At Prsitologi Glolis 5 (3): 160-168, 2014 ISSN 2079-2018 IDOSI Pulitions, 2014 DOI: 1829/idosi.pg.2014.5.3.8571 Comprison of the In vitro Inhiitory Effets of Doses of Tnnin Rih Plnt Extrts nd Ivermetin on Egg Hthility, Lrve Development nd Adult Mortlity of Hemonhus ontortus Zellem Alemu, Yisehk Kehero, Assef Keede nd Adu Mohmmed Jimm University College of Agriulture nd Veterinry Mediine, P.O. Box: 307 Jimm, Ethiopi Astrt: The study report here ws undertken to quntify nd ompre the in vitro inhiitory effets of doses of tnnin rih plnt extrts, ivermetin nd distil wter on egg hthility, lrve development nd dult mortlity of Hemonhus ontortus. Fius syhomorus, Phyllnthus sepilis nd Rhus glutinos speies suspeted with high ontents of ondensed tnnins (CT) were olleted from their nturl hitt in Omo Nd distrit of Jimm zone, south western Ethiopi. The leves of plnt speies were dried t 55 C to onstnt weight, ground to 1.5 mm sieve nd sujeted to CT nlysis. Aqueous etone (70%) ws inorported for the extrtion of plnt smples. The extrts were prepred to otin 0.1ml, ml nd ml from 50 mg /ml stok solution nd ompnied y ontrols: Ivermetin (positive ontrol) nd distill wter (negtive ontrol). The dt were sujeted to two-wy nlysis of vrine following the generl liner model proedure of SAS in 5 3 ftoril rrngement. The CT ontents of the respetive plnts were 120, 110 nd 188 g/kg DM, respetively. Interspeies vritions were signifint for CT onentrtion ross the dosge levels (P<0.001). The men egg inhiition perentge of plnt extrts vried from the lowest inhiition effet for P. sepilis (46-57.33%) to the highest inhiition effet for R. glutinos (59.67-76%) (P<0.001). The lrvl development inhiition of ivermetin ws not found to e superior ompred to tnnin rih extrts (P>0.05).The fstest dult motility ws oserved for different doses of ivermetin nd R. glutinos s ompred to other tretment groups (P<0.001). Among plnts, the fstest nd slowest dult motility rtes were reorded for R. glutinos (1:22h to 0:58h) nd P. sepilis (3:00h to 2:50h) (P<0.001). Yet, the effets of negtive ontrol didn t vry in inhiiting egg hthility, lrvl development nd dult motility (P>0.05). In generl, ll plnts showed nti-h. ontortus tivity in the three-life yle of the prsite nd it ws lso ssoited with the dosge levels nd the onentrtion of CTs ontined in the plnts. Furthermore, sine the nti-hemohus tivity of R. glutinos ws found to e fesile s tht of ivermetin, therefore, this plnt n e used s lterntive nthelmenti for the tretment of Hemonhosis. Key words: Tnnin Hemonhus Contortus; In vitro Ivermetin INTRODUCTION milk, met nd wool prodution [7,8,9]. Sheep quire little immunity to H. ontortus nd remin suseptile Sheep husndry is n importnt omponent of throughout their lives to diseses used y this prsite livestok setor nd is ritil soures of sh inome, [10]. Infetions with H. ontortus our in mny ountries met nd fier for smllholder keepers in different frming throughout the world; however, the highest prevlene is systems [1-3], yet, produtivity of sheep under tropil seen in wrm, moist temperte nd tropil regions [11,12]. settings is severely influened y prsiti infesttion The ontrol of H. ontortus in domesti nimls is [4-6]. Hemonhus ontortus, helminths prsite, plys n widely sed on the use of phrmeutilly derived importnt role in sheep prodution leding to enormous nthelminti drugs. However, the urrent effiy of these eonomi losses through mortlity, weight loss, redued drugs hs een redued, euse of the wrong use nd/or Corresponding Author: Yisehk Kehero, Jimm University College of Agriulture nd Veterinry Mediine, P.O. Box: 307 Jimm, Ethiopi. 160
