledge f the Bukusu peple in western Kenya n tick cntrl and management is evaluated. This knwledge is integrated with the cnventinal knwledge f n-hst behaviur f the brwn ear tick Rhipicephalus appendiculatus Neumann and cnsidered fr develping an effective n-hst tick cntrl and management strategy. The studies include labratry and field evaluatin f strategic deplyment f essential ils with repellent effects designed t intercept fraging and attachment behaviur f R. appendiculatus by masking hst-derived attractants. By cmparing the repellent effects f selected essential ils, the impact f the essential ils with the strngest effect n tick behaviur is evaluated, sthat effective prtectin can be affrded. 'Thesumttalftheknwledge,techniques and skills,whichpeplein aparticulargegraphicarea pssess and enable them t get the mst ut f their envirnment. Mst f these ethnknwledge practices,techniques andskillshavebeenpassed frmearliergeneratinsuncriticallybutindividual menand wmen in eachnew generatin adapt andaddtthisbdy fethnknwledge, techniques andskillsin acnstant adjustment tchangingcircumstancesandenvirnmental cnditins.theyin turnpassnthebdyftheethnknwledge,techniques and skills intact tthenext generatin,in aneffrt tprvide themwith survival strategies. Indigenus knwledge evlves in respnse tthe changing envirnmental cnditins including expsure t mre frmal knwledge systems (CTA, 2008).

8 Chapter Fig... The current and future + predictedprbabilityftheccurrence f the R. appendiculatus in N sub-saharan Africa btained by usingthepredictivespeciesmdel (Erasmus et al., 2002) and the DARLAM climate surfaces mdel: (a) current, (b) future (Adpted frm Olwch et al.,2008) I Presencerecrdsf ' Rhipicq>PutfU5 appendiculctus Predicteddistributinfr (a) current and(b)future (in2030) Gegraphical distributin f Rhipicephalus appendiculatus and related rhipicephalids Rhipicephalid species ccur in Eurasia and nrthern Africa (5 species) and in sub- Saharan Africa (-55 species) (Walker et al., 2003). The distributin f R. appendiculatus and ther rhipicephalids in Africa is by n means cntinuus, even in thse cuntries in which they are knwn t ccur cmmnly (Fig..). Their ccurrence is influenced by several factrs, the mst imprtant f which are climate, vegetatin and hst availability (Nrval et al., 992). Using a predictive species mdel (Erasmus et al., 2002) and DAR LAM climate surfaces mdel (Olwch et al., 2008) R. appendiculatus distributin in sub- Sharan Africa is shwn in Fig... In Kenya, the CLIMEX mdel was used t calculate the ecclimatic index (EI), which shws the cincidence f distributin pattern f the dairy cattle with that f R. appendiculatus (Fig..2).In Fig..2, there is a very clse crrelatin between the EI and the recrded R. appendiculatus distributin in much f the affected areas and this als applies t the affected areas f the African cntinent [frm suthern Sudan thrugh t the suth-eastern cast f Suth Africa (Fig..)] with sme exceptins influenced by lw cattle density and ther herbivre hsts due t tsetse fly infestatin (Perry et al., 990; Randlph, and Rgers, 997). Relative humidity, assciated 2

9 General intrductin Fig..2. TTie distributin f Rhipicephalus appendiculatus, thevectrf Theileria parva, inkenya (a).the distributin f extic beef and dairy cattle breeds (mstpreferred cattle,withhighprductivity,byrural farmers) in Kenya, shwing the percentages by District (b).thetw figures shwthe cincidence fthe distributin pattern f dairy cattle with that f Rhipicephalus appendiculatus, thuspsing greatchallenges n the sustainability f these breeds in ECF endemic znes. The ringed area n the tw maps shws thestudyarea (mdified after Kariuki, 989/ with rainfall and altitude, restricts R. appendiculatus t relatively cl and humid bitpes f less than 30 C daily maximum and at least 400 mm annual rainfall, preferably upland savanna with a vegetatin cver existing f wdlands r grasslands (Yeman and Walker, 967). The extent f R. appendiculatus distributin in the castal regins f Mzambique is, hwever, unknwn and n ppulatins f this species have been reprted in West Africa (Walker et al., 2003). The ccurrence and abundance f R. appendiculatus are affected by the amunt f vegetatin cver (Nrval, 977), the abundance f suitable ruminant hsts (Nrval and Lightft, 982) and acaricides used (Hwell et al, 98). The vegetatin cver affects micrclimate (Minshull and Nrval, 982), which is imprtant fr the survival f the freeliving stages f the tick (Branagan, 973). Where vergrazing and the remval f trees reduce the vegetatin cver, R. appendiculatus tends t disappear (Nrval et al., 992). The species becmes abundant in the presence f hsts that have a lw level f resistance t it (Lightft and Nrval, 98). Prlnged intensive acaricide treatment f livestck can cause lcal eradicatin f R. appendiculatus, but the tick can spread again if cntrl measures are stpped (Nrval et al., 992). 3

10 Chapter Under certain circumstances, R. zambeziensis Walker, Nrval & Crwin replaces R. appendiculatus in several f the htter, drier areas f central and suthern Africa, while R. duttni ccurs nly in Angla and Zaire (Nrval et al., 992). Hwever, nthing is knwn abut the factrs that limit the distributin f R. duttni Neumann. Rhipicephalus appendiculatus shares gegraphical distributin and hst range with a number f ther rhipicephalids such as R. evertsi Neumann, R. lunulatus Neumann, R. muhsamae Mrel & Vassiliades, R. praetextatus Gerstacker, R. pravus Dnitz, R. pulchellus Gerstacker, R. sanguineus Latreille, R. simus Kch, R. senegalensis Kch, R. turanicus Pmerantsev, and R. zambeziensis (Walker et al., 2003). Ecnmic imprtance f tick-brne diseases transmitted by Rhipicephalus appendiculatus Tick-brne infectins f livestck are widespread in Africa and present a greater cnstraint t livestck develpment, particularly imprvement f lcal breeds, than in any ther regin f the wrld. This is largely due t the fact that many different tick-brne infectins ccur n the cntinent, the mst imprtant f which are Theileria parva parva Theiler [East Cast fever (ECF)], T. p. bvis Neitz (January disease r Zimbabwe malignant theilerisis), T. p. lawrencei Neitz [Crridr disease (CD)], T. annulata Dschunkwsky and Luhs [Mediterranean Cast fever (MCF)], Cwdria ruminantium Mshkvski (heartwater), Babesia bigemina Smith and Kilbrne, B. bvis Babes (babesisis) and Anaplasma marginale Theiler (anaplasmsis) (Nrval et al., 992). This prblem is cmpunded by the high susceptibility f freign breeds f livestck being used t imprve livestck prductivity in many African cuntries (Nrval et al., 992). Rhipicephalus appendiculatus is undubtedly the mst ecnmically imprtant tick f the African tick species (Nrval et al., 992; Walker et al., 2003). This is due t the fact that it is a highly efficient vectr f T. p. parva, the pathgen f the mst imprtant and cmplex tick-brne disease, ECF (Theiler, 904; Nrval et al., 992; Dlan, 999). The prevalence f ECF is nrmally restricted t central, suthern and eastern Africa where the cattle hsts, Bs taurus L. and B. indicus L., the tick and the parasite share the same gegraphical lcatin (Nrval et al., 992). The disease causes high rates f mrtality and mrbidity in livestck ppulatins, prductivity lsses in animals that recver and is the cause f exclusin f the much desired extic breeds f cattle f high prductivity frm endemic areas (Kariuki, 989). Rhipicephalus appendiculatus is als an efficient vectr f T. p. lawrencei frm African buffal t cattle, causing Crridr r buffal disease in the latter (Neitz, 955). Theileria parva bvis f cattle and T. taurtragi f eland and cattle are als transmitted by R. appendiculatus (Fivaz et al., 989) as is Ehrlichia bvis f cattle (Matsn, 967; Nrval, 979). The Nairbi sheep disease virus (NSDV) and Kisenye sheep disease virus (KSDV), causing hemrrhagic gastrenteritis and high mrtality in sheep and gats, are transmitted primarily by R. appendiculatus (Bugyaki, 955; Buisch et al., 998). Rhipicephalus appendiculatus can als spread Babesia bigemina, Dhri virus and Thgt virus t bth animals and humans (Jnes and Nutall, 989; Walker et al., 2003). In wild animals such as antelpes, infestatins f R. appendiculatus have caused txicsis prblems (Lightft and Nrval, 98). Heavy infestatins f R. appendiculatus have caused the death f eland calves as a result f bth acute and chrnic anaemia (Lewis, 98). The tick als transmits bacteria, Rickettsia cnri and R. aeschlimanii causing tick typhus (Larisa, 200) and virus causing luping ill (Alexander and Neitz, 935) in humans. 4

11 General intrductin Fig..3.Heavyburdensf Rhipicephalus appendiculatus infestatin. Theticks,havingtakenallavailable spacentheanimal'sears (theirpreferred feeding site),andbecmingfully engrgedwithhstbld (a),get attachedintheneighburhdfpredilectin site,theeyelid (b).fig..3ais aphtgraphbyprf.dr. F. Jngejan, Crdinatr,ICTTD-3 Newsletter,issue29,2006.Fig..3bprducedbyICIPE,Nairbi,Kenya. Heavy infestatins f R. appendiculatus n cattle (Fig..3) may result in severe damage t the attachment sites, a fatal txaemia, suppressin f hst immunity and reemergence f tick-brne diseases may cause Tzaneen disease. It has been shwn that fr each fully engrged female, there is a lss f4.0 gf ptential grwth f cattle (Irvin et al., 996). Tick bites such as shwn in Fig..3 cause cutaneus effects such as fcal dermal necrsis, irritatin, haemrrhage, inflammatry respnse ften invlving esinphils and develpment f wunds that becme infected with bacteria such as Staphylcccus causing lcal cutaneus abscesses r pyaemia (Wall and Shearer, 997). Heavy tick infestatin may als cause significant bld lss, reduced prductivity in terms f pr prductin f milk, meat, hides and skins, reduced weight-gain and restlessness (Nrval et al., 988; Pegram et al., 989a). Fr instance, in an ECF-infested area f Kenya, de Castr et al. (987) recrded a decrease f live weight f cattle f 2.8 kg fr a 200 kg animal. Milk prductin was reduced by 9 gper each engrging female R. appendiculatus in indigenus sanga cattle (Nrval et al., 997). Secndary infectins in wunds caused by tick bites such as infectins f Dermatphilus cnglensis, may result in high mrtality f livestck (FAO, 998). Tick-bite lesins als predispse animals t screwwrm myiasis (Wall and Shearer, 997). In sub-saharan Africa, 76 millin cattle live in the ECF-affected regin cvering an area f abut 56 millin hectares (Mukhebi et al., 99). East Cast fever puts at risk the lives f abut 25 millin cattle in Burundi, Kenya, Malawi, Mzambique, Rwanda, Sudan, Tanzania, Uganda, Zaire, Zambia and Zimbabwe (Nrval et al., 992).The disease has been reprted t cause half a millin deaths f cattle per year in East Africa (VIE, 2002). In Kenya alne, it has been estimated that 50-80% f the natinal cattle ppulatin f abut 0 millin animals, are expsed t tick infestatin, and f these animals % die f ECF each year (Mbg et al, 995; VIE, 2002). A micr-ecnmic analysis cmpleted in 988 n eight large and medium-sized farms, with a ttal f 37,779 head f cattle, in Na- 5

12 Chapter ^_ unfed adults Fig..4. The life cyclef Rhipicephalus appendiculatus displaying the teletrpic type f behaviur (thethree-hstcycle) unfedlarvae kuru District, Central Rift Valley, Kenya, shwed that the cst f acaricides, prductin lsses and lsses due t clinical theilerisis and ther tick-brne diseases amunted t apprximately US$ 55,305 r $3.64 per animal per year (Kariuki, 989). Extic cattle, B. taunts are highly susceptible t ECF with a mrtality f 90-00% while indigenus cattle, B. indicus, suffer a much lwer fatality f abut 0-40% in calves due t acquired immunity (Sutherst et al., 978). Upn recvery, cattle becme reservirs f ECF (Yung et al., 986) as bserved t in wildlife (Yung et al., 98). This, hwever, maintains the disease in cattle ppulatins lnger, particularly where cattle and wildlife share grazing fields with the availability f suitable vectrs (Nrval et al., 992). Besides cattle mrbidity and mrtality as well as severe ecnmic lsses as a result f direct and indirect tick parasitism, stck management als requires large financial inputs. Fr example, csts incurred fr cntrl peratins have been estimated at US$ 7.02/ head/year r US $.08 per hectare (Mukhebi et al., 993). Cuntries expsed t the threat f ECF als face a large financial burden due t imprtatin f acaricides, training f lcal persnnel, treatment f infected animals, maintenance f infrastructure and investments int research n apprpriate slutins t ticks and TBDs. Such ecnmic cnstraints have been estimated t cst millins f US dllars annually in many African cuntries (Mukhebi etal., 993). The bilgy and behaviur f Rhipicephalus appendiculatus Life cycle / Rhipicephalus appendiculatus Ticks are bligate, bld-feeding ectparasites f vertebrates: mammals, birds, reptiles and amphibians, mre particularly mammals and birds. Adults f mst tick species parasitize wild and dmestic artidactyls, perissdactyls, r carnivres (Cumming, 998). Bvine cattle are the main hst f R. appendiculatus, but gats,buffales, elands, waterbucks, 6

13 General intrductin Fig..5. A generalized life cycle f Theileria parvain the animal hst and in the vectr, the Rhipicephalus appendiculatusasunderstd tday, (figure adpted frm the Internatinal Labratry fr Research n Animal Diseases, Annual Reprt,980). Sprzites Sprblast LYMPHOBLAST,>\ Clnal expansin (.. f parasitized cells Mergny W Merzkes Pfrptasms ki erythrcytes Qamnts nyalas, greater kudus and sable antelpes serve as nn-dmestic hsts, while dgs and sheep are als infested. Rhipicephalus appendiculatus becme well adapted t dmestic cattle and can be maintained by all stages feeding n cattle, but immature ticks may feed n smaller antelpes and scrub hares, thus shwing a teltrpic type f behaviur (threehst cycle) with a tendency t the mntrpic type (ne-hst cycle). On cattle, the immature stages f R. appendiculatus attach mainly n the neck and dewlap, the cheeks, eyelids, muzzle and ears. The adult R. appendiculatus prefer t feed n the ear pinna f bvids but nt in the ear canal. In heavy infestatins, adults are als fund arund the eyelids (Fig..3b) and hrns, n the upper neck, in the tail-brush and arund the anus. The teltrpic type f behaviur f R. appendiculatus is summarized in Fig..4. Only abut 2-5% f tick's lifespan is spent n a hst and the majrity f their life cycle is spent n the grund r vegetatin (Branagan, 973). The interactin f the parasite Theileria parva parva with the vectr Rhipicephalus appendiculatus and the vertebrate hst The interactins f the tick, hst and parasite exhibit a cmplex relatinship whse understanding remains key t the successful cntrl and management f tick and TBDs (Nrval et al., 992). The parasite underges sexual develpment in the vectr (definitive hst) and asexual develpment in the mammalian (intermediate hst) hst (Fig..5). The sprzites f T. parva, prduced in large numbers in the acinar cells f the salivary glands f the in- 7

14 Chapter fected tick, are inculated alng with saliva during bld feeding f R. appendiculatus t initiate its asexual cycle f develpment in the mammalian hst. Using specific receptrs, the sprzites rapidly enter target lymphcytes, which becme transfrmed after the Theileria schiznt is frmed. The entry f T. p. parva sprzites int bvine peripheral bld lymphcytes is temperature-dependent and requires the participatin f live and intact sprzites and hst cells (Shaw et al., 99). This prcess invlves a sequence f events (recgnitin and attachment f sprzite t lymphcyte by binding, frmatin f a very clse cntinual junctin between the sprzite and lymphcyte membranes, zippering f sprzite t lymphcyte membrane, separatin f enclsing hst cell membrane frm the sprzite, entry f sprzite int lymphcyte cytplasm and finally the frmatin f an rderly array f hst cell-derived micrtubules arund the sprzite) (Fig..5). The parasite des nt use rhptries and micrspheres fr entry int the hst cell, but uses them t destry the surrunding hst cell membrane after entry int the hst cell (Nrval et al., 992). This prevents the hst cell frm being able t use lyssmal activity against the parasite. The infected lymphcyte is transfrmed int a lymphblast and divides in cnjunctin with the schiznt, giving rise t tw schiznt-infected daughter cells at frequent intervals. This prcess has been termed "parasite-induced reversible transfrmatin" because, if the cells are treated with antitheileria drugs, the transfrmed cells revert t quiescent lymphcytes (Ole-Miyi, 989). Within the infected lymphcytes, schiznts are assciated with micrtubules invlved in spindle frmatin during hst cell divisin (Nrval et al., 992). Clnal expansin f infected lymphid cells ccurs with an apprximate tenfld increase f schiznts every 3 days. Schiznts, traditinally called macrschiznts r Kch's blue bdies, vary in size and in the number f nuclei. Early detectable frms are small with nuclei that, when Giemsa-stained, appear as chrmatic granules. The clnal expansin f the Theileriainfected lymphid cells with the cncmitant destructin f the infected tissues appears t give rise t the main pathgenic effects f the disease (Dlan et al., 984). Cattle that recver frm ECF acquire prtective immunity and becme resistant t re-infectin with a stck f T. p. parva hmlgus (parasite-specific Majr Histcmpatibility Mleculesclass I-restricted cyttxic T-lymphcyte respnses) t that which induced initial infectin, but they may die ifchallenged with a heterlgus stck (McKeever, 200). Rhipicephalus appendiculatus becme infected with T. parva when feeding n an infected hst having pirplasms in erythrcytes (Knnai et al., 2007a). Pirplasm-infected erythrcytes are ingested by ticks f the larval r nymphal stages and underg a sexual develpment cycle in the gut f the replete tick t prduce zygtes, which in turn develp int mtile kinete stages that infect the salivary gland acini f the next instar, the nymph r adult (Fawcet et al., 985). Inthe salivary glands f the tick, the kinete develps int infective sprzites (sprgnic phase) and this repeats the cycle when the infected tick with infective sprzites in its salivary glands takes a bld meal frm a susceptible cattle hst (Fig..5). Theileria parva nly mature and enter the saliva after the tick attaches t a hst fr a cnsiderable perid f 3 4 days (Martin et al., 964). This perid has been recently shwn t be between 24 and 72 hurs (Ochanda et al., 988; Knnai et al., 2007a). Hwever, if envirnmental temperatures are high, infective sprzites can develp in ticks n the grund and may enter the hst within hurs f attachment (Ochanda et al., 988). The time frm entry in the tick t sprzite develpment in the salivary glands is n average 9-20 days fr feeding nymphs and 20-2 days fr adult females (Ochanda et al., 996; Watt and Walker, 2000). Frm day 4 after T. p. parva infectin f cattle by a tick bite, individual schiznts underg mergny t prduce merzites (traditinally called micrschiznts). Merzites

15 General intrductin invade the erythrcytes t becme pirplasms, which may subsequently underg limited divisin als by mergny (Cnrad et al., 986). Behaviur f Rhipicephalus appendiculatus Hst-seeking behaviur f Rhipicephalus appendiculatus Bld-feeding arthrpds such as ticks, have, ver time, develped a cmplex relatinship with their mammalian hsts. Brad variatins ccur in hst-specificity f ticks, duratin and multiplicity f cntacts, and in hst-lcatin behaviur (Gibsn and Trr, 999). The results btained frm analysis f a quantitative data set f 43,65 individual cllectin recrds f ticks in Africa suggest the existence f a spectrum in hst-specificity but with the edges f this spectrum readily demarcated (Cumming, 998). Frm this brad range f a spectrum in hst-specificity, generalists and specialists in hst/prey lcatin can be discerned (Steidle and van Ln, 2003). Nevertheless, what induces hst-seeking behaviur f the vectr and its subsequent finding and selectin f suitable hsts by different tick species has nt been fully understd. This behaviur has been cnsidered either as the result f evlutinary adaptatin prcesses t the hst-derived stimuli (Cupp, 99; Steidle and van Ln, 2003), pathgen-induced behaviur in the vectr, nrmal feeding habits, visual cues, hst fd and r its prducts such as faeces, urine r exuviae (Steidle and van Ln, 2003) r cmbinatins f these factrs. In ther arthrpds such as msquites, the rle f lfactin in hst-seeking behaviur has been explained (Takken, 99), including cues as human breath, bdy durs etc. (Mukabana, 2002). It is assumed that hst-seeking behaviur f ticks is affected by similar cues. The cmbined knwledge demnstrates that the hst-vectr-parasite relatinship is cmplex in nature, and whse pattern f respnses and sequence f behaviural events, particularly f the vectr, have t be clearly understd and strategically integrated in epidemilgical tls in rder t achieve sustainable cntrl and management f vectr-brne diseases. Previus studies indicated that varius attractive hst-derived stimuli (e.g., hst texture, hst skin humidity, hst bdy temperature and chemical factrs (kairmnes/ allmnes/synmnes 2 ) such as skin emanatins, breath, urine and faeces, influence hstseeking behaviur in ticks (Sika, 996). Kairmnes are the main sensry cues used by haematphagus rganisms t find their hsts (Mrdue and Mrdue, 2003). Envirnmental factrs cmplement these kairmnes in influencing hst-seeking behaviur in ticks (Speybreck et al., 2003). Adult R. appendiculatus search their hsts fr a bld meal when they are active early in the day. They becme active under specific sets f temperature, rainfall, humidity, length f the rainy seasn, number f rainy and cludy days, and day length (Pegram et al., 989b). Numbers f adult R. appendiculatus n the hst increase after the nset f the rains (Berkvens et al., 998). The main factr respnsible fr this phenlgy is thught t be day length, where a lng phtperid terminates the state f diapause and induces hst-seeking behaviur in the wet mnths (Madder et al., 2002). Diapause in ticks is cnsidered t be a pre-adaptive behaviur t allw the ticks t survive unfavurable cnditins f a given seasn. Near the equatr, ticks are nn-diapausing and may usually feed thrughut the year and their numbers vary less (Speybreck et al., 2004). 2 Anallmneis achemical substanceprducedbyanrganism,which inducesin amemberfanther species a behaviral r physilgical reactin favrable t the emitter; may be mutualistic r antagnistic. Ifthebenefit isttherecipient the substance isreferred tas akairmne but ifbth rganismsbenefit thenitis asynmne (SbarbatiandOsculati,2006) 9

16 Chapter Habitat Hstanimal I Awayfrm FS Clser tandat FS Statinary/ scanning Drpff <- Qrisuccessfirsearch7 attachment Directinal mvement ' General hst attractant = Feeding site attractant Vlatiles frm feeding ticks j l *^ Hst allmne (Push effect) > Pull effect 0 Behaviuralphases I I Tickactivities FS =Feedingsite Fig..6. Asequencefrientatinbehaviuralactivitiesf Rhipicephalus appendiculatus nthehst. The prcessischaracterizedbydifferent behaviuralpatterns (after Sika,996). Once the weather cnditins becme favurable fr the ticks t becme active, the chemical cues elicit lng-range respnses while the physical cues elicit shrt-range respnses in the hst-lcatin prcesses (Mrdue and Mrdue, 2003). The integratin f physical factrs and kairmnes in the light f the pivtal rle played by envirnmental factrs in influencing hst-seeking behaviurs f ticks has nt been explred in building up tick cntrl device(s) that suit different climatic cnditins. Differential selectin f predilectin feeding sites by Rhipicephalus appendiculatus Once n the hst animal, many tick species will nt prbe until they have arrived at the preferred feeding site and are nt at risk f being remved frm the hst. This preference fr feeding site may serve t avid cmpetitin amngst the tick species feeding n the same hst animal and perhaps inter-species mate cnfusin between clsely related species, thereby enhancing their survival and reprductivity (Chiltn et al., 992). This may be as a result f evlutinary adaptatins f certain tick species t the attractin f specific stimuli riginating frm the predilectin feeding site n the hst animal. Once n the hst animal, even very clsely related species demnstrate predilectin fr feeding at different bdy sites as demnstrated fr R. appendiculatus and R. evertsi (Sika, 996; Wanzala et al., 2004). The mechanisms underlying these species-specific interactins and selectin f the feeding site n the hst have nt yet been understd. Fr instance, adult R. appendiculatus shwed marked preference fr feeding in the inner part f the bvine ear pinna while the immature nes shwed less selectivity by feeding n many ther parts f the hst in additin t the ear pinna (Walker, 974) Aphermne (frm Greek cpepcpher "t bear" + 'puvn "hrmne") is a chemical 'message' secreted externallybyaninsectrtheranimalthrughwhich itcmmunicateswith antherindividualf the same speciesthrugh asense fsmell t influence rtrigger specific natural behaviral rphysilgical respnses (especially fsexualbehaviurs) (VetandDicke, 992).Phermnesare usedtattractmembersftheppsitesexfrreprductin,tmarkfdtrailsrlcatinandterritry,andareused aswarningsralarm systems.each insect (ranimal)hasitswnsetfcmplex chemical phermne, smefwhich havebeen identified and used intrapst mnitr and smetimes cntrl pest ppulatins. In ther cases, phermnes are used t cnfuse r lure insect pests awayfrmtargetcrps.

17 General intrductin Just as during hst lcatin, a wide range f factrs ranging frm physical t chemical are invlved in the feeding site lcatin n the hst by ticks. The rle f semichemicals (phermnes 3, hst kairmnes and sme allmnes and hst-micrbial durs) des nt help the tick t identify a suitable hst and reach it (Snenshine et al., 986), but cntinues while n the hst t help it identify a suitable feeding site (Nrval et al., 989a;Wanzala et al., 2004). The selectin f the feeding site is nt a randm activity r chance effect but is systematically brught abut by a well-crdinated steretyped sequence f behaviural events (Fig..6) elicited by hst-derived semichemicals (Sika, 996). Often, ticks have t travel relatively lng distances n the hst t reach the targeted feeding site (McDwell and Walade, 985).Rutine bservatins shwed that this phase is characterised by different behaviural patterns, which include runs, stps, buts, turns, scanning etc. (Wanzala et al., 2004). The settlement at 'preferred feeding sites' is als related t physical characteristics f the hst (texture, bdy temperature, skin humidity, etc.) as well as chemical factrs (skin emanatins, hst-derived micrbial durs, breath, faeces and urine), which are likely t prvide ptimum cnditins fr the attachment f ticks (Dube and Kemp, 979). Observatins have shwn that ticks tend t avid desiccatin by chsing t ccupy less expsed areas r parts n the hst (Rberts, 97). Micr - envirnmental cnditins specific t certain bdy areas f the hst e.g., the hygrmetric index in the ear cavity, skin temperature and humidity have als been suggested t play a rle in the feeding site preference by certain tick species (Waladde et al., 99). The cmplex drus envirnment met by individual ticks n their way t the preferred feeding site is imprtant in maintaining the riented curse f the ticks. This ffers an durpermeated backgrund (resulting frm multiple secretins and vlatile emissins n the skin surface f bvine hst). Chemical cmpunds in sweat and ther skin secretins, detectable by lfactry and/r tactile receptrs, are believed t facilitate the selectin f suitable feeding sites (Waladde and Rice, 982). Earlier, Balashv (972) reprted electrphysilgical evidence n the detectin f glucse and sucrse by the type A sensilla in the lfactry rgans f the tick. Mre recent studies suggest that different tick species themselves als play a rle in guiding ther ticks f the same species t the suitable feeding site. This is the case in certain Amblymma species where an aggregatin f the ticks at specific feeding sites is supprted by the male-emitted attractin/aggregatin attachment phermne (rthnitrphenl) secreted by attached and feeding ticks, attract cnspecific males, females and nymphs (Maranga et al., 2003). In the majrity f the hard ticks studied s far, feeding females prduce an attractant sex phermne cntaining 2,6-dichlrphenl, which attracts males t the feeding site (Rechav et al., 976). In the case f R. appendiculatus, lfactmetric experiments using swabs impregnated with ear emanatins evked attractin f adults but repelled nymphs and larvae f the same species (Akinyi, 99). In the same study, ear swabs mixed with male tick extracts inhibited respnses f these immature stages. N explanatin was given t accunt fr these results. Likewise, fr R. evertsi, althugh its feeding site prpensity is knwn (arund the anal regin) (Elbl and Anasts, 966), adequate data are lacking t prvide insights int its feeding site selectin and the nature f the semichemical signal(s) invlved. Stimuli present in the hst's anal regin, which attract R. evertsi, are f interest, as in additin t bdy surface vlatiles, and may include effluvium frm the gut and vlatiles frm dung. In additin, the phermnes emitted by male R. evertsi while in the preparasitic as well as in the parasitic phases, may play a significant rle in feeding site lcatin by hst-seeking cnspecifics (Gethe and Neitz, 985). 2

18 Chapter The migratry buts therefre, which R. appendiculatus and a clsely related species, R. evertsi perfrm during search and lcatin f the predilectin feeding sites, are guided by attracting and repelling hst-derived semichemicals in "push" and "push-pull" mdes (Wanzala et al., 2004). With this phermnal and allelchemical (kairmnal/ allmnal) knwledge cupled with that f envirnmental factrs, it is pssible t explre n-hst tick cntrl interventins invlving: (i) tactical use f repellent btanicals near/ arund the feeding sites t cnfuse the ticks (the 'push' tactic), (ii) divert ticks (the 'pull' tactic) t simple traps strategically placed n the hst bdy and cntaminated with a killing agent such as pathgenic fungi r acaricide btanicals, and (iii) cncurrent prtectin f feeding sites with repellents and diversin f ticks t the traps (the "push" and "push-pull" tactics). As much as differential selectin f predilectin feeding sites studies are shwn t be imprtant in the cntrl and management f ticks n the hsts, nly casual attentin has been given t the feeding site lcatin behaviur f relevant arthrpds. Respnses /Rhipicephalus appendiculatus and ther ticks t hst-derived kairmnes Semichemical (kairmnal as well as allmnal) cmmunicatin is quite well knwn and demnstrated in ticks (Yder et al., 993). Hst-derived durs influence n-hst rientatin f the tick and their selectin f feeding sites. Hst-brne kairmnes fall int fur main classes based n their nature and the crrespnding behaviur evked: (i) C0 2 and ther bvine breath vlatiles, (ii) skin emanatins (iii) specific feeding site attractants (hst -derived cues that elicit respnses, which help ticks t lcate a specific feeding site n the hst) as previusly explained and (iv) vlatiles frm excretry prducts (urine and faeces). Carbn dixide (5-0% f expired air f mammalian breath) is amng hst-riginated drants influencing the behaviur f several tick species, either as an attractant (Garcia, 962) r a stimulant (Nrval et al., 989b). Sauer et al. (974) reprted that C0 2 dses f - 8% enhanced hst-seeking behaviur in the American lne star tick, Amblymma americanum. Other cmpunds f interest fund in trace amunts in x breath are acetne, cten-3-l (ctenl) and butanne. A blend f ctenl and C0 2 was fund t enhance catches f tsetse flies (Vale, 980), but these cmpnents didn't evke any attractin in Amblymma hebraeum (Nrval et al., 987). Other detailed studies shwed that C0 2 's rle in hst lcatin by ticks is mre intricate, as elevated C0 2 in cmbinatin with ther stimuli (e.g., heat, dur and temperature) elicit greater respnses frm ticks than when these stimuli were presented singly (Hwell, 975). Carbn dixide has als been shwn t be a phagstimulant fr R. appendiculatus and Amblymma variegatum. Using a 5% CO2 atmsphere, the larvae and nymphs f A. variegatum can be induced t fully engrge in an in vitr system in the ttal absence f a hst animal, achieving engrged weights cmparable t thse achieved n mammalian hsts and with a high multing rate. Rhipicephalus appendiculatus larvae, nymphs and females as well as female A. variegatum can be fed in an artificial system when the C0 2 cncentratin is 7% (Ann., 2008). Ticks lcate their hst by respnding t hst-derived durs. Fr example, adult Ixdes scapularis Say have been shwn t travel >0 m in respnse t hst-derived kairmnal cues (Carrll and Schmidtmann, 996). Stimuli frm hsts serve as a multi-purpse indicatr fr hst prximity, t guide t a ptential hst and t elicit feeding respnses nce the cntact with the apprpriate site has been made (Hwell, 975). Waladde and Rice (982) distinguished tw types f inherent appetence behaviur used by a tick vis-avis its hst. In the questing type, passive ticks wuld await fr a passing hst t cling n. The hunting type, in cntrast, wuld actively mve t a ptential hst and is likely t make use f hst durs fr rientatin, alne r in cnjunctin with ther cues. There is, therefre, a great variatin in hst lcatin behaviur between tick species. In general, the pres- 22

19 General intrductin ence f varius stimuli has made pssible a hierarchy with regard t whether these act at clse, mid- r lng-range distances during the hst-finding prcess as bserved fr sme insect grups (Visser, 986). Attractin and attachment f a tick t hst skin are believed t be influenced chiefly by lfactry stimuli augmented by thermal stimuli (Hwell, 975). Rare cases f visual and/r acustic stimuli have been prpsed (Waladde and Rice, 982). Hst skin emanatins are assumed t functin as kairmnes. Olfactmetric experiments have indicated that many hst parts d nt play a kairmnal rle as swabs f calves frm legs, back, perineum and belly were unattractive t adults, nymphs and larvae f R. appendiculatus (Akinyi, 99). Swabs f calves cllected frm inside the ear, n the inner and upper sides f the ear pinna and arund the ear base, hwever, were attractive. In related labratry and field studies, adult /. scapularis, Dermacentr variabilis Say, 82 and Amblymma americanum Linnaeus, 758 have been shwn t exhibit similar behaviur in respnse t substances rubbed frm their respective hsts and nn-hst animals (Carrll et al., 995). While n the hst, different tick species exhibit varius lcmtry and explratry behaviural patterns, which relate t either mate finding r selectin f feeding sites (Snenshine et al., 986). Fr species such as R. appendiculatus, whse mating takes place n the hst after attachment, females merely mve t feeding sites and remain there until engrgement is cmpleted. The pre-mating behaviur f the male is cmparatively mre intricate. Observatins n D. variabilis and A. americanum shwed that males first d nt mve very much frm their attachment pint (Gladney and Drummnd, 970). It is nly after they becme sexually active fllwing a bld meal that they disperse, smetimes extensively, ver the hst t seek the females (Hamiltn et al., 994). This mate seeking behaviur may be mediated by phermnes, which may als attract unfed cnspecifics f bth sexes t the feeding site. It has been assumed that stimuli frm preferred feeding sites may play a similar rle as attractants. Sme develpments in livestck tick cntrl and management: successes and failures Tick cntrl is practiced in a wide variety f circumstances invlving different tick and hst species. The main reasns fr tick cntrl are t prtect hsts (livestck) frm irritatin and prductin lsses, frmatin f lesins that can becme secndarily infested, damage t hides and udders, txicsis, paralysis, and f greatest imprtance, infectin with a wide variety f disease agents (Nrval et al., 992). Cntrl als prevents the spread f tick species and the diseases they transmit t unaffected areas, regins, r cntinents (Aiell and Mays, 2003). The resultant prduct being the increased livestck prductin manifested in imprved livelihd and ecnmic develpment and grwth f livestck hlders (Mukhebi et al., 99). The cntrl f ticks has been under taken by chemical means, cultural measures including use f btanicals, mechanical, bicntrl and immunlgical techniques. These will be cnsidered belw. Chemical cntrl The first applicatin f ixdicides t cntrl ticks n cattle was made by treating the infested cattle with varius ils-including paraffin but withut much success (Harrisn et al., 973). An effective chemical cntrl f livestck ticks began with the intrductin f arsenical slutins as cattle dips in Suth Africa in 893 and in Australia in 895. The use f chemical ixdicides against livestck ticks has cntinued until tday. In Africa, seven basic acaricide grups have been indiscriminately used t cntrl livestck ticks and these are: (i) arsenicals, (ii) rganchlrines, (iii) rganphsphates, (iv) carbamates, (v) amidi- 23

20 Chapter nes, (vi) pyrethrins and (vii) synthetic pyrethrids (Mitchell, 996). Mde f applicatin f these acaricides have included the use f dip tanks, knapsack hand sprayers, hand dressing, squirting acaricide n predilectin feeding sites f ticks, dusting, etc. (Awumbila, 996). In recent years, several ther methds f acaricide applicatin have been tested, including the slw release f systemic acaricides frm implants and bluses; the slw release f cnventinal acaricides frm impregnated ear-tags; 'pur-ns', which are applied n the backs f livestck and spread rapidly ver the entire bdy surface; and 'spt-ns', which are similar t 'pur-ns' but have less capacity t spread (Nrval, 989). Acaricide applicatin may either be directed against the free-living stages in the envirnment r the parasitic stages n the hsts. In either case, the prblems assciated with the use f acaricides in livestck industry are very challenging withut imminent slutins. Acaricides are cstly and ut f reach f pr rural livestck farmers wh als d nt have sufficient technical knw-hw f managing and handling them (Nrval et al., 992). The high csts f acaricides have becme a majr prblem fr smallhlder farmers wh cnstitute the majrity in the livestck industry n the cntinent f Africa (Kariuki, 996). The cntrl f ixdid ticks by acaricidal treatment f vegetatin has been dne in specific sites t reduce the risk f tick infestatin t susceptible hsts. This methd is hwever nt sustainable and has nt been recmmended fr wider use because f assciated envirnmental pllutin prblems and the high csts invlved in the treatment f large areas (Aiell and Mays, 2003). In additin, inapprpriate drainage f dip liquid causes water pllutin and indiscriminate damage t the fauna and flra in the envirnment. Few quantitative data are available n the impact f these acaricides n flra and fauna but it can be assumed t be substantial at the lcal level (de Haan et al., 996). Acaricides are a health hazard as well. They cause fd pisning (thrugh meat, bld and milk) and residual txicity. Fr instance, rganchlrine prducts have been demnstrated t leave residues in meat and milk (Mitchell, 996). Intensive applicatin f acaricides t livestck creates an enztically unstable disease situatin 4 in the ppulatin (Nrval et al., 992). When tick cntrl breaks dwn, large lsses can ccur. An example f this was in Zimbabwe, where a cmpulsry dipping plicy had been in frce since 94 and when dipping infrastructure brke dwn during the war f independence between 974 and 979, an estimated ne millin cattle died, mainly f tick-brne diseases (Lawrence et al, 980). The ticks have cnsistently shwn themselves t pssess a genetic pl cntaining the ptential t resist a wide range f chemical pisns. This has been cmpunded by illegal cattle mvement, civil unrest in sme areas, pr management and inadequate maintenance f cattle dips and pr use f manufacturers' instructins (Matthewsn, 984; Nlan, 990). It has been shwn that the indiscriminate use f acaricides may affect future tick cntrl as this has the cnsequence f facilitating rapid develpment f tick resistance t the active cmpunds used in the acaricide frmulatins (Fraga et al., 2003). There is therefre a very real danger that unless new acaricides f different chemical structures are frthcming, tick resistance t existing cmpunds will spread. But the prblem is that the develpment f new acaricides is a lng and very expen-sive prcess (Graf et al., 2004) Apre-immune statust tick-brne infectins, s-called endemic stability, ften establishes inindigenuscattlethrugh acntinuuscntactwiththeinfectius agents frm earlyinlife.thisstatusis nrmally remved during excessive applicatin f acaricides. The presence f endemic stability is claimedtbethemainfactr inlimitinglsses,mainlyexpressedintermsflwrabsencefmrtality due t tick-brne infectins in indigenus cattle ppulatins. Cattle that are expsed t Anaplasma, Babesia r Cwdria rganisms early in life dntusually develp the clinical diseaseand are subsequently immune. This frm f age-related immunity des nt ccur with T. parva (Latif, 992).

