Alternation of anthelmintic treatments: A molecular evaluation for benzimidazole resistance in nematodes

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1 Altenation of anthelmintic teatments: A molecula evaluation fo benzimidazole esistance in nematodes Vincent Leignel, Anne Silveste, Jean-Fançois Humbet, Jacques Cabaet To cite this vesion: Vincent Leignel, Anne Silveste, Jean-Fançois Humbet, Jacques Cabaet. Altenation of anthelmintic teatments: A molecula evaluation fo benzimidazole esistance in nematodes. Veteinay Paasitology, Elsevie, 2010, 172 (1-2, pp <bioemco > HAL Id: bioemco Submitted on 13 Aug 2010 HAL is a multi-disciplinay open access achive fo the deposit and dissemination of scientific eseach documents, whethe they ae published o not. The documents may come fom teaching and eseach institutions in Fance o aboad, o fom public o pivate eseach centes. L achive ouvete pluidisciplinaie HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau echeche, publiés ou non, émanant des établissements d enseignement et de echeche fançais ou étanges, des laboatoies publics ou pivés.

2 Veteinay Paasitology Volume 172, Issues 1-2, 27 August 2010, Pages Altenation of anthelmintic teatments: A molecula evaluation fo benzimidazole esistance in nematodes V. Leignel a, A. Silveste b, J.F. Humbet c and J. Cabaet b a Univesité du Maine, Laboatoie Me-Molécules-Santé, F Le Mans, Fance b INRA, UR1282 Infectiologie Animale et Santé Publique, F Nouzilly, Fance c INRA UMR Bioemco, Site de l ENS, Pais, Fance Received 24 Novembe 2009; evised 29 Mach 2010; accepted 20 Apil Available online 28 Apil Abstact The evolution of benzimidazoles (BZ esistance in Teladosagia cicumcincta was investigated in a contolled tial with lambs, submitted to diffeent teatment egimens. Fou paddocks wee seeded with a T. cicumcincta stain constituted by 25% of BZ-esistant nematodes. Ten pemanent lambs wee allocated to each paddock, fom Apil to Novembe in ode to enew the contamination of pastue. Monthly, thee tace lambs wee allocated in each paddock. BZesistant nematode fequency was detemined (PCR diagnosis. The faecal egg count eduction test (pemanent lambs and the numbe of nematodes in lambs wee also detemined (pemanent and tace lambs. Fou diffeent egimens of teatments wee pefomed: contol, levamisole (a non-bz dug, fenbendazole (a BZ dug, and an altenation of levamisole and fenbendazole evey second teatment. The same potocol was epeated on two consecutive gazing seasons, inceasing the numbe of teatments (3 in fist yea and 5 in second yea. The popotions of BZ-esistant nematodes did not change duing all the study in both the contol and the levamisole paddocks, suppoting an equal global fitness of BZ-esistant and susceptible nematodes. Thus, no evesion of BZ esistance is to be expected. In the altenated dug goup and in the BZ teated goup, BZ-esistant nematodes inceased fom 25% to 47% and to 78%, espectively. BZ esistance inceased popotionally to the selective pessue (numbe of BZ teatments. The dug altenation is not a good solution to delay impotantly the evolution of esistance when moe than 25% of nematodes ae BZ-esistant. This study is the fist evaluation of BZ-esistance evolution (using individual genotyping in contolled conditions. It showed that when a monogenic anthelmintic esistance is established at 25% in a sexually epoducing nematode population, it seems to be impossible to pevent its incease even when using limited numbe of BZ teatments. Keywods: Teatment altenation; Anthelmintic; Levamisole; Benzimidazoles; Nematode; Teladosagia 1. Intoduction The esistance against theapeutical poducts (antibiotic, anthelmintic, and insecticide is actually one of the majo contempoay challenges fo health maintenance. The anthelmintic esistance of nematodes constitutes a poblem in small uminants and othe hebivoes in most egions of the wold ([Chatie et al., 2001] and [Kaplan, 2004]. Rae new molecules ae found such as cyclooctadepsipeptides (Von Samson-Himmelstjena et al., 2005, o amino-acetonitile