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The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America: Argentina
veterinary parasitology ELSEVIER
Veterinary Parasitology 62 (1996) 189-197
The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America: Argentina C. Eddi a, j. Caracostantogolo a, M. Pe~a a, j. Schapiro a, L. Marangunich a, p.j. Waller b,,, J.W. Hansen c a Instituto Nacional de Tecnologia Agropecuria, Centro de lnvestigaci6n en Ciencias Veterinarias, 1708 Moron, Buenos Aires, Argentina h CSIRO Division of Animal Health, McMaster Laboratory, Locked Bag 1, Blacktown, NSW 2148, Australia c FAO Animal Production and Health Division, Room C-537, Via delle Terme di Caracalla, 00100 Rome, Italy
Received 8 June 1995; accepted 29 September 1995
Abstract Sixty-five sheep farms in the northern provinces of Buenos Aires, Entre Rios, Corrientes, Cordoba and Sante Fe were used in this survey on anthelmintic resistance. Anthelmintic groups tested were the benzimidazoles, levamisole, the combination levamisole + benzimidazole product and the avermectins. The overall level of resistance was 46% of properties, with resistance to the individual drug groups being 40%, 22%, 11% and 6%, respectively. On a large proportion of farms the resistance recorded was to two, or more, anthelmintic groups. Haemonchus contortus, Ostertagia spp. and Trichostrongylus spp. were the most abundant parasite species recorded. Resistance was greatest in the province of Corrientes where the frequency of treatment is generally high due to the endemic nature of H. contortus. Management practices were also important with resistance greatest on farms where frequent drenching is carded out and on farms carrying only sheep. Keywords: Sheep-Nematoda; Haemonchus contortus; Osteragia spp.; Trichostrongylus spp.; Resistance; Argentina
* Corresponding author at: Division of Animal Production, Pastoral Research Laboratory, New England Highway, Armidale, NSW 2350, Australia. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSD1 0304-401 7(95)00905-1
190
C. Eddi et al. / Veterinary Parasitology 62 (1996) 189-197
1. Introduction
The direct north-south alignment of Argentina, extending from above the Tropic of Capricorn in the north (Provinces of Jujuy and Salta) to Ushuaia in Tierra del Fuego at 54°S (the southernmost town in the world) some 3000 km south, accounts for the extreme temperature variations of this country. Tropical/subtropical conditions prevail in the north-east provinces which receive high levels of rainfall sweeping down from Brazil. The Andean mountain chain exerts a profound influence on rainfall in the rest of the country. Moisture bearing clouds from the Pacific deposit huge amounts of rainfall on the western side of the mountains in central and southern Chile. Immediately over the mountain range in the Andean foothills of Argentina, rainfall is also high ( > I000 mm year-J). However, rainfall declines dramatically within very short distances from the mountains with annual precipitation of 150-200 mm recorded in districts only 300-400 km east of the mountains. Prevailing weather conditions in the vast Patagonian region, south of the Rio Negro, are cold, dry and windy. Average annual rainfall and temperatures for Argentina are shown in Fig. 1. The Argentinian sheep industry is entirely based on permanent grazing on natural pastures. The national sheep population dramatically declined from a peak of approximately 140 million in the 1940s to approximately 45 million in the late 1960s. More recently, the rate of reduction has tended to tail-off with an average annual rate of reduction of 800 000 year -l, from 35 million in 1977 to 24.5 million in 1993. Current estimates suggest that the national flock size has reached a plateau of approximately 25-30 million sheep. Nearly h~lf the sheep population is in Patagonia where the average flock size is about 1500 head (see Fig. 1). There are very few cattle in this area so that sheep outnumber them by more than 50:1. Rainfall in this region is very low, except for very restricted areas on the immediate Andean foothills and in a few mountain valleys. This, coupled with very low temperatures for extended periods during autumn, winter and spring, mean that nematode parasite disease is of little importance in Patagonia, apart from the few mountain valleys and very restricted areas along water courses subjected to periodic flooding. The major sheep breed in this region is the Merino, with Corriedale and Romney Marsh well represented. In contrast to the cold, dry Patagonian region is the other important sheep raising area in the