21 General intrductin As chemical cntrl is still the main methd f tick cntrl wrldwide but has becme unsustainable, it may jepardise the envisaged target f revlutinizing livestck prductin t meet the needs f fd security by Mechanical cntrl: handpicking In certain East African pastral cmmunities, livestck is cmmunally rganized, brught tgether and held in a crutch facility where ticks are picked ff the animals ne by ne and either buried r thrwn int fire (Marina et al., 200). This practice was als cnducted during milking and cleaning f livestck sheds by wmen (Marina et al., 200). Sme ticks, after being picked frm the respective hst animals, were given t chickens/birds at hme as a fd supplement. Hwever, this methd is tedius, time cnsuming and invlves much labur in rder t serve a big herd f cattle and hence is nt sustainable. Hst grming Tday, there exists evidence t strngly supprt the cncept that grming (cleaning the fur f an animal), which ccurs amngst animal cmmunities, is beneficial (Hart, 2000; Park, 2008). In Africa it has been bserved that many species f wild bvids live in tick infested envirnments but usually with lw levels f tick lads. This is the result f a behaviural defence against infestatins with ectparasites such as ticks as was experimentally demnstrated and bserved in free-ranging adult female impala (Mring, 995; Mring et al., 995;). It is reasnable t assume that much f the vulnerability f cattle t ticks reflects a relatively reduced predispsitin t grm, a behaviur that in turn stems frm their derivatin frm Eurpean (and mre recently frm Nrth American) stck, where the envirnment has been relatively free frm ticks (Hart et al., 996). Thusands f years f lw expsure t ticks may have led t a selectin against frequent grming in favur f reducing the cst assciated with frequent grming. The grming in many wild bvids bserved in a number f studies prvide evidence that systemic chemical cues (chemical signals affecting the whle bdy) may be mdulating the grming rate (Hart, 997). Thus, there might be sme way t enhance the grming f Eurpean-derived cattle thrugh apprpriate administratin f bilgical substances that are fund t increase grming in wild bvids. Given the ptential value f grming in the remval f attached ticks, anther pssibility is that cattle might be selectively bred (r genetically engineered) t shw high rates f grming and, hence, be less susceptible t tick-brne diseases. Hwever, befre embarking upn a cstly and time cnsuming breeding prgramme, effective and prtective values f grming fr tick cntrl first needs t be ascertained. In Trinidad, studies by Smith in 974 shwed that grming was effective in reducing the infestatin f cattle with the tick, Amblymma cajennense Fabricius (Smith, 975). In Kenya, adult female R. appendiculatus have been bserved t die as a result f increased grming by cattle because f increased dermal irritatin by the ticks (Essuman et al., 99). There have been studies t shw that f the interspecific and intraspecific determinants that mdulate grming in animals, sme f the intraspecific determinants appear 5 Parasitidsdiffer frmparasites intheirrelatinshipwith thehst. In atrulyparasitic relatinship, theparasiteandhstlivesidebysidewithlittlerndamagetthehstrganismwhiletheparasite takes enugh nutrients t live n andreprduce withut draining thehst's reserves. In aparasitid relatinship, the hst isusually killed after the full develpment fthe ther rganism (parasitid). Thistypefrelatinshipseemstccurnlyinrganismsthathavefast reprductin rates (suchas insectsrmites) (Gdfray, 994). 25

22 Chapter t be mediated by systemic physilgical influences, suggesting there may be ways t imprve grming activity in weak grmers, such as cattle (Hart, 2000). Bilgical cntrl Bilgical cntrl f ticks is the use f natural enemies (parasitids, pathgens, parasites and predatrs) that can reduce the density f the target ppulatin r even eliminate it. The develpment f bilgical cntrl f ticks is mre neglected than that f mst ther animal pests (Rutz and Pattersn, 990) and lags behind that f plant pests by several decades (Samish, 2000). In the literature, hwever, mre than 257 tick bicntrl agents are mentined, cmprising 00 species f pathgens, seven parasitids and 50 predatrs (Samish and Alekseev, 200). The subject has been extensively reviewed by Hu et al. (998) and Samish (2000), wh shed sme light n the way frward and reflect n the previus failures and successes. Only a few studies have as yet been cnducted n pathgens, parasitids 5, and predatrs f ticks. A first remarkable field trial was made with the intrductin f parasitic wasps riginating frm France in the USA in the late 920s and early 930s and in the early 940s in Russia (Hu et al., 998). Hwever, in bth the USA (Smith and Cle, 943) and Russia (Alfeev, 946) they were unsuccessful in cntrlling target tick ppulatins, similar t a previus trial cnducted in the USA between the years 927 and 932 (Cley and Khls, 934). During the past decades, interest in develping bilgical methds fr tick cntrl using birds (Cut, 994), parasitids (Hu et al., 998), entmpathgenic nematdes (Samish, 2000), entmpathgenic fungi, arthrpds (Samish and Alekseev, 200) and bacteria (Hassanain et al., 997) has gained mmentum wrldwide, because f the limited impact fthese rganisms n the envirnment. Hst resistance Cattle hst resistance- a measure f the hst's ability t limit the establishment, grwth rate, fecundity and/r persistence f a parasite ppulatin (Cp and Kyriazakis, 999), has been reprted frm numerus studies that zebu (B. indicus) and sanga (a B. taurus and B. indicus crss-breed) cattle, the indigenus breeds f Asia and Africa, usually carry significantly fewer ixdid ticks than extic Eurpean (B. taurus) breeds f cattle (Utech and Whartn, 982; Aiell and Mays, 2003). It has been shwn that tick infestatin increases as the prprtin f Eurpean genes in an animal increases (Lems et al., 985). Studies have als shwn that the magnitude f lsses due t tick infestatin varies with the gentype f cattle (Lems et al., 985). Within a gentype, lsses per tick unit increase with the number f attached ticks (Pegram et al., 989a; b). The tick resistance f zebu breeds and their crsses is increasingly being cnsidered fr explitatin as a means f cntrl f tick ectparasitic stages n livestck. The intrductin fzebu cattle t Australia has psitively revlutinized the cntrl f B. micrplus n that cntinent as zebu breeds were successfully explited in cattle breeding prgrammes t develp tick resistant cattle breeds that limited the impact f B. micrplus infestatin (Seifert, 984). Use f resistant cattle as a means f tick cntrl is als becming imprtant in Africa, Asia and the Americas (Fraga etal., 2003; Silvaetal., 2007). Hst resistance, reviewed by Latif and Pegram (992), manifests itself as the rejectin f ticks that attach t the hst because f hst-specific physilgical and immunlgical reactins. Highly resistant cattle keep verall tick ppulatins very lw in cntrast t cattle with lw resistance in the same herd that harbur mre ticks in certain seasns (Slmn and Kaaya, 996). Cattle can be ranked fr resistance n the basis f natural tick cunts and abut 0% f cattle ranked as f lw resistance carrying 50% f the ttal tick ppulatin infesting the herd (Latif et al., 99). Cmparisn f the respective tick infesta- 26

23 General intrductin tin n calves and cws shwed that calves had a lwer tick infestatin than cws (Jngejan et al, 987). In mst parts f Africa studied, zebu and sanga cattle are fund t be cnsiderably mre resistant t TBDs (Tatchell and Eastn, 986; Bakheit and Latif, 2002) and the applicatin f intensive dipping with the purpse t increase weight gain is therefre nt justifiable (Nrval et al., 992). The use f naturally tick-resistant cattle bitypes shuld be incrprated in tick cntrl schemes as a means t cntribute t the cntrl f tick infestatins n livestck (Silva et al., 2007). Althugh assessing the levels f hstresistance in different breeds f cattle by selectin, breeding r gene alteratin (de Castr, 99) may take sme time, the feasibility f this methd has been demnstrated at the Internatinal Centre f Insect Physilgy and Eclgy, Kenya (Latif, 992). Vaccinatin A nvel apprach ftick cntrl is t make vaccines against the vectrs rather than against all the individual disease agents they carry (Labuda et al., 2006). Previus studies have shwn this apprach t be feasible (Burke et al., 2005; Labuda et al., 2006). Research has fcused n identifying prteins frm the whle tick macerates, the ticks' salivary glands anddigestive tract as candidate brad-spectrum tick vaccine targets (Trimnell et al., 2005). Effrts have s far yielded ptential vaccines against Bphilus spp. (Willadsen et al., 2006). Prspects f develping similar vaccines against ther ixdid ticks f majr veterinary imprtance have nt been frthcming. Bphilus spp. are ne-hst ticks and shw a marked preference fr bvine hsts, which act as the principal reservir f perhaps the mst imprtant grup f disease agents {Babesia spp.) that Bphilus ticks transmit. By cntrast, mst ther ticks f medical imprtance are three-hst ticks, which infest nt nly cattle but als wild ungulate species. Fr these reasns, vaccines against nn-bphilid ticks may nt be feasible and a develpment f the near future. Anti-tick vaccines, hwever, remain ne f the mst prmising prphylactic measures against tick bites and transmissin f tick-brne pathgens (de la Fuente and Kcan, 2006). Integratin f semichemicals in tick management Unlike in insects where phermnes have been used extensively t cntrl certain crp and rchard pests (Judd and Gardiner, 2005), a semichemical-based strategy has made little headway in the cntrl f ticks and, where attempts have been made, it is nly n trial basis. Fr example, CO2, which is fund in vertebrate breath, acts as a tick kairmne, and has been tested fr sampling and cntrl under different synthetic preparatins such as dry ice, cmpressed gas (Gray, 985). Similarly, sex phermnes have been evaluated in a number f ways. Nrval et al. (989b) evaluated a cmbinatin f CO2 with -nitrphenl (ONP) in the field. Accrding t these wrkers, increased catch f A. hebraeum was btained by the activating effect f C0 2 in synergy with the inherent attractant actin f the phermne. Other attempts have explred the use f phermne/acaricide-treated areas f the hst. This methd prved t be very effective in killing ticks that were lured by the baits (Rechav and Whitehead, 978). A significant prgress in the latter apprach invlves the use f impregnated bjects r decys with a slw release delivery system (Snenshine et al., 992). Anther technique, which has achieved sme degree f effectiveness, is the cnfusant-killing methd tested by Ziv et al. (98). These authrs incrprated the 2,6-DCP-sex attractant phermne in gelatine micrcapsules (applied t hst's fur) and mixed with a pesticide (Prpxur) against the dg tick, Dermacentr variabilis. As a result, a majrity f lured males were killed, while surviving females were left unmated by the induced phermne-permeated backgrund. This successful disruptin f mating was achieved with 5.6 ug/ml f micrencapsulated 2,6-DCP. Mixtures f an acari- 27

24 Chapter cide with an aggregatin phermne r a sex phermne impregnated n plastic decys were amng ther ptins shwing prmise. Hamiltn and Snenshine (989) have patented a decy cated with natural Munting Sex Phermne (MSP) and a pesticide. The decys dispersed n the hair cat f the hst were fund highly lethal t males within 30 minutes and mating attempts by males with decy resulted in 89% f death (Snenshine et al., 992). A mdified methd f the abve was tested by Nrval et al. (992) wh used plastic bands impregnated with cmpnents f the attractant-aggregatin-attachment phermne (AAAP) mixed with the pesticide flumethrin. The pisnus bands were attached t the tails f the cattle and as a result, there was a gd killing f ticks as thse attracted aggregated arund the animal's rear and came int clse cntact with txic chemicals. The cmbinatin f an attractant and acaricide may nt sund as eclgically suitable fr a pesticide-free envirnment. Hwever, since this methd utilizes a behaviural trait that ccurs regularly and predictably, its advantage lies n a reduced amunt f acaricides being used and their tactical applicatin n the hst (Snenshine, 2006). Cultural cntrl Habitat interference and hst remval Habitat interference and hst remval can be directed against bth the free-living and parasitic stages f ticks (Aiell and Mays, 2003). The free-living stages f mst tick species, bth ixdid and argasid, have specific requirements in terms f micrclimate and are restricted t particular micrhabitats within the ecsystems inhabited by their hsts. Destructin f these micrhabitats reduces the abundance f ticks. Alteratin f the envirnment by remval f certain types f vegetatin has been used in the cntrl f A. americanum in recreatinal areas in suth-eastern USA and in the cntrl f Ixdes rubicundus Neumann, 904 in Suth Africa. Cntrl f argasid ticks such as Argas persicus Oken, 88 and A. walkerae Kaiser and Hgstraal, 969 in pultry can be achieved by eliminating cracks in walls and perches, which prvide shelter t the free-living stages. Remval f alternate hsts r hsts f a particular stage f the life cycle can als reduce the abundance f tick species as this may starve ticks t death depending n starvatin perid (Aiell and Mays, 2003). This apprach has ccasinally been advcated fr the cntrl f three-hst ixdid ticks such as R. appendiculatus, Amblymma hebraeum Kch, 844 and /. rubicundus in Africa, and Hyalmma spp in suth-eastern Eurpe and Asia. In Kenya, the Smali cmmunity uses this methd as ne f their tick cntrl strategies (Pers. Cmmun. with Smali pastralists). Rtatin f pastures r pasture spelling has been used in the cntrl f the ne-hst ixdid tick Bphilus micrplus Canestrini, 888 in Australia (Sutherst et al., 979). The methd culd als be applied t ther ne-hst ticks in which the duratin f the spelling perid is determined by the relatively shrt life span f the free-living larvae. Hwever, it has minimal applicatin t multi-hst ixdid ticks r argasid ticks because f the lng survival perids f the unfed nymphs and adults. Burning grazing pastures directly kills ticks, while plughing grazing fields buries them and eventually they die. Ethnknwledge in tick management Traditinal preventive management measures and practices emplyed in livestck tick cntrl and management have lng been used by varius ethnic grups engaged in traditinal animal husbandry. These measures were aimed at reducing the animals' risk f infestatin and have included the fllwing. () Practicing apprpriate herd distributin, in which, 28

25 General intrductin certain areas were used nly by cattle and small ruminants, r nly by camels depending n the varying spread f tick infestatin specific t particular types f animals (Marina et al., 200). (2) Avidance f specific pastures at particular times f the day r year, r thrughut the year. This has invlved areas with a large ppulatin f ticks, msquites and livestck-biting flies during the rainy seasn, which are nly used fr grazing during the dry seasn r nt at all. Areas infested with ticks in varius stages f develpment (e.g., abandned paddcks) are avided fr a number f mnths (Mathias-Mundy and Mc- Crkle, 989). (3) Keeping livestck away frm shady areas that may prvide micrhabitats fr ticks in pastures (Mathias-Mundy and McCrkle, 989). (4) During migratry herding, areas infested with ticks and ther parasites are avided at the times f year when the ppulatin is at its largest (Sutherst 987, Sykes 987). (5) Burning f pastureland suspected t be infested with ticks in varius stages f develpment (West, 965; ITDG and IIRR, 996). (6) Feeding animals n a certain type f sil nutrients t prevent tick infestatin (Marina et al., 200). (7) Hanging a buquet f flwers/leaves at the drs, windws and in the rfs f cattle shade (bma) and grwing certain plant species with repellent prperties arund the bma (ITDG and IIRR, 996). (8) Selective breeding f livestck with particularly high resistance t tick vectrs thus, making it pssible t intrduce a particular livestck species int areas where it culd nt be kept in the past (Sutherst 987). (9) Aviding cmmn grazing grund with wild animals serving as a "reservir" fr pathgensand causing particular tick-brne epiztic diseases and ticks per se (Mathias-Mundy and McCrkle, 989). And (0) Bush clearance t keep the tick ppulatin lw by destrying their micr habitats, particularly thse clse t bmas r frequently visited grazing grunds (Marina et al., 200). In livestck herds infested with ticks, a number f methds are used invlving the release f predatr chickens and birds, kitchen ash mixed with fresh grass, smke directed in the herd r bma and applicatin f varius ethnbtanical preparatins as cncctins, pwder/dust, blus, paste, il etc. In additin, handpicking f ticks was cnducted as a cmmunal cultural practice t reduce tick burden n heavily infested animals (Mathias- Mundy and McCrkle, 989). Of all these methds, applicatins f ethnbtanical prducts play a majr rle in tick cntrl and management. Fr example, in Kenya, the Smali peple take heavily infested herds with ticks t graze in vegetatins with Cmmiphra spp. which have repellent effects and cause ticks t get detached frm their animals (Beentje, 994). This traditinal claim has been cnfirmed bymaradufu (98). Pastralists in the rugged, harsh terrain f nrthern Kenya are increasingly using indigenus medicine rather than mdern veterinary services t care fr the health f their livestck. Instead f relying n regular dipping and spraying with acaricides, which are expensive and ut f their reach r unavailable in such islated areas, the herders use lcally available herbs t cntrl ectparasites, particularly ticks. Surveys and field trials carried ut in the Samburu and Turkana cmmunities f Kenya by the Intermediate Technlgy Develpment Grup-Eastern Africa (ITDG-EA), revealed that these tw pastral cmmunities use a cmbinatin f three traditinal herbal remedies fr tick cntrl, namely, tbacc (Nictiana tabacum L.) mixed with sdm apple {Slarium incanum L.), labai (Psiadia punctulata (DC) Vatke.) mixed with Ale (Ale secundiflra Engl.) and Africa live tree [Olea eurpea (L.) ssp. africana (Vent.) P. Green] mixed with Ilkerereai (Cadia purpurea (G. Piccili) Aitn) The results frm these field trials were prmising, as n tick (R. appendiculatus, R. evertsi, B. declratus and A. variegatum) culd be fund n the cattle with a mixture f either A. secundiflra and P. punctulata r A. secundiflra and O. eurpea 4 days pst-treatment (Akall, 2003). Hwever, in all the field trials, cattle treated with these traditinal anti-tick remedies, had tick reinfestatin starting n the tenth day 29

26 Chapter pst-treatment. Previus wrk at ICIPE invlving the interactin f lcal cmmunities and cnventinal researchers indicated that cmpnents f many plants culd act as allmnes fr sme lcal livestck tick species (Chiera et al., 977; Malnza et al., 992; Mwangi et al., 995a, b; Mwangi, 996; Ndung'u et al., 995; Lwande et al., 999. It was bserved that R. appendiculatuspreferred t ascend drier vegetatin than a fresh green ne, pssibly because f an unknwn allmnal factr(s) (Mwangi et al., 995b). Sme trpical legumes, especially thse f the genus Stylsanthes spp. have been reprted t exude sticky substances frm their trichmes that trap and kill ascending larvae f B. micrplus ticks (Sutherst et al., 982; Zimmerman et al., 984; Sutherst and Wilsn, 986; Wilsn and Sutherst, 986; Sutherst et al., 988; Wilsn et al., 989). In additin t these trichmes, glandular emanatins f Stylsanthes spp. were als fund t kill ticks (Sutherst et al., 982; Nrval et al., 983). Melinis minutiflra has als been identified t have strng antitick prperties (Menendenz, 924; Chiera et al., 977; Thmpsn et al., 978; Mwangi et al., 995a, b). Althugh it has been cnclusively shwn that crude extracts frm a number f plants have acaricidal, repellent and grwth regulating prperties against a variety f livestck tick species, the active cmpunds and blends are yet t be characterized and evaluated in bth labratry and field under varying cnditins in rder t allw their integratin int the strategies f tick cntrl and management. Current strategies and views n tick management Initially the main uses f acaricides were tick eradicatin, preventin f spread f ticks and tick-brne diseases and eradicatin and cntrl f tick-brne diseases. The eradicatin prgrammes were successful in sme eclgically marginal subtrpical areas, such as suthern USA and central Argentina where Bphilus spp. and babesisis were eradicated and suthern Africa where East Cast fever (caused by Theileria parva parva) was eradicated. The prgrammes were less successful in the trpical areas f nrth-eastern Australia, Central America, the Caribbean Islands, and eastern Africa. In the areas where ticks and tick-brne diseases eradicatin prgrammes were nt achieved, the csts f maintaining intensive tick cntrl prgrammes have becme prhibitive. Fr this reasn, integrated cntrl strategies are being encuraged. Frm previus experiences, the future f livestck tick cntrl and management lies in the integratin f the available classical and mdern technlgies (indigenus knwledge, vectr and hst semichemicals, hst-vectr assciatins, tick bicntrl agents, hst grming behaviurs, hst genetics, chemicals, vaccines, eclgical, immunlgical and the management f acaricide resistance), as it seems unlikely that any f the technlgies (as reviewed abve) is rbust enugh t stand alne (Willadsen, 997; Grssard, 998). The effectiveness f these cst-cntainment strategies requires better knwledge f the dynamic assciatins amng the disease agents, their vertebrate hsts, the tick vectrs and the envirnment. Cntrl f tick-brne diseases will require the utilizatin f the principles f endemic stability and develpment f imprved recmbinant vaccines. Strict quarantine measures need t be enfrced in rder t prevent re-intrductin f ticks and tick-brne diseases in cuntries where they have been eradicated. Climatematching mdels, gegraphic infrmatin systems, and expert systems (mdels based n expert knwledge and artificial intelligence) are being used t identify currently unaffected areas in which ticks may establish if intrduced (Aiell and Mays, 2003). Integratin f cmmunity-based ethnbtanicals in tick cntrl and management is increasingly being recgnised as an effective means f livestck tick management at lcal level (ICIPE's livestck tick cntrl and management strategy, ). 30

27 General intrductin Statement f prblem Althugh the applicatin f traditinal knwledge in tick cntrl and management is widespread, every cmmunity has its wn specific and unique knwledge. Fr this knwledge t be useful, each cmmunity therefre, needs t develp its wn knwledge-derived practice within its cultural framewrk. Hwever, as much asthis realizatin exists, a cnsiderable amunt f this indigenus knwledge is fast disappearing, while still in undcumented and unevaluated frms because f: () untimely deaths f persns with ethnknwledge, (2) extinctin f plant and animal species, the surces f the much needed ethnpharmaclgically active substances, (3) encrachment f develpment and mdernizatin, (4) adptin f new lifestyles and educatin systems that d nt embrace indigenus knwledge, (5) intermarriages, (6) changes in farming systems, (7) changes in human settlement patterns, (8) religius beliefs and western faith that label African ethnknwledge as inferir, (9) expsure t freign cultures, which believe that western type f prducts are always superir t dmestic nes, and (0) failure f gvernments/states t supprt and develp a legal framewrk and clear wrking plicy fr sustainable utilizatin f indigenus knwledge (Munyua et al., 998). Much as the afrementined infrmatin reveals the imprtance f indigenus knwledge as well as the dangers and threats facing it, a lt f it is nly knwn t few individuals (healers, traditinal medicine experts, village elders, spiritualists, herbalists, ritualists) in their respective cmmunities (Martin, 2004). The results f sme dcumented and evaluated indigenus knwledge lies n the shelves f libraries and labratries in many institutins and therefre nt many utside peple knw abut it r use it. Effective use f indigenus knwledge and its prducts have been affected by pr frmulatin, packaging, strage, standardizatin and determinatin f ptimal cncentratin and applicatin regimes (Martin, 2004). These prblems are cmpunded by the facts that ethnpractitiners d nt receive frmal training and their infrmal educatin is never systematic, dcumented and standardized and ethically accepted by all stakehlders in the cmmunity. This has raised fears f verdsing and pisning leading t death and ther undesirable side effects amngst the users. Frm a practical pint f view, therefre, identificatin, dcumentatin and scientific evaluatin f ethnbtanicals with anti-tick prperties available t and used by different cmmunities and the way they affect target tick species, may prvide an understanding f the underlying science s that better ways f using effective nes are develped and imprved t benefit the lcal cmmunities (Fig..7). Mrever, the strategic deplyment f anti-tick ethnbtanicals that takes int accunt n-hst behaviur f target tick species, and particularly their feeding site lcatin behaviur, may pen up effective, efficient, simple, accessible and sustainable methds f cntrlling target tick species suitable fr different rural cmmunities with minimal external input. Why study the Bukusu cmmunity in western Kenya? Research in ethnveterinary knwledge is mainly fcused n pastralists. Wrkshps held in Kenya in 996 (ITDG and IIRR, 996) and in India in 997 (McCrkle et al., 997) challenged this fcus and revealed that many arable farming cmmunities such as the Bukusu nce led a pastral life and therefre pssess a large amunt f ethnveterinary knwledge just like the pastralists. Indeed the Bukusu cmmunity has a vast amunt f knwledge abut farming practices, rich pastral vcabulary and the brad variety f legends cnnected with pastral life (Makila, 978). Secndly, during my childhd I watched my late maternal and paternal grandfathers apply several ethnpractices n tick-infested cattle, including the use f plants as 3

28 Chapter STARTING POINT OF OUR RESEARCH PROJECT I On hst (livestck') Tick parasitic stages: 5%f ttal tick ppulatin is always fund n hst where the ticks spend V 2 f theirlifecycle. CONTROL STRATEGIES ANDMECHANISMS FOR LIVE STOCK TICKS (R APPENDICULATUS) Parasitic stagesn the hst Nn-parasitic stages (hstseeking free stages) Off hst Tnpasture/externalenvirnment) Tick hst seeking stages: 95% f ttal tick ppulatin is always fund n pasture where the ticks spend 3 /i f theirlife cycle. Pssible interventin strategies On-hst cntrl devices Off-hst cntrl mechanisms Lcally-based and cmmunity-specific ethnbtanicals t manipulate feeding behaviur ftickswhilenhst Repellent cntrl device Attractive/trap cntrl device Txic cntrl device Cngenital malfrmatin cntrl device Acmbinatin cntrl deviceinvlving: repellent + txic +attractive/trap cntrl device Otheralternativesyet t be develped? Eithercmpleteremval r cnsiderable reductin ftickburdensnhstfr imprved livestckprductin atcmmunity level Cmmunity-based manipulatin f pasture/envirnment/eclgical mdificatin strategies * Repellent grass/pasture * Txic grass/pasture Attractive/trapgrass/pasture Strategicusefgrass/pasture inducing cngenital malfrmatin inlivestck ticks Strategiccmbinatin frepellent +txic + attractive/trap grass/pasture " Otheralternativesyettbedevelped? JJ- Develpment ftick-free pastures/envirnments/eclgical nichesinwhich livestck arefree frm tick infestatin, tick-bme diseases etc. Fig..7. Pssible interplay f factrs suitable fr the develpment f n-hst and ff-hst cmmunitybased integrated tick cntrl and management strategies, which are effective and sustainable under lcal 32

29 General intrductin bilgical acaricides, in the Bukusu cmmunity. These culd be applied as dusting pwders, pastes, juice extracts, and decctin substances. Other plant treatments included blus r infusins r smke. I wuld ften accmpany my grandfathers n their cnsultatin missins in the neighburhd. They were fndly knwn as Abalesi ba efiay, literally meaning 'the maids f the livestck', which translates t mean persns wh prvide primary healthcare t the animals in the village and are cnsulted in majr cases invlving livestck ill-health. As a result, ethnpractices caught my eye. I was fascinated by the rich repertire f traditinal knwledge and techniques applied in almst all spheres f peple's lives, be it in agriculture, healthcare, delivery f children, r in livestck r fisheries. And therefre, the prerequisite familiarity with the research cmmunity when ne is beginning t develp a career in ethnbtany was fulfilled andtk up the challenge (Martin, 2004). Why start first with n-hst and nt ff-hst apprach? This questin stems frm the infrmatin illustrated in the cnceptual framewrk f the prject in Fig..7 as t which fthe tw alternative appraches is apprpriate t start with. One need just t imagine what may happen if we started this research prject with ff-hst tick cntrl and management apprach and we neither kill 95% f ticks always present in the hst envirnment nr have in place apprpriate n-hst cntrl and management strategies and mechanisms! Ticks and tick-brne disease epidemics and related prblems may develp and wipe ut large numbers f herds f cattle within a shrt perid f time. Being guided by ur research questin regarding the type f tick cntrl technlgy that shuld be develped and put in place fr rural livestck farmers t use, we wanted t knw what the lcal cmmunities knw abut tick cntrl strategies and mechanisms (ethnpractices fr tick cntrl and management) and hw what they knw can best fit in an n-hst tick cntrl tactic. Why lcal cmmunity-driven tick cntrl interventin? In flie past, many effective tick cntrl interventins have been implemented and failed. One reasn fr this scenari is that the end users (the rural lcal cmmunity livestck farmers) tgether with their centuries' ld useful ethnknwledge are nt invlved in develping, planning and executin f such interventins. The current prject cnsiders this as a pririty. It will be easier and cheaper t implement any new technlgy fr tick cntrl and management within the lcal cmmunities because f prir familiarity and acceptability f lcal materials and ethn-knwledge used if the apprach is cmmunity-driven. This will make sense nly if the peple themselves tgether with their ethnknwledge are directly invlved in the prject. Secndly, they will learn and train t use their wn ethnknwledge in a better way and will nt require external persnnel t train them n hw t use the technlgy after its develpment. Thirdly, planning and evaluatin f animal health prgrammes require a clear understanding f bth the epidemilgy f the diseases in questin and the livestck prductin systems invlved (Putt et al., 988), which are clearly understd in rural cmmunities wh are the majrity livestck keepers (Kariuki, 996). Such an understanding can nly be achieved thrugh the availability f reliable and up-tdate infrmatin at all stages f the planning and evaluatin prcesses, which invlves clse interactin f rural cmmunities and researchers. 33

30 Chapter Objectives f the studies Main bjective The wrk under taken in this thesis had as its main aim t evaluate ethnpractices affecting livestck ticks frm the Bukusu cmmunity in western Kenya and investigate their ptential, strategic use and, in the lnger term, integratin with the knwledge f n-hst behaviur f the brwn ear tick R. appendiculatus fr develping an effective n-hst tick cntrl and management strategy. Specific bjectives. T survey, dcument and evaluate ptential anti-tick ethnbtanical(s) and ther practices asused by the Bukusu cmmunity in western Kenya (Chapter 2). 2. T extract essential ils frm selected plants, evaluate their effects n adult R. appendiculatus and determine their chemical cmpsitins (Chapters 3and 5) 3. T study the effects f the essential ils at the feeding sites n the navigatin and attachment behaviur f/?, appendiculatus (Chapters 4 and 6). 4. T evaluate an n-hst "push" tactic with the repellent ethnbtanicals fr the cntrl f/?, appendiculatus (Chapters 7and 8). Hyptheses. The selectin f certain plants fr tick cntrl in traditinal rural cmmunities is based n trial and errr prcess, which has led t the discvery f effective ethnbtanicals. Thus, there isan empirical ratinale fr their current use. 2. The lcatin f preferred feeding sites f ticks n the hst is mediated by hstderived semichemicals. It is pssible t manipulate these behaviural patterns thrugh the apprpriate deplyment f selected repellent ethnbtanicals leading t the preventin r reductin f tick bites n cattle. 3. The deplyment f traditinal tick cntrl practices in the current strategies f integrated tick cntrl and management may significantly be imprved if well integrated with scientific knwledge f n-hst tick behaviur. Outline f the thesis Chapter. The general knwledge abut the vectr, the hst and the pathgen is reviewed, while fcusing n the cntrl and management strategies f the vectr, R. appendiculatus. The research questins tgether with the justificatin f the research, bjectives and hyptheses are stated. Chapter 2. In the recent past, research int ethnveterinary knwledge is fcused n pastral and nmadic cmmunities and neglected arable and mixed farming cmmunities. In this chapter, a survey f indigenus anti-tick knwledge f the Bukusu, wh cmbine bth arable and pastral life frms fr ecnmic survival, is presented. Abut 54 candidate anti-tick plant species spread ver 0 genera and 5 families were identified and dcumented, tgether with 0 nn-btanical anti-tick ethnagents. Fllwing an extensive literature search n these plants, eight plants are selected fr labratry and field studies. Chapter 3. This Chapter cmprises labratry assays f essential ils extracted frm the eight selected plants (Tagetes minuta, Tithnia diversiflia, Juniperus prcera, Slaneci manii, Senna didymbtrya, Lantana camara, Securidaca lngepedunulata and 34

31 General intrductin Hslundia ppsita). Repellent effects f these essential ils n adult R. appendiculatus are evaluated using a dual chice climbing assay. The essential ils f tw plants, T. minuta and T. diversiflia with a relatively high repellent effect n adult R. appendiculatus are selected fr an indepth scientific study, cmparisn and chemical characterizatin using GC and GS-MS technlgy. Chapter 4. In this Chapter, I describe the behaviural bilgy f the vectr n tw Friesian steers. On-hst behaviur f adults f tw sympatric tick species, the brwn ear tick (R. appendiculatus Neumann, 90) and the red-legged tick (R. evertsi Neumann, 897), which prefer t feed mainly inside the ears and the anal regins f bvids, respectively, is studied. Up t 95% f R. appendiculatus and 85% f R. evertsi riented tward and lcated their respective feeding sites frm different parts f the hst bdy. An dur-based 'push-pull' pair f stimuli is suggested t accunt fr efficient rientatin and navigatin behaviur f the tw tick species t their respective predilectin feeding sites. It is hypthesized that such cncurrent deplyment f repulsive and attractive cues may be quite widespread amng arthrpds and related rganisms that specialise n specific hsts r micrenvirnments in theperfrmance f their bilgical functins. Chapter 5. A cmparative study is made between a dual-and n-chice assay arrangement n the climbing respnse f adult R. appendiculatus expsed t the essential il f T. minuta. Bth assays shw a significant repellent effect f essential il n climbing behaviur f R. appendiculatus. Hwever, in cntrast t ur predictins, using a n-chice tick climbing apparatus during the assay did nt increase the sensitivity. The dual-chice assay prves a mre sensitive assay than the n-chice assay, althugh the n-chice assay prvides greater residual effects. Chapter 6. In this Chapter I discuss the n-hst behaviur f R. appendiculatus, which was studied in the semi-field experiments t evaluate the repellent effects f the essential ils f T. minuta and T. diversiflia at its predilectin feeding site. This is amngst the few studies in which tick repellent essential ils are evaluated in the presence f hst-derived semichemicals. The disruptin effects f the tw essential ils n the dispersal, rientatin and attachment behaviur f the ticks are cmpared. The previuslysuggested mediatin f specific hst-derived attractive and repellent rientatin and navigatin signals are masked by the essential ils as initially hypthesized. Bth essential ils therefre may ffer ptential fr inclusin in the integrated tick cntrl and management particularly fllwing the labratry and field studies f individual cnstituent cmpunds and selected blends. Chapter 7. In this Chapter, the site n the hst animal and a delivery frmat suitable fr applicatin f essential ils were evaluated. Further, this in viv study als cmpared the repellent ability f the tw essential ils f T. minuta and T. diversiflia in intercepting the mvement f R. appendiculatus tward its predilectin feeding site.fr bth the essential ils, legs + tail sites f essential il applicatin, fllwed by ear smear and then ear tube, had significant effects n rientatin t the hst and attachment site preference f adult R. appendiculatus n the hst animal, in that rder. As treatment f legs + tail is tedius, time cnsuming, and requires mre essential il than the ther tw sites, we recmmended the ear smear site fr treating hst animals with essential ils fr field experiments in Chapter 8. Chapter 8. In this Chapter, I prvide the results f a field study that evaluates the effects f the essential il f T. minuta and T. diversiflia n R. appendiculatus infesting livestck in Bungma District, western Kenya. The ear smear site fr treating hst animals with essential ils as recmmended in Chapter 7 was used t study a "push" tactic fr nhst cntrl f R. appendiculatus in the field. Within -4 days pst-treatment, the number 35

32 Chapter f ticks n animals treated with the essential ils was reduced by mre than half that f the riginal ppulatin. By the 5th day pst-treatment, mre than 75 and 60% f adult R. appendiculatus and ther tick species, respectively, were affected by the essential ils s that they became disldged and drpped ff. A strnger repellent effect was shwn by the essential il f T. minuta than the essential il f T. diversiflia. Bth T. minuta and T. diversiflia essential ils affected several ther less dminant but ecnmically imprtant tick species such as Amblymma variegatum, R. evertsi and Bphilus spp., althugh these tick species have different feeding sites. The results suggests ptential fr apprpriate essential il frmulatins in reducing tick burden and assciated tick-brne diseases amng the resurce-limited livestck farming cmmunity in trpical Africa. Chapter 9. In the final Chapter, I discuss the circumstances surrunding the tick cntrl mechanisms. The discussin fcuses n the questin: why the livestck tick is still a serius pest in livestck industry after mre than 50 years ftick research? Can the integratin f plant-derived repellents in tick cntrl and management strategies prvide a slutin t the chrnic tick-brne prblems in livestck industry? The discussin reflects n the results btained frm this research, pinpinting the general cntributin made and the specific gaps in knwledge and need fr fllw up research and develpment. Relevant cnclusins based n the research results are utlined. 36

33 General intrductin 37

34 Parti Use f ethnbtanicals fr tick cntrl

35 Chapter 2 40

36 Tick cntrl in the Bukusu cmmunity Traditinal knwledge n tick cntrlwithin the Bukusu cmmunity in Bungma District,western Kenya Abstract: In the recent past and currently, research int ethnveterinary knwledge is fcused n pastral nmadic cmmunities and neglected settled arable and mixed farming cmmunities such as the Bukusu. We cnducted a survey f indigenus tick-management knwledge f the Bukusu, wh cmbine bth arable and pastral life frms fr their ecnmic survival, used participatry actin research appraches invlving 272 wmen and men aged between 8 and 8 years. The infrmatin was cllected frm a schl-based questinnaire, grup discussins, and individual interviews as well as discussins with sme key respndents, veterinarians and field staff frm the Ministry f Agriculture, Rural and Livestck Develpment. Traditinally, ticks have been cntrlled and managed using a variety f methds, such as applying ethnbtanicals, hand picking, burning pastures, livestck quarantine, grazing practices, cleaning cattle sheds, burning r burying residues f cattle shed, bird predatin, feeding animals n natural salty sils lcally called 'silng', and applying kersene, sap, fish residues, cattle dung and urine, grease, ash, magadi sda, and sisal juices n cattle. Ethnbtanicals have been applied by a variety f methds, e.g., fumigatin, puring a decctin n the animal, steaming r dusting the animal's skin, hanging plant buquets in cattle sheds, rubbing a blus r paste n the animal, and grazing animals in pasture f identified anti-tick plants. Abut 54 candidate anti-tick plant species spread ver 0 genera and 5 families were identified and dcumented, tgether with 0 nn-btanical anti-tick ethnagents. Frm an extensive literature search, we fund that f the 54 plants dcumented had been scientifically evaluated elsewhere and fund t have either acaricidal, insecticidal r biactive ingredients r were clsely related t plants with acaricidal r tickrepellent prperties. This infrmatin was crucial in prviding leads t identifying suitable acaricidal r tick-repellent btanicals available in different lcal cmmunities and prbable scientific ratinale underlying their use. Intrductin Key wrds: Bukusu, traditinal tick cntrl and management, ethnknwledge, ethnbtanicals, livestck industry, ticks Bungma District is lcated in Western prvince f Kenya between latitude 00 28' and 0 30TSf and lngitude 34 20' and 35 5'E (Fig. 2.). The District ccupies an area f 3074 km 2, with a ppulatin f abut 2 millin peple n the suthern slpes f Mt. Elgn. It lies in an agr-eclgical zne stretching frm trpical alpine znes t lwer mid-land 'Thischapterwillbepublished as:wanzala,w.,takken,w.,pala,a.o.,mukabana,r.w., Hassanali,A.,TraditinalknwledgentickcntrlwithintheBukusucmmunityinBungmaDistrict, westernkenya,whensubmitted. 4

37 Chapter " E -030 N y**** tvy&av! BUTERE/MUMIAS/ \\*M\ N (g> y -9 y...; L_i X-0 7 ' 4km Key: CllectinsitesfplantsamplesinBungmaDistrict (#) andneighburhd (( )) Sampledprimaryschls (-0) used tidentifykeyrespndents Frestedmuntain/bushy hills ( f + + ) Rivers/streams {-^Z^) Fig. 2..ThelcatinfBungmaDistrictinKenya. NtethepsitiningftheDistrictwithintheLake VictriaBasinandtwardsthesuthernslpesfMt. Elgn. zne with tea, wheat/maize-pyrethrum, cffee, sunflwer-maize, sugarcane and cttn plantatins (Martina, 998). The predminant ff-farm vegetatin patterns are riverine frests, rcky frested hillsides, hedgerws, wded grassland relicts, wdlands r clline frest relicts andtree grves whereas thenticeably tree-rich n-farm management units are hme gardens, hmesteads, live fences, cffee- and banana-grves and annual crpping fields (Martina, 200). The lcatin f the District nthe slpes f Mt. Elgn 42

38 Tick cntrl inthe Bukusu cmmunity influences rainfall and mitigates temperatures. Average annual rainfall ranges frm mm, while mean annual temperatures in the suthern parts, away frm the muntain, are abut 2-22 Cand in nrthern areas clser t the muntain are in the range f 5-0 C because f altitude. The District is within the Lake Victria Basin, rising frm 200 m abve the sea level in the West and Suthwest t ver m a.s.l. t the Nrth. The slpes f Mt. Elgn are generally gentle, althugh in sme areas they rise abruptly in the frm f cliffs up t 70 m in height and are cut by deep river grges with frequent waterfalls. The rest f the area cnsists f a gently slping surface falling frm 2 00 m elevatin in the nrtheast t 200 m elevatin in the suthwest. The surface cnsists f wide, nearly flat land separated by shallw river valleys. Several inselbergs and ranges stand abve the general level, frming the Kavujai Hills, Luc h Hills, Sang'al Hills, Mwibale and sme small hills, especially arund Sirisia Divisin. The Nzia River and its tributaries, Chwele, Khalaba, Kibisi, Kuywa, Kimilili, and Lwakhakha mainly drain the District. All the streams, except the Nzia River riginate frm Mt. Elgn (Fig. 2.). Of the 9 ethnic grups f the Luhya peple in western Kenya, the Bukusu cmmunity hlds livestck keeping/farming and livestck per se in high esteem, as livestck is used as a measure f ne's wealth and the animals' bld and meat are required and highly valued in all their traditinal and cultural practices. Examples include funeral ceremnies (where cattle skin is used as a cffin), circumcisin, agricultural festivals, bride wealth exchanges, and during cleansing ceremnies and sacrifices t appease the spirits. In the recent past and currently, research int ethnveterinary knwledge is fcused n pastral and r nmadic cmmunities whereas settled arable and mixed farming cmmunities such as the Bukusu have been neglected. Indeed, almst all f these neglected cmmunities, the Bukusu included, largely led nmadic lifestyles befre settling dwn t either arable, r mixed arable/pastral farming systems (Makila, 978). They therefre have ethnveterinary knwledge that is just as rich as nmadic cmmunities preferred in the ethnveterinary research (ITDG and IIRR, 996), making the Bukusu cmmunity ideal fr current ethnveterinary research. Useful ethnveterinary infrmatin has nt been dcumented t date in many cmmunities such as the Bukusu and is at risk f being lst (Kfi-Tsekp and Kiy, 998). In this study, we discuss the results f a survey cnducted t dcument and analyse anti-tick ethnveterinary practices f the Bukusu cmmunity in western Kenya. The study further helps t demnstrate the link between plant bidiversity and livestck healthcare ethnknwledge systems in the Bukusu cmmunity. The Bukusu cmmunity Bungma is a district including a wide diversity f ppulatins in western Kenya. This is partly due t its establishment as the last stp n the Kenya-Uganda railway in the 920s, rapidly grwing int a cmplex cmmercial and clnial and pst-clnial administrative center alng the railway line (Prt, 2000). A majrity f the residents f Bungma belng t the indigenus Bukusu, wh live near the brder with Uganda, alng the suthern slpes f Mt. Elgn ever since their migratin frm the Sudan/Egypt brder during the secnd half f the last century (Were, 967; Makila, 978). The Bukusu cmmunity is the largest single ethnic grup f the 9 ethnic grups (sub-tribes) f the Luhya peple f interlacstrine Bantu grup f East Africa, with 6.35% f the Luhya ppulatin (Central Bureau f Statistics, 996). The Cmmunity speaks the Bukusu language, whse current diversity f prnunciatins and intnatins, and even the lexical varieties, has been heav-ily influenced gegraphically and with time by immigrants f bth Bantu and Niltic ri-gin (Makila, 978; Wandibba, 998; Rach, 2003). It has been bserved that the Bukusu cntinue t 43

39 Chapter 2 maintain a strng attachment t their traditins, cultures, and superstitins (Crbit and Wanyama, 2004). Hwever, in spite f these bservatins, there is sme evidence that there is a grwing ersin f the value f the Bukusu ethnscientific knwledge (Wandibba, 998; Rach, 2003; Crbit and Wanyama, 2004). This is believed t be the result f a cmbinatin f factrs: () deaths f persns with ethnknwledge, (2) extinctin f plant and animal species and ritual practices, (3) encrachment f develpment and mdernizatin, (4) adptin f lifestyles and educatin systems that d nt embrace ethnknwledge, (5) bias in religius beliefs, (6) perceptin f certain sci-cultural practices as unhygienic, and (7) expenses and risks invlved in sci-cultural practices. Mrever, t date, little written infrmatin is available, little r n infrmatin is being passed n t the current generatin and there is a lack f generalized use f this ethnknwledge acrss the Bukusu cmmunity. This situatin underlies the imprtance f the present study. In the Bukusu cmmunity, plants were histrically and are still highly valued fr nutritinal and medicinal applicatins, educatin, agr-eclgical requirements and functining f ecsystems, ecnmic value and in sci-cultural life (Martina, 998; Mann, 200; UNEP Wrld Cnservatin Mnitring Centre, 2003). Fr centuries, plants have been part f the Bukusu's land-use system. This is demnstrated by the practice f selective clearing f trees, frests, and bushes, leaving useful trees and wdlts t grw in the arable fields fr fdder, timber, fd, medicines, windbreaks, fences/bundaries, supprting crps, shrines, etc. (Martina, 998). A significant prprtin f Bukusu pems, riddles, and prverbs are expressed in assciatin and with symblic meaning f indigenus plants (Martina, 998; Mweseli, 2004), summarising daily life experiences as well as prtraying philsphical bservatins, which elders cnsidered t be essential skills with which t equip grwing children. Sme plants were never used at all and were specifically reserved fr ancestrs, thers fr certain functins nly, while yet thers were used by a certain class f peple and during certain ccasins nly (Martina, 998). These sci-cultural practices and human-envirnment interactins are essential tday fr the recgnitin f ethnbtanical knwledge, and its meaningful utilizatin as well as the develpment f cmmunity-specific cnservatin strategies (Juma, 989). A great deal f infrmatin regarding the use, management, and sci-cultural values f plants is remembered particularly by the lder generatin (45-08 years f age). The livelihd f the Bukusu cmmunity depends slely n the cmbined pastral and arable ecnmies (Martina, 998). Being sedentary pastralists, they had time fr arable farming; wmen and yung girls were knwn t grw millet, Eleusine cracan. Early in the last century, they lived n millet, cassava and small livestck, but nw raise mstly maize, beans, ptates, cassava, bananas and cabbage fr fd and grw sugarcane, cttn, tbacc and sunflwer fr cash. They als keep cattle, gats, sheep, pigs and chickens. In fact, arable farming activities (sugarcane, maize and millet plantatins) have dminated the land use and currently threaten the survival f grasslands, wdlands, bushes and frests, which frm the surce f ethnpharmaclgically active agents upn which ethnknwledge is based (Bisafety News, 2002). Men and yung bys lk after cattle alng the river valleys n patches f grasses alng arable lands, and in grasslands and bushes surrunding frests. Wmen and yung girls are respnsible fr dmestic wrk. Wmen till the land and grw crps fr cnsumptin such as millet, squashes, cassava and ptates. They als lk after calves, sheep, birds and gats. Hwever, the Bukusu custmary rights nly allw the wmen t use land but nt t wn it, despite their significant cntributin t agriculture and animal prductin (Kenya Natinal Archives, ; Nasimiyu, 985; Nangend, 994). It is this divisin f labur that has determined the distributin patterns f a given aspect f ethnknwledge within the Bukusu 44

