Efficacy of Some Trypanocidal Drug Against Trypanosoma equiperdum OVI in Experimentally Infected Mice in Debre Zeit, Ethiopia

Transcription

1 Europen Journl of Biologicl Sciences 7 (1): 7-13, 215 ISSN IDOSI Publictions, 215 DOI: /idosi.ejbs Efficcy of Some Trypnocidl Drug Aginst Trypnosom equiperdum OVI in Experimentlly Infected Mice in Debre Zeit, Ethiopi Beletu Hbte, Abrh Bsrt, Hgos Ashenfi nd Fikru Regss 1 College of Veterinry Medicine, Mekelle University, P.O. Box 231, Mekelle, Ethiopi 2 Addis Abb University College of Veterinry Medicine, P.O. Box 1176, Addis Abb, Ethiopi Abstrct: Trypnocidl drugs remin the principl method of niml Trypnosomisis control in most Africn counties. However, there is growing concern tht future effectiveness my be severely curtiled by wide spred drug resistnce. This study ws therefore, conducted to ssess the efficcy of diminzene diceturte nd cymelrsn ginst Trypnosom equiperdum OVI South Afric strin in experimentlly infected mice. Sensitivity study ws conducted using rnge doses of diminzene diceturte (3.5mg/kg, 7.mg/kg, 14mg/kg nd 28mg/kg) nd cymelrsn (.25 mg/kg,.5 mg/kg 1. mg/kg nd 2.mg/kg) using 5 mice ech being grouped into 1 hving 5 mice ech. Diminzene diceturte t doses of 3.5mg/kg, 7.mg/kg, 14mg/kg filed completely to cure but only 1(2%) mice out of 5 ws survive up to 6 dys treted with diminzene diceturte t dose of 28mg/kg body weight. Cymelrsn t dose of.25mg/kg body weight filed completely to cure mice but dose of.5mg/kg body weight, only 2(4%) mice were cure. All mice treted t higher dose of the cymelrsn 1.mg/kg nd 2.mg/kg body weight cured nd the prsitemi ws not detected for more thn 6 dys. All mice in the control groups showed higher level of prsitemi nd died completely up to 4 dys fter infection. From this study it is indicted tht the minimum curtive dose of cymelrsn required to cure Trypnosom species cusing dourine (South Afric strin) in experimentlly infected mice ws greter thn 1mg/kg. However, further studies should be conducted to know the effect of the drugs on the locl strin of Trypnosom equiperdum like Dodl strin from dourine endemic re in equine from Ethiopi so s to work out the best possible therpeutic strtegies nd/or lterntive control mesures. Key words: Cymelrsn Diminzene Diceturte Ethiopi, Mice T. Equiperdum INTRODUCTION Despite the significnce of horses in the sector of trnsporttion nd griculture to the economy of the World equine popultion is bout million ntion, the tretment ccorded to these species of niml consisting of 44.5 million Donkeys, 57.6 million Horses hs been fr below thn the ttention given to other nd 5.2 Mules [1]. Among this, Ethiopi possesses economic niml species. This cn prtly be due to the 2.75 million horses, 5.2 million Donkeys nd.63 million ge old erroneous concept tht these species re hrdy, mules [2]. Horses hve prominent position in the tolernt nd probbly becuse their met nd milk is not griculturl nd trnsport systems s drft, pck nd edible in Ethiopi [4]. Africn horse sickness, Anthrx, riding nimls. In country where there is less developed Epizootic Lymphngitis, Dourine, Equine piroplsmosis, modern trnsport nd communiction service, the nturl Horse mnge, Rbies, Glnder nd Ulcertive choice rests on the let humn nd pck nimls mode of Lymphngitis re mong the mjor diseses ffecting trnsport, s it hs been the cse in some prts of the horses in Ethiopi [3]. world. Thus, in developing country like Ethiopi, the Trypnosomosis is serious prsitic disese, which contribution of equines in the energy scenrio is of occurs in lrge res of Afric, Ltin Americ, the Middle considerble significnce. The provisions of trnsport Est nd Asi. It ffects most species of domestic through pck nimls, drwing crts, s riding nimls or livestock, mny types of wild nimls nd humn. The txi opertions, lmost certinly contributes more to the most importnt trypnosomes in terms of economic loss ntionl economy [3]. in domestic livestock re the tsetse trnsmitted species Correspondence Author: Abrh Bsrt, College of Veterinry Medicine, Mekelle University, P.O. Box 231, Mekelle, Ethiopi. 7