deivatives (Pichad and Geay, 2008 and they ae not maketed fo lage animals, fo which thee has not been a new class of anthelmintics intoduced in maketplace in almost 25 yeas (Kaplan, We must ely in most places on thee distinct anthelmintic goups with diffeent modes of action: the benzimidazoles (BZ/pobenzimidazoles, the imidazothiazoles/tetahydopyimidines and the macocyclic lactones (Fase et al., Unfotunately, the esistance emegency exists fo these thee dugs ([Sangste and Gills, 1999], [Sangste, 2001], [Kaplan, 2004] and [Bady and Nichols, 2009]. The BZ emain widely used in hebivoes thoughout the wold since they ae cheap and libeate few hamful esidues in the envionment. BZ-esistant nematodes ae efficiently contolled by imidazothiazoles o macocyclic lactones ([Oveend et al., 1994] and [Wauiu et al., 1996]. To maintain the BZ dug efficacy and to contol the evolution of esistance, the altenation of dugs o a simultaneous use of minimum two molecules had been poposed ([Donald et al., 1980], [Pichad et al., 1980], [Dobson et al., 1987], [Walle et al., 1989], [Uhlinge and Kistula, 1992], [Coles, 1994], [McKenna et al., 1996] and [Andews, 2000]. Fomulations pemitting the simultaneous use of two o thee molecules fom the existing thee main goups of anthelmintics ae available in counties such as Austalia (Q-dench, Titon, etc., New Zealand o Uuguay among othes. This solution is not sustainable fom the esidues, envionment and efficacy point of views. An altenation of BZ and anothe dug has been poposed ([Walle et al., 1989], [McKenna, 1990] and [Gimshaw et al., 1996]. Some studies had tested the anthelmintic altenation in expeimental conditions, at a time whee the genetic main mechanisms of esistance to BZ was not known. Futhemoe, evaluation of such altenation should be made in natual conditions since only the paasitic stages (lavae and adult ae submitted to anthelmintic pessues (in a lesse extent aested lavae and the fee-living stages on pastues can be compaed to a sub-population not submitted to selective pessues (in efuge : [Gaba et al., 2006a] and [Kenyon et al., 2009], that would peseve the anthelmintic susceptible alleles. We studied the evolution of BZ esistance in a population of Teladosagia cicumcincta (Nematoda, Tichostongylidae, one of the most fequent tichostongyle in the tempeate climatic zone. Its life cycle pesents a fee-living phase (lavae stages on pastues and a paasitic phase in the small uminant (adult stage. Although seveal mutations wee identified in β-tubulin gene ([Silveste and Cabaet, 2002] and [Ghisi et al., 2007], BZ

3 esistance is mainly confeed by a ecessive point mutation in isotype 1 of the β tubulin gene of T. cicumcincta (Elad and Humbet, 1999 as in most digestive-tact stongyles of uminants. One patially esistant isolate was seeded on fou paddocks and each flock on each paddock has a paticula anthelmintic egimen in ode to evaluate putative evesion (unteated contol, inteaction with levamisole, and the influence of selection pessue with BZ (altenation of teatment of BZ with levamisole o only BZ in condition of natual einfection duing two gazing seasons. 2. Mateials and methods 2.1. Nematode oigin and chaacteisation The T. cicumcincta stain was isolated fom a Fench goat fam in 1996 and maintained in laboatoy conditions duing two geneations. This stain was genotyped fo the BZ esistance by PCR accoding to Humbet and Elad (1997. This stain was genotyped in natual conditions (in fam in 1991 and 1996 (Elad et al., 1999, and befoe this study. All these estimations showed that this stain haboued 25% of BZ-esistant (homozygote nematodes and 75% of BZsusceptible nematodes (25% homozygote SS and 50% heteozygote S (Elad et al., Twenty-five pecent of BZ-esistant nematodes coespond to the lowest level of esistance detected by phenotypic measues (Matin et al., The T. cicumcincta stain was fully susceptible to levamisole Expeimental conditions The expeiment was conducted unde tempeate climate in Nouzilly, in westen Fance duing two consecutive yeas. Rainfalls