40 Tick cntrl in the Bukusu cmmunity cmmunity. Many families live in huses made f mud with straw/grass rfs. The straw/grass rfs prvide fr shade frm the ht sun in the summer mnths and are ttally waterprf fr prtectin in the rainy seasn. A few peple live in cement huses with irn sheet rfs. Bth types f husing suffice and are reasnably cmfrtable. Mst rural hmesteads d nt have electricity r running water. Lighting is with candles r kersenepwered lanterns and water is drawn frm a well n the farm and is either filtered fr drinking r heated fr bathing. Beds usually cnsist f a wden frame with a thick mattress. The significance f archiving and valuing ethnveterinary knwledge The pastral lifestyle was initially the mainstay f the Bukusu ecnmy befre arable farming vertk it, as evidenced tday by the rich archaelgy, nmadic and pastral vcabulary, traditins and scicultural anthrplgy f the Bukusu peple (Makila, 978). Mrever, the cmmunity has well-established ethnveterinary practices in curing, cntrlling and managing livestck diseases and related livestck vectr-brne prblems. A rich histry f anti-tick ethnveterinary practices has evlved t avert tick-related ecnmic lsses and as a way f survival in a tick-prne envirnment (Lewis, 200). Such traditinal applicatins, which have withstd the test f time, have a number f advantages ver mdern equivalents. They are hlistic in actin, cst-effective, easily applied, lcally suitable, easily accessed, envirnmentally friendly and cmmunity-specific (Mathias-Mundy and McCrkle, 989; Hamburger et al., 99; Martin et al., 200). Mrever, these ethnpractices are an integral part f the peple's daily lives and are based n inherent indigenus knwledge develped frm experience gained ver centuries and adapted t lcal culture and envirnment. The knwledge has been acquired thrugh such practical life experiences as bservatins, dreams and visins, friendly exchanges, trade and sales, ceremnies, inheritance, and annual exchange prgrammes f ethnpractitiners. Because this is mainly an ral traditin, ethnknwledge is largely transmitted by wrd f muth frm generatin t generatin (Kkwar, 993), and nly sketchily recrded in bks (Abegaz and Demissew, 998). It is als stred (based n the cllective memry f the cmmunity members) and passed n thrugh sngs, pems, drawings, paintings and stries (Mweseli, 2004). In certain cmmunities, the cllective knwledge is believed t be wned by the ancestrs and kept under the custdy f presently-living elderly men and wmen. There is a danger hwever, that this methd f vesting knwledge in the humans (elderly) can be undermined by their mrtality, thereby causing a lss f imprtant infrmatin fr future generatins. This study therefre was carried ut t survey, dcument and generate a database f anti-tick ethnpractices and knwledge. This resurce may be used t develp a new framewrk fr effective tick management in sub-saharan Africa and the pssible deplyment f sme f the ethnpractices n a wider scale (Alghali, 992). The ethnknwledge generated may be integrated with mdern science t sustainably cntrl and manage livestck ticks f pr livestck farmers. In s ding, value-added knwledge will be prvided back t the cmmunity in the frm f useful prducts (e.g., the develpment f an n-hst tick cntrl device is envisaged) and services, and help ethnpractitiners regain cnfidence in their ethnknwledge and establish links with mdern science fr sustainable use and management f lcal envirnment and its resurces. 45

41 Chapter 2 Materials and methds The surce f anti-tick ethnknwledge Anti-tick ethnknwledge was surveyed frm 3 different surces in the study area. The survey invlved a sample f 272 key respndents f mixed sex and age (8-8 years ld) and was drawn frm a number f surces. These surces included primary schl pupils wh prvided leads t key respndents and lcal schl teachers/educatin fficers wh helped select candidate schls, gave permissin t distribute the questinnaire t pupils, and helped clarify what infrmatin was being asked f pupils. Lcal veterinarians, paraveterinarians, and agricultural extensin fficers respnsible fr prviding extensin services t farmers were als able t identify sme key respndents. We als attended public meetings rganized by lcal administratrs t identify mre pssible respndents. Lcal livestck traders and dealers, as well as individual livestck farmers, cntributed their knwledge n tick cntrl based n their prfessinal and ecnmic activities, whereas cmmunity/village/clan leaders/elders had infrmatin n cmmunal tick cntrl and management prgrammes funded by the Gvernment and NGOs. Lcal ethnpractitiners, including traditinal healers/herbalists/spiritualists/ritualists, frmed a particular subset f knwledgeable peple frm whm key respndents were drawn. Other surces f data included NGOs such as the Cuncil fr Human Eclgy-Kenya, the German Develpment Service and the Bungma Indigenus Trees Cnservatin Club, which had prir experience and clse interactin with lcal livestck farmers and had earlier dcumented part f their ethnveterinary knwledge. Centres knwn fr preparatin f a lcal brew (Busaa) were the mst imprtant meeting pints f ld men and wmen and frmed a venue fr the discussin sessins. Secndary data was btained frm the Bungma District Veterinary Office (DVO) recrds n tick cntrl. All these grups were cnsulted because each was assciated with a specific aspect f ethnknwledge relevant t the study. Keystepstaccessing anti-tick ethnknwledge Cnstitutin f sampling grup The first step was the generatin f apurpsive sample fkeyrespndents frm the 3surces mentined abve. Key respndents are lcal experts r peple in the study area with a prfund knwledge f aparticularissuertechnlgyfinterest (inthiscase, anti-tick ethnknwledge) (Etkin, 993; Waters-BayerandBayer, 994,McCrckleetal., 997). They have amreextensive understanding flcalscialandveterinary-culturalsystemsthan thersinthecmmunity. Apurpsivesamplereferred t aparticular subsetfknwledgeablepepleintheareaftraditinalcntrl and management f livestck ticks. Intensive and extensive cllabratin and interactin with these key respndents was cnsidered t be an effective research strategy (Oakley, 98;Warry, 992). Arandm samplewuldnthavebeen apprpriate fr thissrtfsci-culturalset-up,in which we dependn flk knwledge (Etkin, 993),asnteveryne sampledrandmly mayhave therequiredknwledge (Martin,996). Lcal primary schl pupils: the questinnaire methd This wasthe main methd usedt identify key respndents and btain lcal and remte anti-tick ethnknwledge frmlargely illiteratepeple.theuse fchildren t cllect suchinfrmatin has had precedents in the field (Lans and Brwn, 998;Lans,200),but there isvery littlepublished literatureabut thismethd (Campell,994). Frm alistflcalprimaryschlsatthedistricteducatinoffice (DEO)inBungma, 0schlswereselected frparticipatin inthesurveystudies.theselectinfschlswasbased 46

42 Tick cntrl in the Bukusu cmmunity n variables such as rurality vs. urbanizatin, ethnicity, gender, n-ging teaching activities and gegraphicalspreadf theschlswithinthestudyarea. Inselectedschls,pupilswerevisitedand the basicsf participatryresearch (BaldwinandCervinskas,993)wereexplainedtthem,and the imprtance f their cntributin was stressed. Pupils were asked t interview parents, friends, neighburs,etc.abuttraditinal practicesftickcntrland fill theanswersftherespndentsin a well-structuredquestinnaire.thequestinnairecnsistedf5 questins: () the lcatin where questinnaire is administered, (2) identificatin f the persn being interviewed, (3) type and number f livestck kept by the persn interviewed, (4) what the tick means t the livestck farmer, (5) the kind f tick-related prblems experienced by the livestck farmers, (6) respnses t tick-related prblems, (7) assciatin between ethnremedies and identified tick-related prblem, (8) tick ethncntrl remedies (ethnbtanicals) (9) hw these ethnbtanicals are: harvested, prcessed, and applied, (0) when d livestck farmers apply their ethnremedies, () mde f applicatin f these ethnremedies, (2) mnitring f cases after applicatin and (3) any bservatins made regarding sideeffects f these ethnremedies, (4) any cllabratin sught during tick cntrl, and (5) persnal bservatin f tick infestatin made by the interviewer in the candidate livestck herd f the interviewee. Frm thequestinnaires,keyrespndentswereidentifiedbasingnwhethertheessayand rthequestinnaire indicatedthat arespndenthadptentiallyuseful infrmatin nanti-tickethnknwledge.thismethdwascnsideredveryuseful andrbustbecauseitreducedthe fllwing bias: ()mdellingbias,whichwastheprjectin f theinterviewer'sviewsntthsestudied, (2) strategic bias, which was the expectatin f benefits by the subject, (3) familiar relatinships betweeninterviewerandinterviewee (childrenandparents/neighburs)reducedresistancetthe questining and respnding withrte answers,aswell as utsider bias (Suttn and Orr, 99)and (4) reduces biasfrtheselectin f keyrespndents (Etkin,993; Waters-Bayer and Bayer, 994). Persnal interviews with key respndents Thethird stepwas cnducting theinterviews/discussinswith keyrespndents, fllwed byplant specimenidentificatin andcllectin frm the field andtransprtatin ttheuniversityfnairbi Herbariumfrfurther identificatin andtaxnmicstudies. Theinterviewswereguideddiscussins, semi-structured by amentalchecklistfrelevantpintstcnfirm thevalidityf thequestinnaire infrmatin. Fcus-grup discussins The furth step invlved hlding jint fcus-grup discussins with all stakehlders. A fcusgrup discussin is an explratry discussin designed t btain perceptins n a specific theme frm atarget grup in a nn-threatening envirnment (Krueger, 988;Etkin, 993).Thiskind f grup interactin prduced data and insights that wuld have therwise been less accessible (Mrgan,988). Theinteractin betweenallstakehldersfrmedthecllabrativeandpartici-patry appraisal (PA) appraches utilized tbuild acnsensus andverify thatthe infrmatin frmther intervieweeswasaccuratelyrecrded (ERR,994). Thegrupinteractinalsminimized thebjectificatin ftherespndents asthenlysurcefdata (Oakley, 98).Onepurpsefthisfrmf cllabrativeresearchwastreturn decisinmaking,basedntheretical knwledge,tthecmmunity,ratherthancncedingthisrletthecnventinally-trained expert (Warry, 992). Empwermentfresearchsubjectscantakeplacewhentheryisallwedtinfrmpracticalactinand by returningknwledgefrusetitspintfrigin (Warry, 992),thrugh cnsultative cmmunitybased training wrkshpsandmanualbkletstranslatedintlcallanguages. 47

43 Chapter 2 Cllectin f secndary data The fifth step invlved the cllectin f secndary data n anti-tick plants and ethnpractices frm the District Veterinary Office (DVO). This was fllwed by an extensive literature search n plant taxnmy and ethnbtanical applicatins frm the internet, livestck research institutins, NGOs, East Africa and University f Nairbi Herbaria libraries and labratries. Enumeratins f dcumented plants with effects n livestck ticks An extensive list f ethnbtanicals, including their scientific names as well as vernacular names, grwth habits, family names and ther infrmatin abut their usage can be fund at the end f this dissertatin (Appendix ). The term ethnbtany is used in the cntext f hw peple f a particular culture and regin make use f their indigenus plants: in this case, cmmunities, tribes, clans and sub-clans living in Bungma District. Because f the ethnic diversity amngst cmmunities living in Bungma District, mre than ne vernacular name may be used t refer t a particular plant and/r any ther related plant within a given genus r family. On the ther hand, tw r mre different plant species culd have ne vernacular name depending n their gegraphical lcatins, uses, and assciated ethnic grup(s). Classificatin f grwth life frms and r habits was defined accrding t Yumtetal. (994). Results and discussin Lcal primary schl pupil-lite questinnaire methd Thequestinnairemethdwasinstrumentalinallwingtheparticipatinf acrss-sectinflcal residentsintheresearchsurvey.thishelpedus tbtainmuchuseful infrmatin andensuredthat plants with mre than ne vernacular name were crrectly identified and their uses accuratelyrecrdedthrughutthestudiedcmmunity.inadditin,itallwed acnsiderablenumberfilliterate peple,andthseethnpractitinersandtherknwledgeablekeyrespndentslivinginremteareas t bereached and tmeetthetargets fthesurvey (Suttn and Orr, 99).Many fthesepeple gaveuseful leadingviewpintsasshwnintables Interviews with key respndents and fcus grup discussins Fcus-grup discussins were instrumental in enabling us t transcribe the lcal Bukusu language accurately, especially with regard t lcal plant names and their meanings. This methd f grup discussins furthermre yielded key infrmatin fr the selectin f the anti-tick plants that merit further scientific investigatin. The discussin sessins helped t reslve the cntrversies amng interviewees f multiple names referring t ne plant and vice versa. Fllwing numerus meetings and interactins, a cnsensus was built amngst the discussin grups n the crrect naming f plants and their lcal uses and preparatin. It als became evident that there was need fr cnventinally trained scientists t: () respect the wnership, surce and rigin f ethnknwledge and the needs and sensi-tivities f its hlders (2) take the time t establish a strng, trusting relatinship based n hnesty, penness and cperatin, (3) wrk n prjects f cmmn interest and benefit, (4) cntinuusly fster cmmunicatin between partners, and (5) prvide value-added knwledge back t the cmmunity in the frm f useful prducts (such as reprts) and services, and share equitably with the hlders any benefits arising frm the use f ethnknwledge. Ethnpractitiners shuld be made aware f legislatin prtecting their ethnknwledge base thrugh relevant state ministries, s as t defend the cmmunities' indigenus knwledge frm bipiracy that may threaten it. 48

44 Tick cntrl in the Bukusu cmmunity r- * v t-~ f* > *' <N <N C-l * <N (N O O ~ r- q ^O 0\ * <N ^5»n»n ' S O O O -" -H _< (Nj d d d ri «n CI ^- < d d d d d d d (N <N a d d d -I 3 d d d d d d d e «a- v ^ ~H r in r-; ^ ; ci -^ -si- d d d _J _i f> m <-- e- "2 ^ 00 (3 r- in * rf d d d d v v *",' O K JJ CN m r^t ^-< ^ ^i ^ ^ ^! "". ^H (N (N d d d < O in ^Jd d d >H M m N N d d d ra j c -H _H O -H ( N O H -H O O O f*> c «* «d d -H rt d d d d 3-8 S -a G. O PH GO in y m <N m <N O O O -H -H CO r-h *rt -^t d d d r- J3.tS O "g T3 a u _ " a 2 * a.^ tr W3 II <a " T3 O M c s- -^«U ^ «* m <N <N r*i r*s ~- d CN m d Os --< i O O O, d d m m * ^~ -H^H <=> - - g r* in ^t- ~ d d' d, <t in in *H d d <N - 2 m r- ~ _; _J "> a «s " - B u ri g u 3 5 H J J 3 Si.S g.2?& 4 i m m S _ -Si 4) <J 2 49

45 Chapter 2 Table2.b.Observatinrecrdsbyintervieweesnindividualanimalsftheirherds (n =233) Categries f bservatins Many ticks (> ~20 per animal) Few ticks (~5-20 per animal) Very few ticks (< ~5 per animal) N tick was bserved n any animals in the candidate herd N bservatin was made n individual animals in the candidate herd N f herds bserved Mean N. f ani-mals per herd ± 0±2 8± 7± 0± % f interviewees wh made an bservatin Respnses t questinnaires and results frm the discussin grups Despite the initial cnstraints in accessing anti-tick ethnknwledge, there was sufficient gdwill amng mst f the main cllabrating and participating stakehlders t ensure substantial success in the research prcess. Frm the survey studies, it became apparent that althugh fr a lng time the rle f ethnveterinary medicine and its ptential cntributin t livestck health has been neglected (Kfi-Tsekp and Kiy, 998), a large prprtin f peple still depend n it. This was evidenced by the cmplex cmpsitin f the purpsive sample (n = 272) f key respndents, and their vluminus and high-quality respnses (Tables 2.a and b). Even mre imprtant was the cmmitment and determinatin f all the stakehlders t have their lcal anti-tick ethnknwledge recgnized, dcumented and evaluated. They t were interested in using their ethnknwledge in an imprved and sustainable manner. Frm discussins during ne f the fcus-grup sessins held in Bungma District under the auspices f Bungma Indigenus Trees Cnservatin Club (BITCC) and Bungma Herbalists Assciatin (BHA), it was evident there was cncern that the cmmunity culd lse its rich sci-cultural heritage. This was further cnfirmed by a lack f generalized use f this ethnbtanical knwledge acrss the Bukusu cmmunity (Wandibba, 998; Crbit and Wanyama, 2004; Butey, 2004). There was a cnsensus, therefre, that effrts t cnserve ethnbtanical knwledge f the Bukusu be intensified. Thrughut the clnial era, indigenus knwledge was equated t witchcraft and whlly dismissed and actively suppressed. The practice cntinued, hwever, in a clandestine manner t avid punishment by the clnial administratin and nly after independence did trends twards strengthening natinal and sci-cultural identities began re-emerging (Mubukusu, pers. cmm.). Succeeding gvernments encuraged the integratin f indigenus knwledge int existing health services. Hwever, there was n clear legal framewrk fr its peratin. The cnventially-trained dctrs and varius religius faith grups tk the advantage f lack f legal recgnitin f indigenus knwledge t denunce it. This attitude has encuraged explitatin, withut due recgnitin f indigenus knwledge. Indeed, during ur study, Bungma ethnpractitiners reiterated that they are being explited by malpractices f biprspecting and bipiracy by sme cnventinally trained scientists, sme f whm attempt t lay claims n the ethnknwledge withut giving the indigenus cmmunity prper recgnitin. This behaviur has caused fear amng ethnpractitiners and has cntributed t their reluctance t share indigenus knwledge with freigners. It als accunts fr a discrepancy between knwledge availability and its use, as well as mistrust and dishnesty amng stakehlders (Simn, 998; Hempel, 2002). It is hped that the present study, based n participatry research, will help 50

46 Tick cntrl in the Bukusu cmmunity Table2.2.Tick-bmeprblemsexperiencedbylivestckfarmers aspercentagefrespndents (n =272) Tick-related prblems Livestck diseases per se withut Anaemia Pr perfrmance f the animals Inflicted wunds and damage t skin Irritatin t animals East Cast Fever (ECF) Deaths Weakening animals Anaplasmsis Heartwater General ecnmic lsses Fever Gall sickness (disease) Nn-respndents specificatins % respndents identifying prblems t reinfrce wnership and recgnitin f indigenus ethnbtanical knwledge amngst the Bukusu. Mrever, lcal cmmunity apprval and active invlvement in prjects cnducted by cnventially-trained scientists helps t restre cnfidence in the ethnpractitiners and prvide insights int cnservatin and management f bidiversity and lcal ecsystems in rural settings. Assciated tick and tick-related prblems The Bukusu cmmunity is infrmed and aware f livestck ticks and assciated tick-brne prblems. The cmmunity participants invlved in the survey described t the lcals the nature f ticks and the bjectives f the study in layman's language that was well understd. Frm their respnses, it was clear that the interviewees cnsistently recgnized the parasitic (n-hst) tick stages, which were mre cmmnly seen than the nn-parasitic stages (ff-hst) f ticks (Tables 2.a and b). They bserved ticks n their livestck and categrised the intensity f infestatin (Table 2.b). Results frm Table 2.b crrespnd t tick-related prblems identified in Table 2.2. This cnfirms that every dmestic animal hsts a variety f tick species. Ticks were traditinally recgnised by their clur, size, type f life cycle and n-hst feeding sites and habitat preference (Table 2.a). A cnsiderable prprtin f lcal peple were familiar with the varius ticks that are fund attached n ears, udder, scrtum, tail/anal regin, dewlap/neck, between legs and head. They als recgnized tick and tick-related prblems (T&TRPs) as serius cnstraints t livestck prductin in the study area (Table 2.2). The descriptin f ticks n the hst animals (Table 2.a) tgether with the prblems caused by ticks (Table 2.2) reflected the different tick species available in the study area. Ticks are knwn t cause a number f serius livestck prblems in the study area (Table 2.2). Descriptins f the ticks, including their predilectin feeding sites n their hst animals (Table 2.a) were sufficient t cnfirm their taxnmic status t genus level using vucher specimens frm the Kenya Natinal Museums (KNM) as Rhipicephalus spp., Amblymma spp. and Bphilus spp. A majrity f respndents classified ticks by

47 Chapter 2 Table 2.3. Frequency f ethnmethds used by respndents in tick cntrl and management (n = 272) V Ethnmethds respndents using ethnmethd Handpicking 34 Ethnbtanicals 20 Pasture spelling/ grazing practices 4 Burning pastures 4 Fumigatin with smke 6 Applicatin f kersene, magadi sda, urine, sap, fish resi-dues,, cw dung and grease (alne r mixed) Livestck quarantine 3 Predatin by birds 3 Cleaning cattle shed and burning residues believed t cntain ticks 2 Dusting with ash Table 2.4. Methds f ethnbtanical applicatin (n = 272) Methd f applicatin % f respndents using methd Puring n f the suspensin 74 Fumigatin Rubbing 5 Dusting 5 Anti-tick pastures 2 Steaming Hanging buquet Others their clurs (77.% f the respnses), fllwed by type f life cycle (with 3.% f the respnses) and last by size (with 9.8% f the respnses). Classificatin by type f life cycle is influenced by mdern scientific knwledge, hwever. The highest number f respnses identified the ear (25.4% f respnses) as the mst preferred site f tick attachment n the hst animals (Table 2.a). A cnsiderable prprtin f respndents knew that ticks cause diseases t livestck (6.5%), pr prductivity in affected animals (2.9%), deaths t livestck (6.6%) and sme peple (2.6%) were als aware f ecnmic lsses resulting frm tick infestatin in livestck ppulatins (Table 2.2). The generatin f ethnknwledge thrugh this interactin f cmmunity ethnpractitiners and western trained scientists yielded valuable infrmatin n histric and present patterns in tick infestatin and distributin, in land use ecnmies, bidiversity and ther aspects f lcal ecsystems. On this basis, therefre, sustainable utilizatin and management f lcal resurces can be develped as a value-added return t the lcal cmmunity (Hempel, 2002). Ethncntrl f livestck ticks in the cmmunity While cmmunity-based traditinal knwledge f tick cntrl and management was nt widely shared, it was still an imprtant piece f the puzzle. Understanding lcal cmmunities' ethnknwledge regarding pest management can prvide infrmatin n brad trends such as pest and hst species distributins, abundance and seasnal behaviur patterns f ticks, and as a result, prvide useful infrmatin fr scientists' field wrk (Hempel, 2002). Frm ur survey, it became apparent that ticks have been traditinally cntrlled and managed by a variety f ethnmethds (Table 2.3). All f these ethncntrl strategies cmprised a traditinally integrated system, equivalent t mdern integrated pest 52

48 Tick cntrl in the Bukusu cmmunity management (IPM), aimed at livestck tick cntrl and management in the cmmunity. A number f plants and plant prducts with the ptential t prtect livestck against ticks were dcumented (Appendix ), including anti-tick plants that have been studied in ther labratries. The ethnbtanicals used were applied using a variety f methds (Table 2.4). A detailed literature search n suggested and dcumented anti-tick plants and their prperties are presented in Appendix, as ne f the bases fr selecting the best anti-tick ethnbtanicals that merit mre detailed scientific studies. Integratin f sme f these anti-tick ethnpractices in tick cntrl and management has the ptential t imprve the lcal livestck industry and give the impverished rural ecnmy a fresh impetus. Secndary data and dcumented anti-tick ethnbtanicals In ttal, 54 plants were dcumented and the literature regarding each ne was evaluated in detail (Appendix ). In additin, fur nn-btanical anti-tick agents were dcumented. Based n key respndents' infrmatin, acaricidal, insecticidal, pesticidal and biactivity infrmatin frm literature, eight plant species were selected fr an in-depth scientific studies in the labratry. Hwever, there were many plants mentined several times during the field survey fr which the literature search revealed very little r n leading pesticidal, insecticidal, acaricidal r biactive infrmatin (Appendix ). On the ther hand, a plant species that had been prven in the labratry t have very gd acaricidal prperties may nly have been mentined nce r twice (as in the case f Cleme gynandra). This led t difficulties in selecting the leading anti-tick candidate plant species. Of the eight plants that had already been selected fr labratry studies, tw were re-selected fr detailed scientific studies based n the biassay labratry screening results and their biactive, pesticidal, insecticidal, acaricidal and related taxnmic infrmatin frm the literature search (Appendix ). Many f these dcumented plant species had varius medicinal and cultural applicatins recrded in the literature in ther cmmunities. Frm ur study, the database generated n anti-tick ethnknwledge clearly shwed that there is great ptential fr integratin f anti-tick ethnpractices and knwledge in sustainable tick cntrl and management. Furthermre, the identificatin, dcumentatin, and evaluatin f anti-tick ethnbtanicals and ther anti-tick ethnpractices used by varius cmmunities may fster increased effectiveness and wider acceptance f these ethnremedies by all stakehlders and thereby reduce suspicin against them. A large number f ther plants reputedly wrk as acaricides and insecticides, but reference t their use is difficult t uncver in the scientific literature. Ppular literature, anecdtal evidence, and infrmatin gleaned frm mdern survivalists and seekers f histrical plant knwledge cntain vast amunts f infrmatin. Sme tick ethncntrl applicatins that were fund in the lay literature and mentined by sme respndents but that did nt emerge in a scientific literature search include the use f cw dung, cattle urine, fish residues, sap, and mre than 0 plants in families such as the Lamiaceae, Flacurtiaceae, Acanthaceae and Ebenaceae. Cnclusins The survey f indigenus tick-management knwledge f the Bukusu cmmunity revealed a wealth f infrmatin n plants, plant prducts, and anti-tick ethnpractices having the ptential t cntrl and manage ticks by rural livestck farmers. Evidence frm respndents' infrmatin revealed that sme f the plant uses were brught frm their 53

49 Chapter 2 riginal area/lcatin t Bungma by immigrants. Sme plants and anti-tick ethnpractices had very few ethnknwledge references in the literature; perhaps, they were truly indigenus t the Bukusu cmmunity r perhaps relevant references culd nt be accessed. Thse plants thught t be indigenus t the Bukusu cmmunity and with traditinally claimed effects n livestck ticks numbered mre than 0, in families such as Lamiaceae, Flacurtiaceae, Acanthaceae and Ebenaceae etc. Ritual anti-tick practices cmprised burning pastures, livestck quarantine, types f grazing practices, cleaning cattle sheds, burning r burying residues f cattle sheds, bird predatin, feeding animals n natural salty sils lcally called 'silng', and applying kersene, sap, fish residues, cattle dung and urine, grease, ash, magadi sda, and sisal juices n cattle. Nevertheless, sme f the lcal claims f acaricidal prperties f the ethnbtanicals have been supprted by scientific studies. Elucidatin f scientific ratinale fthese plants and anti-tick ethnpractices, hwever, will prvide mre infrmatin and understanding twards the deplyment f sme f them inthe mst needed integrated tick cntrl and management prgrammes. Acknwledgements Internatinal Fundatin fr Science (IFS) spnsred this wrk under research grant number AB/2782. We greatly appreciate the wrk f Mr. Simn G. Mathenge f Btany Department, The University f Nairbi, Kenya, Mr. Cylus Wekesa Lantana Sinin and Mr. Peter Mungu Ofisi fr plant identificatin in the field. The first authr wish t acknwledge the financial and material supprt received frm the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE) under the Dissertatin Research Internship Prgramme (DRIP). We t als appreciate the c-peratin f Bungma ethnpractitiners, lcal primary schls, Bungma District Veterinary fficer, Bungma District Educatin Officer, lcal administratin (Office f the President f the Republic f Kenya), village/ clan elders and leaders, livestck farmers and Agricultural Extensin Officers, thrughut the study perid. 54

50 Tick cntrl in the Bukusu cmmunity 55

51 PartII Labratry evaluatin f essential ils

52 Chapter 3 58

53 Repellent activities f essential ils Repellent activities fessential ilsf indigenus plants against the brwn ear tick Rhipicephalus appendiculatus Abstract: Essential ils f eight plants (Tagetes minuta, Tithnia clivers if Ha, Juniperus prcera, Slaneci manii, Senna didymbtrya, Lantana camara, Securidaca lngepedunulata and Hslundia ppsitd) were screened fr their repellent activity against the brwn ear tick Rhipicephalus appendiculatus. The essential ils f tw plants, T. minuta and T. diversiflia, which had relatively high repellent effects cmpared t the ther six, were analyzed by gas chrmatgraphy (GC) and gas chrmatgraphy-mass spectrmetry (GC-MS) t establish their chemical cmpsitins. In labratry assays, the essential ils f these tw plants exhibited repellent effects which, at dses f mg and neat il (50 mg), were cmparable t that f the cmmnly-used cmmercial repellent, N,N-diefhyl-3- methylbenzamide (DEET). When the tw essential ils were cmpared, the essential il f T. minuta (RD 50 = mg) manifested strnger repellent effects against R. appendiculatus than the essential il f T. diversiflia (RD 50 = mg). The majr cnstituent f T. minuta was cw-cimene (43.78%), fllwed by dihydrtagetne (6.7%), piperitenne (0.5%), mms-tagetne (8.67%), 3,9- epxy-p-metha-l,8(0)diene (6.47%), P-cimene (3.25%), c/s-tagetne (.95%), (3-caryphyllene (0.84%), bicyclgermacrene (0.62%) and AR-turmerne (0.50%). In the essential il f T. diversiflia, a-pinene ccurred in largest amunt (63.64%) fllwed by P-pinene (5.00%), iscaryphyllene (7.62%), nerlidl (3.70%), tetradecanl (.75%), limnene (.52%), sabinene (.00%), a-cpaene (0.95%), a-gurjunene (0.56%>) and cycldecene (0.54%). The implicatin f these results fr the cntrl f R. appendiculatus is discussed. Intrductin Key wrds: ethnbtanicals, Kenya, Tagetes minuta, Tithnia diversiflia, Asteraceae, essential ils, chemical cmpunds, GC-MS, repellents, Rhipicephalus appendiculatus, dual-chice essay, behaviur In sub-saharan Africa, East Cast Fever (ECF), caused by Theileria parva parva Theiler, 904 and transmitted by the brwn ear tick, Rhipicephalus appendiculatus Neumann, 90, is ne f the majr cnstraints t the develpment f the livestck industry (Nrval et al., 992; Olwch et al., 2008). Of the estimated 2.7 m head f cattle (bth indigenus and extic), 76%is at risk t ECF (East Africa, 2003). The disease is assciated with up t 0% mrtality in zebu calves in ECF endemic areas and can cause up t 00% mrtality in susceptible extic and indigenus breeds (Mbg et al., 995; East Africa, 2003). Cntrl and management f bth vectr and pathgen have cntinued t rely heavily n the applicatin f synthetic chemical acaricides n the hst. This has prved t 'Thischapterwillbesubmittedfrpublicatin as:wanzala,w.,takken,w.,mukabana,r.w.and Hassanali,A.,Repellentactivityfessentialilsfindigenusplantsagainstthebrwneartick Rhipicephalus appendiculatus. 59

54 Chapter 3 be unsustainable in many ways (Nrval et al., 992). The acaricides can eliminate ticks frm the hst, but d nt prevent cntinued re-infestatin frm the surce envirnment, where ticks spend 90% f their life. Fr effective management f ticks, we hypthesize that there must be a mechanism t cntrl ticks n individual hsts as well as in the hst envirnment in rder t prevent hst re-infestatin during grazing. One pssible strategy twards achieving this is the use f tick repellents n the hst and tick-repellent plants in the pasture (envirnment), cmbined with plants that are attractive t ticks surrunding the pastureland s as t develp a push-pull tick cntrl system (Hassanali et al., 2007; Ck et al., 2007). Althugh the prpsed strategy appears cmplex, it may be pssible t achieve in zer grazing/semi-zer grazing livestck farming systems, small scale freerange livestck farming systems and tethering livestck farming systems. Fr thers, such as pastralism and large-scale livestck farming systems, the deplyment f wellfrmulated, cntrlled-release dispensers maybe mre practical. A cmmnly used cmmercial arthrpd repellent, N,N-diethyl-3- methylbenzamide (DEET), is still cnsidered the best available prduct, repelling a wide variety f insects, ticks and mites (Fei and Xin, 2007). In humans, hwever, the repellent may cause insmnia, md disturbances, impaired cgnitive functins, seizures, txic encephalpathy and allergic reactins (Rbbins and Cherniack, 986; Qui et al., 998; Lewis et al., 2000). Thugh DEET is nt expected t biaccumulate, it has been fund t cause cnsiderable envirnmental pllutin (Se et al., 2005). Furthermre, the ability f DEET t disslve sme plastics, rayn, spandex, ther synthetic fabrics, leather and painted r varnished surfaces has led t a search fr alternative repellents. Studies have shwn that plant-based repellents can be cmparable t DEET r even better (Panda, 2004; Trngtkit et al., 2004; Chauhan et al., 2005; Bnd, 2007). The ptential f sme lcal plants and plant prducts fr use as tick repellents n hsts, and tick-repellent plants in grazing areas t prtect livestck frm tick bites has been demnstrated (Menendez, 924; Thmpsn et al., 978; Carrll et al., 989;Wilsn and Sutherst, 990; Webb and David, 2002). One such repellent is the Flea and Tick Granular Repellent, which is made frm essential ils f cedar, cinnamn, mint and lemn grass and has a pleasant dur and can be safely used utdrs fr flea and tick cntrl (Nixalite f America, 2005). Melinis minutiflra (mlasses grass), a trpical grass already in use as livestck fdder (Lersten, 983; Juarez Lagunes et al., 999), cver crp and mulch (Duke, 983) and fr thatching huses (Chiera et al., 977), has been shwn t be txic t ticks (Thmpsn et al., 978) and t repel ticks (Menendez, 924; Hernandez, et al., 990; Mwangi et al., 995b) as well as insects and snakes (Duke, 983). At the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE), a study t develp anti-tick pastures using mlasses grass was undertaken at Mbita and Kuja in Nyanza Prvince, Kenya, t cntrl Amblymma variegatum and R. appendiculatus, vectrs f the livestck diseases heartwater (cwdrisis) and ECF, respectively (Mwangi, et al., 995b). The lcal shrubs Cleme hirta and Gynandrpsis gynandra have been shwn t be pssible tick-repellent pasture plants (Dipelu et al., 992; Malnza et al., 992; Ndung'u et al., 999). Studies have als shwn the tick-repellent prperties f essential ils frm Cmmiphra erythraea and C. myrrh (Maradufu, 98), Cleme mnphylla (Ndung'u et al., 995), Ocimum suave (Mwangi et al., 995a), Cleme hirta (Ndung'u et al., 999) and G. gynandra (Lwande et al., 999) against R. appendiculatus. The practical applicatin f tick-repellent plants and essential ils and their integratin with ther tick cntrl measures either n the hst r in pasture lands culd be a practical and ecnmical way f cntrlling nt nly livestck ticks but ther arthrpd vectrs as well (Gupta and Rutledge, 994; Cpeland et al., 995; WHO, 995). This alter- 60

55 Repellent activities f essential ils native is very suitable because the surce plants and essential ils are lcally accessible and familiar t resurce-pr livestck farmers, meaning that they therefre may nt require the input f external persnnel during their applicatin, as is ften the case with synthetic chemical acaricides. Studies n the frmulatin, mde f actin and pssible txicity f these plant materials have nt yet been dne, hwever. Furthermre, ther prblems may manifest themselves if the plants and their prducts are used n a wider scale. Our bjective in the present study was t identify indigenus plants frm Bungma District in western Kenya as surces f tick repellents with the ptential t reduce cntact between ticks and their livestck hsts. Essential ils were extracted frm these plants and subsequently evaluated n-hstfrtheirabilitytintercept feeding-site lcatin behaviur f the vectr by masking the natural hst durs. Materials and methds Selectin f plants An ethnbtanical survey cnducted in the Bukusu cmmunity in Bungma District, western Kenya, identified 54 plant species having pssible effects n livestck ticks. Each dcumented plant was evaluated in detail (Chapter 2 f this thesis), and based n this evaluatin, eight plant species were selected fr further studies f their repellent effects n adult Rhipicephalus appendicular (Table ). The selectin was based n the fur levels f validity established by Heinrich et al. (992) as fllws: 0. If n infrmatin in the literature supprts the Bukusu traditinal claim fr use, this indicates that the plant might have n effect and hence has n validity. Or may be the plant culd be the true indigenus species f the cmmunity and has nt been evaluated anywhere.. A plant (r anther species f the same genus) which is used in gegraphically distinct areas fr the cntrl f the same livestck tick, attains the lwest level f validity if n further phytchemical r pharmaclgical infrmatin validates its ppular use. The use in mre than ne area increases the likelihd that the plant is active against the tick. 2. If in additin t the ethnbtanical data, phytchemical r pharmaclgical infrmatin als cnfirms its use in the Bukusu cmmunity, the plant is assigned a higher level f validity. Plants in this categry may exert a physilgical actin n the target tick species and are mre likely t be effective remedies in livestck tick cntrl and management than thse atthe lwest level f validity. 3. If ethnbtanical, phytchemical and pharmaclgical data supprt the traditinal use f the plant, it is gruped in the highest level f validity and is mst likely t be an effective tick cntrl and management remedy (Heinrich, et al., 992) and therefre selected (Table ). Plants were cllected frm Bungma District, western Kenya alng the suthern slpes and fthills f Munt Elgn at altitudes ranging frm abut 300 m in the suth t abut m in the nrth. The district is lcated between latitude 0 25'S and 0 53'N and lngitude 34 2 'W and E. All aerial parts f the plants were cllected. The plants were identified in the herbarium at the Schl f Bilgical Sciences, University f Nairbi, Kenya. Vucher specimens f the selected plant species were depsited at the University f Nairbi Herbarium: Tagetes minuta L. (029-BGM-Mwi/2002), Tithnia diversiflia (Hemsl.) A. Gray (05-BGM-Muf/2002), Juniperus prcera Endl. (34-BGM-Elg/2002), Slaneci manii (Hk, f.) C. Jeffrey. (06-BGM-Mwi/2002), Senna didymbtrya 6

56 Chapter 3 ^ 0 > ^ H " T J» > ^ 2 2 p* p D 3 en 3 C 3. fj.- & P P P tn E g. 3> ET* 3 5- S* p. I c P 9 *. P 3 3 a O O CO 3 p-^3 O I t' t CD CTQ fd g t 5 I *<* at fd t* 3 O a 2 3 c 3 D. 3' 9 5' B 3 a* 3 c P 3 O P P a* ' *< 3' fd O O fd P^ 3 3 3t 2 & C fd X 3" 5j a fd 3 en -0 I 3* P fd 5 3> c 3 CL 3' fd 3t 3 fd O P* P < fd ' P O <" fd O 3 T3 O en 2 P* en 3* P fd 5 i-t fd c 3. 5' S- fd St fd 3 I O fd 3* P P ' 2. a fd fd.*? 3 * c p.3 I 3 a. I a < f Se.g ^8-< S-. E. "3 c- ^ ^ fd B 3 * ffife- PS? ft J=. 3 - fd i fd c I 5 C 3 TO *< 5" 3 3 O - S (^ 2 ^3 r s- 3 X a' 0- ^ Eg? I W a CT" 3" t " P O fd O 8S.U 5' CO fd O ' CL. p_ T3 fd 3 " CO 3 S, ' 3 jr g* S 3 ;. Si. fd ^ " " CD 3 5 ^ fd 3 ' 3 ' ' g 3" P 3 VI fd O 3 fd C P cs fl " c p. H > > n 3 H B & r* 3 S^r? S (ra?r j 3 t n 8E. 3' Vi & 3 W 5-3 a G 3 fd a?' Z ft > 3 & TO R fd fd B> S 3 3 D. O. 3 I S^^ O O 3 >< ^ O >-- fd \D " O ja j-j J" - VO >2! S TO 3^ fd fd pa fd 3 ~ Q. E. a K> v\ 3 9 " O "T H- ^O CO fd era fd p 2 ON ^ - 62

57 Repellent activities f essential ils (Fresen.) H.S. Irwin & Bameby (32-BGM-Web/2002), Lantana camara L. (043-BGM- Mwi/2002), Securidaca lngepedunculata Fres. (08-BGM-Mec/2002) and Hslundia ppsita Vahl. (33-BGM-Bul/2002). Extractin f essential ils The plant materials were left in a well-ventilated rm fr -2 weeks befre hydrdistillatin. The materials were cut int small pieces and abut kg f each plant was hydrdistilled using a Clavenger-type apparatus fr 8 h (Clevenger, 928). Pure il was cllected frm every plant int 2ml vials and stred at -20 Cin a freezer until required fr analysis and biassays. Determinatin f the cmpsitin f the essential ils Bth qualitative and quantitative characteristics f the varius essential ils were studied using gas-chrmatgraphy (GC) and gas-chrmatgraphy/mass Spectrmetry (GC-MS) techniques (Thll et al., 2006). The cnstituents f the essential ils were identified by analysis f their mass spectra, direct cmparisn f their mass spectra t the Wiley NBS and NIST databases r library f mass spectra, and c-injectin with authentic standards n the GC. GC analyses were perfrmed with a Hewlett Packard HP 5890A Gas Chrmatgraphy equipped with a flame inizatin detectr (at 230 C). A fused silica capillary clumn (Hewlett Packard, 50 m x 0.22 mm x 0.33 mm CD) cated with methyl silicn (0.3 um film thickness) was used with nitrgen as the carrier gas. All GC analyses were perfrmed in the splitless mde with the injectr temperature at 270 C. The ven temperature was prgrammed frm 60 C isthermal fr 7min, t 20 C at 5 C per min, then t 80 C at 0 C per min, and finally t 220 C at 20 C per min, where it was maintained fr0 min. Peak areas were calculated using a Hewlett Packard 3393 B series integratr and tgether with their GC retentin times, cmpared t thse f authentic samples. GC-MS analyses were perfrmed with a VG Masslab quadruple gas chrmatgraphy-mass spectrmeter. Chrmatgraphic separatins were achieved using a fused silica capillary clumn (Hewlett Packard, 50 m x 0.32 mm ID) cated with Carbwax 20M (0.3 um film thickness) with helium as the carrier gas. All the GC-MS analyses were made in the splitless mde with helium as the carrier gas. The GC clumn was temperature prgrammed as in the case f GC analysis. Cmpunds were identified by their electrn impact (El) mass spectral data, rder f elutin and relative GC retentin times, and by cmparisn ftheir mass spectra and GC retentin times t thse f authentic samples. The cmputer n the GC-MS system recrds a mass spectrum fr each scan and has a library f spectra that can be used t identify an unknwn chemical in the sample. The library cmpares the mass spectrum frm a sample cmpnent with mass spectra in the library. It then reprts a list f likely identificatins alng with the statistical prbability fthe match. Synthetic chemicals Synthetic standard chemicals (authentic samples) used in GC c-injectins were btained frm Sigma Chemical Cmpany, Ple, UK and Aldrich Chemical Cmpany, Gillingham, UK. All the authentic samples used were ver 95% pure. Experimental ticks The ticks used (R. appendiculatus) were btained frm clnies at the Internatinal Livestck Research Institute (ILRI) and bred at ICIPE, Nairbi, Kenya. Rearing cnditins and 63