north-east in the Provinces of Buenos Aires, Entre Rios and Corrientes (see Fig. 1). Annual rainfall in this region is generally high ( > 1000 mm year -l ) and is coupled with warm to hot temperatures. These three provinces collectively account for approximately 33% and 47% of the total sheep and cattle numbers, respectively, in Argentina. The main sheep breeds in these provinces are Merino, Ideal, Corriedale, Romney Marsh and Lincoln. Apart from mixed grazing enterprises with cattle, cropping is an important activity in this region. Joining period is mainly in spring with autumn lambing. Haemonchus contortus is the prime nematode pathogen in the north-east provinces. Trichostrongylus colubriformis, T. axei, Ostertagia circumcinta and O. trifurcata are also prevalent. Major problems of clinical parasitism accompanied by significant mortalities due to H. contortus are regularly encountered in summer and autumn with other parasites assuming dominance in winter and spring. Although the epidemioiogy of
C. Eddi et al. / Veterinary Parasitology 62 (1996) 189-197
191
parasite infection is reasonably well established, there are very few farmers giving regular, strategically planned anthelmintic treatments. Recent surveys of anthelmintic usage in Buenos Aires, Ente Rios and Corrientes provinces indicate that a substantial proportion (approximately 40%) of farmers claim not to use anthelmintics and only recognise parasite disease to be a problem when associated with the clinical manifestations of haemonchosis (C. Eddi, unpublished data, 1992). Those farmers that administer anthelmintic to sheep do so curatively, giving several treatments during summer when they experience deaths from haemonchosis. Recent data indicate that the benzimidazole and levamisole anthelmintics account for 60% of the market with the remainder being the avermectins. Narrow spectrum drug sales are insignificant. Anthelmintic resistance is generally considered to be at a low level in Argentina because of the infrequent use of anthelmintics and the timing, which is mainly curative, i.e. at times when larval numbers on pasture are high. One published report of benzimidazole resistance in H. contortus and N. spathiger has been made (Romero et al., 1992) and a recent limited investigation in 1992 in the Buenos Aires, Corrientes, and Entre Rios provinces detected benzimidazole and levamisole resistance in one out of five farms (C. Eddi, unpublished data, 1992). However, there have been reports from veterinary practitioners of farmers in the humid pampas using frequent, often suppressive treatment of anthelmintics in their sheep flocks. This survey was designed to focus on the sheep regions in the humid north-east region of Argentina where anthelmintic resistance, if present in this country, is likely to have emerged.
2. Materials and methods Seventy-three farms from the provinces of Buenos Aires, Entre Rios, Corrientes, Cordoba and Sante Fe were used in this survey which was conducted between March and July 1994. These farms were selected at random and had a history of anthelmintic usage that was typical of this region, i.e. empirical treatment, usually infrequent and based on the farmers' perception of whether nematode parasites were causing a problem in their flocks. The average farm size was 2400 ha (range 70 to 28 000 ha) which on average received a mean annual rainfall of 950 mm. More than half the properties in the survey were distributed throughout the Buenos Aires province, with approx. 30% located in Corrientes province, and the remainder in Entre Rios, Cordoba and Santa Fe provinces. The majority of these farms grazed sheep often in combination with cattle. Stocking rates varied from 0.5 to 1.0 DSE ha -1 . A questionnaire of anthelmintic usage patterns on farms used in this survey showed that the frequency of treatment varied between 1 to 8 treatments year -1 , with the substituted benzimidazoles (fenbendazole, albendazole and oxfendazole) favoured, but significant use of levamisole and more recently ivermectin was reported. The survey was based on the faecal egg count reduction test (FECRT) (Anonymous, 1989). Sheep used in each trial for each farm were aged between 3 - 6 months and had not been previously treated with anthelmintic. On Day 1 of the trial, faecal samples were
C. Eddi et al. / Veterinary Parasitology 62 (1996) 189-197
192
Provinces
Sheep Distribution
Jujuy Formou
L
Senti
atsmsrcs j~h~nn
o
Mislo noe Fe
Cordoi
ios
Me~za
La
=enos A~r~
> 2000 1999-
[~
•
1000
[]
999 - 500
M
< 499
[]
ena de4 Fuego
Average Annual Temperature
Average Annual Rainfall
Fig. 1. Features of Argentina.