wee 861 and 1032 mm duing the two consecutive gazing seasons, espectively, the latte being ove aveage. Fou adjacent paddocks (7000 m 2 of new pastue constituted of ye-gass and white clove wee seeded with the above descibed T. cicumcincta stain. The paddocks wee fist seeded with T. cicumcincta fom Septembe to Octobe befoe the fist gazing yea with 20 lambs peviously infected with 8000 infective lavae (L3. In Novembe, thee pais of these lambs wee eithe teated with levamisole, fenbendazole o emained unteated. The efficacies wee 99.5% and 83.0%, espectively, fo levamisole and fenbendazole. In Mach, the futue pemanent lambs (naive Romane beed wee infected with 4000 L3 of the same isolate so that they wee exceting eggs at tun out. Ten pemanent lambs wee gazed duing all the gazing season (mid-apil to mid-novembe on each paddock. Pemanent lambs pesent in paddock 1 wee non-teated contols and those on paddock 2 wee teated with levamisole (efficient against BZ-esistant nematodes, Anthelsol. Pemanent lambs fom paddock 3 wee altenatively teated with levamisole (in August duing fist yea, ne and Septembe duing second yea o a benzimidazole, fenbendazole (Panacu, in May and Octobe of fist yea and in May, ly and Octobe in second yea (Fig. 1. Pemanent lambs fom paddock 4 wee teated with fenbendazole. Pemanent lambs wee teated at the manufactue ecommended ovine dose. We thus ealized diffeent selective pocesses on the populations accoding to the anthelmintic egimen (Fig. 1. Both phenotypic and genotypic measues of BZ esistance wee achieved. Teatment efficacy was detemined by the faecal egg count eduction test (FECRT fo the two gazing seasons, on thee occasions (May, Septembe and Octobe fo the fist yea; ne, Septembe and Octobe fo second yea. The estimation of eggs pe gam (EPG was ealized the day of the teatment and 11 days afte teatment and a FECRT was calculated accoding to (Dash et al., 1988, as this fomula takes into account the possible evolution of egg output in contol goup. Dash et al., 1988 fomula elies on befoe and afte teatment evaluation in teated and contol hosts: FECR = 100 (1 [T 2 /T 1 ]/[C 1 /C 2 ] whee T 2 and T 1 ae pe- and post-teatment aithmetic means of the EPG in teated goups and C 1 and C 2 ae pe- and post-teatment aithmetic means of the EPG in the contols. Phenotypic esistance can be measued as 1 FECR. Fig. 1. Anthelmintic teatment pogam applied in the flocks gazing the fou paddocks. BZ, benzimidazoles; Lev, levamisole. At the end of each gazing season, all pemanent lambs wee slaughteed and nematodes counted on an aliquot as indicated in (Gaba et al., 2006b. Fo each paddock, a minimum 50 nematodes pe pemanent lamb wee genotyped to detect the BZ esistance accoding to Humbet and Elad (1997. A total of 1249 T. cicumcincta fom these pemanent lambs wee individually genotyped into BZ-esistant (, BZ-susceptible homozygotes (SS and heteozygotes (S fo this study. In paallel, thee tace lambs wee intoduced in each paddock at monthly o bimonthly intevals (ly August duing the two gazing seasons. Afte one month, each tace lamb was bought indoos fo two weeks and fed on uninfected hay, to let the late ingested lavae matue into adults. The tace lambs wee finally necopsied to collect the paasites. Altogethe, 120 tace lambs wee slaughteed, and 5658 T. cicumcincta (minimum 40 nematodes pe lambs wee genotyped to quantify the fequency of BZ-esistant genotypes Statistical analysis A geneal linea model (GLM was used to compae effects of teatment egimen on egg output, nematode buden and fequency of BZ-esistant nematodes in diffeent paddocks. The GLM use of a hieachical egession analysis technique allows geate flexibility than usual analysis of vaiance by allowing one to combine quantitative and categoical vaiable and to contol fo covaiates. Thus a model, y = a 1 x 1 + a 2 x a i x i + eo is evaluated, whee y is the paamete we want to evaluate (e.g., EPG in elation to othe expeimental factos (x 1 to x i, e.g. paddock, season, yea of expeiment. The value of the model is expessed by a R 2 value and a level of statistical significance p. When the model was significant estimated maginal aveages wee calculated. These aveages wee adjusted fo the covaiates (season and