58 Chapter 3 Fig. 3.. Tick climbing biassay apparatus (placed in a tray measuring 50 x 30 x 5 cm) with water up t.5 cm deep; A, aluminium base; Bi, aluminium rd (26 cm lng x.7 cm in diameter); B 2, 0.8-cm glass tube plugged with wet cttn wl; B 3, filter paper cllar; C, 4.5-cm glass tube plugged with dry cttn wl. The tw aluminium rds, Bi n the aluminium base, A, (5 x 7 cm), were 7 cm apart. The uter tubes, C are held in psitin, 4 cm abve the aluminium base, A by a retrt stand clamp. The five ticks were intrduced n the aluminium base, A, at a psitin marked with a star, 3.5 cm frm the base f the aluminium rds, Bi (mdified frm Brwning, 976). 5 cm 50cm management were as described previusly (Bailey, 960; Irvin and Brcklesby, 970). All experiments were cnducted with newly-emerged adults. Dual chice assay A dual chice tick climbing assay apparatus was used fr screening the essential ils f eight plants fr their tick repellent activities, and further analyses were cnducted n tw f these selected plants, T. minuta and T. diversiflia. The biassay apparatus (Fig. 3.) [described previusly by Wanzala et al. (2004)] made use f a characteristic behaviur f R. appendiculatus: the ticks climb up grass stems and settle fr a perid near the tip f the stem t await any passing ptential hsts (Brwning, 976; Chiera, 985).This experiment was set up at ICIPE, Nairbi, Kenya. An aluminium base f area 05 cm 2 with tw stands f 26 cm in height and 7.0 cm apart was put in a basin f water,.5 cm deep (the water restricts the mvement f the ticks t the aluminium base). Tw sets f glass tubes were used, ne f 4.5 cm (uter ne) and the ther ne 0.8 cm (smaller inner tube) in diameter. A strip f filter paper (Whatmann N 7, 2 cm wide) was stapled t frm a cllar arund the upper parts f each smaller inner glass tubes at a distance f 20 cm frm the aluminium base t prvide the surce f either test durs r pure slvent. One cllar n the pair f the tubes was treated with test dur slutin and the ther ne with the same amunt f pure slvent (dichlrmethane-dcm) t serve as cntrl. After the slvent was allwed t evaprate (abut 0 min.), these tubes were shielded with the wider uter glass tubes frm 4 cm abve the aluminium base t facilitate a relatively unifrm vertical gradient f the test durs alng the 3.7 cm gap between the tw glass tubes. The larger glass tubes als served t shield the smaller nes, in rder t limit the diffusin f the test material and the slvent, and their upper ends were plugged with dry cttn wl. Wet cttn wl n tp f the smaller glass tubes ensured a high relative humidity (>75%) within the clumns. 64

59 Repellent activities f essential ils The test materials and the slvent were dispensed using a calibrated Eppendrf pipette and equilibrated fr 30min befre five adult ticks f mixed ages and sexes were released at the centre f the aluminium base. Prir t each biassay, ticks were kept at high relative humidity (>85% RH) fr 24 h in cntainers with mist cttn wl, s that they were nt dehydrated and as a result wuld have less tendency t drwn in the water surrunding the aluminium base. All biassays were cnducted in a rm kept at 28 ± C and 75 ± 5% RH, which had an exhaust fan running cntinuusly. The assays were run fr h, and the number f ticks abve the filter paper strip n the cntrl glass tube (Nc) and nthe treated glass tube (Nt) were recrded at 5, 30, and 60 minutes. After each test, the apparatus was thrughly cleaned and dried at 00 C. Initial cmparisn f the respnses f ticks in the setup with and withut residual dichlrmethane n bth sides shwed n bias fr either side and n effects f the residual slvent n the adult ticks. Each dse f the test material was tested 20 times; each time with a fresh, naive adult tick. The repellent effect f the essential ils was evaluated accrding t the frmula adpted by Ndung'u et al. (995) and Lwande et al. (999) namely: percentage f repellency (PR) = [(Nc-Nt)/(Nc+Nt)] x 00, where Nt and Nc represent the number f ticks that climbed n r passed the treated and cntrl cllar f filter papers n the glass tubes, respectively. Selectin f tw plants: Tagetes minuta and Tithnia diversiflia Screening f essential ils frm eight plants and selectin f tw fr further studies were based n a cmparative analysis f assay results and ethnic knwledge n tick cntrl. Preliminary studies were cnducted n eight essential ils f plants listed in Table 3.. Tagetes minuta, the plant that shwed the highest tick repellency, was selected fr further assays in rder t select the mst apprpriate dse t be used fr screening the remaining seven plants. Frm the results btained fr tick repellency biassays f T. minuta essential il, a dse f0. mg was chsen. This dse and the neat il (50 mg) were used t screen all eight plants, leading t the selectin f T. diversiflia in additin t T. minuta. Standard cmmercial synthetic arthrpd repellent The repellency biassay results were cmpared with thse previusly btained with DEET. This repellent is a clurless, ily liquid with a slight dur. It is still the best available prduct, repelling a wide variety f insects, ticks and mites and generally lasting lnger than ther repellents (Mansn-Bahr and Apted, 982; Chunge, 986). Previus wrk at ICIPE, tested varius DEET dses and determined their percent repellency against R. appendicular (Table 3.2), with which we cmpared the repellencies caused by essential ils f T. minuta and T. diversiflia. Table 3.2. Mean percentage repellency (± SE) f N,N-diethyl-3-methylbenzamide evaluated in a dual-chice assay against adult Rhipicephalus appendiculatus at the Internatinal Centre f Insect Physilgy and Eclgy, Nairbi, Kenya under the same labratry cnditins as the current studies. Factr Treatment dse (mg) Repellency (%) Categries I ±3.9 II ±3.6 III ±4.5 (Surces: Ndung'uetal.,995; Lwandeetal., 999; Ndung'uetal.,999) rv ±3.6 65

60 Chapter 3 Data management and analysis Data were entered int an SAS database fr analysis. The data (dses) were transfrmed (lgi) and subjected t analysis f variance (ANOVA) (SAS Institute, ). During analysis, percentage repellency (PR) values were cnverted t repellency prbabilities ranging frm 0 t in rder t fit int a prbit mdel. The Tukey HSD test was used t cmpare the mean values f repellency btained fr varius dses f the repellents (SAS Institute, ). Dse-respnse relatinships were determined using prbit analyses and repellent dses (RD) at RD 50 and RD 75 values btained frm the regressin mdel: Prbit [P(dsel)] = p 0 + x P! + Where, p 0 =cefficient f the mdel representing y-intercept, Pi = cefficient f the mdel representing dsel, dsel = Lgi (dse), I = errr term in the data set fthe predictr (regressr) variable (x) and P = repellency prbability. The curves fr the regressin mdels were drawn using R sftware fr Micrsft windws. Results Initial screening f essential ils frm selected plants The essential ils frm eight plants (T. minuta, T. diversiflia, J. prcera, S. manii, S. didymbtrya, L. camara, S. lngepedunculata and H. ppsita) caused repellency against newly emerged R. appendiculatus adults f mixed sexes. The results frm tw different treatment levels [at 0. mg dse and neat il (50 mg)] are shwn in Fig The percent repellencies f neat ils were cmparable t that f the cmmercial arthrpd repellent, DEET, with the exceptin f the essential ils f S. manii, L. camara and S. lngepedunculata. Hwever, at the 0. mg dse, the percent repellencies f the essential ils were belw that f DEET, except that f T. minuta (80.%). 0. mgdse 50 mg dse Fig The repellent effect f essential ils f eight plants in dses f 0. mg and 50 mg (neat il) against Rhipicephalus appendiculatus. Plant species = Tagetes minuta, 2 = Tithnia diversiflia, 3 = Hslundia ppsita, 4 = Slaneci manii, 5 = Lantana camara, 6 = Juniperus prcera, 7 = Senna didymbtrya and 8 = Securidaca lngepedunulata. Fr a given repellent dse, means capped by the same letters are nt significantly different at P<0.000(Student- Newman-Keuls test). 66

61 Repellent activities f essential ils Selectin f tw plants fr dse-respnse studies Tw plants (T. minuta and T. diversiflia) were selected, based n the abve results, shwing relatively high repellent activities against R. appendiculatus. Mrever, these plants were ranked highly by traditinal livestck hlders (Chapter 2and Table.3.). Yield and dse respnse studies f essential il f Tagetes minuta The yield f the essential il f the fresh aerial parts f T. minuta was % w/w. The il is sluble in dichlrmethane (DCM), ether and ethanl and insluble in water (El Deeb et al., 2004). The respnses f R. appendiculatus adult ticks expsed t different dses f T. minuta essential il are shwn in Fig. 3.3a. The repellent effect f the essential il at RD 50 and RD 75 levels is shwn in Table 3.3,whereas the dse-respnse link functin fthe regressin mdel was: Prbit [P(dsel)] = dsel where dsel =Lgi (dse),and P = repellency prbability functin as shwn in Fig. 3.4a. Tagetes minuta 00 >, S 80 ID cu i c dc Dse (mg) Tithnia diversiflia 00 Fig. 33. Mean repellency percentage f different dses f Tagetes minuta (a) and Tithnia diversiflia (b) essential ils against newly emerged adult Rhipicephalus appendiculatus in a dual chice assay. Means with the same letters are nt significantly different at P<0.000 (Student-Newman-Keuls test). 80 «J d d a ft iti T rfi rfl Dse (mg) 67

62 Chapter 3 Table 3.3. Prbit analysis f dse-respnse relatinship f Tagetes minuta and Tithnia diversiflia essential ils at RD 50 and RD 75. Plant species Tagetes minuta Tithnia diversiflia Repellence prbability Repellent dse (mg) Upper cnfidence limit at 95% Lwer cnfidence limit at 95% Yield and dse respnse studies f essential il f Tithnia diversiflia The yield f the essential il f the fresh aerial parts f T. diversiflia was % w/w. The essential il was bserved t be sluble in dichlrmethane (DCM), ether and ethanl and insluble in water. The respnse f newly emerged R. appendiculatus adult ticks expsed t different dses f T. diversiflia essential il are shwn in Fig. 3.3b. The repellent effect f the essential il at RD 50 and RD 75 levels is shwn in Table 3.3, while the dserespnse link functin f the regressin mdel was: Prbit [P(dsel)] = dsel The repellency prbability functin (P) is shwn in Fig. 3.4b. Gas chrmatgraphy analyses f the essential ils f Tagetes minuta and Tithnia diversiflia Gas chrmatgraphy separated the chemical cmpnents in the mixtures f the essential ils f T. minuta and T. diversiflia and the representative spectral utputs are shwn in Figs. 3.5a and b, respectively. The x-axis shws the retentin time (RT), and the y-axis shws the intensity (abundance) f the signal. The RTs prvide the qualitative aspect f the chrmatgram while the chrmatgraphic peak heights r peak areas prvide the quantitative aspect f the analyte. In bth the chrmatgrams, mst chemical cmpnents that shw relatively high peak heights r large peak areas are eluted in the first 30 minutes. There appear t be mre different cmpunds in the essential il f T. diversiflia than in that f T. minuta (Figs. 3.5a and b). Mass spectrscpy f the essential il f Tagetes minuta Individual cmpnents in the sample mixture f the essential il f T. minuta were characterized. One hundred cmpunds were identified in the essential ils. Of these 00 cmpunds, 5% (5 cmpunds) were mnterpenes, 48% (48 cmpunds) were sesquiterpenes and % ( cmpund) was diterpene. Thirteen f 5 mnterpenes were fund in the literature t have repellence prperties, while six f 48 sesquiterpenes were als fund in literature t have sme repellency. Of these 00 cmpunds, cis-cimene ccurred in the largest amunt (43.78%), fllwed by dihydrtagetne (6.7%), piperitenne (0.5%), fraws-tagetne (8.67%), 3,9 epxy-p-metha-,8 (0) diene (6.47%), /?-cimene (3.25%), cis -tagetne (.95%), /?-caryphyllene (0.84%), bicyclgermacrene (0.62%) and ARturmerne (0.50%) in that rder (Table 3.4a). Mass spectrscpy f the essential il f Tithnia diversiflia Individual cmpnents in the sample mixture f the essential il f T. diversiflia were characterized. Fifty cmpunds were identified in this essential il. Of these 50 cmpunds, 40% (20 cmpunds) were mnterpenes, while the rest 60% (30 cmpunds) 68

63 Repellent activities f essential ils Fig The link functin f prbability repellency dse-respnses with Tagetes minuta and Rhipicephalus appendiculatus as explained by the curve generated by a regressin mdel, Prbit [P (dsel)] = dsel (a), while (b) is the link functin f prbability repellency dserespnses with Tithnia diversiflia and Rhipicephalus appendiculatus as explained by the curve generated by a regressin mdel, Prbit [P (dsel)] = dsel. In bth mdels, dsel = Lgi (dse), and P = repellency prbability..0 > 0.6 _ a. ai Dse (mg) O 0.4 « were sesquiterpenes. Twenty-fur percent (2 cmpunds) f the 50 cmpunds and all the mnterpenes were fund in the literature t have sme repellence prperties. Of these 50 cmpunds, a-pinene ccurred in the largest amunt (63.64%), fllwed by P-pinene (5.0%), iscaryphyllene (7.62%), nerlidl (3.70%), -tridecanl (.75%), limnene (.52%), sabinene (.00%), a-cpaene (0.95%), a-gurjunene (0.56%) and cycldecene (0.54%) in that rder (Table 3.4b). 69

64 Chapter 3 00n s * 80 <U O 60 a CIS a 40 p.\ s O-Lp, I^«I ft T+~ t'ppi^a iv i ii l in i iu Retentintime (min) =,^^ -26 A * r O e ce e t L#* ^Tr^TTTTTWt*^! t I'f II>H ' inl ft»i IIIH M i it i i i i j i TTTTITI i i V i i i i i Vti < > Jil.L. j*~?~?*r**y4 rjaul_ Retentin time (min) Fig.3.5.Chrmatgramsgenerated by gaschrmatgraphy fr the essential ils f (a), Tagetes minuta and (b), Tithnia diversiflia. Each f the peaks in thechrmatgram represents the signal created when acmpundelutesfrm the chrmatgraphicclumnintthedetectr. 70

65 Repellent activities f essential ils Table 3.4. GC and GC-MS identified 0majr cnstituent chemical cmpunds in the essential ilf (a) Tagetes minuta and (b) Tithnia diversiflia (a) Peak n. 4 Cmpund cu-cemene Mlecular frmula CiHi 6 RT M + (g/ ml) Base peak 93 Majr peaks 4,79, 05, 2 Relative abundance (%) Identificatin f cmpunds GC-MS C 5 dihydrtagetne CiHigO ,57,69, GC-MS 42 piperitenne CiHi ,9,07, GC-MS 3 4 fr<ms-tagetne 3,9 epxy-p-methal,8(0)diene CiHi 6 0 C0H4O ,67,09, 37, 52 4,79,22, GC-MS C GC-MS 29 /?-cimene CiH ] ,79,05, GC-MS C cw-tagetne /J-caiyphyllene (Trans) bicyclgermacrene AR-turmerne CiH 6 0 C5H24 C5H24 C5 H2Q ,67,09, 52 69,93,33 4,93,36, 6 4,79, GC-MS C GC-MS C GC-MS GC-MS (b) Peak n. 2 Cmpund a-pinene Mlecular frmula C0H6 RT (min) M + (g/ ml) Base peak 93 Majr Peaks 39,77,79, 2, 36 Relative abundance (%) Identificatin f cmpunds GC-MS C 5 /?-pinene C0H ,2, GC-MS C Iscaryphyllene Nerlidl C5H24 C l5 H ,69, 05, 33,6 4,43,93,07, GC-MS GC-MS C 38 -Tridecanl C4H3O , 69, 83, GC-MS C Limnene Sabinene a-cpaene a-gurjunene CiHis ClH!6 C5H24 C5H ,4,53,67, 93 39,4,77,79, 36 4,77,8,93, 9, 6 4,55,9,05, 9,6,6, GC-MS C GC-MS GC-MS GC-MS 47 Cycldecene C2H ,54,8, GC-MS Key M + = Mlecular weight RT = Retentin time (min.) GC-MS = identificatin based n cmparisn f mass spectra in NIST/NBS and Wiley libraries nly GC-MS C = identificatin based n cmparisn f mass spectra in NIST/NBS and Wiley libraries fllwed by a cmparisn with retentin time identical t authentic cmpunds 7

66 Chapter 4 74

67 Hst durs guide ticks t feeding sites Attractive and repellent hst durs guideticks t their respective feeding sites Intrductin Abstract: We have studied n-hst behaviur f adults f the brwn ear tick {Rhipicephalus appendiculatus Neumann, 90) and the red-legged tick (Rhipicephalus evertsi Neumann, 897), which prefer t feed mainly inside the ears and the anal regins f bvids respectively. Bth species were fund t be relatively successful in rienting tward and lcating their respective feeding sites frm different parts f the hst bdy. Our bservatins suggested the peratin f bth avidance (clser t the feeding site f the ther) and attractin (clser t its wn feeding site) respnses f the ticks. In the labratry, dur trapped frm cattle ears attracted.ft. appendiculatus but repelled R. evertsi, whereas that frm the anal regin had an ppsite effect. This dur-based 'push-pull' pair f stimuli may largely accunt fr efficient rientatin behaviur f the tw tick species t their respective feeding sites. We prpse that such cncurrent deplyment f repulsive and attractive cues may be quite widespread amng arthrpds and related rganisms that specialise n specific hsts r micrenvirnments in the perfrmance ftheir bilgical functins. Key wrds: Rhipicephalus appendiculatus, Rhipicephalus evertsi, feeding sites, cattle, repellent and attractive durs, push-pull Bld-feeding arthrpds have evlved a variety f relatinships with their mammalian hsts. Wide variatins ccur in hst specificity, duratin and multiplicity f cntacts, and in hst-lcatin behaviur (Gibsn and Trr, 999). On hst, related species may als demnstrate predilectin fr feeding at different sites. Althugh the signals used by sme bld-feeders t lcate their preferred hsts have been a subject f cnsiderable research, nly casual attentin has been given t feeding site lcatin behaviur f relevant arthrpds. In this study, we reprt n-hst behaviur f adults f tw sympatric tick species, the brwn ear tick {Rhipicephalus appendiculatus Neumann, 90)and the red-legged tick (R. evertsi Neumann, 897), which prefer t feed mainly inside the ears and the anal regins f bvids respectively (Walker, 974). Material and methds All prcedures requiring experimental animals were apprved by ICIPE's Institutinal Animal Care and Use Cmmittee and were perfrmed in cmpliance with guidelines published by Kenya Veterinary Assciatin and Kenya Labratry Animal Technician Assciatin. 'Thischapterwaspublished as:wanzala,w.,sika,k.f.n.,gule,s.andhassanali,a.,2004, Attractiveandrepellenthstdursguideticksttheirrespectivefeeding sites,chemeclgy 4:

68 Chapter 4 Fig. 4.. General rientatins f R. appendiculatus (a) and R. evertsi (b) that initiated mvements frm different releasepints (-6). Ticks The tw tick species used (R. appendiculatus and R. evertsi) were btained frm clnies at the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE), Nairbi, Kenya. Rearing cnditins and management were as described previusly (Bailey, 960; Irvin and Brcklesby, 970). On-hst bservatin studies On-hst behaviur studies used ne f tw Friesian steers (bdy wt. 27^470 kg), with n prir expsure t ticks, held in a crush facility at ICIPE. The respnses f ticks and their navigatin patterns were mnitred frm six different bdy lcatins (Fig. 4.a and b) f Friesian steers representing varying distance frm preferred feeding sites and pssible ar- 76

69 Hst durs guide ticks t feeding sites eas f alightment by the ticks frm their questing psitins n the vegetatin (Brwning, 976). One tick at a time f mixed age and either sex was intrduced at ne f six sites and bserved fr up t 8 hurs. Rhipicephalus appendiculatus were bserved frm t 8.00 hurs and R. evertsi, which is active at night, between 8.00 and hurs. All bservatins were made under dry weather with day temperatures in the range C and night temperatures inthe range C. Each bservatin was replicated 20 times. Odur trapping These were carried ut using adsrbent sachets (4 x 2.5 cm) made up f stainless meshwire (250 mesh) cntaining either activated charcal (0.5 g, 0.2 ug mesh; Chrmpack, Middelburg, The Netherlands) r reverse-phase C 8 -bnded silica (0.2 g, 6-40 urn size; Sigma Aldrich Chemicals, Drset, UK) similar t the prttype described previusly (Gikny et al., 2002). The sachets were held n the inner side f ear pinna r the anal regin with rubber bands fr 2 h. The adsrbent frm each sachet was transferred int a pasteur pipette and eluted with redistilled dichlrmethane (4 ml, > 99.9%). Elutins frm 60 trapping cycles were pled, cncentrated and stred at -20 C until used fr biassays. Fr biassays, an aliqut (2 mg) f the cncentrate was taken up in dichlrmethane (25 ml) t give -80 ppm slutin. Tw-chice climbing assay A set up cnsisting f an aluminium base (5 x 7 x.5 cm) with a pair f aluminium rds (26 cm lng x 0.7 cm in diameter) 7cm apart cvered with glass tubes (0.8 cm in diameter) was used (Fig. 3.). A strip f filter paper (Whatmann N 7, 2 cm wide) was stapled t frm a cllar arund the upper parts f each tube t prvide the surce f test durs. One cllar n the pair f tubes was treated with test dur slutin and the ther with the slvent (dichlrmethane) t serve as cntrl. After the slvent was allwed t evaprate (0 min), these tubes were shielded with wider tubes (4.5 cm in diameter) frm 4 cm abve the aluminium base t facilitate relatively unifrm vertical gradients f the test durs alng the 3.7 cm gap between tw tubes. Wet cttn wl plugs n the tp f these tubes ensured relatively high humidity (>75%) within the clumns. Ticks f mixed age and sex (5) were placed at the centre f the aluminium base and bserved fr 60 minutes. The apparatus was placed in a tray with shallw water, which prevented the dispersal f test ticks frm the base. Initial cmparisn f the respnses f the ticks in the set up with and withut residual dichlrmethane n ne and bth sides, shwed n bias fr either side and n effects f the residual slvent. Each assay was replicated 2 times. The number f ticks that climbed n treated and cntrl clumns were cunted. Mean % attractin r repellency (-ve attractin) was calculated using the frmula, percentage repellence (PR) = [(Nc - Nt)/(Nc + Nt)] x 00, where, Nc = the number f ticks that climbed n the glass rd and r abve the filter paper cllar strip n the cntrl glass tube and Nt = the number f ticks that climbed n and r abve the filter paper cllar strip n the treated glass tube, respectively. Statistical analyses On hst data were analysed by analysis f variance (ANOVA) using the general linear mdel (GLM) prcedure fr SAS fr PC (SAS Institute, ), after lg (n+) transfrmatin. The means were cmpared by Student-Newman-Keuls test (Skal and Rhlf, 995) at P>0.05. Tw-chice assay data were analysed by Student's t-test. 77

70 Chapter 4 Table 4.. On hst respnses f ticks released at different bdy sites f Friesian steers Bdy lcatins (a) R. appendiculatus % respndents Reactin time (h) Initial speed (cm/min) % reach-ing feeding site Time taken t reach feeding site (h).frehead ±0.03a,b 2.00±0.49a,b ±0.66a,b 2.Dewlap ± 0.22 b,c.02 ± 0.25b,c,d ±0.95a,b 3.Fre leg (upper part) ± 0.40c 0.93±0.3c,d 5 (2.28 ± 0.56) 4.Rear leg (upper part) ± 0.25c 0.80 ± 0.3d 20 (5.2 ±.4) 5.Escutchen ± 0.02a 2.2 ± 0.42a ± 0.59a 6. Shulder ±0.0a,b.96±0.43a,b ±0.52a,b (b) R. evertsi. Escutchen 2.Rear leg (upper part) ± 0.0a 2.77 ± 0.2a 0.09 ± 0.06a.79±0.28b,c ± 0.53c 5.7±0.76b,c 3.Fre leg (upper part) ± 0.39b.06 ±0.3c,d ±.02b,c 4.Dewlap ± 0.66c 0.95±0.22d 20 (9.24 ± 0.86) 5. Shulder ± 0.07a 3.04 ± 0.24a ±0.8a,b 6.Back ± 0.06a 2.09 ± 0.40b ±0.7b,c Fr agiven clumn, means fllwed by the same letter are nt significantly different frm ne anther at P>0.05 (Student-Newman -Keuls test). Figures in parentheses in the last clumns were nt included in the statistical cmparisn because f the relatively lw number f the ticks that arrived at the feeding sites. Results and Discussin On-hst bservatins f the ticks shwed a typical sequence f behaviur invlving: (i) a statinary phase at the release pint, which was accmpanied by utstretching f legs and adptin f a psture suggestive f scanning activity; (ii) randm, seemingly explratry mvements; (iii) a clear directinal mvement resulting frm buts f strides and halts, interspersed with mis-turns and readjustments; and (iv) gradual arrestment clser t the feeding site fllwed by attachment. Interestingly, fllwing initial randm mvements mst f the respndents at each release pint riented tward their respective feeding sites (Fig. 4.a and b), althugh, during the bservatin perid, sme appeared subsequently t lse their way. The prprtin f ticks at different sites that initiated mvement (respndents), the average time frm placement n the steer t initial mvements (reactin time), and initial speed were nted. The number f ticks that reached their feeding sites and the time between release and arrival were als recrded (Table 4.a and b). The results shw relatively high rates f successful rientatin f the ticks t their respective feeding sites and suggest the mediatin f specific stimuli in the prcess. We first hypthesized that gradients f vlatile durs frm these sites may prvide the apprpriate rientatin signals. Althugh this wuld accunt fr lwer respnses (prprtin f respndents, their reactin time and initial speed) f ticks depsited n lcatins further away frm the sites (upper rear and fre legs fr R. appendiculatus; upper fre leg and dewlap fr R. evertsi), the behaviur f each species clser t the feeding site f the ther suggested an additinal effect (Table 4.a and b). Fr R. appendiculatus, this isreflected in a relatively large prprtin f the ticks that tk ff frm the upper rear regin within a 78

71 Hst durs guide ticks t feeding sites & appendiculatus t ear dur R. evertsi t anal dur O O O O Dse (ill) fdur slutin OH J R. appendiculatus t anal dur B R. evertsi t ear dur Fig. 4.2 Meanpercentage attractancy rrepellency fearand analvlatiles t R. appendiculatus and R. evertsi in climbingbiassays (*, **and ***indicatestatisticalsignificance atp<0.05,0.0and0.00,respectively). significantly shrt time (0.06 ± 0.02 h frm escutchen, cmpared t 0.72 ± 0.25 h and 0.84 ± 0.40 h frm the upper rear and fre legs, respectively) at relatively high speed (2.2 ± 0.42 cm/min) cmparable t cnspecifics placed n the frehead clse t the ears (2.00 ± 0.49 cm/min). Likewise, the reactin time and initial speed f R. evertsi depsited at the shulder clser t the ear (0.08 ± 0.07 h and 3.04 ± 0.24 cm/min, respectively) were nt significantly different frm thse depsited at the escutchen near the anal regin (0.03 ± 0.0 h and 2.77 ± 0.2 cm/min, respectively). Significantly, ticks f bth species that were placed clser t the feeding site f ther mved away and nne were seen t navigate int the area during the bservatin perid, indicating that lcal stimuli like temperature and/r humidity were unlikely t be primarily respnsible and that the ticks were prbably exhibiting an avidance respnse at the sites. Accrdingly, we mdified ur hypthesis t include the pssibility f cncurrent peratin f bth repellent and attractant effects ('push' and 'pull') in n-hst rientatin behaviurs f the tw tick species. T verify this, we studied the respnses f the ticks in the labratry t dur cllectins frm the ear and anal regin f the steers, respectively. The biassay design ex- 79

72 Chapter 4 plited the well-knwn predispsitin f the ticks t climb up and aggregate n grass stems t await passing hsts (Brwning, 976; Chiera, 985). A chice f tw glass cvered rds, ne with a vertical cncentratin gradient f the test dur and the ther with clean air was ffered t grups f ticks. R. appendiculatus was attracted t the ear vlatiles but repelled by anal vlatiles (Fig. 4.2). On the ther hand, R. evertsi was repelled by the ear vlatiles, but attracted t the anal vlatiles. Thus, the dur cllectins frm the tw sites have ppsite effects n the tw tick species and supprt ur hypthesis n the peratin f bth repellent and attractant effects in the feeding site lcatin behaviurs f these ticks. Hwever, at their respective feeding sites, ther signals may als be invlved in site selectin prcess. Our bservatins shw that clser t their respective feeding sites, the ticks are gradually arrested befre finally attaching fr feeding, suggesting the mediatin f lessvlatile and/r chemtactile stimuli at these sites. The cncept f integrated use f the frces repulsin (r deterrence) and attractin (r stimulatin), i.e., 'push-pull', was previusly prpsed as an efficient and sustainable way f diverting insect pests frm a desired crp t a trap crp (Miller and Cwles, 990). Recently, it was effectively deplyed in reducing damage by stembrers t maize (Zea mays L.) in smallhlder farms in Africa (Khan et al., 200). The present study represents the first demnstratin f the explitatin f the tactic in nature, and suggests that the phenmenn may be quite widespread amng arthrpds and related rganisms, particularly where specialisatin ccurs in their interactins with their hsts r envirnmental niches in functins such as selectin and lcatin f hsts, feeding and vipsitin. Acknwledgements We are very grateful t the technical staff members f Animal Rearing and Quarantine Unit, Behaviural and Chemical Eclgy Department and Bistatistics Unit at the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE), Nairbi, Kenya fr their supprt. The first authr wishes t acknwledge the financial and material supprt received frm ICIPE under the African Reginal fr Pstgraduate Prgrammes in Insect Science (ARPPIS) and the Wageningen University and Research Centre, the Labratry f Entmlgy under PhD Sandwich Fellwship. The authrs wish t thank Mr. Newtn Kmeri Mwanga fr making the illustratins. 80

73 Hst durs guide ticks t feeding sites 8

74 Chapter 5 82

75 The effect f essential il n climbing respnse f R. appendiculatus Dual and n-chice assays n the effect f the essential ilf Tagetes minuta n Rhipicephalus appendiculatus Intrductin Abstract: The effects f the essential il f Tagetes minuta n the climbing respnse f adult Rhipicephalus appendiculatus was studied in dual- and n-chice assays. Bth assays shwed a significant repellent effect f essential il n climbing behaviur f adult R. appendiculatus. Althugh dse-dependent respnses were bserved in bth assays, this was less clear in the dual-chice assay. Cmpared t the dual-chice assay, the n-chice assay underestimated the degree f repellency, particularly f lw dses, implying that this assay may nt be suitable fr screening purpses. T achieve the same repellent effect, a higher dse is required in the n-chice assay. Reasns fr the bserved differences between the dual-chice and n-chice assay are discussed. The dual-chice assay appears t be a mre sensitive assay than the n-chice assay. Key wrds: Rhipicephalus appendiculatus, tick, behaviur, repellency, Tagetes minuta, essential il, dual-chice assay, n-chice assay Ticks are increasingly becming nt nly a big nuisance but a serius animal and human health risk (Tnbak et al., 2006; Vial et al., 2006; Salit, 2007; Jngejan, 2007). Amng prphylactic measures used against them, btanical prducts (repellents, deterrents, txicants, stimulants, arrestants and attractants) have, in the recent past, prven t ffer a sustainable apprach tward integrated tick cntrl and management. In particular, plantbased repellents have been shwn t prtect vertebrates against tick bites (Weldn and Carrll, 2007) and have been recmmended as an effective prphylactic measure against tick bites and/r tick-brne infectin (Schreck et al., 995; Okahl, 996; Jaussaud et al., 200; Jensenius et al., 2004; Rch et al., 2008). In the develpment f repellents, an effective and reliable biassay is essential fr rapid screening f candidate prducts (Dautel et al., 999). The ratinale fr develping a reliable biassay is t help screen large ppulatins f candidate tick repellent btanicals accurately and identify effective nes fr incrpratin int tick cntrl strategies (McMahn et al., Wrldwide, research labratries have develped different assay apparatus t evaluate repellent/attractant prperties f candidate plant prducts against livestck ticks (Dautel et al., 999; Jaensn et al., 2006; Carrll et al., 2003; 2005; Garbui et al., 2006). The assay methds emplyed vary a great deal but generally target the behaviur f ticks during questing fr a hst (Alekseev et al., 2000). Bth chice and n-chice assays are widely used t study the behaviural respnses f arthrpds tward btanicals (repellents, deterrents, arrestants, stimulants and attractants) and synthetic prducts. They are als ppular with studies invlving ther rganisms, in particular insects (Hward et al., 976; 'Thischapterwillbesubmittedfrpublicatin as:wanzala,w.,takken,w.,mukabana,r.w.,and Hassanali,A.,Dualandn-chiceassaysntheeffect ftheessentialilf Tagetes minutan Rhipicephalus appendiculatus. 83

76 Chapter 5 Mndy et al., 998; Papachrists and Stampuls, 2002; Rdriguez-Sana et al., 2006). Fr instance, a dual-chice assay apparatus with the same scientific ratinale as the ne shwn in Fig. 5.a was used t test the ability f a termite t discriminate between tw test chemicals and further shwed that trail phermnes in Reticulitermes virginicus Banks, 907, R.flavipes Kllar, 837 and R. tibialis Banks in Banks and Snyder, 920 were species specific (Hward et al., 976). Basically, three types f assay methds are cmmnly used fr testing tick repellents. First, test substances are applied nt vertebrate hsts, which are subsequently expsed t hungry ticks and the percentage f feeding ticks and that f prtectin affrded are estimated (Slberg et al., 995; Mwangi et al., 995a). Secndly, test material is applied nt a hrizntal r vertical walking path f ticks in the absence f any hst cues and the percentage f ticks entering r passing the treated area is recrded and the prtectin percentage estimated (Mwangi et al., 995a, b; Ndung'u et al., 995). Thirdly, the test material is applied nt a hrizntal r vertical walking path f ticks in the presence f hst cues and the number f ticks entering r passing the treated area is recrded, frm which the degree f prtectin affrded can be calculated (Alekseev et al., 2000). Using vertebrates as experimental hsts is unsuitable fr rutine tests with ticks because f the large number f animals required and because f the time- and cst-intensive prcedures invlved. On the ther hand, assays withut any hst stimuli invlved have the disadvantage that the behaviur-mdifying activity f the tested material in the presence f hst cues remains unknwn (Schreck, 977). Of particular interest t, is tick rientatin behaviur under abitic envirnmental parameters such as relative humidity, temperature and light as described by Okulva (978) in the presence f candidate repellents/attractants and ther hst cues (Alekseev et al., 2000). Therefre, a suitable test system shuld be ne that recgnizes these cnditins. Fr example, in the present study, the test system used tk int accunt relative humidity and temperature, which have been described as attractive hstderived and envirnmental cues that determine the questing behaviur f ticks such as Ixdes ricinus L. (McLed, 935; Lees and Milne, 95; Arthur, 962; Alekseev et al., 2000). Furthermre, the test system examined the repellent activity f the essential il f Tagetes minuta L. during critical behaviural steps fhst finding. Because dual-chice assays mre clsely represent the natural situatin in the field, these are preferred t n-chice assays and are widely used (Ryan, 2002). Fr example, in the USA, a dual-chice filter paper assay was successfully used by Re et al. (2006) t develp a btanical tick repellent (BiUD) against the American dg tick, Dermacentr variabilis Say. Similar t a dual-chice filter paper assay is a vertical assay, in which ticks are allwed t climb a vertical strip f filter paper whse central prtin is treated with a repellent. This methd was als used by Carrll et al. (2003) t cmpare the repellent prperties f N,N-diethyl-3-methylbenzamide (DEET) and 2-methylpiperidinyl-3-cyclhexene- -carbxamide (AI ) and t determine their relative effectiveness against hstseeking nymphs f the blacklegged tick, Ixdes scapularis Say and the lne star tick Amblymma americanum L. In Suth Africa, Nchu et al. (2004) cmpared three types f tick climbing repellent assay methds, that is, a n-chice assay, an avidance assay and a dual-chice assay using essential ils frm T. minuta and Lippia javanica (Burm.f.) Spreng. Althugh mre ticks avided the essential il f L. javanica than that f T. minuta, there was n significant difference in the abilities f the three assays t test the repellency f the tw ils against Hyalmma marginatum rufipeskch ticks. In bth n- and dual-chice assays, there was a significant increase (P<0.05) in repellency with increasing cncentratin fr bth T. minuta and L. javanica essential ils. 84

77 The effect f essential il n climbing respnse fr. appendiculatus In Kenya, the dual-chice assay apparatus has been used t study the repellent effects f sme btanicals against livestck ticks (Malnza et al., 992; Mwangi et al., 995a;b; Ndung'u et al., 995). The assay apparatus was recently mdified by inclusin f wider tubes t shield the inner climbing tubes, avid diffusin f test materials laterally, and facilitate their mre unifrm gradients alng the setup (Chapter 4). Hwever, we realised that in the dual-chice assay, the chice f the first tick t climb the glass tube fitted with either an essential il-treated r cntrl filter paper cllar may influence the final scre f the test as the chice f ne tick may affect that f the thers. The n-chice assay was therefre prpsed, as it des nt have this effect and results frm the essential il treatment and cntrl can be cmpared as independent data sets. In the present paper, we describe experiments t cmpare a dual-chice with a n-chice assay using the essential il f T. minuta as the test substance t examine the behaviur f R. appendiculatus adult ticks. Materials and methds Legal framewrk f animal use All prcedures requiring experimental animals were apprved by ICIPE's Institutinal Animal Care and Use Cmmittee and were perfrmed in cmpliance with guidelines published by the Kenya Veterinary Assciatin and the Kenya Labratry Animal Technician Assciatin (KVA and KLATA, 989). Ticks The tick species used (R. appendiculatus) was btained frm clnies at the Internatinal Livestck Research Institute (ILRI) and transferred t labratries at the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE), Nairbi, Kenya, fr rearing and management. The rearing cnditins and management were as described previusly (Bailey, 960; Irvin and Brcklesby, 970). Extractin f Tagetes minuta essential ils The fresh aerial parts f T. minuta were cut int small pieces and abut kg was hydrdistilled using a Clevenger-type distillatin apparatus fr 8 h (Sereshti and Samadi, 2007). Pure il was cllected int 2 ml- glass vials, sealed and stred at -20 C until required fr analysis and assay studies. The tick climbing assay methds The tw sets f tick climbing assay apparatus used included: -() a duble stranded dualchice (Fig. 5.a) and (2) a n-chice climbing assay apparatus (Fig. 5.b). Duble stranded dual-chice tick repellent climbing assay apparatus A dual-chice tick climbing assay apparatus was used t test fr tick repellency f essential il f T. minuta (Fig. 5.a). The assay apparatus explited the behaviur f the ticks, R. appendiculatus, which climb up grass stems and settle fr a perid near the stem tip t wait fr any passing ptential hsts (Brwning, 976; Chiera, 985), hence the design described previusly (Chapter 4). This experiment was dne accrding t the specificatin set up in the labratry at ICIPE, Nairbi, Kenya. An aluminium base f area 05 cm 2 with tw stands f 26 cm each in height and 7.0 cm apart were put in a basin f water,.5 cm deep (the water restricts the mvement f the ticks t the aluminium base). The tw sets f glass tubes were used, ne f 4.5 cm (uter tube) and the ther ne 0.8 cm (smaller inner tube) 85

78 Chapter 5 5cm Fig. 5..Twbiassay devices t study the impact fputative repellents ntick climbing respnse (mdified frm Brwning, 976).Dual-chice climbing assay apparatus (a).fr details, seefig.3.. Frn-chiceassay (b),thetwaluminiumrds (Bi)nthealuminiumbase,A,wereseparatedbywater.Thefive tickswereintrducedneachf thetwaluminiumbasesat adistancef3.5cm frm thebttmfanaluminiumrd. in diameter. A strip f filter paper (Whatmann N 7, 2 cm wide) was stapled t frm a cllar arund the upper parts f each smaller inner tube at a distance f 20 cm frm the aluminium base t prvide the surce f either test durs r pure slvent. One cllar n the pair f the tubes was treated with test dur slutin and the ther ne with the same amunt f pure slvent (dichlrmethane-dcm) alne t serve as cntrl. After the slvent was allwed t evaprate (fr abut 0 min), these tubes were shielded with wider tubes (4.5 cm d) frm 4 cm abve the aluminium base t shield the inner nes and limit the diffusin f the test material laterally and facilitate relatively unifrm vertical gradients f the durs alng the 3.7 cm gap between tw tubes. The upper ends f larger tubes were plugged with dry cttn wl. Wet cttn wl plugging the tp f the smaller tubes ensured relatively high relative humidity (>75%) within the cl-umns. The test materials and the slvent were dispensed by a calibrated Eppendrf pipette, equilibrated fr 30 minutes and then five adult ticks f mixed ages and sexes were released at the centre f the aluminium base. Prir t each assay, ticks were kept at high relative humidity (RH) (>85% RH) fr 24 h in cntainers with mist cttn wl. All assays were cnducted in a rm f 28 ± C and 75 ± 5% RH. The rm was cntinuusly exhausted f air using a fan. The assays were left t run fr h, during which the number f ticks abve the filter paper strip n the cntrl glass tube (Nc) and n the treated glass tube (Nt) were cunted and recrded after 5, 30, 45 and 60 minutes. After each test, the apparatus was thrughly cleaned and dried at 00 C. Initial cmparisn f the respnses f ticks in the setup with and withut residual dichlrmethane n ne and bth sides, shwed n bias fr either side and n effects f the residual slvent. The repellent effect f the essential il f T. minuta in dual chice assay was evaluated accrding t the frmula adpted by Ndung'u et al. (995) and Lwande et al. (999) namely: percentage f repellency (PR) = [(Nc-Nt)/(Nc+Nt)] x 00, where Nt and Nc represent the number f ticks that climbed n r passed the treated and cntrl cllar f filter papers n the glass tubes, respectively. 86