2 such s Trypnosom congolense, Trypnosom vivx With the wide spred of drug resistnt prsites in to nd Trypnosom brucei [5]. Mechniclly trnsmitted ll trypnosomosis endemic res, development of new trypnosomes such s Trypnosom evnsi nd nti-trypnosml compounds nd drugs to circumvent Trypnosom vivx cuse mjor production losses in the resistnce is urgently needed. However, it ppers respective hosts. unlikely tht new compounds will be introduced into the Dourine is the only form of trypnosomosis cused ner future becuse of lck of interest by the by Trypnosom equiperdum, which is trnsmitted phrmceuticl industry in investing in reserch nd directly from one niml host to nother of the sme development of ntitrypnsoml drugs [15]. Hence, there species without the intervention of n insect vector but it is n urgent need for ssessment of efficcy of the drug trnsmitted lmost exclusively during coitus. The in which representtive number of trypnosomes isolted custive gent of Dourine, T. equiperdum, differs from nd exmined for distribution nd degree of efficcy so s other mmmlin trypnosomes lso in the fct tht is to work out the best possible therpeutic strtegies primrily tissue prsite. The clinicl course of the disese nd/or lterntive control mesures. As prerequisite depends on the pthogenecity of the trypnosome, the for such undertking it is necessry to first obtin resistnce of the breed nd the physicl condition of the informtion on in vivo trypnocidl drug sensitivity nimls. The incubtion period rnges from two weeks till ptterns of the prsites in mice model. Hence, the three months, clinicl signs might be suppressed during present study ws initited nd designed to undertke cold months [6]. Generlly the disese is divided in to studies relted to the ssessment of trypnocidl drug three phses such s primry stge (Genitl Oedem), sensitivity of T. equiperdum OVI, South Africn strin secondry stge (plques nd skin eruptions) nd tertiry with the vilble trypnocidl drugs. Therefore, the stge (Neurologicl signs) [7]. Anemi, cchexi nd objective of the present study ws to ssess the genitl oedem re often seen t post mortem [8]. Dourine trypnocidl drug sensitivity of Diminzene diceturte is strictly limited to the equine. Thus the host rnge is nd Cymelrsn ginst T. equiperdum OVI, South limited to horses, donkeys nd mules under nturl Africn strin in experimentlly infected mice. condition [6]. Dignosis of T.equiperdum, in horse, by stndrd MATERIALS AND METHODS prsitologicl techniques is difficult, owing to the low number of prsites present in the blood or tissue fluids Study Are: The present study ws undertken in nd frequent bsence of the clinicl sign of the moleculr prsitology lbortory of Ethio-Belgium VLIRdisese. Consequently the demonstrtion of UOS funded dourine project which is found inside the trypnosoml ntibodies in the serum hs become the compound of Addis Abb University Fculty of most importnt prmeter in the determining the disese Veterinry Medicine, Debre Zeit, Ethiopi from November sttus of the nimls [9].The principl reson for using 29 to April 21. Debre Zeit is found 47Kms south est serologicl testes of dignosis Trypnosomisis is to over of Addis Abb t (8 44 N nd E) t n ltitude of cme the low level of sensitivity testes in detecting 185 