C. Eddi et al. / Veterinary Parasitology 62 (1996) 189-197
193
randomly collected from ten sheep from a common mob to estimate the nematode egg count at the outset of the trial. A mean egg count of at least 200 epg faeces was a pre-requisite for the test to proceed. On Day 1, 10 sheep were randomly allocated to each of four anthelmintic treatment groups and treated accordingly: Group 1 - Benzimidazole (Albendazole 4.75 mg kg -1, "Valbazen", SmithKline). Group 2 - Levamisole (Levamisole 7.5 mg kg - I , "Nilverm", ICI). Group 3 - Combination (Albendazole 4.75 mg kg- i + Levamisole 7.5 mg kg- ] ). Group 4 - Ivermectin (Ivermectin 0.2 mg k g - i, " I v o m e c " , Merck Sharp & Dohme). Each group was marked with a distinguishing brand and then all sheep were run together. Ten to 14 days after anthelmintic treatment faecal samples were collected from each sheep for post-treatment faecal egg counts. All samples were consigned in cool boxes to the Veterinary Research Centre (CICV-INTA) at Moron, Buenos Aires and all the laboratory procedures were conducted by the same group of trained technologists. Individual sheep faecal egg counts were performed and for each treatment group 2 g of faeces from each sheep was bulked to provide a faecal culture. These cultures were incubated for 7 days at 25°C to allow the development of eggs to infective larvae which were then harvested for larval differentiation into species. Faecal egg counts were expressed as a percentage for each parasite species. Reduction in faecal egg counts, compared with the untreated control group, was analysed for each anthelmintic treatment group according to the RESO statistical package (Anonymous, 1989). Resistance was declared when the reduction in the mean egg count of the treated group was less than 95% and the lower 95% confidence limit of the percentage reduction was less than 90% when compared with those from the untreated group.
3. Results
Mean pre-treatment faecal egg counts of sheep on eight farms selected in the survey were below 200 epg, therefore the test was abandoned on these farms. Of the 65 farms in which the FECRT was performed, the overall level of resistance detected was 46% of properties. The majority of resistance cases was against the benzimidazoles (40% farms), with 22% of farms having populations of worms resistant to levamisole. A small
Table 1 Anthelmintic resistance in H. contortus of sheep in N / E Argentina Province (n = no. farms)
Number (%) farms with resistance Benzimidazole (BZ)
Levamisole (LEV)
Combination (BZ + LEV)
Ivermectin (IVM)
Buenos Aires (n = 32) Corrientes (n = 20) Entre Rios (n = 10) Cordoba (n = 2) Santa Fe (n = 1)
I 18 4 0 1
1 3 1 0 0
0 2 1 0 0
1 0 0 0 0
Total (n = 65)
24 (37%)
5 (8%)
3 (5%)
1 (2%)
C. Eddi et al. / Veterinary Parasitology 62 (1996) 189-197
194
Table 2 Anthehnintic resistance in Ostertagia spp. of sheep in N / E Argentina Province (n = no. farms)
Number (%) farms with resistance Benzimidazole (BZ)
Buenos Aires (n = 32) Corrientes (n = 20) Entre Rios (n = 10) Cordoba (n = 2) Santa Fe (n = I)
1 I1 4 0 0
Total (n = 65)
16 (25%)
Levamisole (LEV) 1 8 4 0 0 13 (20%)
Combination (BZ + LEV)
lvermectin (IVM)
0 3 2 0 0
0 2 I 0 0
5 (8%)
3 (5%)
Table 3 Anthehnintic resistance in Trichostrongylus spp. of sheep in N / E Argentina Number (%) farms with resistance
Province (n = no. farms)
Benzimidazole (BZ)
Levamisole (LEV)
Combination (BZ + LEV)
Ivermectin (IVM)
Buenos Aires (n = 32) Corrientes (n = 20) Entre Rios (n = 10) Cordoba (n = 2) Santa Fe (n = I)
I 5 1 0 0
0 5 1 0 0
0 2 I 0 0
3 0 0 0 0
Total (n = 65)
7 (I 1%)
6(9%)
3(5%)
3(5%)
Table 4 Anthelmintic resistance in sheep flocks in Argentina No. of farms Susceptible
Resistant
% Resistant
A Procince Buenos Aires Corrientes Entre Rios
25 1 5
7 19 5
22 95 50
B. Flock size < 100 100- 500 500-1000 > 1000
4 23 4 3
3 6 2 16
43 21 33 84
27 4
14 21
34 84
30 4
27 8
47 67
C. No. fre~ltmenls year <4 >4 D. Management Mixed with cattle Not mixed
t
C. Eddi et a L / Veterinary Parasitology 62 (1996) 189-197
195
percentage of farms showed parasite resistance to the combination anthelmintic and to ivermectin (! 1% and 6%, respectively). Resistance was present against different drugs in different parasite species on the same farm on 13 occasions (20%). The prevalence of resistance to each of the four anthelmintic classes, in the most common nematode species present, are shown in Tables 1, 2 and 3. Benzimidazole resistance was the most prevalent in populations of H. contortus, but this was also common in Ostertagia spp. which showed similar levels of resistance to levamisole. Trichostrongylus spp. showed only low levels of resistance but to all four anthelmintic groups. Resistance to the combination product and to ivermectin was recorded for all three species. A summary of farmers' responses to the questionnaire is shown in Table 4. Anthelmintic resistance was very high on sheep farms in the Province of Corrientes and on those with large numbers of sheep. There was also a strong association between the presence of resistance and the frequency of anthelmintic treatment. Producers who managed their flocks as separate units from cattle, or were solely sheep farmers also tended to have higher levels of resistance than on farms where mixed grazing with sheep and cattle occurred.