4 yea of expeiment fo tace lambs, and yea fo pemanent lambs. The data did not follow a Gaussian distibution, and wee nepeian logaithm tansfomed (log + 1. Post hoc significance was established with Newman Keuls test. Calculations wee pefomed using Simstat softwae (Péladeau and Lacoutue, Results 3.1. Dynamic of infection based on pemanent lamb egg output and adult nematode buden in tace lambs Nematode buden of pemanent lambs illustate the effects of epeated teatments on nematode establishment afte one yea of infection (Table 1. The numbe of nematodes and the numbe of aested lavae pe pemanent lamb wee not significantly diffeent between paddocks duing the fist and the second gazing season (Table 1. Aested lavae wee thee- to foufold highe in second season in compaison with the fist, in all fou paddocks. These data give a good pictue of infection at the end of gazing season but do not infom on the paasitic pessue all along the gazing season. Convesely the aveage EPG (Table 1 give futhe infomation on paasitic pessue: it was highest in the contol paddock in the second gazing season, as obseved fom nematode budens. Table 1. Aveage, standad deviation (SD and ange of adult nematodes and aested lavae (L4 pe pemanent lamb necopsied in the two consecutive seasons fo the fou paddocks. EPG Paddock 1 (contol Paddock 2 (levamiso le teatment Paddock 3 (altenati on of teatment Paddock 4 (fenbendazo le teatment Fist yea Aveage 168 a* 129 a 141 a 145 a SD Range Second yea Aveage 98 a 58 b 55 b 72 b SD Range Nematode s Adults L4 Adults L4 Adult s L4 Adult s L4 Fist yea Aveage 2188 a 4483 a 1533 a 2860 a 1974 a 8473 a 3249 a 5195 a SD Range , , , , ,833 Second yea Aveage 3411 a 11,636 b 2324 a 9747 b 1524 a 10,464 b 2453 a 14,880 b SD , , , ,701

5 EPG Paddock 1 (contol Paddock 2 (levamiso le teatment Paddock 3 (altenati on of teatment Paddock 4 (fenbendazo le teatment Range , , , ,570 Estimate d maginal aveage * * 2798 a 7714 b 1928 a 6303 b 1749 a 8863 b 2851 a 10,037 b * Simila lettes in a column indicates that no significant diffeence was shown between paddocks and in a line, between yeas, using GLM. ** GLM with yea and paddock as factos. Duing summe, an incease of EPG was obseved in all paddocks fo the two yeas (Fig. 2. In fist gazing season, the aveage egg outputs pe pemanent lambs in the contol paddock (paddock 1 was highe than EPG obseved in all teated paddocks (Table 1, indicating that even when 25% of nematodes ae BZ-esistant, BZ teatment is useful to educe egg outputs and pastue contamination. In second gazing season, aveage egg outputs wee twice highe than in fist yea in paddock 4 (benzimidazole teatment wheeas othe paddocks pesented simila egg outputs (Table 1. The contol paddock had globally highe egg outputs than othe paddocks. Fig. 2. Evolution of aveage egg outputs duing the two consecutive gazing seasons in pemanent lambs of the fou paddocks (1: contol, 2: levamisole, 3: levamisole and benzimidazole, 4: benzimidazole. Figues coespond to estimated maginal aveages, estimated fom GLM: R 2 = 0.33, p = 0.001; significant covaiates: paddock, yea, month, and inteaction between paddock, yea and month. Nematode budens obseved in tace lambs illustate the infection level of each paddock (Fig. 3 and Table 2. The stongest incease of nematode buden was obseved duing the sping of fist yea and the summe of second yea, in all paddocks. The numbe of nematodes in tace lambs was highe in the paddock 1 (contol goup eaching an aveage of 7274 and 8400 nematodes pe lamb in the two consecutive gazing seasons, espectively. No significant diffeence was obseved between the paddocks 2 4 (teated goups in fist gazing season. Convesely, in second yea, the numbe of nematodes in tace lambs was influenced by the anthelmintic egimen administeed to pemanent lambs. In the paddocks 2 (levamisole teatment and 3 (altenating