meters bove se level. The men nnul rin fll is chronic infection. Alterntive strtegies, utilizing nuclic 885.4mm with bimodl distribution. There re lternting cid technologies such s the PCR lso often high dry nd riny seson in the re. The long riny extends sensitivity nd might be of use in the dignosis of from June to September nd contributes bout 84% of the T.equiperpum [1]. totl nnul rinfll. While the dry seson lst from For the tretment of T.equiperdum infection the October to Februry. The short riny seson lsts from sme drug which is used for T.evnsi re vilble. Mrch to My. The men nnul minimum nd mximum Evidence from in vitro drug sensitivity determintion of tempertures re 14 C nd 26.3 C respectively with n T.equiperdum indictes tht Surmin, Diminzen, overll verge of 18.7 C. The men reltive humidity is Quinpyrmine nd Cymelrsn re effective ginst this 61.3 % [16]. trypnosome species [11, 12]. In Ethiopi, Dourine is n endemic problem of horses in the Arsi Ble highlnd Experimentl Animls: Swiss white mice bout 6 weeks where tretment nd control of the disese becomes of ge weighing 2-25 gm were obtined from the difficult. The difficulty is minly in terms of trypnocidl breeding colony of the Ntionl Veterinry Institute (NVI). drug shortge which plys n influencing role in The mice were mintined under stndrd commercil controlling nd prevention of the disese in n endemic pelleted rtion nd wter dlibitum in the lbortory of re [13, 14]. the VLIR-UOS. 8

3 Smple Collection: Approprite blood smple ws control group. Mice in the tretment group were collected regulrly to evlute the level of prsitemi by weighed on digitl blnce prior to dministrtion of cutting the tip of the til of the mice. Whenever lrge the trypnocidl drugs. volume of blood (up to 2ml) is required for preprtion of The mice in the different tretment nd control cryostbiltes crdic puncture ws performed fter group were then being infected with T. equiperdum humnely euthnizing the mice with ether. OVI, South Africn strin dpted to mice with 1-1 trypnosomes per preprtion which is estimted to be Experimentl Design: Totlly 6 mice were used in which trypnosomes per ml blood [17] by intrperitonel 1 (grouped in to two hving 5 mice ech) for dpttion inocultion with.2 ml blood from donor mice tken t of the prsite nd 5 for tretments nd control. Initilly, pek prsitemi. ten (1) donor mice (5 mice for ech drug) were infected with T. equiperdum OVI South Africn strin stbiltes Tretment nd Monitoring: Diminzene diceturte originted from Institute of Tropicl Medicine, Antwerp, (Diminsn, Btch DG/2337 DUIPER WEG 9, 3449 JA Belgium. Before infected the cryostbiltes were woerden, Hollnd) nd Bis (minoethylthio) 4- preserved in liquid nitrogen, following defrosting t room melminophenylrsine dihydrochloride (cymelrsn, lot B temperture or preferbly t 37 C in wter bth. The 9118A, MERIAL-17, rue Bourgelt 69Z Lyon-Frnce) defrosted stbiltes ws then being mixed with were used for tretment of infected mice in the ech Phosphte Substrte Glucose (PSG). The route of tretment group. Diminzene diceturte ws dministered infection of the mice ws intr peritonel route with vi intrperitonel route t doses of 3.5 mg /kg, 7. mg/kg, mximum dose of.2 ml. 14. mg/kg nd 28. mg/kg body weight. Similrly The ten donor mice were checked for prsitemi Cymelrsn ws dministered vi intrperitonel route t dily by tking drop of blood from the tip of the til of doses of.25 mg/kg,.5 mg/kg, 1. mg/kg nd 2. mg/kg the nimls. Once prsitemi gets highly multiplied body weight (Tble 1). The control groups received the within three to five dys, it ws successfully possible to sme mount of sterile distilled wter by intrperitonel serilly pssge