At Prsitologi Glolis 5 (2): 160-168, 2014 widespred pplition of poor qulity syntheti or semi- leves were dried t pproximtely 55 C for 72 hours to syntheti nthelmintis nd onsequently the onstnt weight using ir fored drying oven nd ground development of resistnt prsite strins [13-15]. to 1mm using Wiley mill. Aout 50 mg of ground mterils Moreover, the high ost of these drugs, residul onern were pled in glss eker of pproximtely 25 ml in food nimls nd environmentl pollution hve stirred pity. For eh smple 10 ml of queous etone (70%) up interest in mediinl plnts s n lterntive soure of ws dded nd the eker ws suspended in n ultrsoni nthelminti drugs [16-18]. Hene, the use of indigenous wter th nd sujeted to ultrsoni tretment for plnt preprtions s livestok dewormers is gining 20 minute t room temperture (verge 25 C). The ground s one of the options nd sustinle methods ontent of the eker ws then trnsferred to redily dpted to rurl frming ommunities [19, 20]. entrifuge tues, ooled on ie nd sujeted to Tnnin extrtives from foliges of tropil fodder trees entrifugtion for 15 minute t 3000 rpm [25]. nd shrus n e the est strtegy to use s lterntive strtegy for ontrolling helminthes [21]. Colletion of Adult Prsites nd Egg Reovery Ethno-veterinry surveys onduted so fr in Tehniques: The dult femle prsites of H. ontortus Ethiopi indite tht severl trditionl helers use were olleted from the oms of nturlly infeted lolly ville mediinl plnts for tretment of vrious sheep. The egg reovery test ws performed ording to niml helth prolems inluding tretment of helminth the method desried y Jr et l. [14] where femle infetion [8, 22]. However, very few efforts hve een dult worms were rushed using pestle nd mortr. mde to sientifilly sreen nd evlute the nthelminti After liertion, the eggs were ultured in 250 ml jr effet of ondensed tnnins (CT). Besides, the serh for filled with utolved sheep fees for eight dys t room lterntive tretment for hemil tretments is now temperture. dys y the emergene nd widespred diffusion of resistne to nthelmintis in popultions of Egg Hthility Inhiition: The egg hthility gstrointestinl nemtodes in ruminnts speilly sheep inhiition test ws onduted ording to the nd gots. Therefore, the present study is intended to proedures desried y Coles et l. [26]. Extrts of CTs ompre the effiy of CT-rih extrts of leves of Fius from the three plnt speies were used s the test syhomorus, Phyllnthus sepilis nd Rhus glutinos tretments. Ivermetin nd distill wter were used s with positive nd negtive ontrols ginst Hemonhus positive nd negtive ontrols, respetively. The tril ws ontortus of lol sheep. onduted in 10 ml test tues. In the ssy, pproximtely 170-270 eggs in 1.5 ml of distill wter were pled in eh MATERIALS AND METHODS test tue. The three doses (0.1 ml, ml nd ml) of eh plnt extrt ws dded in to test tues seprtely Desription of the Study Are: The study of tnnin nd the test tues were overed with luminum foil omponent nd in vitro nti-hemonhus ontortus mking 15 to 20 holes for ir irultion nd kept in n tivity of ondensed tnnins (CT) were rried out in inutor t 27 C for 48 hr. The experiment ws repeted niml nutrition nd prsitology lortories, three times. Hthed lrve nd unhthed eggs were respetively, of Jimm University, College of Agriulture then ounted under disseting mirosope t 40 nd Veterinry Mediine (JUCAVM) mpus whih is mgnifition. Therefter, the nti- H. ontortus effiy loted in Jimm ity, south western Ethiopi. Jimm ity of CT ws ssessed y field ontrolled