Veterinary Parasitology, 45 ( 1992 ) 111-116 Elsevier Science Publishers B.V., Amsterdam
111
Haemonchus contortus resistance to ivermectin
and netobimin in Brazilian sheep L.S. Vieira, M.E.A. Berne, A.C.R. Cavalcante and C.A.F. Costa Centro Nacional de Pesquisa de Caprinos (EMBRAPA-CNPC), Caixa Postal D-I O, CEP 62100, Sobral, CE, Brazil (Accepted 9 June 1992)
ABSTRACT Vieira, L.S., Berne, M.E.A., Cavalcante, A.C.R. and Costa, C.A.F., 1992. Haemonchus contortus resistance to ivermectin and netobimin in Brazilian sheep. Vet. Parasitol., 45:111-116. Suffolk, Texel, Hampshire Down and Ile de France sheep from the municipalities of Porto Amazonas, Piraquara and Araucaria in the State of Paran~i, and Bag6 in the State of Rio Grande do Sul were brought to Sobral, State of Cear~i, to be used in a cross-breeding project. On arrival they had clinical signs of nematode parasitosis, and one Suffolk female died. The animals were treated orally with ivermectin (0.2 mg kg -~ ) and fifteen days later with netobimin (20.0 rag kg-1). Neither drug reduced the egg counts (measured in eggs per gram, EPG) significantly, and this suggested that the nematodes in the sheep were resistant to the anthelmintics used. Haemonchus contortus was the species involved. The egg counts were reduced after oral treatment with trichlorfon (100.0 mg kg -~ ). Haemonchus contortus larvae obtained from these animals before trichlorfon treatment and passaged through two nematode-free sheep were used in a further experiment. Twenty 6- to 9-month-old nematode-free Iambs were infected with the H. contortus larvae ( 10 000 per animal) and, after the infection was confirmed, were randomly divided into four groups of five animals. Group I was orally treated with ivermectin at 0.2 mg kg- ~, Group II with oral netobimin at 20.0 mg kg -~, Group III with oral trichlorfon at 100.0 mg kg -I and Group IV was a non-treated control. Egg counts and faecal cultures were taken before dosing on the day of treatment and seven days later when all animals were necropsied and the nematodes were collected from the abomasa and counted. Drug efficacy measured by the reduction in adult H. contortus numbers was 99.9% for trichlorfon, 38.9% for netobimin and 17.6% for ivermectin. The results demonstrated the resistance of this strain ofH. Contortus to ivermectin and netobimin.
INTRODUCTION
Resistance of gastrointestinal nematodes to benzimidazole and imidazole anthelmintics has spread dramatically in recent years (Vieira, 1986). Considering that helminth control in small ruminants is based on strategic mediCorrespondence to: L.S. Vieira, Centro Nacional de Pesquisa de Caprinos (EMBRAPA-CNPC), Caixa Postal D-10, CEP 62100, Sobral, CE, Brazil.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
1 12
L.S. VIEIRA ET AL.