4. Discussion This survey on the prevalence of anthelmintic resistance in nematode parasites of sheep is the first conducted in Argentina. Prior to this work the general perception amongst sheep farmers and their advisers was that resistance was unlikely to be a problem. Anthelmintics appeared to be working satisfactorily and no reports of clinical failure due to drug resistant worms had been made, which in other countries often was the prelude to recognising the presence of widespread resistance (Waller, 1985). Therefore it is of great concern that anthelmintic resistance was recorded in all three of the most important ovine nematode parasites, namely H. contortus, Ostertagia spp., Trichostrongylus spp., to all the broad spectrum anthelmintic formulations. The highest levels of resistance was to the benzimidazoles in H. contortus (37% of farms), but also approximately one-quarter of the sheep farms had Ostertagia spp. resistant to both benzimidazoles and levamisole. Only in Trichostrongylus sp. were the levels of resistance relatively low. Resistance in nematode parasites to levamisole, but particularly to the benzimidazole, anthelmintics is now widespread and often at high levels in sheep flocks throughout the world (for reviews see Prichard, 1990; Waller, 1994). These two separate groups of broad spectrum anthelmintics have now been readily available to farmers for almost three decades and it would come as no surprise that resistance to these drugs would be present in sheep flocks in northern Argentina where anthelmintic treatment is considered to be more prevalent than in the south of the country. However, what is of major concern is that resistance has been recorded in both the combination (BZ + LEV) product and to ivermectin in all three nematode parasite species. It has been shown that the benzimidazole + ievamisole combination restores efficacy where resistance to either one drug exists and therefore has been recommended as a useful strategy to delay the
196
C. Eddi et al./ Veterinary Parasitology62 (1996) 189-197
development of resistance (Anderson et al., 1988). Clearly the combination product cannot be relied upon in northern Argentina to delay the development of anthelmintic resistance in sheep flocks. Ivermectin represents the first of the highly potent, new group of end-ectocides, the macrocyclic lactones. With regard to this group, there has been debate as to whether or not the avermectins and the milbemycins share cross resistance (Shoop, 1993; Kieran, 1994) but recent evidence now indicates that this is in fact the case (Rothwell and Rolfe, 1994 and P.F. Rolfe, J.M. Loughlin and K. Vanhoff, personal communication, 1994). Resistance to ivermectin is now emerging with increasing frequency in sheep flocks throughout the world, particularly in H. contortus, but also in Ostertagia and Trichostrongylus spp. These reports are based on individual farm, or isolate, and relate to only one parasite species. There has been no published survey in which ivermectin resistance has been detected in any of these species, let alone all three species, and therefore the results of this survey must be considered with great alarm. On the basis of precedent (Waller, 1994), clearly this situation will get worse unless effective counter measures are implemented without delay. The prevalence of resistance is not uniform over the survey area. The problem is clearly much greater in the province of Corrientes. This is closely correlated with the overall greater frequency of anthelmintic treatment farmers give to their flocks in this province where H. contortus is recognised as one of the most important disease problems. The problem is also particularly bad on those farms with large numbers of sheep. This may be attributed either to a generally higher stocking rate than on farms with small flocks, necessitating more frequent antheimintic treatment, or secondly, that these farmers are generally more affluent and thus spend more on anthelmintics. There is a tendency that resistance is also higher on farms which do not, or have no opportunity to, mix sheep with cattle. It is well known, certainly amongst veterinary parasitologists, that the nematode parasites of one host are, in general, unsuited to the other. Therefore, farmers are achieving what they consider to be satisfactory worm control of their sheep without having to resort as often to anthelmintic treatment. This survey demonstrates conclusively that anthelmintic resistance is a serious problem in northern Argentina and should be recognised as such by all parties, whether they be farmers, extension workers, research workers and the pharmaceutical industry. Although the problem is not as great as in other regions in South America surveyed concurrently, ameliorative measures need to be implemented with the same degree of urgency. It is hoped that all the above parties will work towards this goal co-operatively in the immediate future.