teatment, nematode populations wee limited to 5000 nematodes in aveage (maximum obseved 9739 nematodes; but in paddock 4 (benzimidazole teatment nematode population eached 6800 nematodes in aveage, eaching a maximum obseved aound 14,600 nematodes, in spite of fenbendazole teatment (Fig. 3 and Table 2. Fig. 3. Evolution of aveage nematode buden duing the two consecutive gazing seasons in tace lambs of the fou paddocks (1: contol, 2: levamisole, 3: levamisole and benzimidazole, 4: benzimidazole. Figues coespond to estimated maginal aveages, estimated fom GLM: R 2 = 0.72, p = 0.001; significant covaiates: paddock, yea, month, and inteaction between paddock, yea and month. Table 2. Aveage, standad deviation (SD adult nematodes pe tace lamb necopsied in the two consecutive gazing seasons fo the fou paddocks. Paddock 1 (contol Paddock 2 (levamisole teatment Paddock 3 (altenative teatment Paddock 4 (fenbendazole teatment Fist gazing season Aveage SD

6 Paddock 1 (contol Paddock 2 (levamisole teatment Paddock 3 (altenative teatment Paddock 4 (fenbendazole teatment Range , Second gazing season Aveage SD Range , ,599 Estimated maginal aveage * 7758 a** 4093 b 4376 b 5068 b * GLM with yea, peiod and paddock as factos, p = ** Simila lettes in a line indicates that no significant diffeence was shown between paddocks using GLM Evolution of esistance in elationship to benzimidazole selective pessue A phenotypic and a genotypic measue of BZ anthelmintic esistance wee achieved with a FECRT and a PCR diagnosis, espectively (Table 3. Convesely to genotyping, the FECR data pesented a less clea evolution of esistance. In paddock 3, the efficacy of BZ dug was unchanged in fist gazing season (switching fom 67% to 69%, although the fequency of BZ-esistant genotypes inceased modeately fom 22% to 31%. In second gazing season, the efficacy of BZ dug switched damatically fom 89% to 19%, although the fequency of BZ-esistant genotypes was unchanged (44.8% to 47%. In paddock 4 (benzimidazole teatment, the BZ efficacy switched fom 69% to 43%, coesponding to the incease of the fequency of BZesistant genotypes fom 21.4% to 52%. Similaly in second yea, BZ efficacy was maintained aound 65% and 27% and the fequency of BZ-esistant genotypes inceased fom 60% to 78%. In paddock 2 (levamisole teatment, the FECRT indicated an impotant vaiability of the levamisole efficacy in two consecutive gazing seasons (anged between 16% and 91% although the T. cicumcincta stain is fully levamisole susceptible (Table 3. BZ FECRT evaluated with Dash et al. fomula was significantly elated to the pecentage of allele ( s = 060; p = on data of paddocks 3 and 4. Table 3. Benzimidazole esistance evaluation (fequency of BZ allele, fequency of BZ nematodes, FECRT in tace lambs and in pemanent lambs, along the two gazing season expeiment. Numbes between backets coespond to standad deviation. ND: not detemined. Fist gazing season Second gazing season Tace lambs Pem anen t Tace lambs Pem anen t May ne ly Au gu st Sept embe Oct obe Octo be ne ly Au gu st Sept embe Octobe Novemb e Nove mbe Paddock 1 Contol % nemato de 26.2 ( (3 22 ( ( ( ( ( ( ( ( (6.4 EPG 213 a ND ND ND 99 ND ND

7 Fist gazing season Second gazing season Tace lambs Pem anen t Tace lambs Pem anen t May ne ly Au gu st Sept embe Oct obe Octo be ne ly Au gu st Sept embe Octobe Novemb e Nove mbe Paddock 2 Levamisole % nemato de 22.4 ( ( ( ( ( ( ( (6 28 ( ( (2 FECRT 59% b ND ND 80% 52% ND 84 % ND 91% 16% ND Paddock 3 Levamisole /fenbendaz ole % nemato de 22 ( ( ( ( ( ( ( ( ( ( (13. 1 FECRT 67% ND ND 85% 69% ND 89 % ND 64% 19% ND Paddock 4 Fenbendazo le % nemato de 21.4 ( ( ( ( ( ( ( ( ( ( (4.6 FECRT 69% ND ND 70% 43% ND 65 % 67% 27% ND % significan ce 1 = 2 = 3 = 4 c 1 = 2 < 3 < 4 FECRT significan ce 1 > 2, 3, 4 1 > 2, 3, 4 1 > 2, 3, 4 1 > 2, 3, 4 1 < 2, 3, 4 1 = 2 = 3 = 4 Full-size table a Estimated EPG. b FEC eduction using Dash et al. fomula. c Significance of diffeence between paddocks (e.g., 1 > 1, 2, 3, 4: contol (paddock 1 has highe EPG than teated goups.