cation with anthelmintics, it can be expected that anthelmintic resistance will represent a major problem in sheep and goat production. Among the gastrointestinal nematodes, H a e m o n c h u s contortus is the one which develops resistance most rapidly as a result of its high biotic potential (Echevarria and Trindade, 1989). The efficacy of ivermectin against H. contortus in sheep has been well-documented by several authors. Swan et al. (1984), testing ivermectin orally at a dose rate of 0.2 mg kg- 1 in sheep, observed an efficacy greater than 90.0% against adult and immature forms of H. contortus. Santiago et al. (1985a) observed that both oral and injectable formulations of ivermectin at a dose rate of 0.2 mg kg-1 in sheep had 100.0% efficacy against H. contortus which was resistant to benzimidazoles. Hembry et al. (1986), Lyons et al. (1986), Robin (1986) and Njanja et al. (1987) also reported high efficacy of ivermectin against H. contortus in sheep. The efficacy of netobimin against H. contortus in sheep is also well-documented. Steel et al. (1985b), working with this anthelmintic in sheep infected with H. contortus strains susceptible and resistant to benzimidazoles, observed an efficacy of 98.6% at a dose rate of 20.0 mg kg -1. Menezes (1988), using helminths from Sobral, State of Ceani, Brazil, observed that ivermectin (0.2 mg kg -1 ) and netobimin (20.0 mg kg -~ ) had an efficacy of 100.0% in the reduction of egg counts in goats and sheep experimentally infected with H. contortus. Duncan et al. ( 1985 ) and Santiago et al. (1985b) also reported high efficacy of netobimin against H. contortus in sheep. Despite ivermectin being available only recently, nematode resistance has already been reported. Carmichael et al. ( 1987 ), working with South African sheep, were the first to report resistance to ivermectin in H. contortus. According to these authors, ivermectin was in use for 18 to 24 months with treatment intervals of 3 to 5 weeks. Also in South Africa, Van Wyk and Malan ( 1988 ) observed H. contortus resistance to ivermectin in sheep after 11 anthelmintic treatments over a period of two and a half years. Echevarria and Trindade (1989), in Bag6, State of Rio Grande do Sul, Brazil, observed resistance to ivermectin in H. contortus from sheep after 32 anthelmintic treatments at a dose rate of 0.2 mg kg-1 over a period of four and a half years. According to these authors the anthelmintic efficacy was reduced to 60.0%. The literature reviewed has not revealed any record of H. contortus resistance to netobimin. The study described in this paper identifies a strain of H. contortus from sheep from the States of Paran~i and Rio Grande do Sul, Brazil, resistant to both ivermectin and netobimin. MATERIALSAND METHODS Nine animals (three Suffolk, two Texel, two Hampshire Down and two Ile de France) were brought to the research facilities of the Brazilian National
HAEMONCHUS CONTORTUS DRUG RESISTANCEIN SHEEP
1 13
Goat Research Center (EMBRAPA-CNPC), Sobral, State of Cear~i, in October 1987 to be used in a cross-breeding project. These animals came from the municipalities of Porto Amazonas, Araucaria and Piraquara in the State of Paranfi, and Bag6 in the State of Rio Grande do Sul. Clinical signs of helminthiasis were observed just after arrival, and one Suffolk ewe died. As a result, the remaining sheep were medicated, orally, with ivermectin at 0.2 mg kg- 1, and 15 days later were treated again, this time with netobimin given orally at 20.0 mg kg- 1. Even after the second medication there was no significant reduction in the number of helminth eggs per gram of faeces (EPG), suggesting nematode resistance to these products. The species involved in the suspected resistance was H. contortus. The egg counts were reduced after one oral administration of trichlorfon at 100.0 mg kg -~ 15 days after the netobimin treatment. After being treated with ivermectin and netobimin without a significant drop in egg counts, and before being treated with trichlorfon, the animals were provided with collecting bags so that faeces for cultures could be collected. The