Acknowledgements This survey was financially supported by the Technical Cooperation Programme administered by the Food & Agricultural Organization of the United Nations.
References Anderson, N.. Martin, P.J. and Jarrett, R.G.. 1988. Mixtures of anthelmintics: a strategy against resistance. Aust. Vet. J., 65: 62-64.
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Anonymous, 1989. Antbelmintic resistance. Report of the Working Party for the Animal Health Committee of the Standing Committee on Agriculture, SCA Technical Report Series No. 28, Canberra. Kieran, P.J., 1994. Moxidectin against ivermectin - resistant nematodes - a global view. Aust. Vet. J., 71: 18-20. Prichard, R.K., 1990. Anthelmintic resistance in nematodes: extent, recent understanding and future directions for control and research. Int. J. Parasit., 20: 515-523. Romero, J., F_,spinosa, G. and Valera, A.R., 1992. Demostraci6n de resistencia al oxfendazole en Trichostrongylidos de ovinos de la zona deprimida del salado. Rev. Med. Vet., 73: 82-86. Rothwell, J.T. and Rolfe, P., 1994. Correspondence - Moxidectin against ivermectin-resistant nematodes - a global view. Aust. Vet. J., 71: 158. Shoop, W.L, 1993. lvermectin resistance. Parasitol. Today, 9:154-159. Waller, P.J., 1985. Resistance to anthelmintics and the implications for animal production. In: N. Anderson and P.J. Waller (Editors), Resistance in Nematodes to Antbelmintic Drugs. CSIRO Division of Animal Health, Glebe, Australia, pp. I - I I. Waller, P.J., 1994. The development of anthelmintic resistance in ruminant livestock. Acta Tropica, 56: 233-243.
Veterinary Parasitology 62 (1996) 189-197
The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America: Argentina C. Eddi a, j. Caracostantogolo a, M. Pe~a a, j. Schapiro a, L. Marangunich a, p.j. Waller b,,, J.W. Hansen c a Instituto Nacional de Tecnologia Agropecuria, Centro de lnvestigaci6n en Ciencias Veterinarias, 1708 Moron, Buenos Aires, Argentina h CSIRO Division of Animal Health, McMaster Laboratory, Locked Bag 1, Blacktown, NSW 2148, Australia c FAO Animal Production and Health Division, Room C-537, Via delle Terme di Caracalla, 00100 Rome, Italy
Received 8 June 1995; accepted 29 September 1995
Abstract Sixty-five sheep farms in the northern provinces of Buenos Aires, Entre Rios, Corrientes, Cordoba and Sante Fe were used in this survey on anthelmintic resistance. Anthelmintic groups tested were the benzimidazoles, levamisole, the combination levamisole + benzimidazole product and the avermectins. The overall level of resistance was 46% of properties, with resistance to the individual drug groups being 40%, 22%, 11% and 6%, respectively. On a large proportion of farms the resistance recorded was to two, or more, anthelmintic groups. Haemonchus contortus, Ostertagia spp. and Trichostrongylus spp. were the most abundant parasite species recorded. Resistance was greatest in the province of Corrientes where the frequency of treatment is generally high due to the endemic nature of H. contortus. Management practices were also important with resistance greatest on farms where frequent drenching is carded out and on farms carrying only sheep. Keywords: Sheep-Nematoda; Haemonchus contortus; Osteragia spp.; Trichostrongylus spp.; Resistance; Argentina
* Corresponding author at: Division of Animal Production, Pastoral Research Laboratory, New England Highway, Armidale, NSW 2350, Australia. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSD1 0304-401 7(95)00905-1
190
C. Eddi et al. / Veterinary Parasitology 62 (1996) 189-197
1. Introduction
The direct north-south alignment of Argentina, extending from above the Tropic of Capricorn in the north (Provinces of Jujuy and Salta) to Ushuaia in Tierra del Fuego at 54°S (the southernmost town in the world) some 3000 km south, accounts for the extreme temperature variations of this country. Tropical/subtropical conditions prevail in the north-east provinces which receive high levels of rainfall sweeping down from Brazil. The Andean mountain chain exerts a profound influence on rainfall in the rest of the country. Moisture bearing clouds from the Pacific deposit huge amounts of rainfall on the western side of the mountains in central and southern Chile. Immediately over the mountain range in the Andean foothills of Argentina, rainfall is also high ( > I000 mm year-J). However, rainfall declines dramatically within very short distances from the mountains