8 In all fou paddocks, BZ esistance was measued monthly (bimonthly in ly August by the PCR diagnosis on adult nematodes, collected afte necopsy of tace lambs. No significant evolution of the fequency of BZ-esistant genotypes was obseved in the paddocks 1 (contol and 2 (afte 8 levamisole teatments duing the second gazing season. The fequency of was well estimated by a GLM (R 2 = 0.87, p = The significant vaiables in model wee paddock, yea, month, and inteaction between paddock, yea and month. The significant diffeences between paddocks wee evaluated with Newman Keuls (see the legend in Table 3. Fequency of BZ-esistant nematodes was maintained aound 25 30%. In paddock 3 whee pemanent lambs wee teated with an altenating anthelmintic egimen (levamisole o fenbendazole, the fequency of BZ-esistant genotypes inceased fom 23% to 47% afte 5 BZ and 3 levamisole teatments, duing the two gazing seasons. The stongest incease of the fequency of BZesistant genotypes was obseved in the paddock 4, whee only fenbendazole teatments wee ealized: BZ-esistant genotypes inceased fom 21% to 78% afte 8 BZ teatments (Table 3. The fequency of BZ-esistant genotypes in pemanent lambs, at the end of the gazing season, was 28% (paddock 1, 26% (paddock 2, 53% (paddock 3 and 61% (paddock 4 in fist yea. Afte the second gazing season, this fequency was unchanged in paddock 1 (22%, in paddock 2 (20% and in paddock 3 (54%, but in paddock 4 the fequency of BZ-esistant genotypes eached 89%. In paddock 2 (levamisole teated goup, levamisole teatment administeed in Octobe fom second gazing yea was inefficient: 16% (Table 3. This is consistent with the known inefficacy of levamisole against aested L4 lavae, which epesent 93% of the whole nematode buden in paddock 2 (Table Discussion Ou main objective was to investigate the evolution of BZ esistance when nematodes wee maintained unde diffeent teatment egimens. In ou expeimental design, the gazing season anged fom Apil May to Novembe, the stocking ate was 16 lambs/ha, the numbe of anthelmintic teatments anged fom 3 (in fist yea to 5 (in second yea, we used fenbendazole (a benzimidazole and levamisole (an imidazothiazole anthelmintics. Although ou expeimental design with one single species was simplified compaed to odinay fam whee lambs ae infected with a community of nematodes, this simplification should not affect the qualitative findings on contol and esistance epoted hee. Few studies wee conducted on dug combination efficiency, and anthelmintic esistance was monitoed by phenotypic methods (FECRT and in vito lethal dose on eggs, see [Coope et al., 1996], [Maingi et al., 2002], [Wigley et al., 2006] and [Sutheland et al., 2008]. Dug altenation studies wee also ae (Uhlinge and Kistula, In the pesent study, we monitoed pecisely evolution of BZ-esistant gene fequency by individual genotyping of nematodes. We used FECRT to estimate phenotypic efficacy and subsequent impact of teatments on pastues contamination. Fo both BZ and levamisole, FECRT yielded vey diffeent estimates of efficacy. Although not elated to anthelmintic esistance, poo efficacy of both dugs has been ecoded peviously, in pat elated to the pesence of T. cicumcincta inhibited lavae which ae less susceptible to eithe imidazothiazoles ([Walley, 1966], [McKenna, 1974] and [Williams, 1991] o BZ (Cabaet et al., 1979 to a lesse extent, as obseved at the end of each gazing season. The iegula efficiency of FECRT to detect esistance has been questioned (Humbet et al., 2001 and we concentated on the fequency of BZ-esistant nematodes to expess BZ esistance. In paddock 1 (contol goup and paddock 2 (levamisole goup, final fequencies of BZesistant and BZ-susceptible nematodes in pemanent lambs wee unchanged at the end of the study (Table 3. Howeve it