79 The effect f essential il n climbing respnse f R. appendiculatus Single stranded n-chice tick repellent climbing assay apparatus Except fr the experimental design, labratry cnditins and specificatins f the singlestranded climbing apparatus were as described fr the dual-chice assay (Fig. 5.b). Tw climbing rds were placed n separate bases at a distance f 27.5 cm within a tray (D) filled with tap water up t.5 cm deep. In each experiment, five newly-hatched and 24-hhydrated adult ticks f mixed sexes were placed at a distance f 3.5 cm frm the base f aluminium rd, B] (Fig. 5.b). Here, the assays were als left t run fr hand the number f ticks abve the filter paper strip n the cntrl experiment glass tube (Nc) and n the treated experiment glass tube (Nt) were cunted and recrded after 5, 30,45 and 60 min. The effect f the essential il f T. minuta was evaluated by cmparing the climbing respnse f ticks n the glass tube fitted with a filter paper cllar treated with dichlrmethane (cntrl) with thse climbing the glass tube fitted with a filter paper cllar treated with the il. Chice f the type and dse(s) f the essential ils used In preliminary dse respnse assays with the essential ils f T. minuta and Tithnia diversiflia (Hemsl.) A. Gray, the frmer shwed a higher repellent effect against adult R. appendiculatus than the latter (Chapter 4). The essential il f T. minuta was therefre selected fr cmparisn assays at the 0.025, 0., and mg dses. In the n-chice essay a dse f2.25 mg f il was als tested. Data management and analysis Climbing assay data Data were entered int a Statistical Prducts and Service Slutins (SPSS versin. fr Windws) spreadsheet database and analysed. A ne-way analysis f variance (ANOVA) and Univariate analysis f SPSS were used t cmpare means between dses and ver time. The means were separated using Student-Newman Keuls test at P = 0.05 (Zar, 996). Using an independent samples t-test, the mean percentage f climbing ticks in the essential il-treated experiment was cmpared with the ticks climbing the cntrl glass tube whse cllar f filter paper was treated with dichlrmethane in the n-chice assay (Dixn and Massey, 969). Results Evaluatin f repellent prperty f Tagetes minuta essential il using a dual-chice assay The results f the dse-dependent respnse f newly emerged adult R. appendiculatus t the essential il f T. minuta in a dual-chice climbing assay apparatus are shwn in Tables 5. and 5.2. Fr a given dse and time f bservatin, there were varying degrees f dse- and time-dependent respnses, respectively. In the first 5 minutes, there was a significant difference between mean percentage f repellencies caused by different dses f essential f T. minuta (P = 0.006). Thereafter, with the exceptin f 45th minute's bservatin (P = 0.036), there was n significant difference between the mean percentage f repellencies caused by different dses f the essential il f T. minuta (P>0.05). There was n significant difference between the mean percentage f repellencies caused by lwer dses f essential il f T. minuta ver time (P>0.05). In the highest dse f essential il f T. minuta ( mg), there was a significant difference between the mean percentage f repellencies caused by the dse ver time (P = 0.07) (Student-Newman-Keuls test) (Table 5.). 87

80 Chapter 5 Table 5.. The mean (± SE) percentage f repellency caused hy Tagetes minuta essential il ver time and at different dses using the dual-chice assay (n = 5). dses (mg) P - values ±5.07bl 5.7±3.7bl 00.0 ±00al ± 4.05al 72.0±2.27al 00.0 ±00al 0.8 Time (min) ±5.24bcl 72.0±2.27bl 00.0 ±00abl ±3.94al 78.0±2.09al 88.0±6.a2 Within a clumn, means with the same letter(s) and acrss a given rw, means with the same number(s) after the letter(s) are nt significantly different at P = 0.05 (Student-Newman-Keuls test), respectively P-values Table 5.2. Overall mean (± SE) percentage f repellency f the essential il f Tagetes minuta btained using a dual -chice assay Dses f essential il f Tagetes minuta (mg) Mean percentage f repellency ±7. lb ± 6.26b ±.69a P-values <0.05 Within a given clumn, means (± SE) with the same letter(s) are nt significantly different at P = 0.05 (Student-New-man-Keuls test). This assay did nt shw a clear trend f time-dependent respnses f adult R. appendiculatus t the essential il f T. minuta il. Hwever, the assay shws a clear trend f dsedependent tick climbing respnses (Table 5.2). Evaluatin f repellent prperty f essential il f Tagetes minuta using a n-chice assay The results reprting the mean percentage f climbing ticks with respect t the dses f the essential il f T. minuta and time f bservatin in the n-chice assay are shwn in Fig These results suggest a time-dependent respnse in which the average number f ticks climbing up the glass tube in the presence f essential il r dichlrmethane is a functin f the time taken fr bservatin, i.e. the percentage f ticks climbing increased with time (Fig. 5.2). Using an independent samples t-test, the mean percentage f climbing ticks in the essential il-treated experiment was cmpared with the ticks climbing the cntrl glass tube whse cllar f filter paper was treated with dichlrmethane. At any ne given time, significantly mre ticks climbed up the cntrl glass tube than the il-treated tube fr every bservatin made at all dses f essential il (P<0.05)(Table 5.3). The mean percentage f ticks climbing the glass tube fitted with an il-treated filter paper cllar decreases significantly with increasing cncentratin f the essential il f T. minuta (P<0.05) (Fig. 5.2 and Table 5.3). At the highest dse f the essential il, the repellent effect n R. appendiculatus was greatest (Table 5.3) and maintained fr a lnger perid f time than with the ther dses (Fig. 5.2d). Hwever, at the lwer dses (0.025 and 0. mg) there was an increasing number f ticks climbing up the essential il-treated glass tube ver time. The gap between the cntrl and essential il-treated experiments widened with increasing cncentratin f the essential il ver time fr all dses except mg. Thus the variables, time, dse and repellent effect f the essential il are interac-

81 The effect f essential il n climbing respnse f R. appendiculatus 00 i a S 60 « mg f essentialil - Cntrl Cntrl Observatin time (min) DC S U * 40 u GO 2 20 ) S 0 i r Observatin time (min) 60 S * ft mgfessentialil b b Observatintime (min) -Cntrl b 00 c S U "? M (3 5 < inn a a mg fessential il b b a a Observatin time (min) Cntrl b a 60 Fig.5.2. Meanclimbingrespnse (%)vertimefdifferent dsesfessentialilf Tagetes minutawithadult Rhipicephcdus appendiculatususing an-chice assay.thefigures frm (a)t (d)represent dsesfessential ilf Tagetes minuta expsed tnewlyemerged Rhipicephalus appendiculatus in an-chice climbingassay (n =5). Freithercntrlressentialiltreatment,themeans (±SE)withthesamelettersarent significantly different frmneantherat P =0.05 (Student-NewmanKeuls test). tive. The trend f mre ticks climbing ver time was als nted in the cntrls (Fig. 5.2) and significantly higher than in the essential il-treated experiment at all bservatin times fr all the dses (P<0.05) (Table 5.3). Between dses f the essential il f T. minuta, mean percentages f ticks climbing the glass tube fitted with a treated cllar f filter paper were significantly different frm each ther (P<0.05) (Table 5.3). With the mg dse f essential il, the mean percentage f ticks climbing the glass tube after the first 5 minutes was significantly different (P = 0.04) frm the subsequent mean percentage f ticks climbing the glass tube thereafter, between 30 and 60 minutes (Fig. 5.2). Within a 60-minute bservatin perid, the mean percentages f ticks climbing the glass tube whse cllar f filter paper is treated with the dses 0. mg, mg and 2.25 mg f the essential il f T. minuta, were nt significantly different frm ne anther at P = 0.087, P = and P = 0.06, respectively (Fig. 5.2). While in the cntrl mre ticks climbed up the glass tube than in the essential iltreated experiment, there were sme significant differences in the percentage f ticks climbing up the glass tube between the bservatin perids (P<0.05) except fr mg dse (P = 0.067). 89

82 Chapter 5 Table 5.3. The mean (± SE) percentage f ticks climbing a glass tube fitted with a cllar f filter paper treated with either the essential il f Tagetes minuta r dichlrmethane (cntrl) in a n-chice assay Mean percentage f ticks climbing a glass tube Dses f essential il f Tagetes minuta (mg) Essential il treatment Cntrl P-vahies ±2.53al 84.0±2.03c2 < ±2.59bl 7.0±2.7b2 < ±3.00cl 73.8±2.39b2 < ±0.35dl 56.8±2.62a2 <0.05 P-values <0.05 <0.05 Within a given clumn, means (± SE) with the same letter(s) are nt significantly different at P = 0.05 (Student-New-man- Keuls test). In a given rw, means (± SE) with the same number(s) after the letter(s) are nt significantly different at P = 0.05 (Student-Newman-Keuls test). 90 Discussin and cnclusin The tw assay methds investigated in this study shwed a significant repellent effect f the essential il f T. minuta, even thugh the il was nt tested in the presence f hstderived stimuli as suggested by Dautel (2004; Dautel and Cranna, 2006). The repellent effect f the essential il f T. minuta in the presence f a live hst was demnstrated, hwever, in Chapters 6, 7 and 8 and we cnclude that this essential il affects adult R. appendicular under varius circumstances. Kschier and Sedy (2003) als shwed a significant repellent effect f the essential ils frm plants within the Lamiaceae family against Thrips tabaci Lindeman using the dual-and n-chice assays. The effect f the il was less in the n-chice assay cmpared t the dual-chice assay, particularly with lw dses, implying that this assay methd may be less suitable fr screening purpses (Ryan, 2002). This is because the n-chice assay may nt be able t identify plants whse essential ils have lw repellent activity and is therefre less discriminatry. In the n-chice assay, high values f the repellent effect cmparable t thse btained in the dual-chice assay were nly achieved with a high dse (2.25 mg) f the essential il f T. minuta. In cntrast t ur predictins, therefre, using a n-chice tick climbing apparatus t study the effects f repellent f the il did nt increase treatment accuracy; instead, it was time cnsuming and the mean repellent effects were cmparatively lw. Althugh the tw experimental set-ups are tw different designs, the mechanism by which these assays exert their influence n tick climbing behaviur, hwever, remains unknwn. The advantage f the n-chice assay is that there is n interactin between the ticks climbing the essential il-treated and dichlrmethane-treated (cntrl) glass tubes, as each is separately ffered t the ticks (as an independent experiment). Because ticks in the treatment with the essential il and in the cntrls are tested independently, the verall result f the assay shuld reflect the true effect f the il n the ticks better than in the dual chice assay, where ticks respnding t the il and cntrl may affect each ther. We realise, that t btain a better estimate f this behaviur, in the n-chice essay the behaviur f the ticks shuld ideally be examined n single individuals as ptential aggregating effects may affect the end result (Snenshine, 2006). Als, it seems better if the distance between the tw climbing set-ups is much larger, r that tests are cnducted in different rms t exclude ptential effects f treatment dur(s). The equatin used in this study t generate the data fr the dual-chice assay des nt, hwever, recgnize (a) the varied interactins invlved such as () influence f the

83 The effect f essential il n climbing respnse fif. appendiculatus test dur frm the treated filter paper cllar and (2) test rganisms that keep n climbing n the cntrl and treated glass tubes and vice versa befre making a final decisin, (b) test rganisms that shw the test material t cnfer either true repellent effects r excitrepellent/irritant effects, (c) test rganisms that drp ff in water and drwn and (d) nnrespnding test rganisms, which were initially part f the entire interactin and riginal ppulatin (n = 5). Althugh these factrs are nt cnsidered in the equatin, they influenced the number f ticks that climbed the glass tube fitted with filter paper cllar treated with either dichlrmethane r the essential il f T. minuta. In a n-chice assay apparatus, the interactin () abve is minimised while interactin (2) is cmpletely remved. Nevertheless, factrs b, c and d abve equally affect the results btained by bth tick climbing assay apparatus. This culd be the reasn why the results shwed the same pattern f increase in repellent effect with increasing cncentratin, as in the results btained by Nchu et al. (2004; 2005). But this small difference emanating frm interactin (2) abve may nt be sufficient t explain the lw values btained when a n-chice assay apparatus alne is used. It is pssible that frm the experimental-design pint f view in bth assays, there might be an aggregatin effect amngst interacting ticks due t phermnes (Snenshine 2006). This phermnal influence may be having an effect n the climbing behaviur and ther intraspecific interactins f R. appendiculatus ticks in bth dual-and n-chice assays (Snenshine, 985). This aggregatin behaviur f R. appendiculatus has als been bserved in the labratry (Brwning, 976) and field (Chiera, 985) and is attributed t phermnes (Snenshine 2006). Pssibly, the phermnal effect may be suppressed by the essential il in bth assays. This suppressin may be greater in the dual-chice assay than in the n-chice assay due t the fact that the tw glass tubes fitted with treated filter paper cllar in the frmer assay are clse t ne anther. This phermnal effect may be remved if ticks are bserved ne at a time and between bservatins, the assay apparatus arerinsed with 99.98% alchl. Althugh the dual-chice assay set up appears t be effective, it requires, during statistical analysis f data generated by the equatin (PR = [(Nc - Nt)/(Nc + Nt)] x 00), a statistical mdel that includes all the existing variatins and factrs mentined in the paragraphs abve in rder that the abslute repellency is estimated with respect t these variatins and factrs. Hwever, in a dual-chice assay, data are pled after emplying the equatin (PR = [(PR) = [(Nc - Nt)/(Nc + Nt)] x 00), and therefre, this des nt put int cnsideratin the time ver which the bserva-tins were made. A n-chice set up generates data that gives mre cmprehensive infrmatin n the behaviur f the ticks in respnse t the essential il f T. minuta ver time and within dses than the dual-chice set up, especially abut the interactin f the variables selected fr analysis. Hwever, the nchice assay des nt simulate a natural field situatin f freedm f chice (Ryan, 2002), thus changing the behaviur f test rganisms, a situatin that makes it a less efficient testing device (Huang et al., 2003; Adebwale and Adedire, 2006). In the n-chice assay, mre ticks climbed the cntrl rd than the essential iltreated rd fr all dses f the essential il f T. minuta ver time, and this difference was significant. This suggests a significant repellent effect f the essential il n climbing behaviur f R. appendiculatus. Over time, the gap between the number f ticks climbing the cntrl and the essential il-treated glass tubes in the n-chice assay widened with increasing cncentratin f the essential il f T. minuta. This implied significant dse-and time-dependent respnses fr. appendiculatus adult ticks t the essential il. Such cnsistent significant respnses were nt btained with the dual-chice assay, except fr dse respnses. Thus, in the n-chice assay, the variables "time" and "repellency" were inter- 9

84 Chapter 5 active. The average number f ticks affected by the repellent effect f the il was a functin f the time f expsure f ticks t the il. In the first 5 minutes, the repellent effect was relatively high and thereafter this reduced with time. The reductin in repellency was greater with lwer dses than with higher nes. The higher dses tended t maintain a higher repellent effect against ticks fr a lnger perid f time than the lwer nes. This trend f the results is cmparable t that btained by Dlan et al (2008) when testing essential il (lemn, picaridin and ntkatne) against /. scapularis in vertical, finger and hrizntal assays. Hwever, these results were different frm the results btained with a dual-chice assay, implying that the tw assays manifest different patterns f behaviural respnses. The trend in which the average number f ticks climbing up the glass tube increased ver time in the n-chice assay was als nted in the cntrls. Whether this trend reflects the natural tick climbing behaviur r nt, is yet t be cnfirmed as the cntrl material (dichlrmethane) was nt shwn t cause any behaviural effects. It is pssible that this behaviur was caused by increasing degrees f nutritinal depletin, resulting in strnger behaviural respnses. It is cncluded that bth assay methds tested in this study prvide baseline data, against which nvel livestck tick repellents can be examined and selected fr field-testing and subsequent develpment. The dual-chice assay prved a mre sensitive assay than the n-chice assay. Acknwledgements This research was jintly supprted by the Internatinal Fundatin fr Sciences, Stckhlm, Sweden and the Organizatin fr the Prhibitin f Chemical weapns, The Hague, The Nether-lands thrugh a grant AB/ The first authr wishes t acknwledge the financial and material supprt received frm the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE) under the African Reginal fr Pstgraduate Prgrammes in Insect Science (ARPPIS) and the Wageningen University and Research Centre, the Labratry f Entmlgy under PhD Sandwich Fellwship. The authrs wish t thank Mr. Newtn Kmeri Mwanga fr illustratins and ICIPE staff fr their technical supprt. T them all, we are very grateful. 92

85 The effect f essential il n climbing respnse f R. appendiculatus 93

86 PartIII Field evaluatin f essential ils

87 Chapter 6 96

88 Effect f essential ils n n-hst tick behaviur Theeffect fessential ilsf Tagetes minuta and Tithnia diversiflia n n-hst behaviur f thebrwn ear tick Rhipicephalus appendiculatus Intrductin Abstract: On-hst behaviur f Rhipicephalus appendiculatus was studied in the field t evaluate the putative repellent effects f essential ils f Tagetes minuta and Tithnia diversiflia at its predilectin feeding site. Oils f bth plants caused a disruptin f rientatin, mvement and attachment behaviur f ticks. Mre ticks drpped ff in the treatments with the tw essential ils than with the cntrl. Treating the ear pinna with the essential il f T. minuta caused the highest percentage f ticks t drp ff the hst bdy. N tick reached the ear pinna treated with the essen-tial il f T. minuta and up t 30% f ticks (frm the frehead release site) reached the ear base. When the ear pinna was treated with the essential il f T. diversiflia, ne tick reached the ear pinna and up t 40% f ticks (frm the dewlap release site) reached the ear base. The results shw that T. minuta repels ticks mre strngly than T. diversiflia. Hwever, bth essential ils ffer pssibilities fr explitatin f ptentially effective and envirnmentally acceptable tls fr n-hst tick cntrl. Key wrds: Rhipicephalus appendiculatus, n-hst rientatin behaviur, predilectin feeding site, cattle, repellent, Tagetes minuta, Tithnia diversiflia, essential ils Ticks and tick-brne diseases (TBDs) are a majr cnstraint fr the develpment f the livestck industry thrughut the trpics. Wrldwide, an estimated 600 millin cattle are expsed t anaplasmsis and babesisis, whereas 200 millin cattle are expsed t theilerisis, all being ecnmically imprtant TBDs. In eastern, central and suthern Africa, East Cast fever (ECF), caused by Theileria parva and transmitted by Rhipicephalus appendiculatus, is cnsidered t be the mst significant tick-brne disease f cattle. The parasite infects cattle as well as buffal (Syncerus cffer) in 2 cuntries f the regin and 25 millin cattle are at risk. One millin cattle are estimated t have died f ECF in 989 alne (Mukhebi et al., 992). Fr many years, the cntrl f ticks and the diseases they transmit has been largely thrugh the applicatin f acaricides as dips, dust, and pur-ns, using ear tags, sprays r systemic acaricides. The repeated applicatin f acaricides prevents transmissin f the parasites and this methd has been used very successfully thrughut sub- Saharan Africa, in cnjunctin with cntrl f animal mvement, quarantine and slaughter 'Thischapterwillbepublishedas:Wanzala,W.,Takken,W.,Mukabana,R.W.andHassanali, A., Theeffect ftheessentialilsf Tagetes minutaand Tithnia diversifliann-hstbehaviurf thebrwneartick Rhipicephalus appendiculatus, whensubmitted 97

89 Chapter 6 f infested animals (Lawrence, 99). Hwever, the develpment f resistance in ticks t successive acaricide cmpunds has been a majr prblem (Nrval et al., 992). This prblem has been cmpunded by the increasing csts f acaricides, illegal cattle mvement, civil unrest, pr management and inadequate maintenance f dips, especially cmmunal nes. The devastating extent f recurring prlnged drught in sme parts f Africa has made many dip tanks nn-peratinal, due t lack f sufficient water t main-tain them. Anther cmplicatin assciated with the use f acaricides is that they are envirnmental pllutants and have been fund t cntaminate milk and meat (Mitchell, 996). Tick cntrl by the use f repellents is cnsidered an alternative strategy (Muthuswami and Nisha, 2006). Sme tick- and insect repellents are available and widely used fr prtectin by humans (Frances and Wirtz, 2005). The use ftpical repellents prvides an effective prphylactic measure against biting arthrpd vectrs and arthrpd-brne diseases at an individual level (Gupta and Rutledge, 994; Hch et al., 995; Nentwig, 2003), especially in areas where suppressin f arthrpd vectrs is nt practical r feasible. Examples f such repellent materials, include, but are nt limited t N,N-diethyl-3- methylbenzamide (DEET), p-menthane-3,8-dil (PMD), permethrin, allethrin, pipernyl butxide, lemngrass il, citrnella il, eucalyptus il, camphr, geranium il, ethyl hexanedil, ethyl butylacetylaminprpinate and hydrxyethyl-isbutyl-piperidine. Repellents cmmnly available t cnsumers cntain the active ingredients DEET, a few repellents cntain permethrin, while nne r very few repellents cntain btanical essential ils (New Yrk State Department f Health (2737/04), 2004). The develpment f btanical essential ils as arthrpd repellents has been n the increase in the recent decade, in rder t replace DEET due t its reprted envirnmental pllutin and txicity in human ppulatin using it (Waka et al., 2004; Se et al., 2005; Jaensn et al., 2006; Kegley et al., 2007; Walschaerts et al., 2007). In the livestck industry, repellents are less cmmnly used than in human health, althugh traditinal livestck wners may use a range f ethnbtanical prducts t prtect their animals frm tick bites (Martin et al., 200; Guarrera et al., 2004; Van de Putte, 2005; Mathias, 2005; Passalacqua et al., 2006; Bnd, 2007). Cmmercial repellent prducts cntaining btanical (plant-based) ils, such thse f geranium, cedar, lemn grass, sy and r citrnella have been available. There is limited infrmatin, hwever, n the effectiveness f btanical ils individually and when cmbined with ther ingredients. Available infrmatin indicates that, cmpared t the effectiveness f DEET r permethrin, btanical essential ils generally d nt prvide the same duratin f prtectin (Cnsumers Unin, 993; 2000). The efficacy f these natural prducts is prly understd, whilst cmmercial prducts are rare. There isneed therefre t fcus research n the develpment f effective and safe livestck tick repellents that can be incrprated in the existing Integrated Tick Management (ITM) fr livestck tick cntrl prgrammes (Munt et al., 999). The life cycle f an ixdid tick ften has a ttal duratin f 6 years and hst attachment may cnstitute less than 2% (VA mnths) f this time. Ticks spend mst f their life cycle away frm their hsts, hiding either in the sil and vegetatin r in the nests f their hsts. Hwever, ticks select habitats with gd pprtunities t encunter a hst fr feeding. Hst-seeking and -recgnitin are tw mst imprtant and challenging activities in the life cycle f ticks. Many ticks, in particular certain Hyalmma, Amblymma, Ornithdrs and Dermacentr species (Snenshine, 99; Ann, 2008), seek their hsts by hunting, whereas thers use an ambushing strategy, e.g. the larvae f Bphilus spp. and mst ixdid ticks (Snenshine, 993; Eckert et al., 2005). Other tick species respnd t vlatile hst emanatins, vibratins, visual cues, radiant heat and tuch with questing be- 98

90 Effect f essential ils n n-hst tick behaviur haviur, e.g. R. appendiculatus (Speybreck et al., 2003). Hwever, little is knwn n hw different hst specificities are encded in the durs. Questing ticks climb up the stems f grass r perch n the edges f leaves n the grund in an erect psture, while the first pair f legs is waved in the directin f the stimuli f the hst passing by. Subsequently, the ticks climb and grab nt the ptential hst bdy using their frnt leg claws. Once n the hst, gustatry and lfactry cues seem t aid the ticks in deciding whether it will remain n the hst. By 'push' and 'push-pull' actin mdes f the hst's vlatiles, ticks may be guided t particular feeding sites (Chapter 4). Sme tick species prefer feeding sites where they are ut f reach f attacks by the grming behaviur f the hsts. On artificial substrates, ticks rientate twards certain hst skin extracts (Akinyi, 99; Sika, 996), but the chemical nature f the directing cues is nt yet knwn. Hwever, gas chrmatgraphycupled electrphysilgy recrdings using different vertebrate durs has shwn that lactne, methylsalicylate, carbn dixide, sulfide, benzaldehyde, 2-hydrxybenzaldehyde, aliphatic aldehydes, 2,6-dichlrphenl, nitrphenl, pentanic acid, 2-methylprpanic acid, butanic acid, ammnia, and 3-pentanne, may be invlved in hst identificatin by the ticks (Ann. 2008). Masking these natural vlatiles and diverting ticks away frm their prspective hsts may be an attractive strategy t incrprate int the existing ITM fr livestck tick cntrl and management. Sme tick species exhibit specializatin in selecting their feeding site n their hsts. Such specificity may serve t maximize survival and reprductin f the species (Chiltn et al., 992). Adult R. appendiculatus have a marked preference fr feeding mainly inside the ears f bvids, whereas their immatures shw less selectivity, and are fund feeding n many parts f the hst bdy in cnsiderable numbers (Walker, 974). A hst-derived dur-based 'push' and 'push-pull' pair f stimuli has been suggested t be respnsible fr the rientatin behaviur f adult R. appendiculatus t its preferred feeding site (Sika, 996; Wanzala et al., 2004). In this study, we investigated the n-hst behaviur f unfed adults f the brwn ear tick R. appendiculatus and assessed the effect f the applicatin f essential ils f T. minuta and T. diversiflia n this behaviur in natural field sites in Bungma District, western Kenya. Material and methds The study site The experiments were cnducted under utdr cnditins at the study site in Bungma District, western Kenya, which lies between latitude 0 25'N and 0 53'N and lngitude 34 2'E and 35 04'E (Fig. 2.). The altitude ranges frm m abve sea level. The mean annual temperature ranges frm 2 t 22 C with variatins in mean maximum and minimum temperature ranging frm 25 t 32 Cand t 3 C, respectively. Rainfall in the district has a bimdal pattern and varies n average frm mm annually. The majr rainy seasn is frm March t July, while the minr ne starts in August and cntinues int Octber. Experimental ticks The tick species used (Rhipicephalus appendiculatus) was btained frm clnies reared in the insectary at the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE), Nairbi, Kenya. Rearing cnditins and handling were as described previusly (Bailey, 960; Irvin and Brcklesby, 970).The newly hatched and 24 h-starved adult R. appendiculatus were transprted in glass vials buried in mist sand frm ICIPE, Nairbi t Bungma (a 99

91 Chapter 6 Fig. 6.. Sites n thehst animal (bvid)usedfrthe studyfn-hstnavigatin behaviur f R. appendiculatustwards the predilectinfeeding site (theear) in the presence f essential ils.marked site =Frehead, 2 = Dewlap, 3 = Freleg, 4 = Rear leg, 5 = Escutchen and 6 =Shulder. distance f abut 500 km), and used within 48 hafter hatching. Behaviural studies Observatin f n-hst behaviur was dne using indigenus zebu steers (bdy weight kg), bught frm livestck farmers near and / r within the study site. The animals had nt been expsed t acaricides r ther chemicals. The animals were held in a crutch facility built at Mwibale. The respnses f ticks and their navigatin patterns were mnitred starting frm six different bdy lcatins n zebu steers, representing varying distance by the bserver (WW) frm preferred feeding sites and pssible areas f alightment by the ticks frm their questing psitins n the vegetatin (Brwning, 976) (Fig. 6.). One tick at a time f mixed age and sex was placed at ne f six sites n the hst animal and bserved fr up t 4 h, between 07:00 and 24:00 hurs (Fig. 6.). All bservatins were made during dry weather with day temperatures in the range f Cand relative humidity ranging between 60-85%. Each bservatin was replicated 20 times, each time using a naive tick. The eventual phermne trail left by each tick (Snenshine, 2006) was remved by wiping the animal with 99% alchl and leaving it t evaprate befre a new tick was placed n the animal. Dispensing f essential il vlatiles Essential il f T. minuta and T. diversiflia had been btained by the hydrdistillatin methd using a Clevenger type distillatin apparatus (Sereshti and Samadi, 2007). Essential ils were diluted t 0% in durless vaseline petrleum jelly (BP-USP 00% Grade) (Unilever, Kenya) skin prtectant. This carrier material cntains n clurs, fragrances r irritants. One ml f the stck 0% frmulatin f essential ils was placed in a 5 cm 3 - Eppendrf tube which was attached by a string t a plastic ear tag n the inner side f the ear pinna. The Eppendrf tube was left pen s that there was cntinuus release f either essential il vlatiles r petrleum jelly. The implicatin was that at all times a dse f 00 ml f pure essential il in 900 ml f vaseline petrleum jelly was expsed t the ticks n 00

92 Effect f essential ils n n-hst tick behaviur the inner side f the ear f the animal. The dur frm the applied essential ils was presumed t gradually cntaminate the ear and its surrunding area. Bth ears were simultaneusly treated and ticks were released frm previusly marked sites (Wanzala et al., 2004) (Fig. 6.). The 20 ticks were released ne at a time and mnitred until the final destinatin was determined. Legal use f experimental animals in the field All prcedures requiring the use f experimental animals in the field were apprved by District Veterinary Officer (DVO) f Bungma District, western Kenya. The imprtance, seriusness and risk-free nature f the prject were further explained t the Bukusu cmmunity by the DVO and agricultural extensin fficers wrking within the study area. The field experiments were perfrmed in cmpliance with guidelines published by Kenya Veterinary Assciatin and Kenya Labratry Animal Technician Assciatin, regarding the ethical use and handling f labratry and farm animals in the filed (KVA and KLATA, 989). Infrmed cnsent was btained frm the livestck farmer vlunteer frm whm we rented field experimental plts. Statistics Data were entered in Excel database structure and then entered int a Statistical Prducts and Service Slutins (SPSS versin 5 fr Windws) database fr analysis. Data fr nhst tick behaviur were analysed by ne-way analysis f variance (ANOVA) and univariate analysis using the general linear mdel (GLM) prcedure fr SPSS. The mean differences were cmpared and separated using Student-Newman-Keuls test at P = 0.05 (Skal andrhlf, 995). Differences between respnses f the ticks under different treatments and at different release pints (lcatins) n the hst bdy were analysed using the Kruskal Wallis H -test (Kruskal and Wallis, 952). Any significant results btained by the Kruskal Wallis H -test, between and within grups f release pints and treatments, were analysed using Wilcxn-Mann-Whitney U-test with Bnferrni crrectin (Wilcxn, 945; Mann and Whitney, 947; Bergmann et al., 2000). Results The steretyped sequence f n-hst rientatin behaviurs fr. appendiculatus revealed a set f sequential activities (particularly in the cntrl experiment). These activities ranged frm an inactive statinary/scanning phase t the nset f erratic mvements that became increasingly rapid and directinal and finally t arrestment at the predilectin feeding site. Althugh variatins ccurred in the duratin f each set f rientatin behaviurs fr every tick bserved, the steretyped sequence f rientatin behaviurs cnsistently ccurred, irrespective f type f treatment, release pint n the hsts' bdy and age, sex and bdy size f candidate ticks. As bserved previusly in a cntrlled envirnment (Chapter 4), these steretyped respnses cmprised runs and strides f varying intensities, alternating with stps, and ccasinally, walk-away and back-up mvements. At the predilectin feeding site, ticks behaved differently, each taking time t start the attachment prcess by muth insertin int the hst integument befre imbibing bld much later. The results f riented respnses and behaviural mvements f R. appendiculatus t the ear treated with the essential ils f T. minuta and T. diversiflia demnstrated a disruptive effect (Tables 6. and 6.2). Bth release pints and treatments (the essential ils f T. minuta and T. diversi- 0

93 Chapter 6 Table 6.. On-hst behaviur f Rhipicephalus appendiculatus released at varius lcatins nzebu steersresultingfrm treatment fthepredilectin feeding siteswith eitherpetrleum jelly (Cntrl)r theessentialilfeither T. minuta (Tm)r T. diversiflia (Td); (n =20). Site f tick release Treatment N. f ticks attached near and r at the ear base N. f ticks attached at the ear pinna N. f ticks attached in the NRP N. f ticks that drpped ff Percentage f nnrespnding ticks 2.Frehead 2. Dewlap 3. Freleg 4. Rear leg 5. Escutchen 6. Shulder Cntrl Tm Td Cntrl Tm Td Cntrl Tm Td Cntrl Tm Td Cntrl Tm Td Cntrl Tm Td 'NRP-NeighburhdfReleasePints (bdylcatins). 2 Theterm 'respnding'referstthepercentagefthettalnumberfticksatdifferent releasepintsthatinitiated anymvementswithinand /raway flia and petrleum jelly) had a significant effect n the respnses f ticks n the hst while searching fr a predilectin feeding site. Fr all the release pints, the number f ticks reaching the ear base [H (2) = 6.086, P = 0.048], ear pinna [H (2) = 4.500, P = 0.00] and thse that drpped ff [H (2) = 8.972, P = 0.0], were significantly affected by treatment. N tick reached the ear pinna treated with the essential il f T. minuta, while nly ne tick reached the ear pinna treated with the essential il f T. diversiflia. Mre ticks drpped ff in the treatment with the tw essential ils than in the cntrl, with the ear pinna treated with the essential il f T. minuta shwing the highest number f ticks drpping ff frm varius release pints (Table 6.). Mre ticks frm the varius release pints reached the base f the ear when the essential il f T. diversiflia was applied n the ear pinna than in the case f the treatment with the essential il f T. minuta (U = 4.5, r = , P = 0.029). The number f ticks frm varius release pints reaching the ear base in the cntrl treatment was significantly different frm the treatment with T. minuta (U = 5.50, r = , P = 0.043) but nt frm the treatment with T. diversiflia (U = 7.5, r = , P = 0.934). The number f ticks reaching the ear pinna was significantly higher in the cntrls than in the treatment with either T. minuta (U = 0.00, r = , P = 0.002) 02

94 Effect f essential ils n n-hst tick behaviur r T. diversiflia (U = 0.500, r = , P = 0.004). Mre ticks were affected by the treatment with T. minuta and T. diversiflia than by the treatment with the petrleum jelly (Table 6.). Hwever, whereas the treatment with T. minuta and T. diversiflia equally prevented ticks frm getting attached at the ear pinna (U = 5.00, r = , P = 0.37), the treatment with the essential il f T. diversiflia did nt prevent the ticks frm getting attached atthe ear base like its cunterpart. The number f ticks that was attached in the neighburhd f release pints (NRP) [H (2) = 2.589, P = 0.274] and that representing the nn-respndent ticks nce placed at the release pints [H (2) = 0.834, P = 0.659], were nt affected by the treatment with petrleum jelly and the essential ils f T. minuta and T. diversiflia. The site f release f the ticks n the hst did affect their directinal behaviur in searching fr a predilectin feeding site (PO.05). The number f ticks reaching the ear base frm release pint was the highest and significantly different frm all ther release pints (P<0.05). Hwever, the number f ticks reaching the predilectin feeding site frm release pint did nt significantly differ frm that arriving frm release pint 2 (P>0.05). The site f release f ticks n the hst animal significantly affected the nn-respnding ticks [H (5) =.533, P = 0.042]. Fr example, the lcatin f release sites 3 and 4 may nt have allwed the ticks placed at these sites t receive sufficient amunts f hstderived attractants r repellents, r applied essential ils f the tw plants. The number f ticks nt respnding frm release site 4 was the highest and significantly different frm all ther release sites (PO.05) (Mann-Whitney U-test) (Table 6.). The site f release f the ticks, the essential il treatment and the interactin f these tw factrs had a significant effect n the behaviural respnses f tick vectrs n the hst (P<0.05) (Table 6.2). Respnse times f ticks released at frehead, escutchen, shulder and dewlap were shrt and nt significantly different (P<0.05) fr all the treatments except fr dewlap in the cntrl. Ticks tk significantly lnger t respnd when released at fre- and rear legs (P<0.05). Fr a specific release pint, the three treatments had the same significant effect n respnse time except fr the freleg and dewlap release pints (Table 6.2). At the freleg, ticks respnded in a significantly shrter time t the treatment with T. diversiflia than in the cntrls. At the dewlap, ticks tk a significantly shrter time t respnd t the treatment with T. minuta and T. diversiflia than in the cntrls; with the respnse time t the treatment with T. minuta being the shrtest. The initial walking speed f ticks at frehead, fre- and rear legs was nt significantly different fr all the treatments. At the dewlap, escutchen and shulder, the initial speed was highest in the treatment with the essential il f T. minuta and lwest in the cntrl except at the escutchen release pint. These differences are statistically significant except fr the treatment with T. diversiflia and T. minuta at the shulder release site (Table 6.2). The time between release f ticks at the varius lcatins and their arrival at the predilectin feeding site was nt significantly different fr all the treatments except fr rear leg and escutchen release pints. Fr bth the rear leg and escutchen release pints, ticks tk significantly much lnger time t reach the predilectin feeding site in the cntrl treatment than in the treatment with the tw essential ils (Table 6.2). Generally, it was bserved that the time it tk ticks t reach the ear pinna and base and becme attached was much lnger than the time t drp ff r attach in the vicinity f the release pints. The effect f this was bserved t be greater in the treatments f the tw essential ils than in the cntrl. Hwever, these differences were nt statistically significant (Table 6.2). 03

95 Chapter 6 the tw essential il treatments, ticks were bserved randmly navigating n the hst bdy and seeming t have accidentally arrived at the predilectin feeding site, as they behaved in a manner that suggested that they were nt affected by a feeding-site-specific cue. The critical key variables f this study, the reactin time, initial speed and time between release and arrival at the predilectin feeding site, were evaluated. Hwever, ther variables, such as the percentage f ticks at different sites that initiated mvement (respndents), the percentage f ticks reaching feeding sites and the percentage f ticks that attached in the neighburhd f release pints, the percentage f ticks that drpped ff after lsing the way, and thse that did nt respnd at all, were als evaluated. By cmparing the percentage f ticks respnding and thse reaching the predilectin feeding site fr all six release pints, the results shwed relatively lw rates f successful rientatin, lcatin and attachment f the ticks t their predilectin feeding site the ear pinna, with the highest numbers cming frm the cntrls fllwed by the animals treated with T. diversiflia essential il and then T. minuta essential il, in that rder. This suggests the masking effect f natural tick attractant stimuli that guide ticks t a predilectin feeding site by the tw essential ils, which again differ in the way they mask. This supprts ur hypthesis that intercepting the tick mvement tward a predilectin feeding site with repellent essential ils f T. minuta and T. diversiflia may prvide a prphylactic mechanism t prtect animals frm tick bites and must be cnsidered as a management strategy. This cntrl strategy wrks at individual level and reduces tick-hst cntact and, subsequently, reduces the chances fr the transmissin f Theileria parva parasite that causes the East Cast fever (ECF) in the hsts (Muthuswami and Nisha, 2006). It is pssible that in the presence f repellent essential ils, even attached ticks may nt sustain the attachment fr the perid f time (between 24 and 72 h) that is sufficient t allw an effective transmissin f the tickbrne pathgen (T. parva) t thehst (Ochanda et al., 988; Knnai et al., 2007b). That the essential il f T. minuta has a higher repellent effect than the essential il T. diversiflia, is manifested in the fact that treatment by the frmer causes the highest percentage f ticks t drp ff the hst frm varius bdy lcatins and that nne f the ticks gt attached t the ear pinna, the predilectin feeding site. A cnsiderable number f ticks in the cntrl became attached t the ear pinna and just a few drpped ff, whereas nly ne tick gt attached t the ear pinna f the animals treated with the essential il f T. diversiflia, where a cnsiderable percentage f ticks drpped ff the hst bdy. Althugh bth essential ils shwed significant effects n R. appendiculatus lcating their preferred feeding site, the essential il f T. diversiflia did nt prevent the ticks frm becming attached t the ear base, like its cunterpart. The data further supprt results btained previusly in Chapter 4 regarding the respnses f ticks depsited n bdy lcatins further away (escutchen, upper rear legs and frelegs) and clse t (frehead and shulder) predilectin feeding sites (Wanzala et al., 2004). Fr instance, althugh release site 5 (escutchen) was relatively furthest remved frm the predilectin feeding site, the ticks riented respnses and behaviural navigatins frm this pint were similar t thse f ticks navigating frm bdy lcatins clser t the ear pinna. Fr these tw regins n the hst animals, there was n significant difference in the mean reactin time, initial speed and time taken t reach the predilectin feeding site. These bservatins imply the previusly nted peratin f bth avidance (clser t nne predilectin feeding sites) and attractin (clser t the predilectin feeding sites) respnses f the ticks (Sika, 996; Wanzala et al., 2004). Hwever, the influence f the essential il can nt be ruled ut. Als the fact that mre ticks respnded in the treatments than in the cntrls, may have been due t the repellent effects f the essential ils.fr instance, frm release pints f the hst animals treated with the essential ils f T. minuta, the ticks' 06

96 Effect f essential ils n n-hst tick behaviur mean reactin time was lwer and initial speed higher than either thse frm the cntrl animals r the animals treated with the essential il f T. diversiflia. Therefre, it is pssible that these essential ils can be used t cnfuse the ticks while n the hst, and adversely affect their feeding habits thrugh which they transmit the etilgic agents f tickbrne diseases. Frm the results presented in the study, we shwed that bth essential ils had repellent effects n adult R. appendiculatus and that the essential il f T. minuta repels ticks mre than the essential il f T. diversiflia. Hwever, bth essential ils may ffer ptential fr incrpratin int integrated tick cntrl and management (ITCM), particularly fllwing the labratry and field studies f individual cnstituent cmpunds and selected blends. Tgether with the essential ils frm plants such as wild basil, Ocimum suave Willd, mlasses grass, Melinis minutiflra Beauv., neem, Azadirachta indica Adr. Juss. and African spiderflwer, Gynandrpsis gynandra (L.) Briq. (Mwangi et al., 995a, b; Ndung'u et al., 995; Malnza et al., 992; Waka et al., 2004; Garbui et al., 2006), they ffer pssibilities f explitatin f this ptential in effective and envirnmentally acceptable methds f tick cntrl mechanisms. Acknwledgements This research was jintly supprted by the Internatinal Fundatin fr Sciences, Stckhlm, Sweden and the Organizatin fr the Prhibitin f Chemical weapns, The Hague, The Netherlands thrugh a grant AB/ The first authr wishes t acknwledge the financial and material supprt received frm the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE) under the African Reginal fr Pstgraduate Prgrammes in Insect Science (ARPPIS) and the Wageningen University and Research Centre, the Labratry f Entmlgy under PhD Sandwich Fellwship. The authrs wish t thank Mr. Newtn Kmeri Mwanga fr illustratins and ICIPE staff fr their technical supprt. 07