fel egg ount is situted t 7 40'N nd 36 50'E ltitude nd longitude, redution test lulted ording to Tylor et l. [27] respetively [23]. nd Coles et l. [26]. The perent fel egg ount redution ws lulted using the following formul: Colletion of Plnt Leves nd Tnnin Extrtion: Three tnnin-rih plnt speies nmely Fius syhomorus, % FECR = ((-)/)*100; where,, EPG pre-tretment nd Phyllnthus sepilis nd Rhus glutinos were olleted, EPG post-tretment from their nturl hitt, Omo Nd distrit of Jimm zone southwestern Ethiopi. The rowse speies were seleted Lrvl Development Inhiition Assy: The lrvl sed on their undne in the re, preferene nd development inhiition test ws onduted with essiility to rowsing livestok s well s dditionl modifition of the tehnique desried y Cost et l. uses other thn livestok feed [24]. The olleted fresh [27]. The CT extrts otined from the three plnt speies 161
At Prsitologi Glolis 5 (2): 160-168, 2014 were used s the test tretments. Ivermetin ws s y 74% for ml dose, it hd lerly oserved in the positive ontrol while untreted eggs either with present study tht tnnin extrts from vrious tnnin rih ivermetin or CT extrts in distilled wter were used s plnts hd ility to inhiit egg hthility to mximum negtive ontrol. After inuting the eggs t 27 C for 76% for ml dose. 24 hours, n liquot of 1.5ml, ontining 100-150 first The egg inhiition pity of doses of tnnin rih stge lrve (L1) of H. ontortus ws mixed with 10 gm of plnts were omprle with ivermetin, the ommeril fees tht olleted from oms of nturl infeted nthelmintis drug (Figure 1). For 0.1ml CT extrt, the sheep. Eh of 0.1ml, ml nd ml of CT extrts ws highest nd lowest perentge egg hthility inhiition dded on sheep fees whih ontining L 1. The test ws reorded for R. glutinos nd distill wter, mterils were inuted for 6 dys t room temperture. respetively (P<0.001). th At the end of the 6 dy the wll of eh up ontining the smple ws thoroughly rinsed with 10 ml of distill Lrvl Development Inhiition: The experiment ws done wter to ollet the lrve. Then one drop of Lugol s with exposure of H. ontortus L 1 through L 3(Tle 2). iodine solution ws dded nd ll (L3) stge lrve were Similr to perent % egg hthility inhiition, the lrvl ounted under stereomirosope. development inhiition effets of the doses of plnt extrts ws lerly ssoited with either tnnin Adult Motility Test: Adult motility test ws performed onentrtion or doses of the plnt extrts, mening, ording to Petersen et l. [28]. Aordingly, 0.1ml, ml lrger volumes (doses) hd the gretest lrvl nd ml of eh of CT extrts ws tken nd diluted development inhiition ility (P<0.001). The lrvl with distilled wter. Iveremtin ws used s positive development inhiition ws rnged from 51.33% t 0.1ml ontrol while distilled wter ws used s negtive ontrol. dose, 62% t ml dose, 73.33% t ml dose for For eh tretment 30 dult prsites were used. The R. glutinos s well s 54.67% t 0.1ml dose, 56.33% t different onentrtions of eh plnt extrts (0.1ml, ml dose, 66.33% t ml dose for P. sepilis. ml nd ml) were pled in petridish. The prsites Anti-Hemonhus ontortus tivity of ivermetin ws were then immersed nd the time for mortlity ws not found to e superior ompred to tnnin rih extrts reorded for eh respetive onentrtion. (Figure 2). The lrvl development inhiition (LDI) perentges Sttistil Anlysis: A two-wy nlysis of vrine ws of 0.1mm of tnnin rih extrts were higher thn followed using the generl liner model (GLM) proedures ivermetin nd distil H2O. However, the LDI ws not of sttistil nlysis system [29]. Differenes were signifintly different for ml exept