seems that autumn peiod is unfavouable to the suvival of lavae since thee is a decease in pecentages of in contol paddock and absence of expected incease of in the paddocks 3 and 4 (Table 3. The latte is in accodance with the findings of Elad et al. (1998 in laboatoy conditions (paametes: pime-infection of naive lambs, cultue fom eggs to lavae at stable tempeatue 23 C, stoage of infective lavae at 4 C duing moe than thee months. This suppots an equivalent global fitness (e.g., including majo life taits of esistant and susceptible nematodes, but some life taits (such as suvival of fee-living stages to low tempeatue may be compensated thoughout the life cycle of T. cicumcincta. This is in accodance with othes findings on Haemonchus contotus (Melo, 2005, and the bette suvival of BZ-esistant lavae to dy climate and the bette suvival of BZ-susceptible lavae to hot tempeatue. The possible evesion of the BZ esistance should be studied futhemoe, exploing diffeential success in life taits of esistant and susceptible nematodes. Ou esults show that altenation of BZ and levamisole succeeded in delaying the selection of BZ esistance at medium tem, in compaison with the exclusive BZ teatment egimen: in paddock 3 (altenation of BZ and levamisole, fequency of BZ-esistant nematodes eached 45% at the end of the expeiment, coesponding to 5 BZ teatments (and 3 levamisole teatments. In paddock 4, the 5th BZ teatment was done in ne fom second yea, and the fequency of BZesistant nematodes was 78%. So, the same numbe of BZ teatment (hee, 5 teatments selected less apidly esistant nematodes in paddock 3 than in paddock 4. Nevetheless, the altenating teatment egimen has a shot lasting effect (Uhlinge and Kistula, In paddock 4, the fequency of BZ-esistant nematodes switched fom 45% to 80% with only one supplementay BZ teatment (administeed in ne of second gazing season. So, in all likelihood, the altenating teatment egimen would esult in such high BZ-esistance fequency duing the subsequent gazing season. In the pesent study, lambs wee set stocked duing the entie gazing season, favouing effects of efuge of fee-living stages pesent on pastues (Van Wyk, Duing the ealy phase of selection, when esistance gene fequency is 0.5%, fee-living stages pesent on pastues may favou the loss of esistance gene by genetic dift (Gaba et al., 2006a. Convesely, the pesent study shows that efuge is not efficient anymoe to delay selection when the fequency of BZ-esistant nematodes eaches high level such as 25% of the population. This study demonstated that when the BZ-esistant nematodes ae pesent at medium level (25% in the pesent study in natual population, no evesion seems to be possible, even if

9 anothe anthelmintic molecule without any coss-esistance is used. Refuge is not efficient enough at medium esistance level to allow the loss of esistant alleles by genetic dift (Gaba et al., 2006a. The altenating teatment with levamisole/fenbendazole seemed efficient to contol adult nematodes, but it did not allow the contol of aested lavae o the delay of BZ-esistance allele selection. As a conclusion, we may ecommend to use BZ until BZ esistance is fully pesent, unde tempeate climate. Fist, even fo medium high BZesistance fequency, BZ teatment allowed a eduction of both nematode buden and pastue contamination. Second, BZ ae boad-spectum anthelmintics allowing Dicocoelium and pulmonay stongyles contol. Thid, BZ ae efficient against susceptible aested lavae, convesely to othe boad-spectum anthelmintics ([Andews, 2000] and [Batley et al., 2004]. When BZ esistance is obseved, dug combination may favou a bette nematode contol than dugs alone (Albonico et al., 2003 but futhe wok is equied to demonstate a eal synegism between dugs (Entocasso et al., Acknowledgements