Trypnosom equiperdum OVI South route in replcement of the tretment. Ech drug ws Afric strin to the next mice then mice were humnely prepred by dissolving the required quntity of ech euthnized to collect blood vi intrcrdil puncture. Ech compound in sterile distilled wter before use ccording time the cryostbiltes were prepred nd kept in liquid to respective mnufcturer instruction nd required dose nitrogen. Two drugs, diminzene diceturte nd of the drug ws dministered in.2 ml of solution for ll Cymelrsn, were studied for their sensitivity on T. tretment groups [18]. Mice were monitored every equiperdum OVI, South Africn strin. In ech of the lternte dy up to 6 dys for the presence of study 25 mice were divided rndomly in to five trypnosomes by microscopic exmintion of wet smers experimentl groups (group I-V) of five mice ech. of til blood. Presence of trypnosomes in the blood The first four groups (I-IV) comprise the tretment groups smer ws considered s indiction of ineffectiveness of (those to be infected nd treted with different doses of the trypnocidl drug used or possibly drug resistnce. diminzene diceturte). The fifth group (group V) served s infected untreted control group. While,the groups Dt Anlysis: Dt were collected, stored properly in from VI toxv infected nd treted with different doses of Micro soft Excel sheet nd then nlyzed using GMP-5 Cymelrsn, while group X keept s infected untreted Sttisticl soft wre [19]. Tble 1: Tretment groups for T. equiperdum OVI South Afric strin in mice using diminzene diceturte nd cymelrsn. Trypnocidl drugs used Tretment groups No of mice per group Diminzene Diceturte (mg/kg bw) Cymelrsn (mg/kg bw) I II III IV V 5 VI VII VIII 5-1. IX 5-2. X 5 =Distilled wter insted of Trypnocidl drug 9

4 Tble 2: Results of diminzene diceturte nd cymelrsn to Trypnosom equiperdum OVI South Afric strin in mice t different dose rnge. Drugs Doses (mg/kg) No. mice treted/relpsed Men relpse intervl in dys ± SD Diminzene diceturte 3.5 mg/kg 5/5 1 ±. 7. mg/kg 5/5 1.2± mg/kg 5/5 1.4 ± mg/kg 5/4 2.5 ± P vlue.2 Cymelrsn.25 mg/kg 5/5 6.6 ± mg/kg 5/3 11. ± mg/kg 5/ Cured 2. mg/kg 5/ Cured P vlue.1 Control Distilled wter!/1 Died fter 3 dys of post infection but fter one dy of prsitemi detection. = Treted /Died RESULTS occurred fter hevy prsitemi development which my lst only 3 to 5 dys fter infection [2]. Diminzene diceturte t doses 3.5mg/kg, 7mg/kg The results from the drug sensitivity showed tht nd 14mg/kg body weight filed completely to cure but Diminzene diceturte t dose rnge of 3.5mg/kg, only 1(2%) mice ws survived up to 6 dys Which 7.mg/kg, 14mg/kg body weight filed completely to cure treted t dose of 28mg/kg body weight (Tble 2). Trypnosom equiperdum OVI South Afric strin due to However, fter 6 dys of dexmethsone inocultion, sever prsitemi. However, only 1(2%) mouse ws prsitemi of dourine cusing Trypnosom spp (South survived up to 6 dys fter tretment with diminzene Afric strin) ws demonstrted in the blood of the diceturte t dose of 28mg/kg body weight. The men mouse. In cse of cymelrsn, mice infected with relpse intervl for the dose rnge of 3.5mg/kg, 7.mg/kg, T.equiprdum OVI South Afric strin nd treted t dose 14.mg/kg nd 28mg/kg body weight used ws 1±., 1.2± of.25mg/kg body weight were filed completely to cure.45, 1.4 ±.55 nd 2.5 ± 1. dys respectiviliy. but 2(4%) of mice treted t dose of.5mg/kg body From other similr studies it ws indicted tht weight survived nd the remining 3(6%) mice were diminzene diceturte is less effective ginst relpsed within 11. ± 1. dys. However, mice treted Trypnosomes of subgenus Trypnozoon thn with cymelrsn t higher doses of 1.mg/kg nd 2.mg/kg T. congolense nd