for P. sepilis deemed signifint when P<0.05, wheres 0.05<P<0.10 (Figure 2). The ml of R. glutinos ws superior in ws onsidered to show sttistil tendeny for inhiiting lrvl development ompred to ll tretment differene. Levene's test ws rried out to detet the groups (P<0.001). homogeneity of experimentl vrines. Bsed on this test, there ws no evidene tht the residuls (i.e., errors) Adult Motility Test: Tle 3 presents dult prsite were not normlly distriuted. mortlities in hours; where the lrger numer of hours indited the slower mortlity rte of the prsite. In other RESULTS words, the lower time indites the fstest motility rte (P<0.001). It ws oserved for ll tretment groups tht Egg Hthility Inhiition: Egg hthility inhiition the dult motility inhiition ws highly ssoited with perentges of plnt extrts were ssoited with the doses of the plnt extrts, i. e, shorter times were doses of CT extrts (Tle 1). The highest vlues of % reorded for smller doses (P<0.001). egg hthility inhiition ws reorded for the lrgest The fstest dult motility ws oserved for different doses of plnt extrts (P<0.001). The men inhiition doses of ivermetin nd R. glutinos s ompred to perentge otined for eh plnt extrt ws other tretment groups (P<0.001, Figure 3). Among plnts, signifintly different with differenes in doses of the fstest dult motility rte ws R. glutinos for dose pplition (P<0.001). Aross the tretments exept distill (0.1ml for1:22h, ml for 1:15h, ml for 0:58h) wter, % egg hthility inhiition ws linked to doses wheres the slowest dult motility rte ws P. sepilis t s well s tnnin onentrtion (P<0.001). Even though dose (0.1ml for 3:00h, ml for 2:53h, ml for 2:50h) ivermetin, positive ontrol, inhiited egg hthility (P<0.001). 162
At Prsitologi Glolis 5 (2): 160-168, 2014 Tle 1: Effets of doses of tnnin rih plnt extrts ivermetin nd distill wter on perent egg hthility inhiition Tretments (T) Dose (ml, D) Men SEM P-vlue (T D) Fius syhomorus 0.1 48.00 5.292 57.33 3.667 63.33 3.667 <0.001 Rhus glutinos 0.1 59.67 3.667 64.67 3.667 76.00 2.000 <0.001 Phyllnthus sepilis 0.1 46.00 8.145 54.00 8.145 57.33 6.888 <0.001 Ivermetin 0.1 50.67 7.965 68.67 1.667 74.00 4.726 <0.001 Distill wter 0.1 15.00 2.000 15.00 2.000 14.00 1.320 <0.175,, mens with different supersripts in the olumn re signifintly different (P<0.001); T, tretment; D, doses; T D, tretment dose intertion Tle 2: Effets of doses of tretments on lrvl development inhiition Tretments Dose (m1) Men SEM P-Vlue (T D) Fius syomorus 0.1 57.67 7.424 59.00 11.269 <0.001 67.33 10.138 Rhus glutinos 0.1 51.33 4.410 62.00 7.000 <0.001 73.33 9.667 Phyllnthus sepilis 0.1 54.67 6.119 56.33 8.007 <0.001 66.33 8.667 Ivermetin 0.1 43.67 4.410 55.67 3.180 <0.001 63.33 6.360 Distil H2O 0.1 27.00 3.215 24.33 5.812 0.455 21.73 8.891,, mens with different supersripts in the olumn re signifintly different (P<0.001); T, tretment; D, doses; T Í D, tretment dose intertion Tle 3: Adult motility inhiitions of tnnin rih plnt extrts, ivermetin nd distill wter Tretments Dose (ml) Men (time) SEM P-vlue (T D) Fius syomorus 0.1 2:59 0.219 2:49 0.259 <0.001 2:41 0.295 Rhus glutinos 0.1 1:22 0.094 1:15 0.079 <0.001 0:58 0.172 Phyllnthus sepilis 0.1 3:00 0.245 2:53 0.2489 <0.001 2:50 0.269 Ivermetin 0.1 0:57 0.219 0:52 0.235 <0.001 0:40 0.250 Wter 0.1 7:00 06 7:00 06 0.784 7:00 06,, mens with different supersripts in the olumn re signifintly different (P<0.001); T, tretment; D, doses; T Í D, tretment dose intertion 163