97 Chapter 7 08

98 Essential ils affect tick rientatin and attachment preference Essential ils f Tagetes minuta and Tithnia diversifliaaffect rientatin t cattle and attachment site preference f the brwn ear tick, Rhipicephalus appendiculatus Intrductin Abstract: The effects f essential ils f Tagetes minuta and Tithnia diversiflia n rientatin behaviur and attachment site preference f newly hatched adult Rhipicephalus appendiculatus n cattle were evaluated. Hst animals were treated at the ear pinna (by smearing the il directly n the ear r suspending a tube cntaining the il n the ear pinna) and legs + tail in semi-field plts. The legs + tail sites f the essential il applicatin shwed the lwest mean percentage f ticks bserved n the bdy f the hst (6.5 ±.9% and 26.0 ± 2.8%) and the highest mean percentage reductin f attaching ticks (76.5 ± 3.9% and 67.0 ± 0.8%) fr the essential ils f T. minuta and T. diversiflia, respectively. The cntrl animals had the highest mean percentage f ticks bserved (93.0 ± 2.%). The ear tube resulted in the highest mean percentage f ticks n the hst (47.5 ± 5.% and 55.8 ± 5.%) and a lwest mean percentage reductin f attaching ticks (44.8 ± 5.%) and 36.5 ± 7.4%) fr the essential ils f T. minuta and T. diversiflia, respectively. Fr bth the essential ils, legs + tail sites f essential il applicatin, fllwed by ear smear and then ear tube, had significant effects n rientatin t the hst and attachment site preference f adult R. appendiculatus n the hst, in that rder. As treatment f legs + tail is tedius, time cnsuming, and requires mre essential il than the ther tw sites, we recmmend the ear smear site fr treating hsts with essential il. Better perfrmance f the tw essential ils might be btained by increasing their cncentratin and by frmulating them in a carrier material that stabilizes the active ingredients. Key wrds: hst-seeking Rhipicephalus appendiculatus, behaviur, semi-field, essential ils, Tagetes minuta, Tithnia diversiflia, repellents, cattle Ticks and tick-brne diseases cntinue t be a majr cnstraint t livestck prductin and develpment, particularly in the trpics (McCsker, 993; Slingenbergh et al., 2002; My and Masika, 2008). The infestatin f ticks n livestck and the resulting tick-brne diseases pse serius sci-ecnmic prblems (Jnssn et al., 998; D'Haese et al., 999; Makala et al., 2003; Peter et al., 2005;Muchenje et al., 2008). The current cnventinal methd f tick cntrl relies mainly n the applicatin f chemical acaricides. Hwever, this methd is assciated with a number f prblems including envirnmental pllutin, chemical residues in fd prducts as well as in wl, 'This chapter will be published as: Wanzala, W., Takken, W., Mukabana, R.W., and Hassanali,A. Essential ilsf Tagetes minuta and Tithnia diversiflia affect therientatin behaviurfthetick, Rhipicephalus appendiculatus, ncattle,whensubmitted 09

99 Chapter 7 develpment f tick resistance and high csts f acaricides. The use f acaricides des nt prevent the ticks already attached frm transmitting the disease-causing pathgens. In additin, acaricide applicatin nly targets 5% f the ttal tick ppulatin in any given envirnment, while 95% f ticks, which are present n the vegetatin, are left unaffected. Adult ticks can survive fr mre than a year withut a bld meal, particularly in dense bush and rainfrest (Lans, 200). These ticks becme a surce fr re-infestatin f hst animals during grazing. A methd and prduct that cntinuusly keep ticks away frm the hsts are therefre desirable. Amng tick cntrl strategies under cnsideratin t that effect are prphylactic measures that prevent cntact between ticks and hst animals, i.e., repellents. Repellents prvide a practical means f prtectin against tick bites and alng with vaccines, they may be a fundamental resurce fr minimizing the transmissin f tick -brne diseases at an individual level (Staub et al., 2002). Repellent cmpunds in varius cmmercial frmulatins are available fr tick bite preventin. There is cncern hwever, abut pssible adverse effects f sme f these cmpunds n human health and nn-target species in the envirnment (Abdel-Rahman et al., 200). Fr these reasns, the develpment fnvel repellents can be f great value. Repellent effects f sme plants and plant prducts n the brwn ear tick Rhipicephalus appendiculatus have been demnstrated (Dipelu and Ndungu, 99; Malnza et al., 992; Mwangi et al., 995a, b; Lwande et al., 999; Ndung'u et al., 999). A 0% cncentratin f the essential il f Ocimum suave in liquid paraffin was fund t prtect rabbits against the attachment larvae f/?, appendiculatus and repelled mre than 70% f adult R. appendiculatus fr 5 days (Mwangi et al., 995a; ICIPE, 998/99). A neem il cncentratin f 25% applied directly t the skin f hsts repelled all stages f R. appendiculatus and shwed sme antifeedant activities (ICIPE, 997). Tagetes minuta and Tithnia diversiflia have been used traditinally t cntrl livestck ticks in Central Kenya (Njrge and Bussmann, 2006). In the present study, an apprach prviding prtectin against R. appendiculatus bites n hsts was explred. The effects f essential ils f T. minuta and T. diversiflia n hst lcatin and attachment site preference f newly hatched adult R. appendiculatus n hsts treated n the ear (smearing il n the ear r suspending a tube cntaining il n the ear) and legs + tail are evaluated. Materials and methds Experimental hst animals A grup fnine indigenus zebu cattle (Bs indicus)was purchased frm livestck farmers in Bungma District, western Kenya. Details f the area, such as the tick ppulatin, cattle diseases, climate and vegetatin are described in Chapter 3 f this thesis. Theilerisis is endemic in the study area and there are large ppulatins f alternative wild animal hsts fr cattle ticks and diseases. The experimental cattle used had been reared under traditinal management in Bungma District. Sme f the animals were purchased during a market day frm the Bukusu livestck farmers as steers aged between I6V2 t 8 mnths, while thers were xen used in the farms fr plughing. Accrding t farmers' recrds, these animals had been immersed weekly in varius types f acaricides used indiscriminately in the area fr the cntrl f ticks. Recrds at Bungma District Veterinary Office (DVO) indicated that acaricides used included rganchlrine cmpunds [lindane (0.5%t 25% v/v), dieldrin (0.55% w/v) and txaphene ( % w/v)], rganphsphate cmpunds [chlrpyrifs (0.02% v/v), chlrfenvinphs (0.05% v/v), cumaphs (0.% v/v), dixathin (0 075% w/v) etc.], carbamate acaricides [e.g., carbaryl (0.2% w/v), prmacyl 0

100 Essential ils affect tick rientatin and attachment preference (0.5% w/v)], pyrethrins (0.02% w/v), pyrethrids (permethrin, decamethrin, deltamethrin, cyhalthrin, cyfluthrin, flumethrin etc.) (0.02% % w/v), and the cmmnly used frma-midine (amitraz (0.025% w/v)]. Experimental ticks The tick species used (Rhipicephalus appendiculatus) was btained frm a clny reared in the insectary at the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE), Nairbi, Kenya. This clny was initially btained frm the rearing unit at the Internatinal Livestck Research Institute (ILRI), Nairbi, Kenya. The clny at ILRI had been established frm cllectins at Muguga in Kiambu District, Kenya and reared under labratry cnditins since 952 using the methds described by Bailey (960). At ICIPE, R. appendiculatus were bred n New Zealand white rabbits as described by Bailey (960). Rearing cnditins and management were as described previusly (Bailey, 960; Irvin and Brcklesby, 970). The newly hatched adult R. appendiculatus were transprted in a clbx n mist sand frm ICIPE t Bungma (a distance f abut 500 km), and used within a 48-h perid after hatching. Essential ils Essential ils f T. minuta and T. diversiflia were btained by the hydrdistillatin methd using Clevenger type distillatin apparatus (Sereshti and Samadi, 2007). Essential ils were diluted t 0% in durless vaseline petrleum jelly (BP-USP 00% Grade) (Unilever Kenya Limited). This carrier material cntained n clur, fragrance r irritants and was used fr cntrl experiments. The essential ils were transprted frm ICIPE t Bungma in a cl bx n ice and stred in a freezer at -4 C until required fr use n hsts. After being taken ut f the freezer, the ils were used within 2 hurs. The frmulatin used in this study was adpted frm the previus assays in the labratry and field at ICIPE while evaluating the essential il f Ocimum suave against R. appendiculatus (Mwangi et al., 995a; ICIPE, 998/99). Experimental pasture plts Adult R. appendiculatus that had been starved fr 24 h were released in a demarcated pasture plt measuring 4 m x 2 m w, between 09:00 and :00 hurs (Fig. 7.). The release time cincided with the time when mst livestck tick species leave their hideut t start hunting fr suitable hsts fr a bld meal (Nrval et al., 992). After this time, when it gets htter, ticks hide under existing vegetatin and/r in burrws t avid desiccatin and subsequent death. Within the demarcated experimental plts, any plants knwn t be used fr prtectin against biting insects (Kkwar, 993) and livestck ticks (Malnza et al., 992; Lwande et al., 999; Ndung'u et al., 995; 999; Mwangi et al., 995a; b) were remved t avid interference with the results. A ditch measuring 30 cm wide x 5 cm deep was dug rund the demarcated plt (Fig. 7.). The ditch was lined with aluminium fil and filled with water t prevent the ticks frm leaving the plt during and after the experiment. The plt was then infested with 200 adult R. appendiculatus. Freshly cllected, tick-free Napier grass was evenly spread ver the entire plt as fdder, t make the experimental animal walk arund the demarcated plt cmfrtably while feeding n this and sme ther grasses fund within the plt. The treated hst, having either knwn levels f tick infestatin r n prir expsure t ticks, was allwed t stay in the plt fr 3 h in rder fr ticks t attach t it and mve t their preferred feeding sites. Three hurs was, in preliminary bservatins, fund t be enugh time fr the ticks t infest the treated animals and reach the preferred feeding site. Ill

101 Chapter 7 Napier grass. Grass Ditch (30cmwideby 5cmdeep) cvered withaluminium filand filled withwater N H 2m Fig. 7..Experimentalpltinwhich200adult Rhipicephalus appendiculatuswerereleased, indicating variusexperimentaltreatments: (a)tubeswithpetrleum jelly (cntrl)ressentialilsf T. minutar T. diversiflia; (b)earstreatedbysmearingpetrleum jelly (cntrl)ressentialilsf T. minutar T. diversiflia; and (c)legsandtailtreatedbysmearingwithpetrleum jelly (cntrl)ressentialilsf T. minutar T. diversiflia. 4m After 3 h, ticks were recvered frm the expsed animal and cunted. The plt was then burnt dwn using paraffin in rder t kill any ticks that had nt climbed n the hst, and thus avid tick infestatin in the study area. A new plt was used fr each f the 27 replicates. Treatments The nine hst animals used were randmly divided int three grups f three, ne fr each f the treatment sites n the hst: (a) Eppendrf tube with il, suspended n the inner side f bth ear pinnae f each animal (ear tube), (b) il smeared n the inner side f bth ear pinnae (ear smear), and (c) il smeared n legs and tail (leg + tail smear) (Fig. 7.). In each grup, ne animal was randmly selected t be treated with petrleum jelly (cntrl), ne was treated with the essential il f T. minuta and the third hst was treated with the essential il f T. diversiflia, bth essential ils diluted t 0% in petrleum jelly. Once used in this rder, the hsts were nt inter-changed until the end f the experimental perid in rder t avid crss cntaminatin. There were three independent replicates fr each treatment. Each replicate was cnducted in its wn plt. Fr each treatment site and within each treatment applicatin, ne animal was used t make bservatins three times successively. Between the replicates, the entire bdy f the animal was thrughly cleaned using 99% alchl. Thrugh cleaning was meant t remve any residues f previus applicatins f petrleum jelly r essential ils and any durs that may have been prduced by previusly attached ticks, which culd have affected the results f successive trials. This precautin was taken because it is 2

102 Essential ils affect tick rientatin and attachment preference knwn that ticks themselves may play a rle in guiding ther ticks f the same species t preferred feeding sites, as has been bserved in Amblymma ticks (Schni et al., 984; Diehl et al., 99; Nrval et al., 99) and ^?. appendicular (Sika, 996). After cleaning, the hst was allwed t stay in the pen fr abut min befre being used again, allwing its fur t dry by evapratin. Recvery f ticks and determinatin f the impact f treatment n tick attachment Ticks were bserved and recvered n the bdy f the hst at five specific sites. The five sites were: ) Head-eyes/nse/ears/hrn-base (Hd), 2) Dewlap (Dp), 3) Frelegs (Fl), 4) Hind legs (HI), and 5) Tail (T). The five sites were chsen as recvery sites fllwing preliminary field bservatins f grazing animals that shwed them t be the mst preferred areas f nt nly alightment by the ticks frm their questing psitins n the vegetatin (Brwning, 976) but als f temprary attachment (Wanzala et al., 2004). The fllwing frmula was used t estimate the percentage f reductin f adult R. appendiculatus fund attached n the hst, caused by each essential il type: (N.ticksreleased-N.attachedin treatment)- %reductinf _ ticksnhst ~ ( N ticks released-n.attachedincntrl), nfl N.ticksreleased x luu Statistical analyses Data f the number f ticks bserved n the bdy f hsts were analysed by ne-way analysis f variance (ANOVA) using the general linear mdel (GLM) prcedure fr SAS system fr PC (SAS Institute, ). The effect f essential ils delivered n hsts at the ear (smear r tube) and legs + tail and the mean percentage f reductin f attaching ticks were cmpared by Students-Newman-Keuls test (Skal and Rhlf, 995) at P = The effects f the essential ils f T. minuta and T. diversiflia n the number f ticks that lcated the hst and attached t it was examined by paired samples t-test statistics using the t-test prcedure f the Statistical Prducts and Service Slutins (SPSS) versin 5 fr windws. A cmparisn f the three essential il applicatin sites was dne using a neway ANOVA. Legal use f field experimental animals All prcedures requiring the use f experimental animals in the field were apprved by District Veterinary Office f Bungma District, western Kenya. The imprtance and riskfree nature f the prject was further explained t the Bukusu cmmunity by the DVO and agricultural extensin fficers wrking within the study area. The field experiments were perfrmed in cmpliance with guidelines published by the Kenya Veterinary Assciatin and the Kenya Labratry Animal Technician Assciatin, regarding the ethical use and handling f labratry and farm animals in the field (KVA and KLATA, 989). Infrmed cnsent was btained frm the livestck farmer frm whm we rented the experimental field plts. 3

103 Chapter 7 Table 7.. The mean (± SE) number and percentage f adult Rhipicephalus appendiculatus ticks bserved n the hst treated n the ear (smear and tube) and legs + tail with petrleum jelly (cntrl) and the essential ils f either T. minuta (Tm) r T. diversiflia (Td) and % reductin in numbers n hsts f the 200 ticks released. Animal cde Essential il Treatment site f the essential il n the hst Mean tick cunts n the hst bdy after 3 h f hst expsure (± SE) Mean % freleased ticks cunted n the hst after 3 h fhst expsure(± SE) % reductin in ticks n hst after 3 h f hst expsure Cet Ces Ctl Tmet Tmes Tmtl Tdet Tdes Tdtl Cntrl Cntrl Cntrl Tm Tm Tm Td Td Td Eartube Ear smear Legs + tail smear Ear tube Ear smear Legs + tail smear Ear tube Ear smear Legs + tail smear 84.7 ±4.67d 72.3 ± 3.8d 86.0 ± 4.6d 95.0±0.2bc 50.3 ± 2.85a 33.0 ± 3.79a.7 ± 0.4c 83.3 ± 9.39b 52.0 ± 5.69a 92.3 ± 2.33d 86.2±.59d 93.0 ± 2.08d 47.5±5.bc 25.2 ±.42a 6.5 ±.89a 55.8 ± 5.07c 4.7 ± 4.69b 26.0 ± 2.84a 44.8±5.0bc 6.0±0.87ab 76.5 ± 3.9a 36.5 ± 7.40c 44.5 ± 5.80bc 67.0 ± 0.76a - nt applicable, as the cntrl values were used t calculate the mean % reductin in ticks n hst in the equatin described in the materials and methds. Fr a given clumn, means fllwed by the same letter(s) are nt significantly different frm ne anther at P =0.05 (t-test, SAS). Results Treatment f hsts had a significant effect n the percentage f the released ticks cunted n their bdies (P<0.05) (Table 7.). Mre ticks were fund n the cntrl hsts than n the animals treated with the essential ils f either T. diversiflia r T. minuta (P<0.05) (Table 7.). The mean percentage f released ticks cunted n the hsts treated with the essential il f T. minuta was significantly lwer than the mean percentage f released ticks cunted n the hsts treated with the essential il f T. diversiflia fr crrespnding treatment sites [t 00 5 (8) = 3.438, P = 0.009]. The site f delivery f the essential il n the hsts had a significant effect n the mean percentage f released ticks cunted n their bdies (P<0.05) (Table 7.). In a grup f the hsts treated with the essential il f T. minuta, the methd f essential il delivery using the ear tube had a significantly higher mean percentage f released ticks cunted n the hst than the ear and legs + tail smears (P<0.05). The legs + tail smear methd had the lwest mean percentage f released ticks cunted n the hst. In the grup f the hsts treated with the essential il f T. diversiflia, the mean percentage f released ticks cunted n the hst were significantly different fr the three methds f delivery f the essential il n the hsts with the ear tube methd having the highest percentage f ticks cunted n the hst while the legs + tail smear methd having the lwest percentage f ticks cunted n the hst (PO.05). Treatment f hsts had a significant effect n the percentage f reductin f ticks n the hst after 3 h f hst expsure (P<0.05) (Table 7.). There was a significant difference in percentage reductin f ticks n the hsts after 3 h expsure between the hsts treated with the essential il f T. minuta and T. diversiflia fr crrespnding treatment sites [t.05 (8) = 3.438, P = 0.009]. The percentage reductin f ticks n the hsts was significantly higher fr animals treated with the essential il f T. minuta than the essential il f T. diversiflia (P<0.05). The cntrl animals had the highest mean percentage f ticks (percentage f ticks released experimentally) (93.0 ± 2.%). The site f treatment n the hsts had a significant effect n the percentage f reductin f ticks n the hst (PO.05). In a grup f the hsts treated with the essential il 4

104 Essential ils affect tick rientatin and attachment preference f T. minuta, the ear tube treatment had a significantly lwer mean percentage f reductin f ticks n the hsts than the legs + tail smear methd but the ear smear treatment was nt significantly different frm the ther tw (P<0.05). In the grup f the hsts treated with the essential il f T. diversiflia, the ear tube and smear treatment sites had a significantly lwer mean percentage f reductin f ticks n the hst than the legs + tail smear treatment site (PO.05). Crrespnding values fr mean percentage reductin f attaching ticks were significantly higher fr the essential il f T. minuta, hwever, than thse fr the essential il ff. diversiflia (P<0.05). Of the three applicatin sites f essential ils, smearing f essential ils f either T. minuta r T. diversiflia n the legs and tail f the hsts caused a significantly greater repellent effects n rientatin t the hst and attachment site preference f adult R. appendiculatus n the hst than treatment f the ear with the tube r smearing the essential ils n the ear (Tables 7.). In bth the grups f hst animals treated with either essential il f T. minuta r T. diversiflia, the ear smear site was secnd t that f smearing essential ils n the legs + tail, and the ear tube third, in terms f preventing ticks frm attaching t the hst at their preferred feeding site (Table 7.). The three sites f applicatin f essential ilshad a significant effect n the mean number and percentage f adult R. appendiculatus bserved n the hsts (P<0.05) (Table 7.). The legs + tail and ear smear sites (with the exceptin f the sites treated with the essential il f T. diversiflia) were nt significantly different frm ne anther but the frmer was significantly different frm the ear tube methd (P<0.05). Site f tick attachment The cntrl hsts, treated with petrleum jelly nly, were used as indicatrs fr identifying preferential sites fr attachment during the bservatin perid and als as areference pint fr cmparing the behaviural effect f the tw essential ils (Table 7.). The cnsideratin was based n the mean number f ticks bserved at different sites n the bdy f the hst mean (Table 7.2). The essential il f T. minuta delivered by the tube n the ear as well as all methds used t deliver the essential il f T. diversiflia, did nt affect the chice f ticks fr any f the five bdy sites (Dp, Fl, HI, T and Hd) (P>0.05) (Table 7.2). This implies that using the three delivery methds, the essential il f T. diversiflia affected the attachment f/?, appendiculatus t the five bdy sites equally, whereas with the essential il f T. minuta, there was a significant difference between the chice f ticks fr bdy sites when using ear smear and legs + tail methds f delivering the essential il (P<0.05) (Table 7.2). During the delivery f the essential il f T. minuta using the ear smear methd, R. appendiculatus preferred attaching at the hind legs and then the fre legs, fllwed by the remaining three sites which were equally selected. When the essential il f T. minuta was delivered by the legs + tail methd, the tail site was the least preferred fr attachment by R. appendiculatus while the remaining fur bdy sites were equally selected. Cmparisn f the main repellent effects f the essential ils f Tagetes minuta and Tithnia diversiflia The mean percentage f tick reductin n the hst by the essential il f T. minuta (60.8 ± 4.9%) was higher than that by the essential il f T. diversiflia (49.3 ± 5.3%). The mean percentage f ticks bserved (calculated as a percentage f the number f ticks released experimentally) n the hst treated with the essential il f T. diversiflia (4.2 ± 4.8%) was higher than that f the essential il f T. minuta (29.7 ± 4.9%). This implies therefre that the essential il f T. minuta had a greater repellent effect n adult R. appendiculatus 5

105 Chapter 7 S w & 5= z. rr >< er R c a. 3 Pf a* S 3> 3 < p. fd 3j j 3 ^ B fci Wl 0. n g-z H. r w w r f i w r w r n u> P H- H- I H- M P ^ ^ H- 5? g * Si H- ON $ ~ P M- la ps *-* ^ U> H- -j H- M- a\ cr" H- ON ON <-/l CT\ I + 3 H- Isj t/i 4^ O. _p_. t * * W t U> H- Wi t IO < t t. i M OO M H- H- H- \ ON i^i t -- J_L f» J_L ^ JL SI ^: a* b 4-5. en P a* p re a a. 3 II ^-* s 4 Si 3" -t» u. U > S-T3 "S - rj '* * C a* fr ss H a- ~4 K> H 3" 3 s "5 n re 3 O ts* re 3" U2 -t K-' CD 0- O ^3 cr a CD 3 re 2 s% ^. 3"?- s» 3- O H 3- O re *_? c s? «O re a a itl _L S re sn re <" OQ O FJ- ffi - Cr. s 3 s 2:& re -~5 y B rr g H" H- H- H- y \ ~ P S* ST< H- B. H ^ e 3 3 rf PH!> 2»' re 2 7 P IS % rere re~ JS. 6

106 Essential ils affect tick rientatin and attachment preference than the essential il f T. diversiflia. The means were cmpared by paired samples t-test and the tw differences were fund significant, t 0.05 (2) = 4.486; P = Discussin and cnclusin In this study, all three treatment sites n the hst gave sme degree f prtectin against hst-seeking R. appendiculatus, but the essential ils f T. minuta and T. diversiflia varied cnsiderably in their effects. Rhipicephalus appendiculatus were mre affected by the essential ils f T. minuta and T. diversiflia when smeared rund the legs + tail f the hst than when applied n the ear by either smearing r suspending a tube cntaining the il n the inner side f the ear. Hwever, treatment f the legs + tail is tedius, time cnsuming, and requires mre essential il, and hence may be unecnmical fr use by the target grup, the rural livestck farmers. Suspending the tube n the ear had the least effect n hst-seeking R. appendiculatus, and its difference with the legs + tail treatment was significant fr bth essential ils (P<0.05). The ear tube culd pssibly ffer slw release f the dur, which wuld make it likely t affect ticks ver a lnger perid f time than the ther tw sites. Hwever, this has yet t be evaluated. Smearing the inner side f the ear f the hst with the essential il seems t ffer the best cmprmise f ease f applicatin and gd repellency, and istherefre the technique that wasused in Chapter 8. In the cntrl hsts (with the exceptin f the ear smear methd), the dewlap was the mst preferred site fr R. appendiculatus attachment fllwed by head, frelegs, hind legs, and tail in that rder. These bservatins, hwever, appear t cntradict the knwn fact that R. appendiculatus ticks have a marked preference fr attachment t the ears and head regin f cattle (Yeman and Walker, 967). This cntradictin may be due t the specific directin t which the dur cming frm the ear initially flws in the highest cncentratin, as this wuld be mre imprtant t the 24 h-starved hst-seeking ticks than tracing the actual surce f the dur. This f curse will depend n the psitining f the hst ear and the directin f the breeze blwing arund the entire ear f the hst. Nevertheless, the dur intensity fluctuates cnsiderably and gradually decreases after several hurs cmpared t the surce and this eventually results in the directinal mvement f ticks t the surce (ear) (Sika, 996; Wanzala et al., 2004). A number f ticks that were bserved n the legs f the hst were fund between the hves. This part f the leg prvides the first sft part f the hst that the newlyhatched, hst-seeking ticks cme int cntact with upn arrival n the hst bdy. These sft parts act as stimulants fr the ticks t start preparing t feed (Sika, 996). This als accunts fr the attachment f mre ticks n the nse, eyelids and base f the hrns, upn alighting n the hst bdy frm their questing psitins n the vegetatin. These parts are als sft and/r cntain the sft waste residues f the hst. By the time ticks were being lcated n the hsts, mst f them were still scanning and therefre had nt mved away frm the entry site. Hwever, the ticks that were remved frm the hst bdy while scanning, in the prcess f attaching, r attached, did nt shw any evidence f feeding; when crushed between white paper twels there was n bld apparent, nr had the females increased in size, a physical manifestatin f feeding. Due t the itching and irritatin caused by tick mvement and attachment n the hst, a number f ticks are remved frm the bdy f the hst using tail, tngue and scratching with the muth, mre particularly n the frelegs and tail (Hadani et al., 977). The hind legs are als used t scratch and remve ticks attached n the head. This may have reduced the number f ticks that were bserved n the bdy f the hst. Frm the previus studies (Sika, 996; Wanzala et al., 2004), there is n dubt that the efficacy f 7

107 Chapter 7 the essential ils was als affected by hst-derived repellents (frm the anal regin) and attractants (frm the ear) and ther bdy emanatins (Akinyi, 99; Sika, 996). Althugh it was hypthesized that the effects f hst-derived repellents and attractants were masked by the essential ils, it is nt yet knwn t what extent this happens. It istherefre pssible that these hst-derived repellents and attractants maybe cntributry factrs t sme f the bserved discrepancies in the results, especially in the cntrls. The efficacy f essential ils was als influenced by several ther factrs, such as their chemical cnstituents, envirnmental cnditins at the time f applicatin, and the persistence and the spread f the active ingredients n the hst bdy (Mwangi et al., 995a). This study has shwn that the essential ils f T. minuta and T. diversiflia may prvide a useful prphylactic means f reducing vectr (tick)-hst (livestck) cntact, thereby reducing the number f tick bites and subsequent tick-brne infectins, physical damage, and resulting secndary infectins. Different frmulatins, particularly thse that stabilize the active ingredients and perhaps higher cncentratins than the ne used (0%) shuld be tested in multi-lcatinal field trials in rder t determine whether these essential ils are apprpriate fr use n a large scale. The use f plant extracts with knwn effects n livestck ticks in this way may be a useful cmplementary r alternative tick cntrl methd t the heavy use f classical acaricides. This may decrease the quantity f txic acaricide residues peridically released in the envirnment. In additin, the essential ils have the added advantage that they may prvide a certain degree f cntrl f flies and ther ectparasites that disturb and bite the target hsts (Tled et al., 2003; Adedire and Akinneye, 2004;Taiw and Makinde, 2005). Acknwledgements This research was jintly supprted by the Internatinal Fundatin fr Science, Stckhlm, Sweden and the Organizatin fr the Prhibitin f Chemical weapns, The Hague, The Netherlands thrugh a grant AB/ The first authr wishes t acknwledge the financial and material supprt received frm the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE) under the African Reginal fr Pstgraduate Prgrammes in Insect Science (ARPPIS) and the Wageningen University and Research Centre, the Labratry f Entmlgy under PhD Sandwich Fellwship. The authrs wish t thank Mr. Newtn Kmeri Mwanga fr making illustratins and ICIPE staff fr their technical supprt. 8

108 Essential ils affect tick rientatin and attachment preference 9

109 Chapter 8 20

110 Essential ils prtect livestck frm infestatin with ticks Essential ils f indigenus plants prtect livestck frm infestatins with Rhipicephalus appendiculatus and ther tick speciesin herds grazing in natural pastures in Kenya 8 Abstract: The effects f essential ils f Tagetes minuta and Tithnia diversiflia n Rhipicephalus appendiculatus infesting livestck in Bungma District, western Kenya, were studied. Frty-five zebu cattle naturally infested with ticks were randmly selected frm 5 herds, three animals frm each. Of the three animals within each herd, ne was treated with g f petrleum jelly (cntrl), ne with g f essential il f T. minuta and ne with gf essential il f T. diversiflia n the inner side f the ear pinna, the preferred feeding site f R. appendiculatus. The tick infestatin n each treated hst animal was mnitred daily fr 8 days by cunting the number f ticks attached t the animals. Within ^4 days psttreatment, the number f ticks n hsts treated with the essential ils was reduced by mre than half that f the riginal ppulatin. By the 5th day pst-treatment, mre than 75 and 60% f adult R. appendiculatus and ther tick species, respectively, were affected by the essential ils s that they became disldged and drpped ff. A strnger repellent effect was shwn by the essential il f T. minuta than the essential il f T. diversiflia. The mean residual prtectin affrded by T. minuta was 2.5 days and fr T. diversiflia 7.9 days. There was n significant difference in the effectiveness f the essential ils between male and female R. appendiculatus. Bth T. minuta and T. diversiflia essential ils affected several ther less dminant but ecnmically imprtant tick species such as Amblymma variegatum, R. evertsi and Bphilus spp., althugh these tick species have different feeding sites. The results suggest the ptential fr essential il frmulatins in reducing tick infestatin and assciated tick-brne diseases amng the resurce-limited livestck farming cmmunity in trpical Africa. Intrductin Key wrds: Rhipicephalus appendiculatus, ticks, repellents, essential ils, Tagetes minuta, Tithnia diversiflia, n-hst field trials, Kenya, cattle The sci-ecnmic imprtance f livestck ticks has lng been recgnized wrldwide and therefre their cntrl has been a pririty fr many cuntries in trpical and subtrpical regins (D'Haese et al., 999; Lds et al., 2000; Rajput et al., 2006). The cntrl f livestck ticks is mainly fcused n the fllwing appraches: chemical (My and Masika, 2008), genetical (Aiell and Mays, 2003; Silva et al., 2007), bilgical (Samishet al., 2004), immunlgical (Willadsen and Kemp, 988; Willadsen et al., 2006) and cultural (Miller, 2004). The limitatins f these appraches in the prgrammes and strategies fr cntrl and management f livestck ticks are well knwn (Am, 992; Slmn and 'ThisChapter will be submitted fr publicatin as:wanzala, W., Takken, W., Mukabana,R.W.and Hassanali,A.Essential ilsfindigenusplantsprtect livestck frminfestatins with Rhipicephalus appendiculatusandthertickspeciesinherdsgrazinginnaturalpasturesinkenya. 2

111 Chapter 8 Kaaya, 996) The applicatin f synthetic acaricides is still the main methd fr cntrl and management f livestck ticks and tick-brne disease wrldwide, albeit affrding nly tick eliminatin frm the hst animal but n preventin f re-infestatin (Chapter ). Furthermre, the acaricides d nt affect the larger ppulatin f free-living livestck ticks existing in the hsts' envirnment (vegetatin), which prvides a resurce fr tick reinfestatin. The disadvantages f using these synthetic acaricides relate t acaricide resistance and envirnmental pllutin (Nrval et al., 992; Aiell and Mays, 2003). Therefre, there has been a wrldwide search fr alternative tick cntrl methds that can be applied either alne r integrated with acaricides and r ther tick cntrl methds (Yung et al., 988; Kaaya, 992). Sme f the alternative methds include the use f tick-resistant animals, behaviral manipulatins f ticks using phermnes, quarantine techniques, habitat mdificatin, anti-tick vaccines and bilgical cntrl (Ghsh et al., 2007). Little data is available in the literature n the use f plants and plant prducts that cntain txic r repellent cmpunds. Hwever, the ptential f sme anti-tick plants in pastures and plant prducts as tick repellents r acaricides n hsts has been repeatedly demnstrated (Sutherst et al., 982; Nrval et al., 983; Carrll et al., 989; Miller et al. 995). At the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE), Nairbi, Kenya, a participatry actin research study t develp anti-tick pastures using mlasses grass was investigated at its Nairbi lcatin and in Kuja area, western Kenya, in an effrt t cntrl Amblymma variegatum Fabricius and Rhipicephalus appendiculatus Neumann, the vectrs f cwdrisis and East Cast fever, respectively (Mwangi, et al., 995b). Neem il frm Azadirachta indica A. Juss. was reprted t inhibit larval and nymphal attachment and feeding, as well as t reduce fecundity, egg hatch, and multing in larvae and nymphs f R. appendiculatus, amng thers (ICIPE, 997). A 25% frmulatin f neem il sprayed n de-ticked zebu cattle grazing n heavily infested pasture reduced infestatins f tick larvae by 37-6%, nymphs by 24 65% and adults by 44-62% fr 5 days. In in viv assays, a 0%) slutin f the essential il f Ocimum suave Willd. inparaffin il was fund t be effective against all immature R. appendiculatus and mre than 70% f adults feeding n rabbits (Mwangi et al., 995a). Three applicatins f the 20% cncentratin f the essential il f O. suave prevented R. appendiculatus attachment by 68.8%. Cleme hirta (Kltzsch) Oliv. and Gynandrpsis gynandra (L.) Briq. have been demnstrated t be pssible tick repellent plants in pastures (Dipelu et al., 992;Malnza et al., 992; Ndung'u et al., 999). In anther study n Rusinga Island, western Kenya, leaves f a lcal shrub, Acalypha fruticsa Frssk., were fund t be attractive t R. appendiculatus under field and labratry cnditins, thus shwing the ptential fr use as a trap plant in tick cntrl strategies (Hassan et al., 994). Althugh achievement f the full ptential f plants in livestck tick cntrl pse challenging research and develpment prblems, the plants' anti-tick prperties s far manifested culd make them suitable cmpnents f an integrated tick cntrl strategy. Cnsiderable research is needed, hwever, t select apprpriate plants and plant prducts, establish their efficacy in the labratry and field under different cnditins and devise efficient prductin strategies t allw fr their practical use at lcal level. In this thesis, the repellent actin f the essential ils f T. minuta and T. diversiflia against the brwn ear tick R. appendiculatus has been demnstrated in labratry and semi-field studies. Cattle, treated with either il, exhibited a significantly high drp-ff rate f n-hst ticks and a significantly reduced number f ticks climbing n the hst frm the grund and vegetatin. In the study described in this chapter, effects f the essential ils f T. minuta and T. diversiflia n the infestatin rate f adult R. appendiculatus ticks are evaluated n zebu cattle within herds fnaturally grazing livestck in western Kenya. 22

112 Essential ils prtect livestck frm infestatin with ticks 34 20'E / 0 \\\ 355'E Fig. 8..ThelcatinfBungmaDistrictinKenya.The field experimentswerelcatedn 5different livestckfarms (indicatedwithstars). Methds and materials Gegraphy f experimental field site The study was carried ut in Bungma District, western Kenya, situated between latitude 00 28' and 0 30' N and lngitude 34 20' and 35 5' E (Fig. 8.). The District ccupies an area f square km. n the suthern slpes f Mt. Elgn, with an altitude ranging frm 200 t m a.s.l. (Backes, 998).The predminant ff-farm vegetatin patterns are riverine frests, rcky frested hillsides, hedgerws, wded grassland relicts, wdlands r clline frest relicts and tree grves, whereas the nticeably tree-rich n-farm management units are hme gardens, hmesteads, live fences, cffee- and banana-grves and annual crpping fields (Backes, 200). Average annual rainfall ranges frm 600 t mm, and mean annual temperatures in the suthern areas are abut 2 t 22 C, whereas in the nrthern areas clser t the muntain, they are in the lwer range f 5 t 0 C because f the altitude. Identificatin f the site fr field experiments within Bungma District The study site cmprising 5 livestck herds was identified based n data frm the Bun- 23

113 Chapter 8 gma District Veterinary Office (DVO). The DVO receives reprts frm regulated and mnitred lcal public markets within Bungma District, where animals are traded fllwing registratin and examinatin f their health status (including livestck tick infestatin). Additinal limited reprts regarding the livestck tick infestatin situatin in the District were received frm individual livestck farmers (DVO, pers. cmm.). A preliminary survey within this area cnfirmed the DVO's reprt that the site was mre heavily infested with livestck ticks than any ther area within the District. Essential ils used in the study The essential ils used were extracted frm T. minuta and T. diversiflia as described in Chapter 4 f this thesis. Petrleum jelly (BP-USP 00% Grade; Unilever, Kenya) was used in the cntrl experiment and in the frmulatin f the essential ils. The essential ils were mixed with petrleum jelly as a 0% frmulatin, adpted frm previus biassay results in the labratry and wrk at ICIPE (ICIPE, 998/99; Mwangi et al., 995a). The essential ils were transprted frm Nairbi t Bungma (a distance f abut 500 km) in a clbx and stred at -4 Cuntil applied n the selected hsts within a 2 h perid. Treatment f experimental animals and fllw-up bservatins In each selected herd, three animals were chsen fr the experiment and marked with cde numbers using a plastic tag and marker pen. One gram f petrleum jelly, cntaining the essential il f either T, minuta r T. diversiflia, was applied t the inner side f the ear pinna f the animal and its behaviural effect cmpared with the cntrl treatment f petrleum jelly nly. The treated animals were mnitred fr their tick infestatin each day fr 8 days (a perid exceeding re-infestatin time), in cllabratin with livestck farmers. We recrded R. appendiculatus ticks f bth sexes as well as ticks f ther species. The experimental animals in any given herd led their nrmal daily life except that they were nt subjected t any frms f acaricide applicatin within the study perid. Tick cllectin and identificatin Ticks cllected frm each experimental animal were placed in 99% ethanl in glass vials, kept in a cl bx andbrught t the labratry f ICIPE in Nairbi fr identificatin. Tick samples were cllected accrding t the predilectin feeding sites as well ther places n the hst, as identified by the farmer. Vucher specimens were btained frm the Labratry f Entmlgy, Schl f Bilgical Sciences, University f Nairbi, Kenya. Ticks were identified fllwing the descriptins f Hgstraal (956a, b), Matthysse and Clb (987), Okell-Onen et al. (999) and Walker et al. (2003). Sme tick specimens culd nt be identified. Data management and statistical analysis Data cllected were analysed using Statistical Prducts and Service Slutins (SPSS versin 5 fr Windws). Preventin f tick re-infestatin n hsts and reductin percentage f tick infestatin n the animals due t treatment with each type f essential il were analysed using ne-way analysis f variance (ANOVA) and univariate analysis f SPSS. Significant differences between tick cunts n the animals fllwing the three treatments, essential ils f either T. minuta r T. diversiflia and the cntrl, were evaluated using the Kruskal Wallis test (H) (Kruskal and Wallis, 952) at a = 0.05 level f significance. Lgarithmic transfrmatin (Lg 0 (tick cunts + ) (Zar, 996), was applied t the data and the transfrmed data subjected t a paired sample t-test t evaluate a generally-bserved trend in the field that the number f male ticks was higher than that f female ticks n the ani- 24

114 Essential ils prtect livestck frm infestatin with ticks Table 8.. The mean (± SE) tick cunts n day zer n hsts naturally infested with ticks and selected fr different treatments befre start f experiment (I = thse hsts selected fr treatment with petrleum jelly (as cntrl), II = thse hsts selected fr treatment with 0% essential il f Tithnia diversiflia in petrleum jelly f Tagetes minuta, and III = thse hsts selected fr treatment with 0% essential il in petrleum jelly) (n = 45). Tick species I R. appendiculatus (c?) 32.3 ± 8.9a R. appendiculatus (?) 3.3 ± 8.60a Other tick species 32.2 ± 2.07a 'Valueswithin arwwerentsignificantly different (P =0.05). Mean (± SE) tick cunts ' II 38.7 ± 8.47a 23.2 ± 2.92a 38.7 ± 2.39a III 24.7 ± 4.59a 24. ± 4.73a 2.9 ±.09a mals at a = 0.05 level f significance. Legal use f experimental animals in the field All prcedures requiring the use f experimental animals in the field were apprved by the District Veterinary Office (DVO) f Bungma District, western Kenya. The imprtance f the prject was further explained t the Bukusu cmmunity by the DVO and agricultural extensin fficers wrking within the study area thrugh rganized wrkshps and lcal sub-chiefs and chiefs' meetings in the entire District. The field experiments were perfrmed in cmpliance with guidelines published by the Kenya Veterinary Assciatin and Kenya Labratry Animal Technician Assciatin, regarding the ethical use and handling f labratry and farm animals in the field (KVA and KLATA, 989). Infrmed cnsent was btained frm the vlunteer livestck farmers whse animals and farms we used. Results Tick infestatin n zebu cattle at the start f the experiment Althugh experimental animals came frm different farms and were randmly sampled, the results indicate that the initial tick infestatin (i.e., the average numbers f adult male and female R. appendiculatus and ther tick species n the hsts) were nt significantly different between animals (P>0.05) (Table 8.). At the start f the experiment, mre males than females f adult R. appendiculatus were fund n the hst (t.s (44) = 4.325; P<0.05). Tick infestatin f experimental animals after treatment On cntrl animals treated with petrleum jelly nly, tick infestatins increased n average by 60%during the 8-day experimental perid due t natural infestatins. Treatments with the essential ils f T. minuta and T. diversiflia resulted in the drp f tick infestatin levels n hst animals ver time. This drp was significant in the treatment with the essential il f T. minuta (P<0.05) and nt in the treatment with the essential il f T. diversiflia (P>0.05) (Figs. 8.2a and b). In the treatment with the essential il f T. minuta, the tick infestatin level significantly drpped up t -4 days pst-treatment, after which it increased again due t tick re-infestatin (P<0.05). The Student Newman-Keuls pst hc criterin indicated that fr hsts treated with the essential il f T. minuta, tick infestatins f the first -2 days pst-treatment and the last days 5-8 fllwing re-infestatin fr males and the -3 days pst-treatment and the last days 2-8 fllwing re-infestatin fr females, were nt significantly different (P>0.05) (Figs. 8.2a and b). Between days 3 and 4 pst-treatment, the mean tick infestatin levels n hsts were significantly lwer than the ther days (P<0.05) (Figs. 8.2a and b). Fr hsts treated with T. diversiflia and petr- 25