with annual precipitation of 150-200 mm recorded in districts only 300-400 km east of the mountains. Prevailing weather conditions in the vast Patagonian region, south of the Rio Negro, are cold, dry and windy. Average annual rainfall and temperatures for Argentina are shown in Fig. 1. The Argentinian sheep industry is entirely based on permanent grazing on natural pastures. The national sheep population dramatically declined from a peak of approximately 140 million in the 1940s to approximately 45 million in the late 1960s. More recently, the rate of reduction has tended to tail-off with an average annual rate of reduction of 800 000 year -l, from 35 million in 1977 to 24.5 million in 1993. Current estimates suggest that the national flock size has reached a plateau of approximately 25-30 million sheep. Nearly h~lf the sheep population is in Patagonia where the average flock size is about 1500 head (see Fig. 1). There are very few cattle in this area so that sheep outnumber them by more than 50:1. Rainfall in this region is very low, except for very restricted areas on the immediate Andean foothills and in a few mountain valleys. This, coupled with very low temperatures for extended periods during autumn, winter and spring, mean that nematode parasite disease is of little importance in Patagonia, apart from the few mountain valleys and very restricted areas along water courses subjected to periodic flooding. The major sheep breed in this region is the Merino, with Corriedale and Romney Marsh well represented. In contrast to the cold, dry Patagonian region is the other important sheep raising area in the north-east in the Provinces of Buenos Aires, Entre Rios and Corrientes (see Fig. 1). Annual rainfall in this region is generally high ( > 1000 mm year -l ) and is coupled with warm to hot temperatures. These three provinces collectively account for approximately 33% and 47% of the total sheep and cattle numbers, respectively, in Argentina. The main sheep breeds in these provinces are Merino, Ideal, Corriedale, Romney Marsh and Lincoln. Apart from mixed grazing enterprises with cattle, cropping is an important activity in this region. Joining period is mainly in spring with autumn lambing. Haemonchus contortus is the prime nematode pathogen in the north-east provinces. Trichostrongylus colubriformis, T. axei, Ostertagia circumcinta and O. trifurcata are also prevalent. Major problems of clinical parasitism accompanied by significant mortalities due to H. contortus are regularly encountered in summer and autumn with other parasites assuming dominance in winter and spring. Although the epidemioiogy of
C. Eddi et al. / Veterinary Parasitology 62 (1996) 189-197
191
parasite infection is reasonably well established, there are very few farmers giving regular, strategically planned anthelmintic treatments. Recent surveys of anthelmintic usage in Buenos Aires, Ente Rios and Corrientes provinces indicate that a substantial proportion (approximately 40%) of farmers claim not to use anthelmintics and only recognise parasite disease to be a problem when associated with the clinical manifestations of haemonchosis (C. Eddi, unpublished data, 1992). Those farmers that administer anthelmintic to sheep do so curatively, giving several treatments during summer when they experience deaths from haemonchosis. Recent data indicate that the benzimidazole and levamisole anthelmintics account for 60% of the market with the remainder being the avermectins. Narrow spectrum drug sales are insignificant. Anthelmintic resistance is generally considered to be at a low level in Argentina because of the infrequent use of anthelmintics and the timing, which is mainly curative, i.e. at times when larval numbers on pasture are high. One published report of benzimidazole resistance in H. contortus and N. spathiger has been made (Romero et al., 1992) and a recent limited investigation in 1992 in the Buenos Aires, Corrientes, and Entre Rios provinces detected benzimidazole and levamisole resistance in one out of five farms (C. Eddi, unpublished data, 1992). However, there have been reports from veterinary practitioners of farmers in the humid pampas using frequent, often suppressive treatment of anthelmintics in their sheep flocks. This survey was designed to focus on the sheep regions in the humid north-east region of Argentina where anthelmintic resistance, if present in this country, is likely to have emerged.