At Prsitologi Glolis 5 (2): 160-168, 2014 Fig. 1: Comprisons of doses of tnnin rih plnt extrts, ivermetin nd distill wter ginst % egg hthility,, inhiition ( different supersripts for % egg hthility inhiition, P<0.001) Fig. 2: Lrvl development inhiition tivities of tretment groups ( different supersripts for % lrvl development,, inhiition, P<0.001) 164
At Prsitologi Glolis 5 (2): 160-168, 2014,,,d Fig. 3: Effets of doses of plnt extrts, ivermetin nd distill wter on dult motility inhiition (hour, different supersripts for % dult motility inhiition, P<0.001) DISCUSSION Results from the in vitro trils n e influened nd onfounded y the presene, in the plnts, of other Different vlues of egg hthing inhiition (Tle 1) unknown iotive sustnes together with differenes in reltion to ondensed tnnin soures ould e in nutritionl vlues in ddition to the different extrtion ttriuted to vrition in geneti ftors nd iohemil solvents. Even if ivermetin (positive ontrol) inhiited tivity of the plnts. This ould e lso ssoited with egg hthility y 50.67-74%, ondensed tnnin the onentrtion of ondensed tnnin in plnt speies. extrted from Rhus glutinos hd more effiient Moln et l. [4] onfirmed the effetiveness of different (59.67-76%) in egg inhiition. This is lso in line with the plnt soure tnnins ginst egg hthing inhiition findings of Min et l. [35) who reported the effet of pity. Condensed tnnins n omplex with the sheth tnnins in egg hthing inhiition of H. ontortus. proteins of nemtodes, whih hve high proline ontent The signifint vritions in lrvl development tht prevent exshethment nd interlte with DNA inhiition of CTs (Tle 2) might e in onurrene with synthesis of prsites [30]. However, the ext the onentrtion of tnnins in eh plnts speies. This mehnisms of tion of these metolites remin osure ould e lso ttriuted to tnnin s pity to ind nd ould differ depending on the prsite, its stge of proteins nd operte through severl mehnisms. development nd possily, the iohemil hrteristis Condensed tnnins my ind to the utile of lrve of the plnt speies [31] while some studies hve shown whih is high in glyoproteins nd use their deth [36]. the effets of tnnins due to their ility to intert nd The lrvl inhiition effet of tnnins, third stge of protet degrdtion of ruminl proteins [32], others hve lrve (L3), on H. ontortus ws reported y Brru et l. demonstrted diret toxi effets of tnnins on nemtodes [37]. [33]. Findings from similr studies n therefore vry The result of the urrent study on lrvl inhiition widely depending on the predominnt mode of tion. pity ws lso supported with the work of Moln et l. It hs lso een suggested tht hemil struture of [4] who reported signifint redution of lrvl ondensed tnnins fr s iologil responses re development (L 1 to L 3) in CT treted H. ontortus. The onerned [35]. results of present study lso gree with findings of 165
At Prsitologi Glolis 5 (2): 160-168, 2014 Hoste et l. [30] who pointed out the most onsistent nti-lrvl effet of tnnins extrted from plnts with highest tnnin ontent. Hoste et l. [30], Ademol et l. [36] nd Min et l. [35] hd lso reported positive redution effet of tnnin in lrvl development of H. ontortus in their studies. Tnnins my ret diretly with dult worms y tthing to their skin, using them distress, or in diretly y improving protein nutrition of the host nd deresing the immune system [35]. The dult motility rte ws ssoited with CT onentrtion s well s plnt speies (Tle 3). Tnnin rih plnts perhps represent n lterntive option to ontrol nemtodes like H. ontotus nd the result of this study is in lose onformity to Mx et l. [32], Mihrete et l. [6] nd Verm et l. [37] who reported on the