115 Chapter a Male Rhipicephalus appendiculatus isa JJ 60 s «40- O J3 % 20- a ^ 00' x> u 2 «40-20' 0- i /. *C b u a M a ^ [ < J\ '., U \c» r a T i a A ; f b & ^ T h NJ I ) b b b * r r i i t t $ r c c b / ; T C C C X. T C T * * * * * * * * b a x r a be a a > T lib Stf* S IS Observatin perid (days) ' be at"ab - ab be be be -Tm -T4 -C 200 i b Female Rhipicephalus appendiculatus is H -T» -Td -C S IS Observatinperid (days)

116 Essential ils prtect livestck frm infestatin with ticks i 20- S s «00- C ick burden S 8 4*» c a e > t ;a. i. v e «i\ Other livestck species i u ) \ i \ l * i. * * ) : * < b, :! t ^ * * a. ^ a ab J - ^ ^ L * «* a a» t _ : te t,._/ / * * i ( * ab., a. ab> ab^r a - v. te : be r ( be a. a ^i a. S.. a.!. ^ ' >a a ^ -L a a -Tm -Td -C n - i i i i i i i i i Observatin perid (days) Fig. 8.2.mean (±SE)percentage ftickinfestatin nthehststreatedwith essential ilf Tagetes minuta (Tm), Tithnia diversiflia (Td) rpetrleumjelly (C) (cntrl) and mnitred daily fr 8days. The effectsf thesetreatmentswereevaluatedn (a)male Rhipicephalus appendiculatus, (b)female Rhipicephalus appendiculatus and (c)therlivestcktick species.oneachday,valuescappedwiththesamelettersarent significantly different at a = 0.05 (Student-Newman Keuls H-test). The asterisk (*) n the standard errr bars indicates that the mean percentage ftick infestatin levels fthe hstswere significantly lwerthan the initial tick infestatin levels fllwing treatment at P = 0.05 level f significance (Student-Newman Keuls H-test; n =45). leum jelly (cntrl), hwever, there were n significant differences f mean tick infestatin levels between days n hsts (P>0.05) fr male and female R. appendiculatus and ther tick species. There was a cntinuus and gradual drp in tick infestatin up t day 8 n the hsts treated with T. diversiflia fr bth male and female R. appendiculatus and ther tick species but this drp was nt significant (P>0.05) (Figs. 8.2a and b). The percentage f reductin f tick infestatin was higher in the treatment with essential il f T. minuta than in that f T. diversiflia. Hwever, the repellent effect f the tw essential ils caused similar patterns f tick respnses (Fig. 8.2). In bth essential il treatments, there was a phase f reductin f tick infestatin lasting 3 4 days with T. minuta and 6-7 days with T. diversiflia, and the effects f bth essential ils reached an ptimum between 3-5 days and 6-7 days pst-treatment, respectively (Fig. 8.2). Tick reinfestatin started between -2 days pst-treatment in the hsts treated with T. minuta, whereas in the hsts treated with T. diversiflia, re-infestatin started at 9 days pst- 27

117 Chapter 8 treatment. Mre ticks re-infested hsts treated with T. diversiflia than thse treated with T. minuta fr male and female R. appendiculatus and ther tick species. Mrever, there was a greater variatin in tick infestatin levels after re-infestatin f the hsts treated with T. diversiflia than thse treated with T. minuta. Tick infestatin levels fllwing reinfestatin were maintained at a lwer level than they were initially fr the hsts treated with T. minuta cmpared t thse treated with T. diversiflia. In all the cntrl treatments, there was a mderate but gradual increase in tick infestatin fthe animals (Fig. 8.2). During the bservatin perid, mre males than females f adult R.appendiculatus were fund n the hsts [t 0.s (494) = 4.252; P<0.05].This male/female rati was maintained up t the end f the experiment [t 0.05 (854) = 7.49; PO.05]. Male R. appendiculatus succumbed t the repellent effects f the tw essential ils mre quickly than their female cunterparts, with the essential il f T. minuta shwing a strnger repellent effect than the essential il f T. diversiflia (Figs. 8.2a and b). Specifically, residual efficacy time f T. minuta was 8days fr female R. appendiculatus (Fig. 8.2b) and 2 days fr male R. appendiculatus (Fig. 8.2a). Tagetes minuta cnsistently kept the levels f male R. appendiculatus significantly lwer than thse f the cntrl during 7 days, while this essential il nly kept the female R. appendiculatus infestatin lwer than cntrls during 9 days. Fr T. diversiflia, these values were days and 6 days fr males and females, respectively. In the treatment with T. minuta, significant re-infestatin started days pst -treatment fr female R. appendiculatus, whereas fr male R. appendiculatus, it started 4 days pst-treatment. With T. diversiflia, re-infestatin started 8 days pst-treatment fr female R. appendiculatus and 2 days later fr males. Other livestck tick species were als affected by treatments with the essential ils f T. minuta and T. diversiflia. Essential ils f T. minuta and T. diversiflia kept the infestatin levels f ther livestck tick species significantly lwer than the cntrl fr and 5 days, respectively. Re-infestatin started and 8 days pst-treatment with T. minuta and T. diversiflia, respectively (Fig. 8.2c). Prtectin f hst animals with essential ils f Tagetes minuta and Tithnia diversiflia Essential ils f T. minuta and T. diversiflia prtected the experimental animals frm tick re-infestatin n average fr 2.53 days (range 4-8 days) and 7.87 days (range 4-2 days) pst treatment, respectively (Table 8.2). Hst animals treated with the essential il f T. minuta were als significantly prtected frm re-infestatin with ther tick species during 2 days pst-treat-ment (P<0.05) (Fig. 8.2c). The duratin f prtectin frm tick reinfestatin n the hsts by the tw essential ils f T. minuta and T. diversiflia was significantly different [t 0.s(4) = 4.336, P = 0.00], with essential ils frm T. minuta giving the strngest prtectin. Discussin and cnclusin Results btained frm this study cnfirm the previus labratry results utlined in Chapters 4 and 6 and semi-field results summarized in Chapters 7 and 8 f this thesis, in which essential ils f T. minuta causes a strng and cnsistent repellent effect n R. appendiculatus. The effect f essential il frm T. diversiflia, althugh significant, is less strng cmpared t that f T. minuta. The tw essential ils shwed a significant difference in their repellent effects n R. appendiculatus and n ther tick species. The essential il f T. minuta repels a larger prprtin f R. appendiculatus and mre quickly than that f T. diversiflia. In the treatment with the essential il f T. minuta, an ptimum effect is at- 28

118 Essential ils prtect livestck frm infestatin with ticks Table 8.2. Observed pst-treatment time (days) ver which individual hsts treated with the essential ils f Tagetes minuta and Tithnia diversiflia were prtected frm tick re-infestatin n 5different farms (n = 30). Pst-treatmenttime (days)verwhichhstswereprtected frm tick re-infestatin Farm Hststreatedwithessentialilf Tagetes minuta Hststreatedwithessentialilf Tithnia diversiflia Range MeaniSE 2.53 ± ±0.6 tained within a shrter perid f time and maintained fr lnger than in the treatment with the essential il f T. diversiflia. The results n duratin f prtectin shwed that reinfestatin started early in hsts treated with the essential il f T. diversiflia cmpared with thse treated with the essential il f T. minuta. These results suggest that the essential il f T. minuta may cntain cmpunds that cnfer a lnger lasting residual efficacy than the essential il f T. diversiflia. The mean prtectin time cincided with the acaricide dipping regime f -2 weeks fr indigenus zebu animals and nce per week fr pure- and crss-breed animals (Nrval et al., 992). Althugh acaricide dipping is expensive in terms f labur input and cst and can cause side effects, it effectively cntrls all the ticks n the cattle, thus maintaining a tick-naive ppulatin f cattle fully susceptible t mre than ne tick species (McCsker, 993). Mrever, effective tick cntrl cntributes t tick-brne disease cntrl, and eventually t a healthy livestck ppulatin. Hwever, 00% cntrl f ticks n livestck may lead t a cmplete lss f immunity t vectr-brne pathgens and this is a disadvantage, because the accidental expsure t ticks and tick-brne pathgens can ptentially cause a destructive situatin in susceptible animals. This was nce witnessed in Zimbabwe during the civil war between 975 and 980 (Nrval et al., 992). By cntrast therefre, the advantage f repellents is that they d nt create a tick-naive ppulatin f cattle. The rate f reductin f tick infestatin was greater than the rate f build-up f the tick infestatin in animals treated with T. minuta, while it was the reverse in animals treated with T. diversiflia. This further cnfirms the strnger repellent effect f the essential il f T. minuta than that f T. diversiflia. This may explain why tick infestatin levels fllwing re-infestatin n the animals treated with the essential il f T. minuta were 29

119 Chapter 8 maintained at a lwer level than they were previusly, while the tick infestatin levels fllwing re-infestatin n the hsts treated with the essential il f T. diversiflia reached a higher level than they were befre. The results btained in this study were cmparable t thse btained elsewhere in Kenya with a 0% frmulatin f the essential il f O. suave in liquid paraffin as well as a 25% frmulatin f neem il against attaching larvae and adults f R. appendiculatus (ICIPE, 998/99; Mwangi et al., 995a). Results f recent field tests f natural repellents (5% ntkatne and carvacrl) against Ixdes ricinus L. by Dlan et al. (2008) were als cmparable t the results btained frm the present study. By the 5th day pst treatment, mre than 75 and 60% f adult R. appendiculatus and ther tick species were affected by the essential ils f T. minuta and T. diversiflia, respectively. The tw essential ils nt nly affected the target species R. appendiculatus but als ther livestck tick species such as A. variegatum, R. evertsi Neumann and Bphilus spp. This brad-spectrum repellent biactivity against livestck ticks may have imprtant implicatins fr the practical use f the essential ils as tick repellent prducts fr a variety f ecnmically imprtant tick species fund in the hst's envirnment. These findings warrant further research t establish t which extent each livestck tick species is affected by each type f essential il. This apprach will facilitate a mre rapid incrpratin f the essential ils int integrated tick cntrl strategies. This may be a suitable apprach fr mst African livestck farmers, wh ften lack the resurces fr apprpriate and sustainable tick cntrl. Mrever, shuld future studies prve these tw plants (T. minuta and T. diversiflia) t have the abilities t repel ticks in the field like Melinis minutiflra Beauv. (Mwangi et al., 995a; Fernandez- Ruvalcaba et al., 2004) and G. gynandra (Malnza et al., 992), then further studies are needed t evaluate hw they can be incrprated int livestck pastures as tick repellent plants. As the essential ils prtected the hsts against tick infestatin fr several days nly, there is a need t stabilize the active ingredients s as t make them prvide lnger prtectin times in the field, cmparable t substances such as pyrethrids, which give2vi mnths' prtectin when impregnated in ear tags (Yung et al., 985). A mre stable frmulatin, which wuld cntrl the individual cmpunds' rate f vaprizatin is needed as this culd ffer cntrlled-release f repellent vlatiles and mre lng-lasting prtectin. Furthermre, the perfrmance f the ils may be affected by envirnmental factrs such as strng sunlight, relative humidity and /r wind as well as activities f the target hst animals (Carrll, 2007). Thus, imprvement f the frmulatin may als need t address ways f dealing with these cnstraints, e.g., micr-encapsulatin f the ils t prtect the active ingredients. The treated animals freely interacted with ther animals in the experimental herd and envirnment (W.W., pers. bs.). Whether r nt the treated animals cnferred sme prtectin t nn-treated animals by virtue f their presence in any ne given herd was nt evaluated and is nt knwn. Hwever, it may be interesting t investigate this questin, as it was recently established with sme tsetse flies' repellents at ICIPE (Saini and Hassanali, ). Whether the bserved increase in tick infestatin in the cntrl animals in all 5 herds was caused by an envirnmental factr r by the presence f repellent-carrying animals within the herds is nt knwn. If the increase is hypthesized t be due t the presence f repellent-carrying animals, then we wuld have expected a decrease in tick infestatin levels in the cntrl hsts during the re-infestatin perid but this trend did nt appear. It wuld be interesting t mnitr tick infestatin levels n the hsts in anther set f cntrls (negative cntrls) away frm the ptential influence f repellents. The phenmenn f the biased male/female rati f R. appendiculatus and ther 30

120 Essential ils prtect livestck frm infestatin with ticks tick species in favur f males agrees with previus reprts n sex ratis f bld-feeding ticks (Lndt et al., 979; Kaiser et al., 982; Mwangi et al., 985). Several pssibilities t explain this phenmenn can be given. One, a substantial prprtin f female ticks may engrge faster than males and drp ff the hst r be preferentially rejected by the hst. Alternatively, mre male than female ticks may succeed in finding a hst. As bserved by Hgstraal (956a, b) and Kaiser et al. (982), it may be pssible that male ticks remain n the hsts lnger than the female ticks. There may als be a rle fr phermnes in the biased male/female rati f R. appendiculatus and ther tick species n the hsts Recent studies shw that in the majrity f hard ticks, feeding females prduce a sex phermne cntaining 2,6-dichlrphenl, which attracts males t the feeding site (Rechav et al., 976; Snenshine, 2006). In additin t the daily activities f hsts, weather, grass status and amunt f vegetatin als affect tick ppulatin survival bth n the hst and in the envirnment (Wilkinsn, 957; Thmpsn et al., 978; Davey et al., 982; Barnard, 986; Thadeu et al., 989;Mwangi et al., 995b; Fernandez-Ruvalcaba et al., 2004).But it is nt clear whether these factrs may preferentially affect ticks at sex level. As male ticks significantly succumb t repellent effects f the essential ils mre than their female cunterparts, we expected this sex rati t change in favur f female ticks fllwing essential il treatment but surprisingly it did nt. It is nt clear whether r nt this sex rati phenmenn crrespnded t the nrmal bilgical behaviur f ticks: feeding and mating, fllwed by females drpping ff the hst bdy t lay eggs (Snenshine, 99; Am, 992). Establishing facts abut this phenmenn is imprtant in livestck tick cntrl and management strategies, particularly in ur studies as it can lead t the develpment f an effective repellent dse fr bth female and male ticks. This infrmatin is als crucial in develping effective n-hst tick cntrl mdels. The tw essential ils f T. minuta and T. diversiflia are pssible candidates t be cnsidered fr integratin int livestck tick management prgrammes in the study area and in ther areas with similar eclgical cnditins. The essential ils f T. minuta and T. diversiflia may peridically be applied alne (at intervals f 7-0 days) r integrated with acaricides and/r ther alternative tick cntrl methds suitable in that particular envirnment, as previusly described fr the essential il f O. suave (Mwangi et al., 995a). Hwever, fllw-up studies t knw what happens t the surviving ticks ff-hst are recmmended. Acknwledgements This research was jintly supprted by the Internatinal Fundatin fr Sciences, Stckhlm, Sweden, and the Organizatin fr the Prhibitin f Chemical Weapns, The Hague, The Netherlands thrugh a grant AB/ The first authr wishes t acknwledge the financial and material supprt received frm the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE) under the African Reginal fr Pstgraduate Prgrammes in Insect Science (ARPPIS) and the Wageningen University and Research Centre, the Labratry f Entmlgy under PhD Sandwich Fellwship. The authrs wish t thank Mr. Newtn Kmeri Mwanga fr illustratins, ICIPE staff fr their technical supprt, Bungma District Veterinary Office and the vlunteer livestck farmers whse animals and farms were used. 3

121 Chapter 9 32

122 Summarizing discussin General discussin and cnclusin Despite mre than 50 years f research n tick cntrl and management, livestck is still strngly affected by ticks and tick-brne diseases in Africa. The situatin is nt yet imprving, as tick-brne zntic diseases are n the rise nt nly in livestck but als in humans (Tnbak et al., 2006; Vial et al., 2006; Jngejan, 2007). In additin, majr tick cntrl prgrammes have cme t an end withut achieving much success and climatic changes caused by glbal warming are psing new challenges (Jngejan, 2006; Uilenberg, 2006). New methds fr cntrlling and managing ticks that g beynd the previus and current nes need therefre t be explred. Many previus studies have fcused n cntrl and management f ticks n the hst withut prviding effective slutins t the chrnic prblems caused by ticks t the livestck industry. The wrk f this thesis has examined the strategic use f plant-derived essential ils as ptential repellents fr n-hst cntrl f the brwn ear tick Ripicephalus appendiculatus. It was fund that essential ils f Tagetes minuta and Tithnia diversiflia, tw plants widely present in western Kenya, affect the behaviur f R. appendiculatus (and ther tick species) and affrd prtectin against tick infestatin fr a perid f apprximately 2 weeks under field cnditins. Key results f this research have indicated hw ethnbtanical repellents can be identified and tested, hw they affect tick behaviur, and hw they can be pssibly used fr livestck tick cntrl and management. Several ther questins need t be answered befre the research presented in this thesis can be effectively applied: () Hw easy and sustainable is the integratin f lcally-derived repellents in tick cntrl and management prgrammes by livestck farmers? (2) What are the advantages and disadvantages f using repellents t cntrl and manage livestck ticks? (3) Can livestck ticks develp resistance t repellents as they have dne t acaricides? (4) Can repellents prvide a sustainable slutin t the chrnic prblems caused by ticks t the livestck industry? This Chapter discusses these questins with relevance t the results reprted in this thesis and where pssible, gaps in knwledge are highlighted and recmmendatins are made that are expected t lead t imprved cntrl and management f livestck ticks. Identificatin f sustainable surces f tick repellents Ptential tick repellents can either be f natural rigin frm plants and animals r synthetic. Synthetic repellents tend t be mre effective and/r lnger lasting than 'natural' repellents (Cllins et al., 993; Fradin and Dax, 2002). Hwever, sme plant-derived repellents are either cmparable t r smewhat better than synthetic repellents (Fradin and Dax, 2002; Jaensn et al., 2005; 2006). Of the synthetic repellents, N,N-diethyl-3-methylbenzamide (DEET) is widely used and remains the principal arthrpd repellent tday (Gupta and Rutledge, 989; Carrll et al., 2004; Katz et al., 2008).Other imprtant cmmercially available synthetic repellents in use include: -piperidinecarbxylic acid, 2-(2-hydrxyethyl)-, -methylprpylester, picaridin (Nentwig et al., 2002), Insect-Repellent 3535 (Thavara et al., 200) and l-(3-cyclhexen-l-yl-carbnyl)-2-methylpiperidine (Carrll et al., 2004). Each f these 33

123 Chapter 9 new ingredients have sme advantages ver DEET and their efficacy appears t be gd based n labratry and field studies (Debbun et al., 2005). In many studies, these labratry-derived cmpunds with knwn repellent efficacy and prperty are taken as standards against which candidate repellents are cmpared. Fr example in Chapter 4 f this thesis, the results f the labratry assays f the repellent effects f the essential ils f T. minuta and T. diversiflia against adult R. appendiculatus at their highest dse f mg were cmparable t that f DEET. These cmparisns give a measure f the repellent ability f the candidate substance(s) and prvide an pprtunity f decisin making as whether t cntinue develping the candidate substance(s) as ptential repellent(s). The fcus f this study was n the evaluatin f the effects f the plant-derived chemical repellents n livestck ticks (R. appendiculatus). Glbally, mre than 50 natural repellents are in use, with the mst cmmn nes being citrnella, eucalyptus, lemn leaves, peppermint, lavender, cedar, canla, rsemary, pennyryal, and cajeput. Althugh any f these repellents might be f interest in prtectin f livestck against tick infestatin, these are all "new" prducts t the Bukusu cmmunity and intrducing these essential ils in the cmmunity wuld nt be better than the failed and abandned tp-dwn apprach in agricultural research and develpment (Whyte, 98). In this study, a bttmup apprach was adpted in rder t invlvethelcalcmmunityindecisin-making,basedn traditinal knwledge, rather than cnceding this rle t cnventinally-trained experts (Warry, 992;Etkin,993). Chapter 2 f this thesis describes hw the surce plants fr the essential ils were identified, while in Chapter 3, an explanatin is given n hw the plants were selected frm a grup f 54 plants described in Chapter 2 and evaluated fr their repellent effects against adult R. appendiculatus. The selectin f eight plants fr essential il extractin and labratry testing was based n: () infrmants' knwledge n hw they use the plants fr livestck tick cntrl and (2) a nn-experimental validatin prcedure (Lans, 200) (Chapter 3). The nn-experimental methd allws fr the separatin f plants used fr cultural reasns frm thse with anti-tick prperties (Brwner et al., 988; Heinrich et al., 992;Lans et al., 2007),thus facilitating the fcus f the research subject. Develping scientifically acceptable and standardized tests fr screening the biactivity f candidate cmpunds is imprtant fr develping effective prducts. A standard screening technique t cmpare behaviural effects f candidate repellents with thse f knwn repellent cmpunds is lacking. Labratries may develp their wn methds fr testing the repellent effects f candidate substances based n the cntext in which the term "repellency" is understd (Schreck, 977). Repellents may keep the arthrpds away frm their target hst animals (true repellents) (Metcalf and Metcalf, 982), r elicit a behaviural respnse that causes them t mve away frm the hst animals after cntact (excitrepellents r irritants) (Chu et al., 997). True repellents are highly vlatile cmpunds that are detected by the lfactry receptrs f the arthrpds and impede cntact between them and their hsts. Recent research has demnstrated that in arthrpds, repellents may act in the same way as true lfactry cmpunds and that repellent behaviur is a respnse t the excitatin f the repellent-specific lfactry neurn (Davis, 985; Syed and Leal, 2008; Lgan et al., 2008). The first step tward experimental validatin f the Bukusu ethnknwledge is described in Chapter 3 f this thesis. Eight plants were selected fr essential il extractin and labratry testing, was based n: () infrmants' knwledge n hw they use the plants fr livestck tick cntrl (Chapter 2) and (2) a nn-experimental validatin prcedure (Lans, 200; Heinrich and Gibbns, 200; Lans et al., 2006) (Chapter 3). The essential ils were screened fr their repellent effects against adult R. appendiculatus and 34

124 Summarizing discussin the results cmpared t a cmmnly used cmmercial arthrpd repellent, DEET (Ndung'u et al., 995). The essential ils f the tw plants, T. minuta and T. diversiflia, having relatively strng repellent effects cmpared t the ils f the ther plants, were selected fr an in-depth scientific study. The results frm these assays demnstrated repellent effects f the essential ils f the plants, thus prviding justificatin fr their use fr tick cntrl and management. In Chapter 5, the repellent effects f the essential il f T. minuta n the climbing respnse behaviur f adult R. appendiculatus in dual-and n chice assays were cmpared. Althugh the dual-chice assay prved a mre sensitive assay than the nchice assay, the design f neither assay was, hwever, ideal fr apprximating the repellent effect f a candidate substance. A study f ther methds used fr testing behaviural effects f repellent substances n tick species revealed that the methds varied greatly in their experimental design, mde f expsure f ticks t the test materials, time between treatment and expsure f ticks t the test materials and the number f experimental ticks expsed t the test material (Schreck, 977; Hadani et al., 977; Mathewsn et al., 98; Staub et al., 2002; Garbui, 2008). Als, the equatins used t calculate the degree f repellency were varied making a cmparisn between results btained by different methds difficult. In additin, the ptential rle f aggregatin phermnes affecting tick behaviur may have affected the climbing assay as well (Snenshine, 985; Snenshine et al., 982; 992). Furthermre, the n-chice assay setup des nt simulate a natural field situatin f freedm f chice while the equatin used t generate the data in the dual-chice assay, has several limitatins (Chapter 5). An experimental design that wuld allw the bservatin f ne tick at a time withut being affected by all the factrs utlined in Chapter 5is recmmended. Aspects f tick behaviur affecting develpment f n-hst tick cntrl device Understanding all aspects f the behaviural bilgy f arthrpds in the presence f their hsts is the basis fr the develpment f effective cntrl and management strategies f any pest, nt nly ticks. Varius aspects f tick bilgy that characterise its life cycle have been studied (Snenshine, 99; Dipelu, 990; Am, 992). Unfrtunately, few f these studies address the questin f hw the knwledge gained may be used in tick cntrl and management strategies, and nne f them addresses the questin f hw sme ticks (e.g., R. appendiculatus and R. evertsi) lcate specific feeding sites while n the hst animal. Factrs that determine vectr-hst interactins and initiate tick-feeding prcesses are critical t pathgen transmissin, yet, this appears t be the least well understd aspect f tick behaviural bilgy. Knwledge n species-specific tick behaviur, such as differences in preferred n-hst feeding sites between clsely related tick species (Iffe-Uspensky et al., 997; Chapter 4), cntributes t the develpment f anti-tick interventins targeted at a specific tick species. This can imprve the efficacy f tick cntrl by aviding expenditures fr the cntrl f nn-target species (Am, 992). In Chapter 4 f this thesis, nhst behaviural bilgy f tw sympatric tick species (R. appendiculatus and R. evertsi) was studied in rder t understand and cmpare the navigatin behaviurs f the tw tick species t their respective feeding sites n the hst. T lcate the feeding site n hsts, adult ticks, R. appendiculatus and R. evertsi explit the cncept f integrated use f repulsive frces (away frm the preferred feeding site) and attractive frces clse t the preferred feeding site t prvide the apprpriate rientatin signals and mvement (Chapter 4). Interestingly, the tw species have evlved t feed far away frm ne anther n the same hst with R. appendiculatus preferring t feed at the ear while R. evertsi prefers t feed at the anal regin (Sika, 996; Wanzala et al., 35

125 Chapter 9 them expensive. The plant-derived repellents may be disadvantaged as peple may prefer grwing fd crps t surce plants f repellents n their limited arable lands. In additin, expsure f repellents t ambient cnditins (slar radiatin, temperature fluctuatins, rainfall, humidity etc.) may influence their effectiveness a great deal (Mwangi et al., 995; Chapter 8). Develpment f tick resistance t repellents The develpment f arthrpd resistance against repellent cmpunds such as pyrethrids has been reprted (Pennetier et al., 2007). Ticks have develped resistance t the repellent effect f permethrin, which acts as a true repellent as well as an excitrepellent/irritant (Metcalf and Metcalf, 982; Hibbard and Bjstad, 989) and cntact acaricide (ISW-TBE, 2008; Bayer Healthcare, 2008). While DEET remains the mst brad-spectrum and effective arthrpd repellent develped t date (Katz et al., 2008), recent reprts als shw that the chief malaria-carrying msquit, Anpheles albimanus, in the United States is becming resistant t it (Ngan, 2008). This is an indicatin that ticks culd als develp resistance t this cmpund. If the use f repellents is widespread and effectively rganised, selectin f a gene cnferring resistance t repellents might be favured rapidly, leading t a high degree f resistance. Hwever, if nt all farmers wuld use repellents, r if a prprtin f each herd is left untreated, it might be pssible t pstpne r even avid the develpment f resistance, certainly if ticks als feed n ther hst animals such as wildlife. Furthermre, as these essential ils are cmpsed f many cmpunds acting tgether in synergism t exert the repellent effect (Knight and Nrtn, 989), the develpment f resistance may be avided. Repellents as a sustainable slutin t the chrnic prblems caused by ticks t the livestck industry Repellents have been in use fr the cntrl f arthrpds fr many years (Dethier, 956). They have becme a ppular methd fr btaining prtectin frm biting arthrpds. Studies have shwn, hwever, that the risk f cntracting an infectin transmitted by a given vectr when using an effective repellent against that vectr ges dwn significantly (Schwantes et al., 2008). Repellents wrk in a unique way, different frm any ther methds used fr vectr cntrl and management. True repellents rarely kill the target vectr arthrpds, instead, the vectrs are just kept at bay (Metcalf and Metcalf, 982). In exerting their effects, repellents interfere with mating and vipsitin respnses as well as feeding (Hcking, 963). Nt all target arthrpd species are equally affected by a given repellent and within a given species, nt all rganisms are affected by the same dse f a recmmended repellent. Repellents als d nt select between the pathgen(s)-infected and nn-infected hst-seeking arthrpds. Repellents may nt be able t prevent bld uptake frm the hst r injectin f saliva int the hst by the feeding vectr. In either way, there may be transmissin f pathgens frm the hst t the vectr and vice versa. Hwever, this may nly be pssible fr instances f pathgen transmissin that take a shrt perid f time during the vectr-hst cntact but may nt be pssible when the vectr-hst cntact perid is quite lng as a prerequisite fr transmissin t take place. In the case f R. appendiculatus and cattle, it takes between 24 and 72 h f tick feeding befre pathgens are transmitted t the hst animal (Ochanda et al., 988;Knnai et al., 2007b). Ticks may nt be able t withstand the effects f an effective repellent fr this perid f time. Thugh sme f the abve factrs may appear t undermine the rle f repellents in the cntrl and management f vectrs and vectr-brne diseases, the fact that repellents significantly minimise the risk f cntracting a disease (Strickman et al., 200), suggest that it is wrth- 38

126 Summarizing discussin while investing in repellent research and develpment. Results frm Chapters 3-8 (with the exceptin f Chapter 4) and previus studies (Bnd, 2007) indicate that it is pssible t keep ticks away frm their apprpriate hsts using repellents. Hwever, if tick repellents are t be cnsidered as an ptin fr prviding prtectin against n-hst tick infestatin, the fllwing three questins need t be addressed adequately: () As repellents neither kill nr cause any harmful effects t the ticks, will the ticks starve t death r find an alternative hst in the presence f the repellents? (2) If an alternative hst will be sught, what kind f hst will it be? (3) In the event f the develpment f a new parasitic relatinship, what will be its pssible nature and hw abut the invlvement f the zntic disease transmissins? These questins shuld be reslved as part f a lng-term strategy f repellents fr preventin f tick infestatin in livestck. Hwever, repellents may be the main tl available t prvide prphylactic measures against infestatins f R. appendiculatus and the resulting tick-brne diseases (Gupta and Rutledge, 994). There is a need, therefre, t have a crdinated effrt amng varius research grups assciated with the science f arthrpd repellents t investigate () the mde f actin f repellents, (2) dses f repellents needed t generate threshld level respnses and (3) cntrlled release delivery mechanisms. This may help in understanding the mechanisms by which repellentswrk under varying cnditins, thus leading t the develpment f better prducts and t their wider applicatin. It is envisaged that this apprach in turn may revlutinize the develpment f repellents fr prtectin at an individual level and their use in vectr cntrl (Hcking, 963). The availability f frmulatins that stabilise the active ingredients in a cntrlled release strategy f the repellent dur s that they remain effective fr a lnger perid f time, may be a way tward develping effective repellents (Wn-Ja et al., 2007). The interceptin f tick rientatin twards predilectin feeding sites and subsequent attachment as a means fr n-hst tick cntrl The in-viv and in-vitr studies in this thesis have shwn that tw sympatric tick species, R. appendiculatus and R. evertsi, shw marked preferences fr different feeding sites n the same hst animal (Sika, 996; Chapter 4). These studies further demnstrated that the feeding site preference behaviur f the tw tick species was mediated by hst semichemicals emanating frm the predilectin feeding sites (Sika, 996; Chapter 4). Our nhst bservatins and labratry assays in Chapter 4 suggested the peratin f bth avidance (clser t the feeding site f the ther) and attractin (clser t its wn feeding site) respnses fr. appendiculatus and R. evertsi. This pair f hst attractive (frm feeding sites) and repulsive (frm nn-feeding sites) durs may be the majr chemrientatin mechanism utilised by the tw tick species t lcate predilectin feeding sites in a "push-pull" mde, althugh nn-lfactry cues may als play a rle (Waladde et al., 979; Sika, 996; Chapter 4). This push-pull mde seemed t ensure that the ticks were unlikely t mve in the wrng directin n the hst and enhanced the prbability f rienting tward their respective predilectin feeding sites. While the stimulus frm ne site acted as a strng kairmne (attractant) t the tick species that fed atthat site,it acted as an allmne (repellent) against the ther tick species, which did nt feed there (Sika, 996; Chapter 4). The mechanisms invlved in predilectin feeding site lcatin are nt yet clearly understd and prbably, the full explanatin f this push-pull mechanism may await the characterisatin f the chemical cmpsitin f hst dur in rder t understand its rigin, mlecular structure, and mde f actin. Frm the quantitative data presented by Cumming (998), R. appendiculatus preferred bvid hsts t any ther hsts, fr unexplained reasns. As utlined in Chapter, 39

127 Chapter 9 hst identificatin and selectin is dependent n physical parameters such as hst mvement (hst-derived vibratins), hst breath, and cntact, and envirnmental parameters such as light, temperature and humidity (Am, 992). Althugh Waladde (987) stated that stimuli emitted by the hsts culd be sensed by the ticks thrugh thermreceptrs and lfactry receptrs, hst specificity and feeding site lcatin are asyet unexplained factrs. Therefre, whether r nt chemical cues emitted by the hst are invlved in hst selectin, has nt yet been fully understd, unlike fr msquites, where hst-specific semichemicals have been identified as behaviur-mediating cues (Takken and Knls, 999). Furthermre, the rle f the parasites in bth infected tick vectrs and hsts in hst selectin by the ticks is als nt yet clearly understd. There is a need, therefre, t undertake behaviural studies n the interactin between ticks and hst-vlatiles in rder t understand the specific chemical cues invlved in hstpreference (Cumming, 998). My wn unpublished data and that f thers (A. Hassanali, pers. cmm.) prvided evidence fr sme hst-attractive chemical cues fr predilectin feeding site lcatin riginating frm micr-rganisms (bacteria) living inside and arund the hst ears,and nt frm the hst itself. This prbably explains why Cumming (2000) fund that hst distributins d nt limit the tick species ranges f mst African ticks and that the hst per se, des nt explain the evlutinary histry f African ticks. This is a cmplex sequence f beneficial and nn-beneficial evlutinary relatinships that warrant further investigatins in rder t understand hw these might be used fr the develpment f n-hst tick cntrl and management strategies. The pssibility f intercepting tick rientatin pathways tward predilectin feeding sites n the hst was shwn in Chapters 6 and 7. The interceptin f these chemrientatin pathways n the hst animals may prvide an individual-based tick cntrl and management ptin that best fits the resurce-limited livestck farming system f trpical Africa. This was the fcus f the current study, which culminated in Chapter 8with a field test in cllabratin with lcal livestck farmers. The results f the present study have prvided a basis fr the develpment f an n-hst tick cntrl and management strategy using plant-based allmnes and hst-based semichemicals at an individual level. Devices laded with tick repellent frmulatins may be strategically deplyed n the hst and replaced after -4 days fr the essential il f T. minuta and 8days fr the essential il f T. diversiflia. The results f the field study described in Chapter 8, suggest that behaviural manipulatins f tick-hst interactins may represent an effective and envirnmentally acceptable strategy fr tick cntrl and management at an individual level. Cnclusins I have endeavured t give specific cnclusins derived frm the analysis f the results presented in this thesis in accrdance with the bjectives stated at the beginning f the studies. My first bjective, t dcument and evaluate ptential anti-tick ethnpractices used by the Bukusu cmmunity in Kenya, resulted in the establishment f a list f 54 plants used in many different ways as repellents. Of these, I selected the eight mst likely candidates fr experimentatin, based n a nn-experimental validatin prcedure (Chapter 2). Using these eight plants as a starting pint, my secnd bjective was t extract their essential ils, determine their chemical cmpsitins, and evaluate their effects n adult R. appendiculatus. The essential ils f T. minuta and T. diversiflia turned ut t be the mst prmising f the eight in terms f their effects n ticks, and were further used in the experiments n tick behaviur (Chapter 3). The essential il f T. minuta had a higher 40

128 Summarizing discussin prprtin f mnterpene cmpunds than the essential il f T. diversiflia, which had a higher prprtin f sesquiterpenes than the essential il f T. minuta. Objective 3 was t study the effects f the essential ils n hst selectin, n-hst navigatin and attachment behaviur ir. appendiculatus (Chapters 6 and 7). We shwed that essential ils f T. minuta and T. diversiflia caused a disruptin f rientatin, walking and attachment behaviur f R. appendiculatus n the hst. Fr bth essential ils, the legs + tail sites f applicatin, fllwed by ear smear and then ear tube, had significant effects n rientatin t the hst and attachment site preference f R. appendiculatus, in that rder. The ear smear methd is recmmended fr treating hsts with essential ils, but cncentratins and frmulatins f the ils may need t be imprved in rder t stablilise their active ingredients. My furth bjective was t evaluate a "push" tactic with the repellent ethnbtanicals fr n-hst cntrl f R. appendiculatus in the field using the ear smear site fr treating hst animals with essential ils as recmmended in Chapter 7. Within ^ days pst-treatment, the number f ticks n animals treated with the essential ils was pushed ff the hst bdy by mre than half the riginal ppulatin. By the 5 th day psttreatment, mre than 75 and 60% f adult R. appendiculatus and ther tick species, respectively, had drpped ff the hst. A strnger pushing effect was shwn by the essential il f T. minuta than the essential il f T. diversiflia. The results suggest the ptential fr the essential ils t be incrprated in the n-hst "push" and "push-pull" strategy fr the cntrl f R. appendiculatus and assciated tick-brne diseases amng the resurce-limited livestck farming cmmunity in trpical Africa. In additin t the fur majr bjectives f my study, tw ther aspects f tick behaviur were studied. The first ne was t cmpare n-hst behaviur f tw sympatric tick species, R. appendiculatus and R. evertsi (Chapter 4). Each species f tick shwed specific behaviural sequences in the navigatin t their preferred feeding sites. Finally, we cmpared a dual-chice with a n-chice assay, using the essential il f T. minuta as the test substance n the climbing respnse behaviur f R. appendiculatus (Chapter 5). The dual-chice assay prves a mre sensitive assay than the n-chice assay, althugh the n-chice assay prvides greater residual effects. 4

129 Appendix 42

130 Appendix is S 3 > O <N i «; -3-2 if _s m m s & 3 ^ 3 P. v 5 J3 ^2 Q a as ^ i > a 3 3- tu 3 O 8 w C O JS e 3 ed _M & 0 s 5 S & s CD S.a * 5 >^ si u * ' <H s > «2 ^ a s 3 fa* 3 S3 E S a b.s3 t 3 ^ c/i Ills * s * a" 3 I "S s f fe S E E a s g w 2 ^? s a n,. 43 >% ^ a. i>a 3 b' H O ^ i? _ ti >^"T3 M 3 n cs 2 O B a g >> I S? I 3 " 3 TS =3 ^

131 Appendix ea u. e t> U-l T3 9 c X O.O CN m ymus idt, 20 P, 200 Ann Schm MTD sl tf & \* - g tn a s CO ea ^ 8 K N ON =2 8> r S -= s w J? a a ~ * S3 M s C ^ S u a u " 3-5 -O U rt a <.a 60 c <- ^2i: "- ea >^ 2> ^ & in ' <^ * < OV po j «O - e i _J 5 C3 CN (N m < 5 3. > 3 e B e = 9 = &<

132 Appendix ON ON m n «: - ON -S 2S< T3 «f- «- rt tf "3 O 2 & 3a S-2 s u. X 2P J ON 2 \ ON " ~ ON W tf " «eg r-ri c/i e Eq act "a «2 CQ 03 U s a c - 3 c 8. w ^ R y s a 6 B W a r^a s -e ^5S S 3 rg 2

133 Appendix 85 f i rt-s _ ce 3y<s i i s \p OS (S si; tf &* " "3 -?! ed g (N "S <, ed* " g «a n "8 j S ed 9 <N ed TTI «a ^S a u y B! N uis ll :w : P» ffl CQ a c 6 5a 8 < e T3 C O. >> ed 5 u 3 9- II U a! IS 8 CO P-.