2. Materials and methods Seventy-three farms from the provinces of Buenos Aires, Entre Rios, Corrientes, Cordoba and Sante Fe were used in this survey which was conducted between March and July 1994. These farms were selected at random and had a history of anthelmintic usage that was typical of this region, i.e. empirical treatment, usually infrequent and based on the farmers' perception of whether nematode parasites were causing a problem in their flocks. The average farm size was 2400 ha (range 70 to 28 000 ha) which on average received a mean annual rainfall of 950 mm. More than half the properties in the survey were distributed throughout the Buenos Aires province, with approx. 30% located in Corrientes province, and the remainder in Entre Rios, Cordoba and Santa Fe provinces. The majority of these farms grazed sheep often in combination with cattle. Stocking rates varied from 0.5 to 1.0 DSE ha -1 . A questionnaire of anthelmintic usage patterns on farms used in this survey showed that the frequency of treatment varied between 1 to 8 treatments year -1 , with the substituted benzimidazoles (fenbendazole, albendazole and oxfendazole) favoured, but significant use of levamisole and more recently ivermectin was reported. The survey was based on the faecal egg count reduction test (FECRT) (Anonymous, 1989). Sheep used in each trial for each farm were aged between 3 - 6 months and had not been previously treated with anthelmintic. On Day 1 of the trial, faecal samples were
C. Eddi et al. / Veterinary Parasitology 62 (1996) 189-197
192
Provinces
Sheep Distribution
Jujuy Formou
L
Senti
atsmsrcs j~h~nn
o
Mislo noe Fe
Cordoi
ios
Me~za
La
=enos A~r~
> 2000 1999-
[~
•
1000
[]
999 - 500
M
< 499
[]
ena de4 Fuego
Average Annual Temperature
Average Annual Rainfall
Fig. 1. Features of Argentina.
C. Eddi et al. / Veterinary Parasitology 62 (1996) 189-197
193
randomly collected from ten sheep from a common mob to estimate the nematode egg count at the outset of the trial. A mean egg count of at least 200 epg faeces was a pre-requisite for the test to proceed. On Day 1, 10 sheep were randomly allocated to each of four anthelmintic treatment groups and treated accordingly: Group 1 - Benzimidazole (Albendazole 4.75 mg kg -1, "Valbazen", SmithKline). Group 2 - Levamisole (Levamisole 7.5 mg kg - I , "Nilverm", ICI). Group 3 - Combination (Albendazole 4.75 mg kg- i + Levamisole 7.5 mg kg- ] ). Group 4 - Ivermectin (Ivermectin 0.2 mg k g - i, " I v o m e c " , Merck Sharp & Dohme). Each group was marked with a distinguishing brand and then all sheep were run together. Ten to 14 days after anthelmintic treatment faecal samples were collected from each sheep for post-treatment faecal egg counts. All samples were consigned in cool boxes to the Veterinary Research Centre (CICV-INTA) at Moron, Buenos Aires and all the laboratory procedures were conducted by the same group of trained technologists. Individual sheep faecal egg counts were performed and for each treatment group 2 g of faeces from each sheep was bulked to provide a faecal culture. These cultures were incubated for 7 days at 25°C to allow the development of eggs to infective larvae which were then harvested for larval differentiation into species. Faecal egg counts were expressed as a percentage for each parasite species. Reduction in faecal egg counts, compared with the untreated control group, was analysed for each anthelmintic treatment group according to the RESO statistical package (Anonymous, 1989). Resistance was declared when the reduction in the mean egg count of the treated group was less than 95% and the lower 95% confidence limit of the percentage reduction was less than 90% when compared with those from the untreated group.
3. Results
Mean pre-treatment faecal egg counts of sheep on eight farms selected in the survey were below 200 epg, therefore the test was abandoned on these farms. Of the 65 farms in which the FECRT was performed, the overall level of resistance detected was 46% of properties. The majority of resistance cases was against the benzimidazoles (40% farms), with 22% of farms having populations of worms resistant to levamisole. A small
Table 1 Anthelmintic resistance in H. contortus of sheep in N / E Argentina Province (n = no. farms)
Number (%) farms with resistance Benzimidazole (BZ)
Levamisole (LEV)
Combination (BZ + LEV)
Ivermectin (IVM)
Buenos Aires (n = 32) Corrientes (n = 20) Entre Rios (n = 10) Cordoba (n = 2) Santa Fe (n = 1)
I 18 4 0 1
1 3 1 0 0
0 2 1 0 0
1 0 0 0 0
Total (n = 65)
24 (37%)
5 (8%)
3 (5%)
1 (2%)
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Table 2 Anthehnintic resistance in Ostertagia spp. of sheep in N / E Argentina Province (n = no. farms)
Number (%) farms with resistance Benzimidazole (BZ)
Buenos Aires (n = 32) Corrientes (n = 20) Entre Rios (n = 10) Cordoba (n = 2) Santa Fe (n = I)
1 I1 4 0 0
Total (n = 65)
16 (25%)
Levamisole (LEV) 1 8 4 0 0 13 (20%)
