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Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland Sarah Murri a,d , Gabriela Knubben-Schweizer b , Paul Torgerson c , Hubertus Hertzberg d,∗ a Ambulatory Clinic and Herd Management Unit, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland b Clinic for Ruminants with Ambulatory and Herd Health Services at the Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-University Munich, Sonnenstrasse 16, 85764 Oberschleissheim, Germany c Section of Epidemiology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, 8057 Zurich, Switzerland d Institute of Parasitology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 266a, 8057 Zurich, Switzerland
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Article history: Received 4 October 2013 Received in revised form 18 February 2014 Accepted 24 February 2014
Keywords: Goats Gastrointestinal nematodes Eprinomectin Anthelmintic resistance Switzerland
a b s t r a c t Eprinomectin (EPN) is a member of the avermectin class of compounds and the only anthelmintic registered for goats in Switzerland with a zero milk withdrawal period. The aim of the present study was to identify the actual efficacy of EPN in an area with a higher density of goat enterprises. Forty-three randomly chosen farms from canton Berne were investigated. At least eight goats were investigated on every farm. Conditions for inclusion in the study were the absence of anthelmintic treatment during the previous six weeks and a pooled faecal sample showing a mean faecal egg count (FEC) higher than 600 epg faeces. Pre- and 14–16 days post-treatment samples were individually collected directly from the rectum. Animals were treated with the recommended dose of EPN (1 mg/kg body weight) after taking the pre-treatment samples. Efficacy of EPN was tested with the faecal egg count reduction test (FECRT) and faecal cultures were performed on every farm from pooled faeces samples before and after treatment. Additionally the farmers completed a questionnaire. None of the gastrointestinal nematode populations of the 43 investigated farms were susceptible to EPN at the required level. The mean egg count reduction was 40%. None of the typical risk factors, such as production type, stocking rate, animal traffic and quarantine measures showed an association with the level of eprinomectin resistance. It can be concluded with 80% certainty that the prevalence of EPN resistance on goat farms is at least 95% in canton Berne. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Anthelmintic resistance of gastrointestinal nematodes (GIN) has become a globally important issue for the goat and sheep industry (reviewed by Kaplan, 2004; Jabbar
∗ Corresponding author. Tel.: +41 44 635 85 16; fax: +41 44 635 89 07. E-mail address: [email protected] (H. Hertzberg).
et al., 2006). In Switzerland benzimidazole resistance is widespread in GIN of small ruminants (Hertzberg et al., 2000). After the detection of the first case of avermectin (AVM) resistance in goats in Switzerland (Schnyder et al., 2005), a further study, focussing on Boer goats and Dorper sheep showed that AVM resistance seems to be widespread in GIN of these breeds (Artho et al., 2007). Eprinomectin (EPN) is a member of the avermectin class of compounds and the only anthelmintic registered for
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Please cite this article in press as: Murri, S., et al., Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.052
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goats in Switzerland with a zero milk withdrawal period, making it desirable in lactating animals (Dupuy et al., 2001). Targeted selective treatment (TST) is seen as an important development to slow down the development of anthelmintic resistance (reviewed by Kenyon et al., 2009). Integrated in a larger study on the implementation of the TST-approach, the present study identified the efficacy of EPN in an area with a high density of goat enterprises. Canton Berne, representing the Swiss midland, as well as the (pre-)alpine region was chosen as study region, as approximately 25% of the total Swiss goat farms (7190) are located in that area.
2. Materials and methods Farms in canton Berne with at least eight goats were randomly contacted by phone. Addresses were provided by the extension and health service for small ruminants (ESSR). Conditions for inclusion in the study were the absence of anthelmintic treatment during the previous six weeks and a mean faecal egg count (FEC) higher than 600 epg faeces. This was determined by a pooled faecal sample provided by the owners. Owners were asked to complete a questionnaire including general farm management, housing systems, animal movements, worm control practices and anthelmintic usage. Pre- and 14–16 days post-treatment samples were individually collected directly from the rectum. Pre-treatment faecal samples were collected from eight animals and post-treatment samples were taken from the six animals with the highest pre-treatment FECs. Because of Poisson errors removing these low counts improves the accuracy of the FECRT. For example, Coles et al. (2006) recommend removing all counts of less than 150 epg from the analysis. Animals were treated with the recommended dose of eprinomectin pour-on solution (1 mg/kg body weight, Eprinex® Pour-On, Biokema). The anthelmintic was administered with the commercially available applicator along the dorsal line, from the withers to the tail head, parting the coat to obtain direct contact with the skin. To avoid underdosing, body weight was visually estimated for every goat individually by two experienced persons, and then an additional 20% of the estimation was added to calculate the individual administered dose. Strongyle nematode FECs were determined with a sensitivity of 50 epg using the modified McMaster technique according to Schmidt (1971). Efficacy of EPN was tested with the FECRT, based on the recommendations of the World Association for the Advancement of Veterinary Parasitology (Coles et al., 1992; reviewed by Coles et al., 2006), but using the statistical analysis of the faecal egg counts according to Torgerson et al. (2005). Due to the generally small flock size, instead of including a control group the mean FECs before and 14–16 days post-treatment were compared. Faecal cultures (Eckert, 1960) were performed on every farm from pooled faeces samples before and after treatment, using 4 g faeces from each animal. 100 third stage larvae (L3) were differentiated according to MAFF (1986) and Levine (1968) at 400× magnification to determine the genera present.
Relationships between the farm specific parameters and the resistance level (based on the FECR values) were tested for significance using a generalised linear model with a negative binomial distribution assumed for the egg counts. The significance level was determined at p = 0.05. All calculations were undertaken in R (R Development Core Team, 2013), using the MASS package (Venables and Ripley, 2002). 3. Results Based on the detected resistance status the survey was terminated after investigation of 43 farms, sampled from August 2011 to November 2012. A total of 258 goats were included in the study. The investigated farms had a flock size between eight and 67 animals and were located in all parts of the canton (Fig. 1) at altitudes between 400 and 1300 metres above sea level. The Saanen and Chamoisee were the most frequent breeds (21% and 14% respectively); in many cases more than one breed per farm were kept (37%). The reduction of the mean faecal egg count after treatment was less than 95% on any of the investigated farms (Table 1). In two farms the reduction was between 90 and 93% with lower confidence intervals below 90%. Thirtynine farms (91%) showed reductions lower than 90%. The mean level of egg count reduction was 40%. Thus it was concluded that there was an 80% certainty for the presence of anthelmintic resistant GIN on at least 95% of goat farms in canton Berne. Differentiation of third-stage-larvae in the pretreatment larval cultures demonstrated that Haemonchus contortus is the predominant species on 33 farms (77%). Trichostrongylus spp. and Teladorsagia spp. were dominant on eight and one farm(s) respectively. For one farm (no. 25) evaluation was not possible due to immature larvae. In post-treatment larval cultures, H. contortus was the dominant resistant species on 41 (95%) farms. In two cases Trichostrongylus spp. was dominant. In addition small proportions of resistant Teladorsagia spp. were detected in 18 farms. In 35 out of 43 farms (81%) goats were kept for milk production and in eight farms (19%) for meat production and breeding purposes. No significant difference was found between milk-producing and non-milk-producing farms with respect to the level of anthelmintic resistance. On all farms, goats had regular access to pasture, but duration of grazing was highly variable. There was no evidence of an association between reported stocking rate and the degree of resistance. Cattle were kept on 25 of the 43 farms (58%), but only 13 farms (30%) kept their goats at least partially on the same pastures as cattle. Sharing pastures with cattle was not associated with the level of resistance. In 12 of 43 farms (28%) all goats (16%) or part of the flock (12%) were grazed alone or with goats from up to six other farms on alpine pastures during the summer. There was no association between transhumance and the level of eprinomectin resistance. Four of 43 farmers (9%) stated that they undertook quarantine measures when integrating new animals into their flocks. Forty-four percent of the farmers answered that parasitic infection represent a
Please cite this article in press as: Murri, S., et al., Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.052
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Fig. 1. Distribution of the investigated farms (white dots) in canton Berne, Switzerland.
problem on their farm. Less than 3 annual anthelmintic treatments were performed by 22 farmers (51%), whereas 21 farmers (49%) treated their animals three or more times per year; no difference was found between these categories with respect to the actual level of resistance (p = 0.91). Eprinomectin had been used previously in 37 of 43 (86%) farms. Farmers relied mainly on macrocyclic lactones and the benzimidazoles (72%), although several farmers no longer use benzimidazoles (28%) because of suspected or confirmed ineffectiveness. Additionally many farmers (44%) use levamisole (Endex® 8.75%, Novartis) off label. 4. Discussion The FECRT indicated that in none of the 43 farms was there sufficient efficacy of EPN against GIN. From these results the greater than 95% prevalence of EPN resistance in canton Berne was estimated with a certainty of 80%. This was much higher than expected and indicates a major EPN resistance problem, especially with respect to H. contortus. With this level of resistance, it would be futile to continue the use of EPN to control GIN in goats in this region and alternative approaches to the control of these parasites must be utilised. In Europe cases of AVM resistant GIN in goats were described in Scotland by Jackson et al. (1992) and subsequently in Denmark and the Netherlands (Maingi et al., 1996; Eysker et al., 2006). The first evidence of AVMresistance in Switzerland was reported 2005 from a Boer goat flock (Schnyder et al., 2005) with an efficacy of ivermectin of 61%, and later confirmed in a further study (Artho et al., 2007), where in 7 and 8 of 24 Boer goat farms AVMresistance was respectively confirmed or suspected. In both
studies the predominant resistant parasite species was also H. contortus. It was suspected that resistant GIN were imported with Boer goats from South Africa to Switzerland between 1999 and 2004. Subsequently the regulations were changed so that only embryos are allowed for importation. During the last decade many farmers bought Boer goat sires and used them to improve the beefiness of their dairy breeds, a practice which most likely favoured the spread of resistant GIN populations. Scheuerle et al. (2009) reported two single cases of EPN resistance in goats in Switzerland and Southern Germany with efficacies of EPN of only 17.4% and 27.5%. All of the participating farms in the present study have a history of using other macrocyclic lactones such as ivermectin, so it is likely that AVM-resistance was present at the time of introduction of EPN into the Swiss market. This would also be an explanation for the finding of EPN resistance on six farms that have never previously used eprinomectin. In Brazil a similar situation was found for Anglo-Nubian goats that were treated previously with ivermectin and moxidectin, leading to a GIN population consisting mainly of H. contortus that was unresponsive to EPN on its first use (Souza Chagas et al., 2006). Eprinomectin as a pour-on formulation has been available in Switzerland for use in cattle since 1999 and in addition was registered in 2004 for goats at 1.0 mg/kg body weight (twice the dose recommended for cattle). The optimal route of administration of EPN has since been debated. Silvestre et al. (2007) found no significant differences in FECR if goats had been shaved to facilitate the absorption of the drug but they recommended oral rather than topical application. Lespine et al. (2003) examined the efficacy of administering EPN by subcutaneous injection, finding this to be 2.5 times more efficient than the usual
Please cite this article in press as: Murri, S., et al., Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.052
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Table 1 Pre- and post-treatment mean faecal egg counts (epg) for every farm, analysis according to Torgerson et al. (2005); in the case of statistically significant reduction, lower and higher confidence intervals are given in brackets (CIs). Percentage of GIN larvae genera/species detected in coprocultures of pooled faecal samples after treatment. Farm ID
Mean epg before treatment (CIs)
Mean epg after treatment (CIs)
Mean reductions (CIs)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
622 (425–941) 970 (652–1185) 1439 (978–2184) 1401 (997–2006) 2530 (1570–4074) 673 (463–1012) 1273 (840–1992) 2452 (1772–3349) 1223 (882–1780) 1281 (903–1916) 1079 (676–1926) 1414 (928–2239) 1361 (809–2375) 917 (679–1245) 551 (391–787) 569 (416–814) 467 (311–720) 1092 (841–1435) 1825 (1380–2427) 2118 (1432–3123) 556 (398–796) 1499 (1142–1998) 716 (480–1116) 2528 (1458–4274) 2008 (1249–3249) 1022 (759–1399) 1147 (769–1794) 1642 (896–3054) 853 (707–1034) 2478 (1609–3760) 1349 (1034–1765) 1281 (912–1827) 1353 (609–3065) 1023 (733–1463) 965 (559–1786) 878 (555–1490) 1220 (803–1931) 647 (423–1016) 2004 (1059–3824) 668 (469–952) 2207 (1341–3598) 1445 (1029–2089) 1059 (885–1234)
315 (207–479) 64 (28–130) 1301 (943–1816) 565 (340–1143) 3082 (1922–4638) 968 (622–1551) 345 (195–741) 1640 (890–3097) 550 (219–1795) 135 (76–245) 116 (64–210) 237 (61–1813) 325 (113–1441) 458 (257–995) 360 (205–762) 468 (334–656) 264 (160–531) 249 (113–841) 1533 (885–2737) 2920 (1530–5159) 394 (233–767) 1161 (703–2034) 362 (140–1424) 793 (417–1751) 1059 (605–1981) 279 (172–451) 918 (662–1309) 731 (468–1230) 676 (556–827) 2601 (1344–4706) 924 (366–2538) 1440 (870–2484) 2374 (1512–3677) 182 (96–499) 204 (89–817) 589 (339–1190) 510 (192–1814) 688 (427–1216) 659 (454–999) 249 (164–385) 1163 (591–2525) 623 (450–855) 658 (521–806)
49 (10–72) 93 (84–97) NS 59 (14–78) NS NS 73 (37–87) NS NS 90 (79–95) 89 (77–95) NS NS NS NS NS NS 77 (22–90) NS NS NS NS NS 68 (17–86) NS 73 (51–85) NS 56 (3–79) NS NS NS NS NS 82 (46–92) 79 (9–92) NS NS NS 67 (31–84) 63 (36–79) NS 57 (30–73) 38 (20–53)
Larval differentiation post treatment (%) Haemonchus 89 52 92 51 92 99 83 71 83 28 62 100 91 84 79 72 93 94 53 100 97 96 97 90 96 87 99 94 96 100 100 89 87 88 96 94 100 94 98 100 98 82 0
Trichostrongylus 10 38 8 45 4 1 15 25 11 69 36 0 9 16 19 28 7 5 47 0 0 4 3 9 4 12 1 5 4 0 0 11 13 12 4 5 0 6 1 0 2 13 97
Teladorsagia 1 10 0 4 4 0 2 4 6 3 2 0 0 0 2 0 0 1 0 0 3 0 0 1 0 1 0 1 0 0 0 0 0 0 0 1 0 0 1 0 0 5 3
NS = no statistically significant reduction.
pour-on administration because of higher bio-availability of the drug. EPN was frequently used as an endectocide on 86% of the farms in this study. Because of the convenient route of administration it is both very popular for lactating animals and for goats kept for meat-production or breeding (75%). So it is unsurprising there were no differences in the level of resistance found between dairy and non-dairy goat farms. Additionally it is still very common to treat all the animals simultaneously: 77% of the participating farmers indicated that they followed this procedure. This procedure raises the selection pressure for anthelmintic resistance genes in GIN. Underdosing as a consequence of underestimating the body weight is considered as an important factor accelerating the development of anthelmintic resistance (Edwards et al., 1986). Farmers rarely own scales and estimating
bodyweight accurately is difficult (Warriss and Edwards, 1995). In addition it is known that farmers, despite the specific recommended dose for goats, use the cattle dose of EPN to treat their goats, because the scale of the applicator is designed for cattle. A wide spectrum of answers was obtained by the farmers concerning the intensity of grassland use; some of them were beyond the possible range. Therefore, no further evaluation of this factor was done. Animal movements are regarded as epidemiologically important factors for the dissemination of resistant GIN populations (reviewed by Silvestre et al., 2002). In Switzerland the tradition of grazing animals from different farms together on pre-alpine and alpine pasture during summer months increases the risk for the dissemination of resistant GIN populations. Due to the high prevalence of EPN resistance, it is not possible to
Please cite this article in press as: Murri, S., et al., Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.052
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identify any risk factors associated with its development in the present study. For delaying the spread of anthelmintic resistance, strict quarantine measures for new arriving animals are necessary (Coles, 2005). Only 4 of the 43 participating farmers (9%) implemented quarantine measures on their farms. Although these measures will now be useless in this area with respect to EPN-resistance, they still may have an important effect with respect to the introduction of multiresistant populations. Beside quarantine measures, regular monitoring of the farm-specific efficacy of anthelmintics by FECRT is recommended (Hertzberg and Bauer, 2000). Of the participating farms only 6 (14%) have ever undertaken a FECRT. This situation shows that crucial procedures for slowing down the development and spread of anthelmintic resistance are not implemented in practice. Besides AVM resistance, little scientific evidence is available about the prevalence of resistance against the remaining classes of anthelmintics in Switzerland. Hertzberg et al. (2000) reported a benzimidazole resistance level of 75% in GIN populations of sheep and goats which will limit the use of benzimidazoles in the majority of Swiss farms. For other European countries similar and even higher levels are reported (Hertzberg and Bauer, 2000). Currently, levamisole is the only remaining drug which can be used in Swiss dairy goat farms in which AVM and benzimidazoles fail to work. Levamisole-resistant GIN have been reported for small ruminants from several countries in Europe, e.g. from Denmark, France, Scotland and Ireland (Maingi et al., 1996; Bartley et al., 2004; Paraud et al., 2009; Good et al., 2012). Up to now levamisole resistance has not been detected in Switzerland. Levamisole is registered for sheep in Switzerland but only available in a combination containing triclabendazole (Endex® 8.75%, Novartis). The results of our and other studies confirm the urgent need of implementing new integrated parasite control measures in the management of small ruminants. Promoting strategies as targeted selective treatment to provide refugia (Van Wyk, 2001) and to treat only the animals benefitting the most from treatment, offer a promising option to slow down the development of resistant GIN populations. Furthermore increasing effort in the education of both veterinarians and farmers, concerning the goals and consequences of anthelmintic usage is strongly needed. Conflict of interest The authors declare that there was no conflict of interest at any point of time. Acknowledgements The support of the goat farmers participating in the study is greatly acknowledged. We want to thank Biokema for providing eprinomectin for this study. Special thanks go to the extension and health service for small ruminants (ESSR) for providing support during the whole study and for their effort on consulting farmers. Finally we highly acknowledge the Federal Veterinary Office (Berne) for funding this study.
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Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland Sarah Murri a,d , Gabriela Knubben-Schweizer b , Paul Torgerson c , Hubertus Hertzberg d,∗ a Ambulatory Clinic and Herd Management Unit, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland b Clinic for Ruminants with Ambulatory and Herd Health Services at the Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-University Munich, Sonnenstrasse 16, 85764 Oberschleissheim, Germany c Section of Epidemiology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, 8057 Zurich, Switzerland d Institute of Parasitology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 266a, 8057 Zurich, Switzerland
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Article history: Received 4 October 2013 Received in revised form 18 February 2014 Accepted 24 February 2014
Keywords: Goats Gastrointestinal nematodes Eprinomectin Anthelmintic resistance Switzerland
a b s t r a c t Eprinomectin (EPN) is a member of the avermectin class of compounds and the only anthelmintic registered for goats in Switzerland with a zero milk withdrawal period. The aim of the present study was to identify the actual efficacy of EPN in an area with a higher density of goat enterprises. Forty-three randomly chosen farms from canton Berne were investigated. At least eight goats were investigated on every farm. Conditions for inclusion in the study were the absence of anthelmintic treatment during the previous six weeks and a pooled faecal sample showing a mean faecal egg count (FEC) higher than 600 epg faeces. Pre- and 14–16 days post-treatment samples were individually collected directly from the rectum. Animals were treated with the recommended dose of EPN (1 mg/kg body weight) after taking the pre-treatment samples. Efficacy of EPN was tested with the faecal egg count reduction test (FECRT) and faecal cultures were performed on every farm from pooled faeces samples before and after treatment. Additionally the farmers completed a questionnaire. None of the gastrointestinal nematode populations of the 43 investigated farms were susceptible to EPN at the required level. The mean egg count reduction was 40%. None of the typical risk factors, such as production type, stocking rate, animal traffic and quarantine measures showed an association with the level of eprinomectin resistance. It can be concluded with 80% certainty that the prevalence of EPN resistance on goat farms is at least 95% in canton Berne. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Anthelmintic resistance of gastrointestinal nematodes (GIN) has become a globally important issue for the goat and sheep industry (reviewed by Kaplan, 2004; Jabbar
∗ Corresponding author. Tel.: +41 44 635 85 16; fax: +41 44 635 89 07. E-mail address: [email protected] (H. Hertzberg).
et al., 2006). In Switzerland benzimidazole resistance is widespread in GIN of small ruminants (Hertzberg et al., 2000). After the detection of the first case of avermectin (AVM) resistance in goats in Switzerland (Schnyder et al., 2005), a further study, focussing on Boer goats and Dorper sheep showed that AVM resistance seems to be widespread in GIN of these breeds (Artho et al., 2007). Eprinomectin (EPN) is a member of the avermectin class of compounds and the only anthelmintic registered for
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Please cite this article in press as: Murri, S., et al., Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.052
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goats in Switzerland with a zero milk withdrawal period, making it desirable in lactating animals (Dupuy et al., 2001). Targeted selective treatment (TST) is seen as an important development to slow down the development of anthelmintic resistance (reviewed by Kenyon et al., 2009). Integrated in a larger study on the implementation of the TST-approach, the present study identified the efficacy of EPN in an area with a high density of goat enterprises. Canton Berne, representing the Swiss midland, as well as the (pre-)alpine region was chosen as study region, as approximately 25% of the total Swiss goat farms (7190) are located in that area.
2. Materials and methods Farms in canton Berne with at least eight goats were randomly contacted by phone. Addresses were provided by the extension and health service for small ruminants (ESSR). Conditions for inclusion in the study were the absence of anthelmintic treatment during the previous six weeks and a mean faecal egg count (FEC) higher than 600 epg faeces. This was determined by a pooled faecal sample provided by the owners. Owners were asked to complete a questionnaire including general farm management, housing systems, animal movements, worm control practices and anthelmintic usage. Pre- and 14–16 days post-treatment samples were individually collected directly from the rectum. Pre-treatment faecal samples were collected from eight animals and post-treatment samples were taken from the six animals with the highest pre-treatment FECs. Because of Poisson errors removing these low counts improves the accuracy of the FECRT. For example, Coles et al. (2006) recommend removing all counts of less than 150 epg from the analysis. Animals were treated with the recommended dose of eprinomectin pour-on solution (1 mg/kg body weight, Eprinex® Pour-On, Biokema). The anthelmintic was administered with the commercially available applicator along the dorsal line, from the withers to the tail head, parting the coat to obtain direct contact with the skin. To avoid underdosing, body weight was visually estimated for every goat individually by two experienced persons, and then an additional 20% of the estimation was added to calculate the individual administered dose. Strongyle nematode FECs were determined with a sensitivity of 50 epg using the modified McMaster technique according to Schmidt (1971). Efficacy of EPN was tested with the FECRT, based on the recommendations of the World Association for the Advancement of Veterinary Parasitology (Coles et al., 1992; reviewed by Coles et al., 2006), but using the statistical analysis of the faecal egg counts according to Torgerson et al. (2005). Due to the generally small flock size, instead of including a control group the mean FECs before and 14–16 days post-treatment were compared. Faecal cultures (Eckert, 1960) were performed on every farm from pooled faeces samples before and after treatment, using 4 g faeces from each animal. 100 third stage larvae (L3) were differentiated according to MAFF (1986) and Levine (1968) at 400× magnification to determine the genera present.
Relationships between the farm specific parameters and the resistance level (based on the FECR values) were tested for significance using a generalised linear model with a negative binomial distribution assumed for the egg counts. The significance level was determined at p = 0.05. All calculations were undertaken in R (R Development Core Team, 2013), using the MASS package (Venables and Ripley, 2002). 3. Results Based on the detected resistance status the survey was terminated after investigation of 43 farms, sampled from August 2011 to November 2012. A total of 258 goats were included in the study. The investigated farms had a flock size between eight and 67 animals and were located in all parts of the canton (Fig. 1) at altitudes between 400 and 1300 metres above sea level. The Saanen and Chamoisee were the most frequent breeds (21% and 14% respectively); in many cases more than one breed per farm were kept (37%). The reduction of the mean faecal egg count after treatment was less than 95% on any of the investigated farms (Table 1). In two farms the reduction was between 90 and 93% with lower confidence intervals below 90%. Thirtynine farms (91%) showed reductions lower than 90%. The mean level of egg count reduction was 40%. Thus it was concluded that there was an 80% certainty for the presence of anthelmintic resistant GIN on at least 95% of goat farms in canton Berne. Differentiation of third-stage-larvae in the pretreatment larval cultures demonstrated that Haemonchus contortus is the predominant species on 33 farms (77%). Trichostrongylus spp. and Teladorsagia spp. were dominant on eight and one farm(s) respectively. For one farm (no. 25) evaluation was not possible due to immature larvae. In post-treatment larval cultures, H. contortus was the dominant resistant species on 41 (95%) farms. In two cases Trichostrongylus spp. was dominant. In addition small proportions of resistant Teladorsagia spp. were detected in 18 farms. In 35 out of 43 farms (81%) goats were kept for milk production and in eight farms (19%) for meat production and breeding purposes. No significant difference was found between milk-producing and non-milk-producing farms with respect to the level of anthelmintic resistance. On all farms, goats had regular access to pasture, but duration of grazing was highly variable. There was no evidence of an association between reported stocking rate and the degree of resistance. Cattle were kept on 25 of the 43 farms (58%), but only 13 farms (30%) kept their goats at least partially on the same pastures as cattle. Sharing pastures with cattle was not associated with the level of resistance. In 12 of 43 farms (28%) all goats (16%) or part of the flock (12%) were grazed alone or with goats from up to six other farms on alpine pastures during the summer. There was no association between transhumance and the level of eprinomectin resistance. Four of 43 farmers (9%) stated that they undertook quarantine measures when integrating new animals into their flocks. Forty-four percent of the farmers answered that parasitic infection represent a
Please cite this article in press as: Murri, S., et al., Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.052
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Fig. 1. Distribution of the investigated farms (white dots) in canton Berne, Switzerland.
problem on their farm. Less than 3 annual anthelmintic treatments were performed by 22 farmers (51%), whereas 21 farmers (49%) treated their animals three or more times per year; no difference was found between these categories with respect to the actual level of resistance (p = 0.91). Eprinomectin had been used previously in 37 of 43 (86%) farms. Farmers relied mainly on macrocyclic lactones and the benzimidazoles (72%), although several farmers no longer use benzimidazoles (28%) because of suspected or confirmed ineffectiveness. Additionally many farmers (44%) use levamisole (Endex® 8.75%, Novartis) off label. 4. Discussion The FECRT indicated that in none of the 43 farms was there sufficient efficacy of EPN against GIN. From these results the greater than 95% prevalence of EPN resistance in canton Berne was estimated with a certainty of 80%. This was much higher than expected and indicates a major EPN resistance problem, especially with respect to H. contortus. With this level of resistance, it would be futile to continue the use of EPN to control GIN in goats in this region and alternative approaches to the control of these parasites must be utilised. In Europe cases of AVM resistant GIN in goats were described in Scotland by Jackson et al. (1992) and subsequently in Denmark and the Netherlands (Maingi et al., 1996; Eysker et al., 2006). The first evidence of AVMresistance in Switzerland was reported 2005 from a Boer goat flock (Schnyder et al., 2005) with an efficacy of ivermectin of 61%, and later confirmed in a further study (Artho et al., 2007), where in 7 and 8 of 24 Boer goat farms AVMresistance was respectively confirmed or suspected. In both
studies the predominant resistant parasite species was also H. contortus. It was suspected that resistant GIN were imported with Boer goats from South Africa to Switzerland between 1999 and 2004. Subsequently the regulations were changed so that only embryos are allowed for importation. During the last decade many farmers bought Boer goat sires and used them to improve the beefiness of their dairy breeds, a practice which most likely favoured the spread of resistant GIN populations. Scheuerle et al. (2009) reported two single cases of EPN resistance in goats in Switzerland and Southern Germany with efficacies of EPN of only 17.4% and 27.5%. All of the participating farms in the present study have a history of using other macrocyclic lactones such as ivermectin, so it is likely that AVM-resistance was present at the time of introduction of EPN into the Swiss market. This would also be an explanation for the finding of EPN resistance on six farms that have never previously used eprinomectin. In Brazil a similar situation was found for Anglo-Nubian goats that were treated previously with ivermectin and moxidectin, leading to a GIN population consisting mainly of H. contortus that was unresponsive to EPN on its first use (Souza Chagas et al., 2006). Eprinomectin as a pour-on formulation has been available in Switzerland for use in cattle since 1999 and in addition was registered in 2004 for goats at 1.0 mg/kg body weight (twice the dose recommended for cattle). The optimal route of administration of EPN has since been debated. Silvestre et al. (2007) found no significant differences in FECR if goats had been shaved to facilitate the absorption of the drug but they recommended oral rather than topical application. Lespine et al. (2003) examined the efficacy of administering EPN by subcutaneous injection, finding this to be 2.5 times more efficient than the usual
Please cite this article in press as: Murri, S., et al., Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.052
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Table 1 Pre- and post-treatment mean faecal egg counts (epg) for every farm, analysis according to Torgerson et al. (2005); in the case of statistically significant reduction, lower and higher confidence intervals are given in brackets (CIs). Percentage of GIN larvae genera/species detected in coprocultures of pooled faecal samples after treatment. Farm ID
Mean epg before treatment (CIs)
Mean epg after treatment (CIs)
Mean reductions (CIs)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
622 (425–941) 970 (652–1185) 1439 (978–2184) 1401 (997–2006) 2530 (1570–4074) 673 (463–1012) 1273 (840–1992) 2452 (1772–3349) 1223 (882–1780) 1281 (903–1916) 1079 (676–1926) 1414 (928–2239) 1361 (809–2375) 917 (679–1245) 551 (391–787) 569 (416–814) 467 (311–720) 1092 (841–1435) 1825 (1380–2427) 2118 (1432–3123) 556 (398–796) 1499 (1142–1998) 716 (480–1116) 2528 (1458–4274) 2008 (1249–3249) 1022 (759–1399) 1147 (769–1794) 1642 (896–3054) 853 (707–1034) 2478 (1609–3760) 1349 (1034–1765) 1281 (912–1827) 1353 (609–3065) 1023 (733–1463) 965 (559–1786) 878 (555–1490) 1220 (803–1931) 647 (423–1016) 2004 (1059–3824) 668 (469–952) 2207 (1341–3598) 1445 (1029–2089) 1059 (885–1234)
315 (207–479) 64 (28–130) 1301 (943–1816) 565 (340–1143) 3082 (1922–4638) 968 (622–1551) 345 (195–741) 1640 (890–3097) 550 (219–1795) 135 (76–245) 116 (64–210) 237 (61–1813) 325 (113–1441) 458 (257–995) 360 (205–762) 468 (334–656) 264 (160–531) 249 (113–841) 1533 (885–2737) 2920 (1530–5159) 394 (233–767) 1161 (703–2034) 362 (140–1424) 793 (417–1751) 1059 (605–1981) 279 (172–451) 918 (662–1309) 731 (468–1230) 676 (556–827) 2601 (1344–4706) 924 (366–2538) 1440 (870–2484) 2374 (1512–3677) 182 (96–499) 204 (89–817) 589 (339–1190) 510 (192–1814) 688 (427–1216) 659 (454–999) 249 (164–385) 1163 (591–2525) 623 (450–855) 658 (521–806)
49 (10–72) 93 (84–97) NS 59 (14–78) NS NS 73 (37–87) NS NS 90 (79–95) 89 (77–95) NS NS NS NS NS NS 77 (22–90) NS NS NS NS NS 68 (17–86) NS 73 (51–85) NS 56 (3–79) NS NS NS NS NS 82 (46–92) 79 (9–92) NS NS NS 67 (31–84) 63 (36–79) NS 57 (30–73) 38 (20–53)
Larval differentiation post treatment (%) Haemonchus 89 52 92 51 92 99 83 71 83 28 62 100 91 84 79 72 93 94 53 100 97 96 97 90 96 87 99 94 96 100 100 89 87 88 96 94 100 94 98 100 98 82 0
Trichostrongylus 10 38 8 45 4 1 15 25 11 69 36 0 9 16 19 28 7 5 47 0 0 4 3 9 4 12 1 5 4 0 0 11 13 12 4 5 0 6 1 0 2 13 97
Teladorsagia 1 10 0 4 4 0 2 4 6 3 2 0 0 0 2 0 0 1 0 0 3 0 0 1 0 1 0 1 0 0 0 0 0 0 0 1 0 0 1 0 0 5 3
NS = no statistically significant reduction.
pour-on administration because of higher bio-availability of the drug. EPN was frequently used as an endectocide on 86% of the farms in this study. Because of the convenient route of administration it is both very popular for lactating animals and for goats kept for meat-production or breeding (75%). So it is unsurprising there were no differences in the level of resistance found between dairy and non-dairy goat farms. Additionally it is still very common to treat all the animals simultaneously: 77% of the participating farmers indicated that they followed this procedure. This procedure raises the selection pressure for anthelmintic resistance genes in GIN. Underdosing as a consequence of underestimating the body weight is considered as an important factor accelerating the development of anthelmintic resistance (Edwards et al., 1986). Farmers rarely own scales and estimating
bodyweight accurately is difficult (Warriss and Edwards, 1995). In addition it is known that farmers, despite the specific recommended dose for goats, use the cattle dose of EPN to treat their goats, because the scale of the applicator is designed for cattle. A wide spectrum of answers was obtained by the farmers concerning the intensity of grassland use; some of them were beyond the possible range. Therefore, no further evaluation of this factor was done. Animal movements are regarded as epidemiologically important factors for the dissemination of resistant GIN populations (reviewed by Silvestre et al., 2002). In Switzerland the tradition of grazing animals from different farms together on pre-alpine and alpine pasture during summer months increases the risk for the dissemination of resistant GIN populations. Due to the high prevalence of EPN resistance, it is not possible to
Please cite this article in press as: Murri, S., et al., Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.052
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identify any risk factors associated with its development in the present study. For delaying the spread of anthelmintic resistance, strict quarantine measures for new arriving animals are necessary (Coles, 2005). Only 4 of the 43 participating farmers (9%) implemented quarantine measures on their farms. Although these measures will now be useless in this area with respect to EPN-resistance, they still may have an important effect with respect to the introduction of multiresistant populations. Beside quarantine measures, regular monitoring of the farm-specific efficacy of anthelmintics by FECRT is recommended (Hertzberg and Bauer, 2000). Of the participating farms only 6 (14%) have ever undertaken a FECRT. This situation shows that crucial procedures for slowing down the development and spread of anthelmintic resistance are not implemented in practice. Besides AVM resistance, little scientific evidence is available about the prevalence of resistance against the remaining classes of anthelmintics in Switzerland. Hertzberg et al. (2000) reported a benzimidazole resistance level of 75% in GIN populations of sheep and goats which will limit the use of benzimidazoles in the majority of Swiss farms. For other European countries similar and even higher levels are reported (Hertzberg and Bauer, 2000). Currently, levamisole is the only remaining drug which can be used in Swiss dairy goat farms in which AVM and benzimidazoles fail to work. Levamisole-resistant GIN have been reported for small ruminants from several countries in Europe, e.g. from Denmark, France, Scotland and Ireland (Maingi et al., 1996; Bartley et al., 2004; Paraud et al., 2009; Good et al., 2012). Up to now levamisole resistance has not been detected in Switzerland. Levamisole is registered for sheep in Switzerland but only available in a combination containing triclabendazole (Endex® 8.75%, Novartis). The results of our and other studies confirm the urgent need of implementing new integrated parasite control measures in the management of small ruminants. Promoting strategies as targeted selective treatment to provide refugia (Van Wyk, 2001) and to treat only the animals benefitting the most from treatment, offer a promising option to slow down the development of resistant GIN populations. Furthermore increasing effort in the education of both veterinarians and farmers, concerning the goals and consequences of anthelmintic usage is strongly needed. Conflict of interest The authors declare that there was no conflict of interest at any point of time. Acknowledgements The support of the goat farmers participating in the study is greatly acknowledged. We want to thank Biokema for providing eprinomectin for this study. Special thanks go to the extension and health service for small ruminants (ESSR) for providing support during the whole study and for their effort on consulting farmers. Finally we highly acknowledge the Federal Veterinary Office (Berne) for funding this study.
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