Vincent Leignel was funded by a PhD gant fom the Fench Ministy of Reseach. Additional financial suppot was povided by Biotechnocente and Hoechst Company (now Intevet. Refeences Albonico et al., 2003 M. Albonico, Q. Bickle, M. Ramsan, A. Monteso, L. Savioli and M. Taylo, Efficacy of mebendazole and lévamisole alone o in combination against intestinal nematode infections afte epeated tageted mebendazole teatment in Zanziba, Bull. Wold Health Ogan. 81 (2003, pp View Recod in Scopus Cited By in Scopus (79 Andews, 2000 S.J. Andews, The efficacy of levamisole, and a mixtue of oxfendazole and levamisole, against the aested stages of benzimidazole-esistant Haemonchus contotus and Ostetagia cicumcincta in sheep, Vet. Paasitol. 88 (2000, pp Abstact PDF (50 K View Recod in Scopus Cited By in Scopus (5 Batley et al., 2004 D.J. Batley, F. Jackson, E. Jackson and N. Sagison, Chaacteisation of two tiple esistant field isolates of Teladosagia fom Scottish lowland sheep fams, Vet. Paasitol. 123 (2004, pp Aticle PDF (114 K View Recod in Scopus Cited By in Scopus (28 Bady and Nichols, 2009 H.A. Bady and W.T. Nichols, Dug esistance in equine paasites: an emeging global poblem, J. Equine Vet. Sci. 29 (2009, pp Aticle PDF (170 K View Recod in Scopus ename=656&_oiginpage=aticle&_zone=at_page&_tageturl=http%3a%2f%2fwww.scopus.com%2finwad% 2Fcitedby.ul%3Feid%3D2-s %26patneID%3D10%26el%3DR3.0.0%26md5%3D74f40452f30e0d0ee63ea940c043365d&_acct=C &_vesion=1&_useid= &md5=f7e316bd68dbe3e2ffd8faff1bd7f5e5Cited By in Scopus (0 Cabaet et al., 1979 J. Cabaet, H. Ouhelli and A. Dakkak, Efficacité compaée du fenbendazole et du tétamisole su les helminthes paasites du mouton au Maoc. II. Helminthes du tube digestif, Rec. Med. Vet. 155 (1979, pp View Recod in Scopus Cited By in Scopus (3 Chatie et al., 2001 C. Chatie, F. Soubiac, I. Pos, A. Silveste, J. Hubet, C. Couquet and J. Cabaet, Pevalence of anthelmintic esistance in gastointestinal nematodes of daiy goats unde extensive management conditions in southwesten Fance, J. Helminthol. 75 (2001, pp View Recod in Scopus Cited By in Scopus (20 Coles, 1994 G.C. Coles, Contol of anthelmintic esistant nematodes. In: G.C. Coles, F.H.M. Bogsteede and S. Geets, Editos, Anthelmintic Resistance in Nematodes of Fam Animals Repot of a Semina Oganised fo the Euopean Commission, Bussels, 8 9 Novembe 1993 (1994, pp Coope et al., 1996 N.A. Coope, P.F. Rolfe, J.E. Season and K.L. Dawson, Naphthalophos combinations with benzimidazoles o levamisole as effective anthelmintics fo sheep, Aust. Vet. J. 74 (1996, pp Full Text via CossRef View Recod in Scopus Cited By in Scopus (2 Dash et al., 1988 K. Dash, K. Hall and I.A. Bage, The ole of aithmetic and geometic wom egg counts in faecal egg count eduction test and in monitoing stategic denching pogams in sheep, Aust. Vet. 65 (1988, pp Full Text via CossRef View Recod in Scopus Cited By in Scopus (59 Dobson et al., 1987 R.J. Dobson, D.A. Giffiths, A.D. Donald and P.J. Walle, A genetic model descibing the evolution of levamisole esistance in Tichostongylus colubifomis, a nematode paasite of sheep, IMA J. Math. Appl. Med. Biol. 4 (1987, pp View Recod in Scopus Cited By in Scopus (16 Donald et al., 1980 A.D. Donald, P.J. Walle, R.J. Dobson and A. Axelsen, The effect of selection with levamisole on benzimidazole esistance in Ostetagia spp. of sheep, Int. J. Paasitol. 10 (1980, pp Abstact PDF (1016 K View Recod in Scopus Cited By in Scopus (5 Elad et al., 1999 L. Elad, J. Cabaet and J.F. Humbet, PCR diagnosis of benzimidazolesusceptibility o -esistance in natual populations of the small uminant paasite, Teladosagia cicumcincta, Vet. Paasitol. 80 (1999, pp Aticle PDF (228 K View Recod in Scopus Cited By in Scopus (52

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