T. vivx. This could be ttributed body weight were completely cured from the infection to the reltively rpid excretion of the drug [5]. with no relpse t lest for more thn 6 dys. All mice in Moreover, diminzene diceturte cn not cross the the control groups showed higher level of prsitemi blood brin brrier nd somtic tissue. Due to this nd died completely up to 4 dys fter infection. Hence, fct, it cnnot be the curtive drug for trypnosomes s per this study the minimum curtive dose (MDC) of with tissue ffinity such s T. equiperdum [11, 12]. The cymelrsn required to cure Trypnosom equiperdum relpse of T. equiperdum fter 6 dys post diminzene OVI from experimentlly infected mice ws recorded to be diceturte therpy could be ssocited with the relese more thn 1mg/kg. of the trypnosomes from tissues inccessible by the drug. DISCUSSION Although diminzene diceturte probbly exerts its ction t the level of the kinetoplst DNA, this hs not Blood from infected horses produce disese very been proven in vivo nd other mechnisms of ction rrely when inoculted directly in to helthy mice. cnnot be excluded [21]. Similrly the moleculr bsis of Therefore, in the current study the infection ws first resistnce to diminzene in trypnosomes is not cler. The estblished in mouse using lredy prepred ccumultion of diminzene ws mrkedly reduced in cryostbiltes of the prsite from horse nd then it ws rsenicl-resistnt T. brucei brucei owing to ltertions in inoculted in to study mice groups. Prsite ppered in the nucleoside trnsporter system. However, there might the blood of study mice with in 2 or 3 dys nd deth be other resistnce mechnisms [11]. 1

5 The result from the drug sensitivity study showed tretment. Yet, currently stmping out strtegy is pplied tht only single shoot of cymelrsn t dose of 1.mg/kg by the OIE with slughtering of CFT positive horses nd 2. mg/kg body weight becomes effective tretment where tretment is prohibited [3]. However, it is not for Trypnosom equiperdum OVI South Afric strin in economiclly fesible to pply strict test nd slughter experimentlly infected mice with no relpse for t lest policy of the OIE to control dourine t lest in developing two months. However, cymelrsn t doses rte of country like Ethiopi. From the present study mice.25mg/kg body weight filed to cure the infection but in infected with T.equiperdum OVI South Afric strin were cse of.5mg/kg only 2(4%) mice survived. In lower cure fter being treted with cymelrsn thn those doses of cymelrsn t.25mg/kg nd.5mg/kg body treted with diminzene diceturte. Therefore, weight men relpse intervls were between 6.6 ± 1.67 nd cymelrsn cn be recommended s choose of tretment 11. ±1. dys. in controlling strtegy of the studied Trypnosomes Cymelrsn however, ws found to be ineffective species. ginst Trypnosom equiperdum in mice t doses of.25mg/kg nd.5mg/kg body weight. The inbility of this CONCLUTION drug to cler prsitemi in mice s well s domestic niml t the stndrd dose of.25mg/kg for T.evnsi Trypnocidl drugs remin the principl method of hs supported by previously reports. For instnce niml Trypnosomisis control in most Africn cymelrsn ws ineffective in buffloes treted t doses counties. However, there is growing concern tht future rnging from.5mg/kg to 3mg/kg [22], in gots treted t effectiveness my be severely curtiled by wide spred dose of.3mg/kg [23], in mice treted t dose of.25 nd drug resistnce. The present study ws done in.5mg/kg [24] nd in cttle treted t dose of.5mg/kg ccordnce with the im of ssessing the Trypnocidl [25]. drug efficcy of diminzene diceturte nd cymelrsn Cymelrsn ws found to be very effective ginst ginst T.equiperdum OVI South Afric strin in T. equiperdum, T. evnsi