nthelminti tivity of CTs in prsiti redutions. Severl uthors [38-42] were lso dislosed the improvement of the produtive nd reprodutive performne of smllholders' herivore livestok through indigenous plnt preprtions s livestok dewormers. In onlusion, these findings onfirmed the effetiveness of nti-h. ontortus tion of ondensed tnnins s ivermetin, ommeril syntheti drug. However, more investigtion is required y using seril dilution test in further to llote the effetive nd effiient dose. The min dvntges of using in vitro tests/ssys to sreen the nti-prsiti properties of the plnts nd plnt extrts re low osts nd rpid turnover, whih llow sreening lrge numer of plnts. In spite of the dvntges of in vitro tests on evlution of nthelminti tivity of plnts, onsidertions should e lso tken in mind tht potentil iotive sustnes used in vitro my not lwys orrespond to in vivo iovilility. Therefore, in vitro ssys should lwys e ompnied y in vivo trils when used to vlidte nthelminti tivity of plnt remedies. Thus, it would e neessry to hieve some in vivo toxiologil nd prsitologil studies using ondensed tnnins. As finl point, euse the nti-hemohus tivity of Rhus glutinos ws found to e fesile s tht of ivermetin, therefore, this plnt n e used s lterntive nthelmenti for the tretment of Hemonhosis. ACKNOWLEDGEMENT The uthors would like to thnk the Jimm University nd VLIR-UOS institutionl university oopertion (IUC-JU) progrm for providing reserh filities nd finnil support. REFERENCES 1. Gethun, L., 2008. Produtive nd Eonomi performne of Smll Ruminnt prodution inprodution system of the Highlnds of Ethiopi. Ph.D. disserttion. University of Hohenheim, Stuttgrt-Hoheinheim, Germny. 2. FAO, 2009. Profile for Climte Chnge. Food nd Agriulture Orgniztion of the United ntions Rome, Itly 3. Yisehk, K., J.L. De Boever nd G.P.J. Jnssens 2014. The zootehnil effet size of polyethylene glyol supplementtion vries mong tnnin-rih plnt speies in zeu ulls (Bos indius). J. Anim. Physiol. Anim.l Nutrit., 98(4): 417-423. 4. Moln, A.L., R.A. Alexnder, I.M. Brookes nd W.C. MN, 2000. Effets of n extrt from sull(hedysrum oronrium) ontining ondensed tnnins on the migrtion of three sheep gstrointestinl nemtodes in vitro. Proeedings of the New Zelnd Soiety of Animl Prodution, 60: 21-25. 5. Mrkos, T., 2006. Produtivity nd helth of indigenous sheep reeds nd rossreds in the entrl Ethiopin highlnds. PhD disserttion. Deprtment of Anim Breeding nd Genetis, Fulty for Veterinry Mediine nd Animl Sienes, Swedish University of Agriulturl Sienes (SLU), Uppsl, Sweden. 6. Mihrete, B., G. Tilhun, K. Yisehk nd A. Mesele, 2011. In-vitro nthelminti tivity of ondensed tnnins from Rhus glutinos, Syzygium guineenses nd Alizi gummifer ginst sheep Hemonhus ontortus. Glo. Veteri, 6: 476-484. 7. Troell, K., P. Wller nd J. Hoglund, 2005. The development nd overwintering survivl of free- Living lrve of Hemonhus ontortus in Sweden. J. Helminthol., 79: 373-379. 8. Egule, T., G. Tilhun, A. Deell, A. Fleke nd E. Mkonnen, 2007. Hemonhus ontortus; in vitro nd in vivo nthelminti tivity of queous nd hydro-loholi extrts of Heder helix. Experim. Prsitol., 116: 340-345. 9. Mehlhorn, H., 2008. Enylopedi of Prsitology, rd 3 edn. Springer, New York 10. Kpln, R.M., J.M. Burke, T.H. Terrill, J.E. Miller, W.R. Getz, S. Moini, E. Vleni, M.J. Willims, L.H. Willimson, M. Lrsen nd A.F. Vtt, 2004. Vlidtion of the FAMACHA eye olor hrt for deteting linil nemi in sheep nd gots on frms in the southern United Sttes. Veterinry Prsitol., 123: 105-120. 166
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