H. contortus larvae harvested from the cultures were inoculated into two nematode-free sheep for further multiplication. The resultant larvae from these two sheep were used to infect 20 lambs of 6 to 9 months of age. These experimental lambs from an undefined breed had been shown to be nematode-free before artificial infection. Each lamb was inoculated orally with 10 000 infective larvae. When patency of the H. contortus infection was confirmed by faecal analysis, the 20 experimental lambs were randomly divided into four groups of five animals. Group I was treated orally with ivermectin at 0.2 mg kg-1; Group II with oral netobimin at 20.0 mg kg-1; Group III with oral trichlorfon at 100.0 mg kg- 1 and Group IV served as a non-treated control. Egg counts and larval cultures were carded out on samples taken immediately prior to treatment and 7 days after treatment when all animals (treated and control groups) were necropsied and the nematodes from the abomasa were collected, identified and counted. The anthelmintic efficacy of each treatment was calculated on the basis of the percentage reduction in egg counts and from a comparison of treated and control adult nematode counts. A standard error (S.E.) was calculated for each percentage efficacy, as recommended by Clark and Turton ( 1973 ). The percentage efficacy minus 2 S.E. units gives the minimal percentage efficacy of the anthelmintic tested with a P
111
Haemonchus contortus resistance to ivermectin
and netobimin in Brazilian sheep L.S. Vieira, M.E.A. Berne, A.C.R. Cavalcante and C.A.F. Costa Centro Nacional de Pesquisa de Caprinos (EMBRAPA-CNPC), Caixa Postal D-I O, CEP 62100, Sobral, CE, Brazil (Accepted 9 June 1992)
ABSTRACT Vieira, L.S., Berne, M.E.A., Cavalcante, A.C.R. and Costa, C.A.F., 1992. Haemonchus contortus resistance to ivermectin and netobimin in Brazilian sheep. Vet. Parasitol., 45:111-116. Suffolk, Texel, Hampshire Down and Ile de France sheep from the municipalities of Porto Amazonas, Piraquara and Araucaria in the State of Paran~i, and Bag6 in the State of Rio Grande do Sul were brought to Sobral, State of Cear~i, to be used in a cross-breeding project. On arrival they had clinical signs of nematode parasitosis, and one Suffolk female died. The animals were treated orally with ivermectin (0.2 mg kg -~ ) and fifteen days later with netobimin (20.0 rag kg-1). Neither drug reduced the egg counts (measured in eggs per gram, EPG) significantly, and this suggested that the nematodes in the sheep were resistant to the anthelmintics used. Haemonchus contortus was the species involved. The egg counts were reduced after oral treatment with trichlorfon (100.0 mg kg -~ ). Haemonchus contortus larvae obtained from these animals before trichlorfon treatment and passaged through two nematode-free sheep were used in a further experiment. Twenty 6- to 9-month-old nematode-free Iambs were infected with the H. contortus larvae ( 10 000 per animal) and, after the infection was confirmed, were randomly divided into four groups of five animals. Group I was orally treated with ivermectin at 0.2 mg kg- ~, Group II with oral netobimin at 20.0 mg kg -~, Group III with oral trichlorfon at 100.0 mg kg -I and Group IV was a non-treated control. Egg counts and faecal cultures were taken before dosing on the day of treatment and seven days later when all animals were necropsied and the nematodes were collected from the abomasa and counted. Drug efficacy measured by the reduction in adult H. contortus numbers was 99.9% for trichlorfon, 38.9% for netobimin and 17.6% for ivermectin. The results demonstrated the resistance of this strain ofH. Contortus to ivermectin and netobimin.
INTRODUCTION
Resistance of gastrointestinal nematodes to benzimidazole and imidazole anthelmintics has spread dramatically in recent years (Vieira, 1986). Considering that helminth control in small ruminants is based on strategic mediCorrespondence to: L.S. Vieira, Centro Nacional de Pesquisa de Caprinos (EMBRAPA-CNPC), Caixa Postal D-10, CEP 62100, Sobral, CE, Brazil.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
1 12
L.S. VIEIRA ET AL.