nd T. brucei brucei, in horses, experimentl infected mice. As study result indicte tht cmels, bufflo, gots nd pigs [11, 22, 23, 26]. The cymelrsn t dose rte of 1mg/kg nd bove ws found rsenicl compound contins the trivlent rsenic element highly effective thn diminzene diceturte considering with mrkedly rective rsenoxide group. The presence the toxicity of the drug if over dosed. Regrding niml of rsenoxide confers the physicochemicl bility of lipid welfre this would be big step forwrd, becuse it cn solubility tht llows pssge cross the blood brin relese n lrming sign to the developed countries tht brrier (BBB) [27]. The rsenicl compound melrsoprol pply the OIE s test nd slughter policy. To combt the reveled the remrkble bility to cross the BBB nd kill problems ssocited with trypnosomes, further reserch Trypnosom brucei gmbiense nd Trypnosom into existing drugs is prerequisite for their optiml usge brucei rhodesiense prsites residing in the CSF. in the overll effort of improving niml helth nd It ws clerly evident tht cymelrsn ws quite productivity through control of trypnosomisis. effective ginst T.evnsi infection in cmels t stndrd Therefore, in vitro ssys should be performed to recommended dose rte of.25mg/kg body weight [28]. detect drug resistnce ginst the vilble isoltes of This ineffectiveness of the drug for the present study Trypnosom equiperdum from equine rering re of probbly confirms the suggestion mde previously [23] Ethiopi where dourine is endemic nd mjor problem.the tht the recommended dose might hve been pplied current strtegies of OIE ginst dourine erdiction strictly for the tretment of cmels only but the higher progrmmes should be reconsidered, since there could be doses re needed to tret T. evnsi in other nimls nd some effective drugs ginst Trypnosom equiperdum. mice. By the sme scenrio in the present study the mice Besides, The toxicity of the cymelrsn should further be were treted under dosge where the metbolic weight of studied in Ethiopi before being imported nd distributed cmels versus mice should be considered. it in endemic re to control dourine. The proposed mechnism of ction of the drug cn be explined in terms of the very strong binding ACKNOWLEDGEMENTS chrcteristics of cymelrsn with trgets on the prsite known s thiol contining enzymes like glycerol-3- This investigtion received finncil support from phosphte dehydrogense (G3PDH) [29]. Ethio-Belgium VLIR-UOS funded dourine project nd free Hence, it cn be recommended to the OIE to replce ccess of lbortory fcilities, technicl nd kind support the current strtegy of erdiction by n pproprite drug from Addis Abb University College of Veterinry 11

6 1. FAO, 23. Globl Livestock production nd Helth Atls (GLIPHA) on line. Avilble on http;// www. Fo org/ 17/3/ CSA, 23. Ethiopin Agriculturl Smple Enumertion, 21/22: Centrl Sttistic Authority, Federl Democrtic Republic of Ethiopi. 3. FAO, Animl production nd Helth Yerbook, Food nd Agriculturl Orgniztion of the United Ntion. Rome. 4. Feseh, G., Use of equine Ethiopi. In; proceedings of the Fourth Ntionl Livestock Improvement Conference, Addis Abb, IAR, Ethiopi, Mullign, H.W., 197. The Africn Trypnosomisis pp. (Mullign, H.W. ed.) London: George Allen nd Unwin Ltd, Stephen, L.E., Trypnosomisis Veterinry Perspective. Pergmon Press, Oxford, Hore, C.A., The Trypnosomes of Mmmls. Zoologicl Monogrph. Blck well Scientific Publictions, Oxford nd Edinburgh, UK. 8. OIE, 21. Dourine. Prt 2. Section 2.5. Chpter In: Mnul of stndrds of dignostic tests nd th vccines, 4 edition, OIE Publictions, Pris, Bishop, P., P.F. Re, L.P. Phipps, R. Boid nd A.G. Luckins, Trpnosom equprdum detection of Trypnosoml ntibodies nd