134 Appendix Kaayaetal., 995 ICIPE Annual Reprt, 998/99 Regassa, m <N 3 4= O Kaayaetal., 995 ICIPE Annual Reprt, 998/99 Regassa, 2000 Muhammad et al., 999 Schmidt, 2003b Aburashed et al.,2003 Kaayaetal., 995 ICIPE Annual Reprt, 998/99 Regassa, 2000 O <^ - al Kaayaetal., 995 ICIPE Annual Reprt, 998/99 Regassa, O 3 B J= u Kaayaetal., 995 ICIPE Annual Reprt, 998/99 Regassa, 2000 CN 3 u CO Kaayaetal., 995 ICIPE Annual Reprt, 998/99 Regassa, <N TJ 43 O CO c CD O! T3 cd O i a u O 5" 2 J 2 H m H CQ H m H m H «H CQ U- u CQ" H" S a Pi ~~ B 2 *^ J3 ^ s 2 * q «i ^ Q O > > > > > > 43 CO 43 /.0 s 4= t/> 2 a 43 2 c >, c - u & g.0 C/3 a a s I -s ff l-l <U 3! CD O > g 8 8 u Pi u a ' 43 3 S 3 b a. -Si "S3 s 5 2 x S w -S N 0, i4 u cd 4= Id ~ "8 % s >> cd 3 -j Z.3,3 00 <! " a s a <3 «. 2 a U O cd u cj cd ' e 4= LLl»- l-i O O s >> cd -3-3 z 3 3 a 3.a 3.a&> M -C! > a,, S 4= -g 5 U CD e 43 u u I g 5-8 -a S > cd 3-3 z 5 5 CL. * ' "3 S a O CD Cd 43 I" H a u c -Si a <j 8 U cd CJ 43 & I s cd fflj 3 3 a. en -C) b ed (D a -2 "a3 2 s d u fees c S "^> : g &43» s

135 Appendix 3?.2 ^ E > > >» > 'V > 'V > > > > z

136 Appendix Z 3 «2S3 l <S s : ^ & i ON i s2-5?-' «i 3 a t «ON 55 <N NO ON ^ ON Neupane, Berger, 95 Krschel, J3 m => X ON 2? ON \ 2 ^ ON - r < N > ffi -i -a H «S " 3S u «BJ t-- Ov ON 00 t-. ON ON O B es d «T3 TJ M K 3 C pi S3 ffl ,! ^ 3, J- "O * 'V v > v > ill n «S I i a -3 II N 5 ^ g c 5-2fel w G as q ^ S S 3 a.a ^ W H If Is s I I?» 5 II S~ IS a I'd -5 t: E*. a

137 Appendix a N If R a a a S2 p 5 S'B 5-2 I U S H 2^.3 S Z ffl.3 60 J3.3 «3 z OS ON ON ON,_rO\ j>j> M? J& I B 6 a» N yg «a a a a S3 "O a a 53 &.S ^ a lill» a * >- 2 >, a Hi S &H n a a-e a a-ei h 3 u <0 UI J3 53 ^ ^ W W 3 - B S * 5 s (J «n > a "it u a ~ _ * CO X ^ ' 60 («^ PH. Q Pi < P a 8 * I-. a m '8 "H S ^> 3 5 p T) b * M > H T) ^ a> b ti,m 0 S b 60

138 Appendix OS ON _-.2 2> 3. s 2 J u- H O" ffl 9 <u a M S3 ta, ^ "S si??; S U 3 a.2? ^ fl «K ^ H rt fl h - ^ «i JI u ffi BC H - a -a il3 T3 < S 2 r<* a - r 2 8 E td O ^ j - S B0 Tt3 "=3 " 3 I"? s II J S <* O m S "3 -O en.^ *- a <^ g <u a s'al'a 4> rt w > gji N (i. 2 P 00 S a I a - 2 ~> 3 J<! O. a 3 & CO M ^ 3 s < ^ «SS- #a ^ E S :e -, 'S } J-3 I-a ^ R2 s ifl^. a 2 ">.2 a <*- > > C fi <L) <> cu tj PL, H 3 C2 ^5 0< H s U ca U «O 3 O 5 S 3. «O ^ ^ ^ -^ a a _ 2 <4) M & ' " ^ «-2.Q y.a -^ Q «a K- -S < r Ja 2 a u R 3 J3 hfl r/l S '& =3 s; J c m g) g I'l

139 Appendix X <N 3 a 3 z explre/sahel/sahelvegetatin l.html as retrieved n 9 August, Getahun, 976 Tyang et al., 995 Adyetal., 997 Zemede and Tadesse, 200 r- \ 3 0\ ~n < a 2 ^> M pa OO! u Getahun, 976 Berger, 994 Annymus, 200Id i e S3 e z (4H. en ii Puyvelde (van) et al., 985 Hines and Eckman, 993 Regassaa, 2000 IT) GO ON 3 3 PH H z" ffl m PH" H OH t4-t a PH'CQ < _H PH"PQ" < P-T u PH" H" 3 Pi a. I a. u "3 O J P. " 03 &? < 3 3- J "8. a & J «s 5 3 g " CD & C T3 =& g.s~2 S " ^.s u 'a O C «3 ^5 (U 3 <D PH 03 sc- Q»- " i M 3 y g S O trt 00 (L)?? «S "M 3 2 ra hj W HH Sw. " i <4H 03 t B _«3 l-a <@s g i v > > > > > > > > > > > > > > > > > > > > > > v J. 5.. > > si S3,0 (H u S s a ^ /3 U i Xi S u PC fix) x in (5 ed <u 's 'a. 3 CO «I * >!. a S 3 5 c a ^.5 3 cd <D a 55 J3 u (2 <!, a 8 «.0.a cd <3 I t '+-» 33 t/3 /3-2 S a S.- '«V O 2 03 'm "S g -3 a & S S -4 ^ Q I Q C 2 S 5 O U 03 I e z 6 3 S 5 CD u 03 c w > 5 S -i.2 «J 3 v I s < 55, S t S 5 2 >> < pa 2 '5. g 00 u ll pa S 6 i S t s < PH.a t 2 S a ^ is u 2 < S. N a K C «03 03 > zzc J 3 K i O 2 -< si "3 c zsg-da UH' M 55, 'S i O 0) 30 (L> J i S 8 3 u <u 2 s i C 03 2 ^ 2 <ri th O PH

140 Appendix in ON 73 c 3 O 3 «^> 60 PH Pi Puyvelde (van) et al., 985 Hines and Eckman, 993 Regassaa,2000 Stckbauer, ON c ^<N T3 03 ^-< K U en > Si Smith, 969 Duke-Elder and MacFaul 972 Schmidt, 2003 Aubreville, 936 Dalziel 937 Irvine 96 Cassady et al., 990 Schmidt, 2003c Wdland, 997 Irvine, 96 Schmidt, 2003c The Wrld Bank Grup, 2003 Erah et al., 2003 SO r- Os a a Matzigkeit, 990 Mwangi, 996 IDTG and IIRR, 996; Cremlyn, 978 Dipelu and Ndungu, 99 Juliette de Ba Levy, 99 Stll, 988 Berger, 994 Adyetal., 997 Mansingh and Williams, 998 Stll, 988 Berger, 994 Adyetal., 997 Mansingh and Williams, 998 Regassa, 2000 f H u 03 PH T-O) X X u s 3 O 6 & 3 3a " 5-a I s! 0 "8 ' O ID 03 *- >S 2 e B 3 S CD 03 H 0- B CD (A 03 a> T3 2 c - 3 a ««.. <L» M O- "2 i CD 0 e t* t ^ 0 g «3 8 a J2 J O P,J3 M 60 «9 ^ c" g u te"3. S'S 00 J JJ c c 3 Pi M « * ^ v * v > > v > > > > > > > * v > 'V > >» (D H CD CD H H O 0 CD CD CD t-< H S CD B J >> P 2 % CD Q 2 c s 3 SJ CD 03 CD O O m CD 03 CD 2 2 "t "3 Q 3 cj CD 03 0 (73 I S 00 <e <u J3 u B 0 s 03 Cfl t-< 0 PH i. 3 <* 3 CD 3 O as.g I 2 O (/: O s z s S ja 0, n CD 03 I w *W3 s 03 z I. s a c SB CD 03 CD O 03 a I 2 z > L«O 8» a c a M >i 3 Si 3 s O CD 03 CD a 03 a " 03 a pa s S S O U O '5. S s S 3 'i.

141 Appendix w a < t-- : 3 S S 2 ^ n i S O w a c X d> ^ J r- 00 <N - S 5 ^ «O fa a 2 Os X - ^ s ^ ^ -Jr "M u a! ^ "S «-C- ON 60 3 a> C. d u t3s2 ^O ^» w 2 rf 2l ~~-,g '5c «ID w S <d M BH B! SB O ei B2 03 I s. s O i I 5a Kl is *5» JS-a s^ 5" 2 q x> 00 c Oil M JS a c eg V 'V > 'V > v 'V > > U a I S 3; 3, I-" s CD <3 "3 S -S-??i CT If K <] S t/3 a n s; 'M K m ^ Q? >

142 Appendix Stanerand Butique, R. 937 Watt and Breyer-Brandwyk, 962 Glver etal., 966 Lemrdant, 97 Van Puyvelde et al., 977 Lindsay andhepper, 978 Harjula, 980 Gelfand et al., 985 Kayngaand Habiyaremye, 987 Heine and Heine, 988 Baerts and Lehmann, 989 Gachathi, 989 Plygenis-Bigendak, 990 Kkwar, 993 Tedesse, 994 He et al., 2000 Araya, 200 He et al., 2002 Nyazema, 2002 s 2! OO s rt OS ^ S S u OS OS CD? OS OS OS c" c cd Kaynga and Habiyaremye, 987 Cunningham, 990, 995 Maundu etal., 200 Michael, 2003 SO Os Os T3 9 Q z Adamantidis, 956 Plygenis-Bigendak, 990 Kkwar, 993 a. 03 PH f PH" PH t" u PH" m <J PH" m ffl Whle plant (used t make smke andhanging buquet) u el < 2 a u c ^ CO s a. '6 < a> <u CO ^ O ^* CO a. c <4-. cd CD S s cd P3 ih M c /rv IS a! a CD < v > > ^ > > > > > u CD H CD CD <D E- CD u t-h H <u CO cd S3 J u 3 CO cd cd cd <D O cd 6 e > cd 3 8 'V, u cd CD 03 CL> O 03 a 3 CD Cd 0> cd a 9 CO fflj 0> > > cd 3 e cd T3 S a c I 3 J2 C cd Ji Jl 3 B 3 l >. ±& cd > a >> S (a 5.g 3 in "5 Q I is W3 5 a a c 3 "5 s J- s CO -C CD S CU ft? ^ ^^ <* C4H* (J a S ^ E?> 5 «J3 s s g la _p 5 B g g a. S c«s» a> a CO ^ O T3 «O P3 2 < s S c 8 a 6 Q

143 Appendix is a <Z CO M.da 5? -, g * c 2..S S «U- ^ i «8 3 "3 O Q CO pa "2 3 2 C 3 <D 3 < J= =. 2Q5 c m a ~s 0) g g p S On 2 ".-' - _ S «3 -g «N «M^3«ON _ t3 "!!» O " " O ~ «< * ia w at i-l O T3 VH S-a u is. c c 2 E T3 3 0) <D > 3 - _ s-^ & a 3 X5 is -a M S O S X u 3 C 53 -a u a. S» s M 2 B 5J 8 5< 2 S S «* 00 eg * M3 cs H3 'V'V'V'V'V'V'V'V -ij ^ ' a, s I 3 ts t> G OT c ^ >. ^ 3 ffl e 3 ^ 3 3 e ^"M 2S rrl s ^.5 ^ O a ^ 3 ct ^

144 Appendix Cunningham and Mbenkum, 993 Schippmann, 200 Wrld Agrfrestry Centre, 2003b USDA, ARS, Natinal Genetic Resurces Prgram,2004 CQ OH" CO s.2 % -a 3 c/> t < <*H f* Os Os W c 2 m Vlfva andpatcka,2003 Stedman, Wilcx & Fllett C., 942 OH Wehmeyer, 966 Fx and Nrwd-Yung, 982 Taylr, 986 Phillipsn, 995 Saka and Musnthi, 994 Mizrahi et al., 2002 u- U Kristensen, ethipianplants/shinasha.html Getahun, 976 pa in Os Os a S H Os Os u B i > H ON ON "ed "S (D H ON ON H ON ON "S (U tu > H IT) ON ON *e3 (D H CJ in ON ON Xi B u H en ON ON i S s b s O Os.s ^ BH" 8 OS Os ^ t «s O.3 m >» O A CQ. 'C ss.2 O vj > 5 "O S <L> Qs u noh <Ji St > CO ed <D C "S, u (4- Cd 03 cd u > >» > > > v > > > > v > > > > > > - s (/3 J3 05 C/5 05 CO e e e 0J 03 > a 03 O ai 03 a> 2 'S u 2 'S ed 60 O J E 03 HJ 4> ed <u ed a ed u ed ed B ed u ed O ed s ed <U ed HJ ed ed s ed CD ed <U O ed s ed _ ed ed s 3 cd <D O ed 5 B Si C ~3 e " 3 B O O DO Z.sl 'S 3 c«3 <S O a & a O S 3 C3 Z ed B B 3 B 5.*' X a "53 a a <J O R S s O a «B 3 J a K "S b t X CO g«a -8 C» -2; i i ed? > H-H* *' X Q ^j. Si a s K (3 e 0 c 2 K > I # a c 2 s* -^ S) CQ i S 5r a a > a -5 ed ffl s a ^5 ^.3 03 > O M s-a g -a hj ^) a a. O

145 Appendix m ON ON C y J3> S a- 5 ON ON a W a r/l a> 2 _f c/t If ^_ u g CO > El Tahir et al., 998 El and Satti, 999 Hussain et al., 999 Bhatt etal., 200 Kumar, 2003 m 00 O BH t S3 8-. c z ON ON? ej CO OS - 3 if?^ s S CO t» Os Os E? CO ON ON? CD pa ON ON u? CD 03 Hmer etal., 990 Hmer etal., 992 Dagneetal., 993 Hines and Eckman, 993 van Grinsven et al., 999 Lukwa etal., 200 ON ON i a W a t» c 3 tn e IB t CJ s C 03 CD T3 CD a u N ON ON ON "a u s l-h Malnza et al., 992 Dipelu et al., 992 Trt and Hassanali, 997 Lwande et al., 999 m a >ri ON ON ON ON ON *3 *«00 5} a - ZZ U UH CO CO CO CO c"«c" PH CO < CQ H CD <D V) 5a - «S 2 3 <D W) 5a ^ 03 O 2 ed CD w 03 O > S < 55 a CD in CD 03 CD <n CD 03 CD CD 5a 03 CD CD O c > > > v > > > * > > > > > v 8 CD CD CD CD CD CD CD f5 CD CD CD CD «3 CD 8 CD U 5 I E 3 U 8 3 U CD 2 d Z I CD 03 CD O g t O d Z CD -2 i-i O c B CD 03 CD CJ -2 O U CD 03 (D O 03 "S O CO a! CD 03 CD O O s S CD 03 CD O 03 O g W i i 03 -i CD 03 CD O 03 t/3 CD 03 CD O 03 "S & 60 J3 J5 V CJ 6 ^^ LIJ St* M 8 d. CO J l en (a t c Si ; W id *l *a s Z 5. >> NS <D? N N i a e a a "3 S "3 K d Q O X u, «3CO S? CD -Q > S 3 0- S a.c "5 a $ a (/I a.a : 8 S a a X W P a a e 3 e 5 a c*3 > 5 ^> a a d- c CO K a s a

146 Appendix ft w i? 2 ri D.3 & m ON "O ON e 5 3S nj s O rt c w & > u fi 4> * s T) 3 tti «y > y v y y I? S.2 u.a t * I S"3 e I, H < -a & a ai 3 e i c I &'l.s u _s 33-3 a e i ^ ^ 3 O rt S «5 Sj u eel ^ *: yi *! 2 S. ^ c 3f5 & = I- S J) 5. S S & ti ft 6» & 5 3 Q H <> O I a

147 Appendix 2 CO a <^ 2 g <N " > <->! i> - S? O e <D u m w PQ S B ^ PQ -T ""' *- u 3 a " «S a ^S-N si. fcc? -^ Ml OS IS- ^-si PH tn 2 J..- S P3 2,O\0\ 9 CA u (N O <N O *ffl fl> v-i as id u q a i4 PQ <> > 6 J w "8 «a j? «t» 3 "2 rt G C «t??j2 Is PD 2 8-P a I I 3 U e w s " l 2 il 3 6 I e S ' 5'S.S <3 55, a S? 5 gp % Q Q

148 Appendix c S H

149 Appendix 2 62

150 Appendix 2 Appendix2. Backgrund infrmatin ntwselected plants fr an in-depth scientific study Tagetes minuta L. Origin and mrphlgy /Tagetes minuta Tagetes minuta (Asteraceae)) is an erect annual herb, grwing t a height f -2 m (Fig.A2.). The plant resembles the garden marigld (T. erecta L). Wild marigld, as it is cmmnly knwn, is a prblematic weed f pastures and numerus crps in East and Suth Africa, Suth America, and Australia (Sule, 993). The undersurface f the leaves bear a number f small, punctate, multicellular glands, rangish in clr, which exude a licrice-like arma when ruptured. Glands may als be fund n the stems and invlucre Fig. A2.. Tagetes minutal. (Asteraceae)plantshwing (a)theyungand (b)thematureaerialparts. Flwersare hermaphrdite (havingbthmaleandfemale rgans)andareinerect clustersattheendsfthebranches.flwer heads arenshrt stalks.leaves are slightlyglssy green,andarepinnately dissected int 4t 6pairsfpinnae. Leafmarginsare finely serrate.heads numerus,usually in flat-tpped cymes,fur rfive fused invlucrecylindricalbractssurrundeachhead (b),8-2mmhigh,apex3-5-tthed yellw-rangerayflrets usually 3 perhead, rays -2 mmlng;disk flrets usually 3-5perhead, and 0 t 5 yellw-range diskflrets percapitula,crllas are2.5mmlng;lngerpappusscales2-3mmlng,thethersare mmlng.thedarkbrwnachenes (c) are0 t 2mmlng,with apappus f t 4tiny scalesand 0t 2retrsely serrulate awns,which are t 3mmlng (Adptedfrm Sule, 993;Wagneretal.,999). 63

151 Appendix 2 bracts. Fur r five fused invlucre bracts surrund each head. The seeds als have an unpleasant dur and can reduce the value f grain harvests when they are mixed in. The genus Tagetes belngs t the Asteraceae family and cmprises 56 species, 27 f them annuals and 29 perennials. Tagetes spp. are grwn all ver the wrld as multipurpse plants (Sule, 993; Vasudevan et al., 997). Of these, T. minuta, T. erecta, T. patula and T. tenuiflia are the mst cmmn (Vasudevan et al., 997) with T. minuta being the mst studied species. Tagetes minuta is a suitable essential il-bearing species and therefre cultivated in several cuntries such as Egypt, France, Mrcc, Brazil, USA and India fr its applicatins in the perfumery, flavuring and pharmaceutical industries (Chalchat et al., 995). Ethnbtanic uses f Tagetes minuta Tagetes minuta has a lng histry f human interest and is used as beverage, cndiment, rnamental, medicinal decctin and in ritual practices (Rees, 87; Mrtn, 98; Sule, 993). The plant's leaves, stems, and flwers are used. In recent years, there has been an increasing interest in the use f T. minuta by indigenus peple in India (Anjaria, 989). Wild marigld is smetimes an alternative hst fr Sclertinia sclertirum, a fungal pathgen that can infect a variety f crps (Sule, 993). The New Wrld peples have been using T. minuta as a beverage, a medicinal tea, and a cndiment since pre-clumbian era (Rees, 87). The lcal names f T. minuta vary by regin, ethnicity and/r by dialects. A beverage is prepared frm T. minuta by steeping a "half-handful" f the dried plant in ht water fr 3-5 minutes. The beverage may be cnsumed warm r cled, and may be sweetened t individual taste (Neher, 968).Fr medicinal use, a decctin made by steeping a "duble handful" f the dried plant in biling water fr 3 t 5 minutes is used as a remedy fr the cmmn cld; including upper and lwer respiratry tract inflammatins, and fr digestive system cmplaints; stmach upset, diarrhea, and "liver" ailments. In many native regins f the wrld, the plant is used in ppular as anthelmintic, diuretic, antispasmdic and t treat stmach and intestinal diseases (Amat, 983). The plant's decctin is cnsumed warm, and may be sweetened t individual taste (Cavanilles, 802; Pardi, 959; Neher, 968). Tagetes minuta is used as a cndiment in Chile and Argentina. It is ppular in rice dishes and as a flavuring in stews. In nrthern Chile suic (a preparatin f T. minuta that enhances flavur f fd) is s highly prized that many peple cllect wild plants t dry a sufficient supply t last the winter (Sule, 993). Leaf infusins and extracts frm Tagetes spp. have been used in flk medicine t treat intestinal and stmach diseases and sme f them have been fund t have biactivity against Gram psitive and Gram negative micrrganisms (Tereschuk et al., 997; Brussalis et al., 999). Tagetes minuta is ften referred t as a weed in many parts f the wrld including Suth America (Cabrera, 97). Many Latin American farmers wh d nt practise industrialized agriculture will leave plants f T. minuta in their fields. This secnd crp is beneficial in several ways: first, rapid grwth f T. minuta quickly shades ut ther plant species that may be f less use t the farmer, secnd, it can be harvested fr persnal use, r fr sale in city markets; and third, it has been reprted t aid in the retentin f humidity in the field (Jimenez-Osrni, 99). Tagetes minuta is cmmercially grwn and harvested fr its essential ils, which are used in the flavur and perfume industry as "Tagetes Oil" (Lawrence, 993). The il is als a majr cmpnent in mst majr fd prducts, including cla beverages, alchlic beverages, frzen dairy desserts, candy, baked gds, gelatins, puddings, cndiments, and relishes (Leung, 980). Brazil is ne majr prducer f T. minuta fr Tagetes Oil (Craveir et al., 988). Wrldwide prductin fthe il was arund.5 tnnes in 984 (Lawrence, 985a;b). 64

152 Appendix 2 In Kenya, an infusin f T. minuta is used fr the treatment f snake bites in the Lu and Kamba cmmunities (Owur and Kisangau, 2006) and prtectin against msquit bites in the tribes f western Kenya (Seyum et al., 2002). Eclgy /Tagetes minuta Tagetes minuta is native t temperate grasslands and mntane regins f suthern Suth America (McVaugh, 943), including Argentina (Espinar, 967), Chile (Reiche, 903), Blivia (Perkins, 92), Peru and in the Chac regin f Paraguay (Herrera, 94). Starting with the Spanish cnquest f Suth America, it was intrduced int Eurpe (Jrdan and Ocana, 955), Asia (Cherpanv, 98), Africa (Hillard, 977), Madagascar (Humbert, 923), India (Ra et al., 988), Australia (Webb, 948) and Hawaii (Hsaka and Thistle, 954). Tagetes minuta is nw a widespread weed in Africa, Suth Eurpe, Suth Asia and Australia (Cabrera, 97). In Kenya, T. minuta is naturalized primarily in arable lands as a weed ever since its intrductin frm Suth America in the 920s (Stadler et al., 998). Tagetes minuta is ften fund grwing in disturbed areas during early successinal stages. This affinity fr disturbed sites has allwed the species t clnize many areas arund the wrld (Sule, 993). This nxius, rapidly grwing armatic herb frms a dense grund cver at higher elevatins ( m). The seeds cling t the hair f animals and are dispersed by bth dmesticated and wild animals. Twards the end f its grwing seasn, the aerial parts f T. minuta dry up and may easily be destryed by fire, but new clnies are frmed rapidly in the fllwing seasn frm seeds depsited in sil. Chemtypes /Tagetes minuta The cmpsitin f the essential ils f many species f Tagetes has been reprted previusly (Machad et al., 994; Lawrence, 2000; Stjanva et al., 2000) and shwed significant differences in their cmpsitin (Lawrence, 985b; Graven et al., 99; Zygadl et al., 993; De Fe et al., 998; Senatre and De Fe, 999). The Tagetes species can be unambigusly differentiated by the chemical cmpsitin f their essential ils (Rss et al., 98; Hethelyi et al., 987; 989; Cres et al., 989). The GC and GC-MS analysis f T. minuta ils indicates that plant rgan, stage f grwth, sil, climate, chemtypes, sunlight, latitude and methds f extractin all affect the cmpsitin f the il (Hethelyi et al., 987; Lawrence and Reynlds, 992; Chalchat et al., 995; Dagher et al., 999;Gil et al., 2000). Tagetes minuta is rich in secndary cmpunds, including acyclic, mncyclic and bicyclic mnterpenes, sesquiterpenes, flavnids, thiphenes, and armatics (Rdriguez and Mabry, 977). There is evidence that the secndary cmpunds in Tagetes are effective deterrents f numerus rganisms, including: - fungi (Chan et al., 975), including thse pathengenic t humans (Camm et al., 975), bacteria (Grver and Ra, 978), rund wrms in general (Lewe, 974), trematdes (Graham et al., 980), nematdes (Grainge and Ahmed, 988), and numerus insect pests thrugh several different mechanisms (Jacbsen, 990; Saxena and Kul, 982; Maradufu et al., 978; Saxena and Srivastava, 973). Many clsely-related plant secndary cmpunds have demnstrated medicinal value in humans (Kennewell, 990;Krlkvas and Burckhalter, 976). In Argentina, dihydrtagetne, a-phellandrene, limnene, -cymene, as well as the ismers f p-cimene, tagetne and tagetenne, were the majr cnstituents f T. minuta essential il (Gil et al., 2000). In Saudi Arabia, GC and GC-MS analyses cnfirmed the presence f tagetne (.52%), 5-ctyn-4-ne,2,7-dimethyl (.52%), Prpanedinitrile,dicyclhexyl- (0.45%) and 2-pinen-4-ne (8.03%) t be the main cmpnents with lesser amunts f l-acetxy-p-menth-3-ne (0.7%) and 9-ctacenamide(Z) (0.48%) (EL- Deeb et al., 2004). In India, the freshly distilled T. minuta il cntained cimene 54.97%, 65

153 Appendix 2 and dihydrtegetne 32.58%as majr cnstituents (Singh et al., 992),while in ther studies (Z)-tagetne, (Z)-p-cimene, dihydrtagetne, (Z)- and (E)-cimenne were fund as majr cnstituents (Chalchat et al., 995; Bansal et al., 999). In Egypt, the main cmpnents f the T. minuta essential il were mnterpenes f which trans- and cz's-tagetne were present in 52.3 % % (Mhamed et al., 2002). In Iran, the GC and GC-MS analysis revealed the main cmpnents t be a-terpinel (20.8%), (Z)-P-cimene (7.7%), dihydrtagetne (3.7%), (E)-cimenne (3.3%), (Z)-tagetne (8.4%) and (Z)-cimenne (6.%) (Mghaddam et al., 2007). In ther studies, ut f ne hundred and fur chemical cmpnents, the majr cnstituents f T. minuta essential il were tagetne, E/Zcimenne, E/Z-cimene, germacrene, limnene, trans-anethle and dihydrtegetne (Hethelyi et al., 986c). Frm these analyses, it culd be seen that the cmpsitin f the ils varied accrding t a number f factrs: the harvesting lcatin (Cravelr et al., 988; Chalchat et al., 995), grwth stage (Hethelyi et al., 986b), plant parts (Hethelyi et al., 986a; Chalchat et al., 995), sil type and nutrient status (Graven et al., 99) and chemtypes (Gil et al.,2000) etc. The bilgical prperties f the essential il /Tagetes minuta Validatin f sme f the flklric claims has shwn that T. minuta cntains cmpunds that have a wide range f biactive prperties. Z-P-cimene and dihydrtagetne, which are cnstituent cmpunds f the essential il f T. minuta, were fund t be antiviral, active against carnatin ring spt (CaRSV) and carnatin vein mttle viruses (CaVMV) (Okiga and Rajamannan, 997; Singh et al., 2002). The essential il f T. minuta generally affect a variety f micrbial rganisms (Rss et al., 98; Hethelyi et al., 989 Suza et al., 2000; Senatre et al., 2004). Allelpathic activities f Tagetes spp, particularly thse against nematdes (Melidgyne spp), have been reviewed (Akhtar and Malik, 2000; Kkalis-Burelle and Rdriguez-Kabana, 2006). Nematcidal activity f T. minuta rts is attributed t thienyls while the bicidal cmpnents f the essential il frm flwers and leaves are terpenids (Vasudevan et al., 997). Dihydrtagetne and Z-P-cimene islated frm T. minuta il shwed strng nematicidal activity against eggs and juveniles f Melidgyne incgnita with dihydrtagetne shwing a higher level f txicity than Z-Pcimene (Adekunle et al., 2007). Sme evidence suggests that the secndary cmpunds f T. minuta are inhibitry t parasitic rt nematdes and ther micrbes (Sule, 993). While cartenid pigments frm Tagetes are useful in fd clring, the vlatile ils f T. minuta are used as flavur cmpnents in fd prducts and as perfumes, and have a suppressive bilgical activity against sme insects and pathgens (Vasudevan et al., 997). Tithnia diversiflia (Hemsley) A. Gray Origin and mrphlgy /Tithnia diversiflia Tithnia diversiflia is a member fthe sunflwer family, Asteraceae. Tithniawas named fr Tithnus, a legendary Trjan lved by the dawn gddess Es, wh turned him int a grasshpper. Tithnia diversiflia is native f Mexic and Central America and is cultivated fr its beautiful flwers and enrmus size. The genus ccurs thrughut Middle America and the West Indies and has becme naturalized arund the trpics. Depending n the gegraphical lcatin f the plant, it may be either annual r perennial, 2-3 m in height with upright and smetimes ligneus stalks in the frm f wdy shrubs (Fig. A2.2). The plant is mstly leafless n its lwer parts. The leaf arrangement is helically alternate. The typical leaf is 5-30 cm lng and has a minute rughness (scabridity) n the upper surface. The whitish petile (acuminate) is fringed halfway with blade tissue at the terminus f 66

154 Fig.A2.2. Tithnia diversiflia (Hemsley)A.Gray (Asteraceae) (Mexican sunflwer/wild sunflwer), (a)shws flralpartsinearlystageswith budsand (b)shws flral partsinlatestages.leaves:mstlysubvate,acute,serrate,0-40 cm lng, lng (2-0 cm) petilate, simple r mstly 3-7-lbed with acute serrate lbes, smewhat glandular, slightlygrayishbeneath,puberulentnbthsurfaces;petileslightlybilbedatbase.itispubescentnthelwerside, which appears greyish. Leaf veins areparallel. Peduncles (b):are 0-30 cmlng; heads large and shwy, 5-5cm wide; invlucre fbracts in 4 series;ray-flrets abut 2-5,with yellw ligules t 6cm lng. Flwers: similarin appearance tthecmmn sunflwer but smaller(a).the flwer disc is abut 3cmdiameter withyellwpetals,4-6 cm lng. Each mature stemmaybear several flwers at thetp fthe branches. Seeds (b) and (c):wind, waterand animals easily disperse the light seeds. Achenes: cuneate, 4-angled, appressed-puberulent, 5 mm lng, 2-awned which three whitish midveins vascularize a mitten-like blade with three r five prminent lbes (Kkwar, 994). Occasinally, new shts pssess unlbed leaves. The plant's flwers are a favurite f bees and are frmed by an range-yellw inflrescence cmpsed f many small flwers crwded tgether. Arund the perimeter, -3 ray flrets (ligulate flwers) frame tubular disk flrets, which smell like a daisy (Fig. A2.2c). After pllinatin, the inferir vary f each disk flret develps as a hairy, gray, flattened, dry, ne-seeded fruit (an achene) hidden by papery, brwn-tipped bracts that, at maturity, are arranged int a hemispherical mund (Kkwar, 994). Ethnbtanic use f Tithnia diversiflia By etymlgical descriptin, the Kenyan cmmn ethnic names include amng thers, maruru, maua and amalulu (fr Luhya), maua makech (fr Lu) and amaua amarr (fr Kisii), all implying that the plant is bitter t the taste. The African farmers have many uses fr the plant, the mst ppular use being as an rganic fertilizer fr vegetable crps in either cmpst r a tea frm (Jama et al., 2000). Traditinally, Kenyans use it fr rnamentals, livestck feeds, wind breaks, envirnmental cnservatin (fr bth sil and water cnservatin), hney prductin, curing f fire-cured tbacc, fuel wd (dry stumps), live fences, bundary demarcatin and as a medicine in the frm f leaves' infusin fr cnstipatin, stmach pains, indigestin, sre thrat and liver pains and diarrhea in livestck (Kkwar, 993). Ady et al. (997) reprted that farmers wrking with the Kenya Wdfuel and Agrfrestry Prgramme (KWAP) identified T. diversiflia as a ptential insecticide t cntrl termite infestatins in farms and hmes. One farmer's experiment with tea frm either fresh leaves r the ash f T. diversiflia, Cassia siamea and C. spectabilis applied t 67

155 Appendix 2 affected trees, prvided prtectin frm termites fr up t 45 days. Anther farmer, wh had a prblem with undergrund termites, made a slutin based n fermented extracts f T. diversiflia and Melia azedarach, which cntrlled the pests when the cncctin was made and pured int the termite mund. After tw years f research, farm results shwed the mst effective treatment t be a slutin made frm T. diversiflia, Vernnia amygdalina and Agave sisalana (Ady et al., 997). Nt nly did this slutin cntrl termites, but it als cntributed t sil fertility (Nagarajah and Nizar, 982;Wanjau et al., 997).By using such lcal resurces, ne avids the need t purchase the hazardus synthetic chemical pesticides. Mrever, farmers were reprted as being enthusiastic abut the use f Tithniagreen manure and its effectiveness (Ady et al., 997). In Nigeria, there are ral reprts amng herbal medicine practitiners linking T. diversiflia with the treatment f menstrual pain (Owyele et al., 2004). In additin, T. diversiflia extracts are used in the treatment f wunds (Riingeler et al., 998) and diabetes mellitus (Takanashi, 998). And recently, in western Kenya, T. diversiflia has been discvered as a very imprtant rganic fertilizer (Wanjiru, 2003; SACRED Africa, 2007), enhancing the availability f Phsphrus t crps, which led t its recmmendatin fr bimass transfer technlgies in Kenya (Gerge et al.,2002 a;b). In Mexic, the place f rigin f the plant, it is used t treat sprains, bne fractures, bruises and cntusins. Peple in Mexic grw the plant in their gardens and use it fr relieving dermatlgical prblems (Heinrich, 998a). In the Lwland Mixe, it is used rally t treat malaria and ther frms f fever and tpically t treat hematmas and muscular cramps (Heinrich et al., 992;Heinrich, 996; Heinrich et al., 998b). It is als used as a liniment in Yucatan (Batchelder, 200). These medicinal uses may result frm the similarity f the flwer heads f this species t the nes f Eurpean arnica (Arnica mntana L.). This assumptin is crrbrated by its ppular names: Arnica de la mntana and arnica. Berlin and Berlin (996) listed T. diversiflia as an imprtant remedy fr gastrintestinal cmplaints and it is cited as an anti-inflammatry and as treatment fr wunds and skin eruptins. In suthern China, peple use T. diversiflia t treat skin diseases (such as athlete's ft), night sweats, as a diuretic, hepatitis, jaundice and cystitis. In Taiwan, the plant is sld in herbal medicine markets as an infusin t imprve liver functin while in Thailand and Japan, it ishighly regarded as an rnamental plant. Eclgy /Tithnia diversiflia In Kenya, T. diversiflia is fund grwing in Western and Central Prvinces as well as castal regins and parts f the Rift Valley, between latitude: FO'N and lngitude: 38 0'E. It is a bushy perennial weed and a valuable green manure (Thijssen et al., 993;Wanjau et al., 997). It was intrduced in Kenya frm Central America as an rnamental and escaped frm cultivatin and nw grws as a wild plant n the fields, in hedges, alng radsides and n wasteland/disturbed areas, nt nly in Kenya, but als elsewhere in the wrld (Akbundu and Agyakwa, 987; Smith, 99; Space and Flynn, 2000). Althugh smetimes cultivated (Kendall and van Huten, 997), T. diversiflia, is nw a pantrpically distributed weed. Chemtypes f Tithnia diversiflia The chemical cmpsitin f essential ils frm T. diversiflia has been previusly described (Lamaty et al., 99; Ku and Chen, 997;Elufiye and Agbedahunsi, 2004a; Mrnkla et al., 2007). Fr instance, Mrnkla et al. (2007) fund leaf il t be cmprised f an abundance f a-pinene (32.9%), (3-caryphyllene (20.8%), germacrene D (2.6%), (3-68

156 Appendix 2 pinene (0.9%) and, 8-cinele (9.%). Germacrene D (20.3%), P-caryphyllene (20.%) and bicyclgermacrene (8.0%) characterized the il f the flwer while a number f aliphatic fatty acids and a diterpenid cmpund, sandaracpimaradiene, that were present in the flwer, culd nt be detected in the leaf il (Mrnkla et al., 2007). The bilgical prperties f the essential il frm Tithnia diversiflia The validatin f sme f the flklric claims have shwn that T. diversiflia cntains cmpunds that have a wide range f biactive prperties, namely, cyttxic (Wu et al., 200), anti-malarial (Elufiye and Agbedahunsi, 2004b), anti-inflammatry (Rungeler et al., 998; Owyele et al., 2004), ptential cancer chempreventive (Gu et al., 2002), antiamebic (Tna et al., 998), antiviral activity against human immundeficiency virus type- (Cs et al., 2002), anti-diarrheal (Tna et al., 999), anti-amebic and spasmlytic activities (Tna et al., 998, 2000), and analgesic prperties (Owyele et al., 2004). Tagitinin C, an anti-plasmdial sesquiterpene lactne, has been islated frm the aerial parts f T. diversiflia fr develpment (Gffin et al., 2002; 2003). (-)-Germacrene D, a chemical cnstituent f the essential il f T. diversiflia, increases attractin and vipsitin by the tbacc budwrm mth Helithis virescens (Mzuraitis et al., 2002). Cmpunds islated frm the aerial parts f T. diversiflia shwed cyttxic activity against HL-60 leukemia cellswith IC 50 values ranging frm 0.3 t 3.0 um (Kurda et al., 2007). Tithnia diversiflia has als been reprted as a ptential candidate fr biinsecticide preparatins against Callsbruchus maculatus (Cleptera: Bruchidae) (Adedire and Akinneye, 2004). 69

157 References 70

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208 References 22

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210 Samenvatting Samenvatting Ditprefschrift beschrijft de effecten van verscheidene etnbtanische plantenextracten p het gedrag van de teek Rhipicephalus appendiculatus (bruine rteek), de belangrijkste verdrager van East Cast fever (ECF) p rundvee in Centraal- en Ost-Afrika. Gebruik makend van participatieve nderzeksmethden, de traditinele kennis ver tekenbestrijding nder de bevlking van de Bukusustam uit West Kenia werd verzameld met het del te achterhalen welke plantensrten gebruikt wrden m teken, die een bedreiging vr de veestapel vrmen, te bestrijden. Dit resulteerde in een lijst van meer dan 50 plantensrten vallende nder 0 genera en 5 plantenfamilies. Een selectie van 8 plantensrten uit deze lijst werd gebruikt m etherische lien te maken, welke vervlgens in het labratrium gebruikt werden in gedragsexperimenten met teken. Uit deze eerste experimenten kwamen de planten Tagetes minuta (geelgren afrikaantje) en Tithnia diversiflia (Mexicaanse znneblem) naar vren als het meest geschikt m verder getest te wrden. Dr middel van twee-keuze-experimenten in het labratrium werd vastgesteld dat etherische lien afkmstig van T. minuta f T. diversiflia een dsisafhankelijke, afsttende werking p het klimgedrag van de teek hebben. Op stieren bleek dat R. appendiculatus een vrkeur hebben m zich aan de binnenzijde van de ren vast te hechten. Behandeling van de ren van stieren met het extract van zwel T. minuta als T. diversiflia resulteerde in een significante afhame van tekenbeten in het r. Deze afname werd k in een veldexperiment gevnden, waar de extracten naast een afname van R. appendiculatus k vr een significante afname van andere tekensrten p de runderen zrgden. Tevens bleek uit veldexperimenten dat T. minuta een sterkere werking p alle aangetrffen tekensrten heeft dan T. diversiflia. De in dit prefschrift beschreven resultaten tnen de ptentie van etherische lien van planten vr de bestrijding van R. appendiculatus en andere tekensrten, bijvrbeeld met een "push-pull" methde, en daarmee gepaard gaande dr teken verdraagbare ziektes in veehuderijsystemen in trpisch Afrika. 223

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212 Acknwledgements Acknwledgements Althugh the jurney t the PhD title was lng, frustrating and tiresme, can nw lk back n a lng rad f develpment f a scientific career with pride rather than with regrets. Kindly accept my aplgies if d nt mentin yur kind cntributin in this bk, fr the space is regulated and limited but d really value yur input t this wrk. This wrk has been cmpleted in the span f ten years and surely a great many peple have cntributed t its develpment in many special and different ways. I have interacted with many peple either directly r indirectly discussing research ideas, funding prblems, experimental design, results and their presentatin, statistics etc. This teamwrk mainly cmprised, prmtrs (Prfs, van Lenteren, Takken and Hassanali), cauthrs, fellw PhD students, my students, wrkers at the Schlarship Netwrk Centre, Nairbi, bistatisticians, Kenya gvernment fficials, ethnpractitiners and livestck farmers in Bungma District, western Kenya, the Labratry f Entmlgy stimulating family and Kenyans at Wageningen UR, my parents, my brthers, my sisters, my wife etc. T them all, am very grateful fr the fruitful interactins in a multi -trphic cntext. T my parents, Mr. Charles Wanzala Osundwa and Mrs. Phanice Masakhwe Wanzala, thank yu mst sincerely fr starting the PhD prject in my early life. Bth f yu were determined t see every child in the family receive quality educatin. Yu went thrugh hard times lking fr schl fees but the curse was wrthwhile. Mama, yu were a n nnsense wman t jke arund with cncerning educatin issues and the whle village new abut it! Thank yu mama, can nw understand why yu had t cane me nce fr running away frm schl. The cmbined effrts f parenting and schl administratin have nw yielded sme gd fruits, the thesis yu are nw reading! Last but nt least, wish t thank my brthers (Jacksn, Bejamin, Jseph and Cylus) and sisters (Emily, Gladys and Hildah) fr their lve, cnsistent supprt, cncern and encuragement thrughut the study perid. Yu did supprt me and my family a lt, thank yu fr this. T my family, thank yu fr yur kind supprt, lve and understanding the pain f tiling twards this gal. Thank yu fr yur interest, mtivatin, trust and particularly fr putting everything in perspective thrughut the perid f my PhD studies. Let this thesis remain a legacy leavebehind fr yu and future generatins t cme. 225

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214 Curriculum vitae Curriculum vitae Wycliffe Wanzala was brn n August in Mumias District, western Kenya. He started frmal educatin at Namamba Primary Schl (Musamba village, Matungu, Mumias District) where he attained a Certificate f Primary Educatin (C.P.E.) in 983. He prceeded t Lubinu Bys Secndary Schl (Mumias District) fr his rdinary level f educatin ('O' level) and btained a Kenya Certificate f Educatin (K.C.E.) in 987 with Divisin. In 988, he jined Nakuru High Schl (Nakuru District) fr his advanced level f educatin ('A' level) and btained a Kenya Advanced Certificate f Educatin in 989.In 990 he jined the Faculty f Science, Cllege f Bilgical and Physical Sciences, Chirm Campus, University f Nairbi and btained a BSc degree (Zlgy, Btany and Meterlgy with a BSc dissertatin in Applied Entmlgy) in 994. In 993/95, hewrked as a labratry technician in the Zlgy Department, University f Nairbi under Prf. Richard W. Mwangi and in 995/97 he was prmted t the psitin f Research Assistant/C-rdinatr (in Applied Entmlgy) in the Faculty f Science fr a prject between the University f Nairbi and Kenya Agricultural Research Institute (KARI). In 995, he entered the MSc class fr Medical Parasitlgy fllwing the award f a schlarship by the German Academic Exchange Service (DAAD) and graduated in 999. Between 996 and 997 he wrked n his MSc thesis n veterinary public health, immunlgy and epidemilgy f human and bvine taenises at the Natinal Veterinary Research Centre (NVRC), Muguga, KARI. On st June, 2000, he jined the Behaviural and Chemical Eclgy Department (BCED) f the Internatinal Centre f Insect Physilgy and Eclgy (ICIPE) as an Intern researcher in preparatin fr his PhD prgramme and studied n-hst tick behaviural eclgy. Between March and August, 200 he was a visiting scientist at the Department f Internatinal Animal Health, Freie Universitat, Berlin, Germany. Frm January 2002-current he became the Principal Investigatr (PI) f an ethnveterinary prject (cntrl and management f livestck ticks) funded by the Internatinal Fundatin fr Science (IFS), Sweden at ICIPE and the University f Nairbi, Kenya. In 2004 he was awarded a sandwich PhD fellwship by the executive bard f Wageningen UR, the Netherlands and in the same year, 2004, he became the funding Directr f The Schlarship Netwrk Centre, Nairbi and Cmmunity Research Assciate at Matungu Rural Herbal and Research Centre, Kenya. In March 2005 he was appinted Hnrary Lecturer, Schl f Bilgical Sciences, University f Nairbi, Kenya. In July, 2005, ICIPE ffered him a pstgraduate research training fellwship award under the African Reginal Pstgraduate Prgramme in Insect Science (ARPPIS). In August, 2005, he was appinted a Part-time Lecturer at the Department f Natural Sciences, Faculty f Science, The Cathlic University f Eastern Africa (CUEA), Kenya. In August, 2006, he was appinted Part-time Lecturer, Christ the Teacher Institute fr Educatin, St. Mary's University f Minnesta, USA, Nairbi Campus, Kenya. On st September, 2008 he returned t Wageningen UR t cmplete his PhD studies fllwing the award f the secnd sandwich PhD fellwship by the executive bard fwageningen UR. After defending his PhD thesis, Wanzala will g back t Kenya t cntinue with his research n ethnknwledge and its applicatin t the cntrl and management f livestck ticks and ther ecnmically imprtant parasites affecting humans. He will als be teaching at the university, CUEA/Christ the Teacher Institute fr Educatin, St. Mary's University f Minnesta, USA, Nairbi Campus. His research wrk will be based at the Schl f Bilgical Sciences, University f Nairbi. 'crrespndence:sundwal@yah.cm rwwanzala@htmail.cm 227