cation with anthelmintics, it can be expected that anthelmintic resistance will represent a major problem in sheep and goat production. Among the gastrointestinal nematodes, H a e m o n c h u s contortus is the one which develops resistance most rapidly as a result of its high biotic potential (Echevarria and Trindade, 1989). The efficacy of ivermectin against H. contortus in sheep has been well-documented by several authors. Swan et al. (1984), testing ivermectin orally at a dose rate of 0.2 mg kg- 1 in sheep, observed an efficacy greater than 90.0% against adult and immature forms of H. contortus. Santiago et al. (1985a) observed that both oral and injectable formulations of ivermectin at a dose rate of 0.2 mg kg-1 in sheep had 100.0% efficacy against H. contortus which was resistant to benzimidazoles. Hembry et al. (1986), Lyons et al. (1986), Robin (1986) and Njanja et al. (1987) also reported high efficacy of ivermectin against H. contortus in sheep. The efficacy of netobimin against H. contortus in sheep is also well-documented. Steel et al. (1985b), working with this anthelmintic in sheep infected with H. contortus strains susceptible and resistant to benzimidazoles, observed an efficacy of 98.6% at a dose rate of 20.0 mg kg -1. Menezes (1988), using helminths from Sobral, State of Ceani, Brazil, observed that ivermectin (0.2 mg kg -1 ) and netobimin (20.0 mg kg -~ ) had an efficacy of 100.0% in the reduction of egg counts in goats and sheep experimentally infected with H. contortus. Duncan et al. ( 1985 ) and Santiago et al. (1985b) also reported high efficacy of netobimin against H. contortus in sheep. Despite ivermectin being available only recently, nematode resistance has already been reported. Carmichael et al. ( 1987 ), working with South African sheep, were the first to report resistance to ivermectin in H. contortus. According to these authors, ivermectin was in use for 18 to 24 months with treatment intervals of 3 to 5 weeks. Also in South Africa, Van Wyk and Malan ( 1988 ) observed H. contortus resistance to ivermectin in sheep after 11 anthelmintic treatments over a period of two and a half years. Echevarria and Trindade (1989), in Bag6, State of Rio Grande do Sul, Brazil, observed resistance to ivermectin in H. contortus from sheep after 32 anthelmintic treatments at a dose rate of 0.2 mg kg-1 over a period of four and a half years. According to these authors the anthelmintic efficacy was reduced to 60.0%. The literature reviewed has not revealed any record of H. contortus resistance to netobimin. The study described in this paper identifies a strain of H. contortus from sheep from the States of Paran~i and Rio Grande do Sul, Brazil, resistant to both ivermectin and netobimin. MATERIALSAND METHODS Nine animals (three Suffolk, two Texel, two Hampshire Down and two Ile de France) were brought to the research facilities of the Brazilian National
HAEMONCHUS CONTORTUS DRUG RESISTANCEIN SHEEP
1 13
Goat Research Center (EMBRAPA-CNPC), Sobral, State of Cear~i, in October 1987 to be used in a cross-breeding project. These animals came from the municipalities of Porto Amazonas, Araucaria and Piraquara in the State of Paranfi, and Bag6 in the State of Rio Grande do Sul. Clinical signs of helminthiasis were observed just after arrival, and one Suffolk ewe died. As a result, the remaining sheep were medicated, orally, with ivermectin at 0.2 mg kg- 1, and 15 days later were treated again, this time with netobimin given orally at 20.0 mg kg- 1. Even after the second medication there was no significant reduction in the number of helminth eggs per gram of faeces (EPG), suggesting nematode resistance to these products. The species involved in the suspected resistance was H. contortus. The egg counts were reduced after one oral administration of trichlorfon at 100.0 mg kg -~ 15 days after the netobimin treatment. After being treated with ivermectin and netobimin without a significant drop in egg counts, and before being treated with trichlorfon, the animals were provided with collecting bags so that faeces for cultures could be collected. The H. contortus larvae harvested from the cultures were inoculated into two nematode-free sheep for further multiplication. The resultant larvae from these two sheep were used to infect 20 lambs of 6 to 9 months of age. These experimental lambs from an undefined breed had been shown to be nematode-free before artificial infection. Each lamb was inoculated orally with 10 000 infective larvae. When patency of the H. contortus infection was confirmed by faecal analysis, the 20 experimental lambs were randomly divided into four groups of five animals. Group I was treated orally with ivermectin at 0.2 mg kg-1; Group II with oral netobimin at 20.0 mg kg-1; Group III with oral trichlorfon at 100.0 mg kg- 1 and Group IV served as a non-treated control. Egg counts and larval cultures were carded out on samples taken immediately prior to treatment and 7 days after treatment when all animals (treated and control groups) were necropsied and the nematodes from the abomasa were collected, identified and counted. The anthelmintic efficacy of each treatment was calculated on the basis of the percentage reduction in egg counts and from a comparison of treated and control adult nematode counts. A standard error (S.E.) was calculated for each percentage efficacy, as recommended by Clark and Turton ( 1973 ). The percentage efficacy minus 2 S.E. units gives the minimal percentage efficacy of the anthelmintic tested with a P