ntigen by enzyme linked immunosorbent ssy. br. Vet., 151: Luckins, A.G., Method of dignosis of Trypnosomsis in livestock wild niml. Rev., 71: Zhng, Z.Q. nd T. Bltz, in vivo nd in vitro sensitivity of T. evnsi nd T. equiperdum to Diminzene, Surmine, Melcy, Quinpyrmine nd Isometmidium. Act Trop., 5: Brn, R. nd Z.R. Lun, Drug sensitivity of Chinese T.evnsi nd T.equiperdum isoltes. Vet. Prsitol, 52(1&2): Clusen, P.H., S. Chulvun, R. Sodnomdrj, M. Greiner, K. Noeckler, C. Stk, K.H. Zessin nd E. Schein, 23. A field study to estimte the prevlence of Trypnosom equiperdum in Mongolin horses. Vet. Prsitol., 115: Europ. J. Biol. Sci., 7 (1): 7-13, 214 Medicine. The uthors lso extend specil 14. Hgos, A., 25. Serologicl nd prsitologicl cknowledgement to Dr. Fuf Dwo, Dr. Ycob Hilu nd survey of dourine (Trypnosom equiperdum) in Mr. Alemu senior lbortory technicin for their selected site of Ethiopi. MSc Thesis, Addis Abb professionl support. University, Fculty of Veterinry Medicine, Debre Zeit, Ethiopi. REFERENCES 15. Gutteridge, W.E., Existing chemotherpy nd its limittions. British Medicl Bulletin, 41: NMSA, 23. Ntionl Metrologicl service Agency, Addis Abb, Ethiopi. 17. Murry, M., P.K. Murry nd W.I.M. McIntyre, An improved technique for the dignosis of Africn Trypnosomisis. Trns. R. Soc. Trop. Med. Hyg., 71: Eisler, M.C., J. Brndt, B. Buer, P.H., Clusen, V., Delespux, P.H. Holmes, A. llemobde, N.M. Mchil, H. Bwmbo, J. McDermott, D. Mehlitz, G. Murill, J.M. Ndung u, A.S. Peregrine, I. Sidibe, L. Sinyngwe nd S. Geerts, 21. Stndrdiztion tests in mice nd cttle for the detection of drug resistnce in tsetse trnsmitted trypnosomes of Africn domestic cttle. Vet. Prsitol., 97: SAS, 22. Sttisticl Anlysis system Institute. User s Guide. JMP Design of Experiment, Version-5. SAS Institute Inc. Cry, NC, USA., Wtson, A.E., 192. Dourine in Cnd : History, Reserch nd Suppression. Dominion of Cnd Deprtment of Agriculture, Helth of Animls Brnch, Ottw, Cnd. 21. Peregrine, A.S. nd M. Mmmn, Phrmcology of Diminzine: review.act Trop., 54: Lun, Z.R., Z.P. Min, D. Hung, J.X., Ling, X.F., Yng nd Y.T. Hung, Cymelrsn in the tretment of buffloes nturlly infected with Trypnosom evnsi in South Chin. Act Tropic, 49: Zweygrth, E., J. Ngernw nd R. Kminsky, Preliminry observtions on the efficcy of mel Cy (Cymelrsn) in domestic nimls infected with stocks of Trypnosom brucei brucei nd T b. evnsi. Tropicl Medicl Prsitology, 43: Syklim, M., J. Ysud nd A. Hshimoto, Preliminry efficcy tril of Cymelrsn in mice rtificilly infected with Trypnosom brucei isolted from dog in Zmbi. The Jpnese. Vet. Res.h, 43: Pyne, R.C., I.P. Suknto, S. Prtoutomo, T.W. Jones, A.G. Luckins nd R. Boid, Efficcy of Cymelrsn in Friesin Holstein clves infected with Trypnosom evnsi. Trop. Ani. Helth nd Prod., 26:

7 26. Zweygrth, E. nd R. Kminsky, 199. Evlution of 29. Andrew, J.N., 23. Aresenicl (melrsoprol), n rsenicl compound (Mel Cy, Cymelrsn) ginst pentmidine nd surmin in the tretment of humn susceptible nd drug-resistnt Trypnosom brucei Africn Trypnosomosis. Prsitol Res., 9: brucei nd T.b. evnsi. Trop Med Prsitol, 3. Zblotskij, V.T., C. Georgiu, T.D. Wl, P.H. Clusen, 41(2): F. Cles nd L. Tourtier, 23. The current 27. Peppin, J. nd F. Milord, The tretment of chllenges of dourine: Difficulties in differentiting humn Africn trypnosomisis. Adv. Prsitol., Trypnosom equiperdum within the subgenus 33: Trypnozoon. Rev. Sci. Tec. Off. Int. Epiz., 28. Mus, M.M., A.M. Abdoon, B.T. Nsir, Y.I. Slim, 22(3): A.Y. Abdel-Rhmn nd A.M. Shommein, Efficcy of melrsoprol in the tretment of nturl chronic Trypnosom evnsi infection in cmels in the Sudn. Révue D'Elevge et de Médicine Vétérinire des Pys Tropicux, 47: