Research in Veterinary Science 96 (2014) 133–138
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Assessing resistance of ivermectin and moxidectin against nematodes in cattle naturally infected using three different methodologies Welber Daniel Zanetti Lopes a,⇑, Weslen Fabricio Pires Teixeira a, Gustavo Felippelli a, Breno Cayeiro Cruz a, Willian Giquelin Maciel a, Vando Edésio Soares a,b, Thais Rabelo dos Santos a, Lucas Vinicius Shigaki de Matos a, Flávia Carolina Fávero a, Alvimar José da Costa a a CPPAR, Animal Health Research Center, Faculdade de Ciências Agrárias e Veterinárias, UNESP, Via de acesso prof. Paulo Donatto Castellani, s/n CEP: 14884-900, Jaboticabal, São Paulo, Brazil b UNICASTELO – Universidade Camilo Castelo Branco, Brazil
a r t i c l e
i n f o
Article history: Received 17 June 2013 Accepted 1 November 2013
Keywords: Anthelmintic Cattle Ivermectin Moxidectin Resistance
a b s t r a c t The objective of this study was to evaluate the accuracy of the faecal egg count reduction test (FECRT) and the faecal egg count efficacy test (FECET) to assess the resistance status of ivermectin (630 lg/kg) and moxidectin (200 lg/kg), using the controlled efficacy test as a reference, and whether the results of the EPG are equivalent to the efficacy results from the parasitological necropsies. Two experiments were conducted. The results demonstrate that it was not possible to demonstrate that the EPG values were equivalent with the ivermectin and moxidectin efficacy obtained by parasitological necropsies, mainly if the phenomenon of parasites resistance is not advanced in a determined field population. Maybe the FECET technique would be possibly better than the FECRT. The high anthelmintic efficacy of 200 lg/kg moxidectin, in naturally infected cattle, against field population of nematodes that are resistant to 630 lg/kg ivermectin, was observed in this study. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The livestock industry has worldwide socioeconomic significance. However, production problems have occurred due to the damage caused by helminths, especially in young animals. Souza et al. (2008) described that the main impact of helminths infection on livestock production are delayed growth and even animal mortality in the most susceptible categories (animals from 8 to 12 month of age). Controlling these cattle parasites is a key factor in improving production, but the combat efforts undertaken in most cattle farms are performed incorrectly through the excessive and disordered use of therapeutic approaches; this usage in turn increases the production costs and accelerates the process of selection of parasite resistance, which represents a significant, if not the most significant, sanitation issue in animal production (Fiel et al., 2001). Studies from different authors and various countries have primarily reported the resistance of Cooperia and Haemonchus to avermectins (Anziani et al., 2004; Rangel et al., 2005; Borges et al., 2008; Souza et al., 2008; Lopes et al., 2009, 2013; de Graef et al., 2012). The occurrence of residues from these drugs in animal products should ⇑ Corresponding author. Tel.: +55 (16) 32021720. E-mail addresses: [email protected], [email protected] (W.D.Z. Lopes). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.11.001
also be noted (Sindicato Nacional da Indústria de Produtos para Saúde Animal – SINDAN, 2008). Various definitions about resistance are described in the literature. In accordance with Vercruysse et al. (2001), a nematode strain is considered resistant when the efficacy of a given formulation, calculated by using its geometric means, is less than 90%. Furthermore, the VICH (2001) GL12 (Anthelmintics: bovine) highlights the importance of considering the significant difference between the treatment and control groups at 5% probability level. Recent studies have indicated that the arithmetic mean should be used to diagnose whether a helminth strain is resistant to a given formulation (Dobson et al., 2009; Vercruysse et al., 2011). Recent guidelines from the W.A.A.V.P. [World Association for the Advancement of Veterinary Parasitology] for assessing the efficacy of anthelmintic formulations prepared for ruminants and horses (Geary et al., 2012) report that resistance can be attributed to a population of helminth species showing a substantial reduction in efficacy (680%) when treated with an anthelmintic that had previously demonstrated an efficacy above 95% against that species. The main therapeutic drugs used to control endoparasitic infections in cattle belong to chemical groups including the imidazothiazoles, salicylanilides, organophosphates, benzimidazoles, macrocyclic lactones and phenolic substitutes. Given the convenience of administration, the groups most frequently administered by injection to control bovine helminths are the macrocyclic
134
W.D.Z. Lopes et al. / Research in Veterinary Science 96 (2014) 133–138
lactones, followed by the imidazothiazoles and benzimidazoles (Borges et al., 2008). Macrocyclic lactones are derived from the fermentation of an actinomycete fungus found in the soil, Streptomyces avermitilis. The avermectins belonging to this group (a: without + verm: worm + ect: ectoparasite + in: pharmaceutical product) are ivermectin, abamectin, doramectin, eprinomectin, selamectin. The milbemycins, which also belongs to this group, are compound by moxidectin and milbemycin oxime. The moxidectin stands out in the milbemycin group because its mechanism of action and resistance is presumed to be equal to that of the other avermectins. However, recent studies have indicated that its mode of action is similar, but not identical to the avermectins (Prichard et al., 2012). The main objective of the present study was to evaluate the accuracy of the faecal egg count reduction test (FECRT) and the faecal egg count efficacy test (FECET) to assess the resistance status of ivermectin (630 lg/kg) and moxidectin (200 lg/kg), using the controlled efficacy test (worm counts) as a reference, and whether the results of the EPG are equivalent to the ivermectin and moxidectin efficacy results from the parasitological necropsies. The second objective was to investigate whether both ivermectin-resistant field populations showed side-resistance
Efficacy percentage ¼
release formulation of 630 lg/kg 3.15% ivermectin (Ivomec GoldÒ, Merial Animal Health, Ltd.), both applied subcutaneously. A third group was kept as a control (saline solution, administered subcutaneously). The 40 cattle from both experiments were transferred and held in suspended freestalls at the CPPAR [Animal health research center (Centro de Pesquisas em Sanidade Animal)] at the FCAV [School of agricultural and veterinary sciences (Faculdade de Ciências Agrárias e Veterinárias)] of the UNESP [‘‘Júlio de Mesquita Filho’’ São Paulo State University (Universidade Estadual Paulista ‘‘Júlio de Mesquita Filho’’)], Jaboticabal, São Paulo, Brazil, to prevent helminthic reinfection throughout the experimental period. 2.2. Fecal collection and parasitological necropsies To assess the percentage of helminth load reduction in the animals in both studies, strongylid EPG counts (Gordon and Whitlock, 1939) were performed for each individual animal at the 2nd, 4th, 6th, 8th, 10th, 12th and 14th days post-treatment (DPT). The reduction and efficacy percentages were calculated according to the equations below:
Mean EPG of the control group at day X Mean EPG of the treatment group at day X 100 Mean EPG of the control group at day X
Reduction percentage ¼
Mean EPG of the group at day zero Mean EPG of the group at day X 100 Mean EPG of the group at day zero
against moxidectin, under controlled conditions. 2. Materials and methods 2.1. Study animals The present study was performed from January to July 2012 in two farms. Experiment I was performed in the same farm, located in the center-west region of Minas Gerais, Brazil, in which populations of Haemonchus placei, Cooperia punctata, Cooperia spatulata, Trichuris discolor and Oesophagostomum radiatum had already been described as resistant to 3.15% ivermectin at a dose of 630 lg/kg (Borges et al., 2008; Lopes et al., 2009; Felippelli, 2012). Sixteen naturally infected calves aged from 6 to 12 months were selected through counts of nematode eggs per gram of feces (EPG P500) using the method described by Gordon and Whitlock (1939). The animals were randomly sorted into two groups of eight cattle each by the mean of three consecutive EPG counts (days 3, 2 and 1). After the random draw, eight animals were treated subcutaneously with 200 lg/kg moxidectin (CydectinÒ, Zoetis Animal Health.) in a volume of 1 mL/50 kg body weight. Eight other animals were kept as controls (saline solution, administered subcutaneously). Cattle from the south region of Minas Gerais were used in experiment II. Twenty-four calves aged six to twelve months that were naturally infected with gastrointestinal nematodes were selected and divided into three groups of eight animals each, according to their EPGs. After a random draw, the first group of cattle was treated with 200 lg/kg moxidectin (CydectinÒ, Zoetis Animal Health, Ltd.) and the second group with high dose in a sustained
In both experiments I and II, all animals (control and treatment) were necropsied at the 14th DPT. The digestive system was separated by double ligature marks in different anatomical segments (abomasum, duodenum, jejunum, ileum, cecum, colon and rectum). The contents of each segment were fixed in 10.0% formaldehyde and heated to 80 °C. The abomasums were individually subjected to digestion with a pepsin hydrochloric acid solution. Each abomasum was placed in a 1% pepsin solution. The volume, by weight, of this solution was at least three times the weight of the mucosa. The mucosal material was digested in the digestive solution in a water bath at 37–40 °C for no longer than 4–6 h (Wood et al., 1995). The other organs (lungs and liver) were visually examined to determine the total of possible number of helminths present (Wood et al., 1995; Vercruysse et al., 2000). The volume, by weight, of this solution was at least three times the weight of the mucosa. 2.3. Helminth species identification A 10% aliquot from the total content of each segment was retained for examination and an estimation of the parasite load. The helminths were collected by using a magnifying glass, and the generic and specific identifications of the helminths were performed using a stereoscopic microscopic (magnification 100– 400), according to the taxonomic criteria described by Levine (1968), Costa (1982), Ueno and Golçalves (1998), and Achi et al. (2003). A drop or two of lactophenol was added to facilitate identification.
135
W.D.Z. Lopes et al. / Research in Veterinary Science 96 (2014) 133–138
2.4. Data analysis
made regarding the efficacy results or statistical analyses of the studied formulation for the aforementioned helminth species. Using the results from the post-treatment EPG counts, the high values of reduction and/or efficacy found with moxidectin (200 lg/kg) through the EPG counts were consistent with the efficacy results obtained through the parasitological necropsy. The statistical analyses of the EPG counts, whose results are expressed in Table 2, reinforce such conclusions. On the other hand, although the results of the EPG counts have been consistent with the parasitological necropsy, if only the FECRT technique would be used, certainly the resistance of O. radiatum adults detected in the parasitological necropsy would not be diagnosed by EPG techniques (Tables 1 and 2). Seven species were identified in experiment II: H. placei (adult), H. placei (L4), C. punctata (adult), C. spatulata (adult), C. pectinata (adult), T. axei (adult), O. radiatum (adult), O. radiatum (L4) and T. discolor (adult) (Table 3). The moxidectin (200 lg/kg) was effective, reaching efficacy values (arithmetic means) greater than 90% against H. placei adults (98.70%), H. placei L4 (99.16%), C. punctata (93.80%), C. spatulata (100.00%) and T. axei (100.00%). The ivermectin (630 lg/kg) showed high efficacy (P90%) only against C. spatulata (90.05%) and T. axei (93.82%) (Table 3). Low or no anthelmintic efficacy was found for either formulation against immature (L4) and adult forms of O. radiatum (Table 3). The moxidectin, administered subcutaneously, exhibited low anthelmintic efficacy against adult and/or immature (L4) stages of O. radiatum in both experiments (I and II) (Tables 1 and 3). The total numbers of H. placei (adults and L4), C. punctata, C. spatulata, T. axei and Oesophagostomum (adults) in cattle treated with the formulation containing moxidectin were significantly lower (P 6 0.05) than those in the control group. The total numbers of nematodes found in the group treated with 3.15% ivermectin were significantly lower (P 6 0.05) than the ones found in the control group for H. placei (adults), C. punctata, C. spatulata, T. axei and O. radiatum adults (Table 3). When comparing only the treatment groups (ivermectin and moxidectin), animals treated with 200 lg/kg moxidectin demonstrated significantly lower numbers
The EPG counts were log transformed [ln (EPG counts +1)] prior to the analysis. The data were analyzed using a linear mixed model for repeated measures. The model included the fixed effects of the treatment, day and interaction treatment versus the day of study. The efficacy of each drug was calculated for each nematode species in each date based on the arithmetic and geometric means. For the latter calculations, the counts were first log transformed [ln (x + 1)], according to the VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products) guidelines (Vercruysse et al., 2000). Given the relative helminth counts, the statistical analysis was performed using a generalized linear mixed model with treatment-fixed and block random effects and a block-treatment interaction (SAS Institute, 1989–1996). All hypotheses were tested at a 0.05 level of significance. 3. Results The following helminth species were identified in the first experiment: H. placei (adult), H. placei (L4), H. similis (adult), C. punctata (adult), C. spatulata (adult), C. pectinata (adult), Bunostomum phlebotomum (adult), Trichostrongylus axei (adult), O. radiatum (adult), O. radiatum (L4) and T. discolor (adult) (Table 1). In the nine species identified, subcutaneous treatment with moxidectin exhibited 100% efficacy against C. spatulata. Against H. placei, C. punctata and C. pectinata adults and T. axei, the studied formulation resulted in efficiencies (arithmetic means) of 97.84%, 98.51%, 98.34% and 99.79%, respectively (Table 1). However, moxidectin (200 lg/kg) showed low efficacy (55.67%, Table 1) against the adult forms of O. radiatum. The total number of H. placei (adults), C. punctata, C. pectinata, C. spatulata and T. axei, in cattle treated with moxidectin (200 lg/kg) were significantly (P 6 0.05) lower than those recorded in the control group (Table 1). The low number of cattle in the control group parasitized by immature forms (L4) of H. placei (three animals), H. similis (two animals), B. phlebotomum (one animal), O. radiatum (one animal) and T. discolor (two animals), did not permit conclusions to be
Table 1 Mean counts of helminth species (complete sample) collected from cattle belonging to the control and treated groups; results of variance analysis of helminthes and efficacy percentage in experiment I. Helminths
b c d
T02: moxidectinb (200 lg/kg)
ANOVA
Mean counta/range
Mean counta/range
Value of Fc
Pr < Fd
Haemonchus placei (adult)
4019.13
Haemonchus placei (L4) Haemonchus similis Cooperia punctata
41.75 2.25 8923.88
Cooperia pectinata Cooperia spatulata Bunostomum phlebotomum Trichostrongylus axei Oesophagostomum radiatum (adult) Oesophagostomum radiatum (L4) Trichuris discolor a
T01: control (saline solution)
Efficacy (arithmetic mean)
Classification of strain moxidectin
671– 10,741 0–170 0–12 103– 37,702 0–772 0–386 0–10 3–2970 0–1070
A
86.88
0–223
B
36.83
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Assessing resistance of ivermectin and moxidectin against nematodes in cattle naturally infected using three different methodologies Welber Daniel Zanetti Lopes a,⇑, Weslen Fabricio Pires Teixeira a, Gustavo Felippelli a, Breno Cayeiro Cruz a, Willian Giquelin Maciel a, Vando Edésio Soares a,b, Thais Rabelo dos Santos a, Lucas Vinicius Shigaki de Matos a, Flávia Carolina Fávero a, Alvimar José da Costa a a CPPAR, Animal Health Research Center, Faculdade de Ciências Agrárias e Veterinárias, UNESP, Via de acesso prof. Paulo Donatto Castellani, s/n CEP: 14884-900, Jaboticabal, São Paulo, Brazil b UNICASTELO – Universidade Camilo Castelo Branco, Brazil
a r t i c l e
i n f o
Article history: Received 17 June 2013 Accepted 1 November 2013
Keywords: Anthelmintic Cattle Ivermectin Moxidectin Resistance
a b s t r a c t The objective of this study was to evaluate the accuracy of the faecal egg count reduction test (FECRT) and the faecal egg count efficacy test (FECET) to assess the resistance status of ivermectin (630 lg/kg) and moxidectin (200 lg/kg), using the controlled efficacy test as a reference, and whether the results of the EPG are equivalent to the efficacy results from the parasitological necropsies. Two experiments were conducted. The results demonstrate that it was not possible to demonstrate that the EPG values were equivalent with the ivermectin and moxidectin efficacy obtained by parasitological necropsies, mainly if the phenomenon of parasites resistance is not advanced in a determined field population. Maybe the FECET technique would be possibly better than the FECRT. The high anthelmintic efficacy of 200 lg/kg moxidectin, in naturally infected cattle, against field population of nematodes that are resistant to 630 lg/kg ivermectin, was observed in this study. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The livestock industry has worldwide socioeconomic significance. However, production problems have occurred due to the damage caused by helminths, especially in young animals. Souza et al. (2008) described that the main impact of helminths infection on livestock production are delayed growth and even animal mortality in the most susceptible categories (animals from 8 to 12 month of age). Controlling these cattle parasites is a key factor in improving production, but the combat efforts undertaken in most cattle farms are performed incorrectly through the excessive and disordered use of therapeutic approaches; this usage in turn increases the production costs and accelerates the process of selection of parasite resistance, which represents a significant, if not the most significant, sanitation issue in animal production (Fiel et al., 2001). Studies from different authors and various countries have primarily reported the resistance of Cooperia and Haemonchus to avermectins (Anziani et al., 2004; Rangel et al., 2005; Borges et al., 2008; Souza et al., 2008; Lopes et al., 2009, 2013; de Graef et al., 2012). The occurrence of residues from these drugs in animal products should ⇑ Corresponding author. Tel.: +55 (16) 32021720. E-mail addresses: [email protected], [email protected] (W.D.Z. Lopes). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.11.001
also be noted (Sindicato Nacional da Indústria de Produtos para Saúde Animal – SINDAN, 2008). Various definitions about resistance are described in the literature. In accordance with Vercruysse et al. (2001), a nematode strain is considered resistant when the efficacy of a given formulation, calculated by using its geometric means, is less than 90%. Furthermore, the VICH (2001) GL12 (Anthelmintics: bovine) highlights the importance of considering the significant difference between the treatment and control groups at 5% probability level. Recent studies have indicated that the arithmetic mean should be used to diagnose whether a helminth strain is resistant to a given formulation (Dobson et al., 2009; Vercruysse et al., 2011). Recent guidelines from the W.A.A.V.P. [World Association for the Advancement of Veterinary Parasitology] for assessing the efficacy of anthelmintic formulations prepared for ruminants and horses (Geary et al., 2012) report that resistance can be attributed to a population of helminth species showing a substantial reduction in efficacy (680%) when treated with an anthelmintic that had previously demonstrated an efficacy above 95% against that species. The main therapeutic drugs used to control endoparasitic infections in cattle belong to chemical groups including the imidazothiazoles, salicylanilides, organophosphates, benzimidazoles, macrocyclic lactones and phenolic substitutes. Given the convenience of administration, the groups most frequently administered by injection to control bovine helminths are the macrocyclic
134
W.D.Z. Lopes et al. / Research in Veterinary Science 96 (2014) 133–138
lactones, followed by the imidazothiazoles and benzimidazoles (Borges et al., 2008). Macrocyclic lactones are derived from the fermentation of an actinomycete fungus found in the soil, Streptomyces avermitilis. The avermectins belonging to this group (a: without + verm: worm + ect: ectoparasite + in: pharmaceutical product) are ivermectin, abamectin, doramectin, eprinomectin, selamectin. The milbemycins, which also belongs to this group, are compound by moxidectin and milbemycin oxime. The moxidectin stands out in the milbemycin group because its mechanism of action and resistance is presumed to be equal to that of the other avermectins. However, recent studies have indicated that its mode of action is similar, but not identical to the avermectins (Prichard et al., 2012). The main objective of the present study was to evaluate the accuracy of the faecal egg count reduction test (FECRT) and the faecal egg count efficacy test (FECET) to assess the resistance status of ivermectin (630 lg/kg) and moxidectin (200 lg/kg), using the controlled efficacy test (worm counts) as a reference, and whether the results of the EPG are equivalent to the ivermectin and moxidectin efficacy results from the parasitological necropsies. The second objective was to investigate whether both ivermectin-resistant field populations showed side-resistance
Efficacy percentage ¼
release formulation of 630 lg/kg 3.15% ivermectin (Ivomec GoldÒ, Merial Animal Health, Ltd.), both applied subcutaneously. A third group was kept as a control (saline solution, administered subcutaneously). The 40 cattle from both experiments were transferred and held in suspended freestalls at the CPPAR [Animal health research center (Centro de Pesquisas em Sanidade Animal)] at the FCAV [School of agricultural and veterinary sciences (Faculdade de Ciências Agrárias e Veterinárias)] of the UNESP [‘‘Júlio de Mesquita Filho’’ São Paulo State University (Universidade Estadual Paulista ‘‘Júlio de Mesquita Filho’’)], Jaboticabal, São Paulo, Brazil, to prevent helminthic reinfection throughout the experimental period. 2.2. Fecal collection and parasitological necropsies To assess the percentage of helminth load reduction in the animals in both studies, strongylid EPG counts (Gordon and Whitlock, 1939) were performed for each individual animal at the 2nd, 4th, 6th, 8th, 10th, 12th and 14th days post-treatment (DPT). The reduction and efficacy percentages were calculated according to the equations below:
Mean EPG of the control group at day X Mean EPG of the treatment group at day X 100 Mean EPG of the control group at day X
Reduction percentage ¼
Mean EPG of the group at day zero Mean EPG of the group at day X 100 Mean EPG of the group at day zero
against moxidectin, under controlled conditions. 2. Materials and methods 2.1. Study animals The present study was performed from January to July 2012 in two farms. Experiment I was performed in the same farm, located in the center-west region of Minas Gerais, Brazil, in which populations of Haemonchus placei, Cooperia punctata, Cooperia spatulata, Trichuris discolor and Oesophagostomum radiatum had already been described as resistant to 3.15% ivermectin at a dose of 630 lg/kg (Borges et al., 2008; Lopes et al., 2009; Felippelli, 2012). Sixteen naturally infected calves aged from 6 to 12 months were selected through counts of nematode eggs per gram of feces (EPG P500) using the method described by Gordon and Whitlock (1939). The animals were randomly sorted into two groups of eight cattle each by the mean of three consecutive EPG counts (days 3, 2 and 1). After the random draw, eight animals were treated subcutaneously with 200 lg/kg moxidectin (CydectinÒ, Zoetis Animal Health.) in a volume of 1 mL/50 kg body weight. Eight other animals were kept as controls (saline solution, administered subcutaneously). Cattle from the south region of Minas Gerais were used in experiment II. Twenty-four calves aged six to twelve months that were naturally infected with gastrointestinal nematodes were selected and divided into three groups of eight animals each, according to their EPGs. After a random draw, the first group of cattle was treated with 200 lg/kg moxidectin (CydectinÒ, Zoetis Animal Health, Ltd.) and the second group with high dose in a sustained
In both experiments I and II, all animals (control and treatment) were necropsied at the 14th DPT. The digestive system was separated by double ligature marks in different anatomical segments (abomasum, duodenum, jejunum, ileum, cecum, colon and rectum). The contents of each segment were fixed in 10.0% formaldehyde and heated to 80 °C. The abomasums were individually subjected to digestion with a pepsin hydrochloric acid solution. Each abomasum was placed in a 1% pepsin solution. The volume, by weight, of this solution was at least three times the weight of the mucosa. The mucosal material was digested in the digestive solution in a water bath at 37–40 °C for no longer than 4–6 h (Wood et al., 1995). The other organs (lungs and liver) were visually examined to determine the total of possible number of helminths present (Wood et al., 1995; Vercruysse et al., 2000). The volume, by weight, of this solution was at least three times the weight of the mucosa. 2.3. Helminth species identification A 10% aliquot from the total content of each segment was retained for examination and an estimation of the parasite load. The helminths were collected by using a magnifying glass, and the generic and specific identifications of the helminths were performed using a stereoscopic microscopic (magnification 100– 400), according to the taxonomic criteria described by Levine (1968), Costa (1982), Ueno and Golçalves (1998), and Achi et al. (2003). A drop or two of lactophenol was added to facilitate identification.
135
W.D.Z. Lopes et al. / Research in Veterinary Science 96 (2014) 133–138
2.4. Data analysis
made regarding the efficacy results or statistical analyses of the studied formulation for the aforementioned helminth species. Using the results from the post-treatment EPG counts, the high values of reduction and/or efficacy found with moxidectin (200 lg/kg) through the EPG counts were consistent with the efficacy results obtained through the parasitological necropsy. The statistical analyses of the EPG counts, whose results are expressed in Table 2, reinforce such conclusions. On the other hand, although the results of the EPG counts have been consistent with the parasitological necropsy, if only the FECRT technique would be used, certainly the resistance of O. radiatum adults detected in the parasitological necropsy would not be diagnosed by EPG techniques (Tables 1 and 2). Seven species were identified in experiment II: H. placei (adult), H. placei (L4), C. punctata (adult), C. spatulata (adult), C. pectinata (adult), T. axei (adult), O. radiatum (adult), O. radiatum (L4) and T. discolor (adult) (Table 3). The moxidectin (200 lg/kg) was effective, reaching efficacy values (arithmetic means) greater than 90% against H. placei adults (98.70%), H. placei L4 (99.16%), C. punctata (93.80%), C. spatulata (100.00%) and T. axei (100.00%). The ivermectin (630 lg/kg) showed high efficacy (P90%) only against C. spatulata (90.05%) and T. axei (93.82%) (Table 3). Low or no anthelmintic efficacy was found for either formulation against immature (L4) and adult forms of O. radiatum (Table 3). The moxidectin, administered subcutaneously, exhibited low anthelmintic efficacy against adult and/or immature (L4) stages of O. radiatum in both experiments (I and II) (Tables 1 and 3). The total numbers of H. placei (adults and L4), C. punctata, C. spatulata, T. axei and Oesophagostomum (adults) in cattle treated with the formulation containing moxidectin were significantly lower (P 6 0.05) than those in the control group. The total numbers of nematodes found in the group treated with 3.15% ivermectin were significantly lower (P 6 0.05) than the ones found in the control group for H. placei (adults), C. punctata, C. spatulata, T. axei and O. radiatum adults (Table 3). When comparing only the treatment groups (ivermectin and moxidectin), animals treated with 200 lg/kg moxidectin demonstrated significantly lower numbers
The EPG counts were log transformed [ln (EPG counts +1)] prior to the analysis. The data were analyzed using a linear mixed model for repeated measures. The model included the fixed effects of the treatment, day and interaction treatment versus the day of study. The efficacy of each drug was calculated for each nematode species in each date based on the arithmetic and geometric means. For the latter calculations, the counts were first log transformed [ln (x + 1)], according to the VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products) guidelines (Vercruysse et al., 2000). Given the relative helminth counts, the statistical analysis was performed using a generalized linear mixed model with treatment-fixed and block random effects and a block-treatment interaction (SAS Institute, 1989–1996). All hypotheses were tested at a 0.05 level of significance. 3. Results The following helminth species were identified in the first experiment: H. placei (adult), H. placei (L4), H. similis (adult), C. punctata (adult), C. spatulata (adult), C. pectinata (adult), Bunostomum phlebotomum (adult), Trichostrongylus axei (adult), O. radiatum (adult), O. radiatum (L4) and T. discolor (adult) (Table 1). In the nine species identified, subcutaneous treatment with moxidectin exhibited 100% efficacy against C. spatulata. Against H. placei, C. punctata and C. pectinata adults and T. axei, the studied formulation resulted in efficiencies (arithmetic means) of 97.84%, 98.51%, 98.34% and 99.79%, respectively (Table 1). However, moxidectin (200 lg/kg) showed low efficacy (55.67%, Table 1) against the adult forms of O. radiatum. The total number of H. placei (adults), C. punctata, C. pectinata, C. spatulata and T. axei, in cattle treated with moxidectin (200 lg/kg) were significantly (P 6 0.05) lower than those recorded in the control group (Table 1). The low number of cattle in the control group parasitized by immature forms (L4) of H. placei (three animals), H. similis (two animals), B. phlebotomum (one animal), O. radiatum (one animal) and T. discolor (two animals), did not permit conclusions to be
Table 1 Mean counts of helminth species (complete sample) collected from cattle belonging to the control and treated groups; results of variance analysis of helminthes and efficacy percentage in experiment I. Helminths
b c d
T02: moxidectinb (200 lg/kg)
ANOVA
Mean counta/range
Mean counta/range
Value of Fc
Pr < Fd
Haemonchus placei (adult)
4019.13
Haemonchus placei (L4) Haemonchus similis Cooperia punctata
41.75 2.25 8923.88
Cooperia pectinata Cooperia spatulata Bunostomum phlebotomum Trichostrongylus axei Oesophagostomum radiatum (adult) Oesophagostomum radiatum (L4) Trichuris discolor a
T01: control (saline solution)
Efficacy (arithmetic mean)
Classification of strain moxidectin
671– 10,741 0–170 0–12 103– 37,702 0–772 0–386 0–10 3–2970 0–1070
A
86.88
0–223
B
36.83