Combination (BZ + LEV)
lvermectin (IVM)
0 3 2 0 0
0 2 I 0 0
5 (8%)
3 (5%)
Table 3 Anthehnintic resistance in Trichostrongylus spp. of sheep in N / E Argentina Number (%) farms with resistance
Province (n = no. farms)
Benzimidazole (BZ)
Levamisole (LEV)
Combination (BZ + LEV)
Ivermectin (IVM)
Buenos Aires (n = 32) Corrientes (n = 20) Entre Rios (n = 10) Cordoba (n = 2) Santa Fe (n = I)
I 5 1 0 0
0 5 1 0 0
0 2 I 0 0
3 0 0 0 0
Total (n = 65)
7 (I 1%)
6(9%)
3(5%)
3(5%)
Table 4 Anthelmintic resistance in sheep flocks in Argentina No. of farms Susceptible
Resistant
% Resistant
A Procince Buenos Aires Corrientes Entre Rios
25 1 5
7 19 5
22 95 50
B. Flock size < 100 100- 500 500-1000 > 1000
4 23 4 3
3 6 2 16
43 21 33 84
27 4
14 21
34 84
30 4
27 8
47 67
C. No. fre~ltmenls year <4 >4 D. Management Mixed with cattle Not mixed
t
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percentage of farms showed parasite resistance to the combination anthelmintic and to ivermectin (! 1% and 6%, respectively). Resistance was present against different drugs in different parasite species on the same farm on 13 occasions (20%). The prevalence of resistance to each of the four anthelmintic classes, in the most common nematode species present, are shown in Tables 1, 2 and 3. Benzimidazole resistance was the most prevalent in populations of H. contortus, but this was also common in Ostertagia spp. which showed similar levels of resistance to levamisole. Trichostrongylus spp. showed only low levels of resistance but to all four anthelmintic groups. Resistance to the combination product and to ivermectin was recorded for all three species. A summary of farmers' responses to the questionnaire is shown in Table 4. Anthelmintic resistance was very high on sheep farms in the Province of Corrientes and on those with large numbers of sheep. There was also a strong association between the presence of resistance and the frequency of anthelmintic treatment. Producers who managed their flocks as separate units from cattle, or were solely sheep farmers also tended to have higher levels of resistance than on farms where mixed grazing with sheep and cattle occurred.
4. Discussion This survey on the prevalence of anthelmintic resistance in nematode parasites of sheep is the first conducted in Argentina. Prior to this work the general perception amongst sheep farmers and their advisers was that resistance was unlikely to be a problem. Anthelmintics appeared to be working satisfactorily and no reports of clinical failure due to drug resistant worms had been made, which in other countries often was the prelude to recognising the presence of widespread resistance (Waller, 1985). Therefore it is of great concern that anthelmintic resistance was recorded in all three of the most important ovine nematode parasites, namely H. contortus, Ostertagia spp., Trichostrongylus spp., to all the broad spectrum anthelmintic formulations. The highest levels of resistance was to the benzimidazoles in H. contortus (37% of farms), but also approximately one-quarter of the sheep farms had Ostertagia spp. resistant to both benzimidazoles and levamisole. Only in Trichostrongylus sp. were the levels of resistance relatively low. Resistance in nematode parasites to levamisole, but particularly to the benzimidazole, anthelmintics is now widespread and often at high levels in sheep flocks throughout the world (for reviews see Prichard, 1990; Waller, 1994). These two separate groups of broad spectrum anthelmintics have now been readily available to farmers for almost three decades and it would come as no surprise that resistance to these drugs would be present in sheep flocks in northern Argentina where anthelmintic treatment is considered to be more prevalent than in the south of the country. However, what is of major concern is that resistance has been recorded in both the combination (BZ + LEV) product and to ivermectin in all three nematode parasite species. It has been shown that the benzimidazole + ievamisole combination restores efficacy where resistance to either one drug exists and therefore has been recommended as a useful strategy to delay the
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development of resistance (Anderson et al., 1988). Clearly the combination product cannot be relied upon in northern Argentina to delay the development of anthelmintic resistance in sheep flocks. Ivermectin represents the first of the highly potent, new group of end-ectocides, the macrocyclic lactones. With regard to this group, there has been debate as to whether or not the avermectins and the milbemycins share cross resistance (Shoop, 1993; Kieran, 1994) but recent evidence now indicates that this is in fact the case (Rothwell and Rolfe, 1994 and P.F. Rolfe, J.M. Loughlin and K. Vanhoff, personal communication, 1994). Resistance to ivermectin is now emerging with increasing frequency in sheep flocks throughout the world, particularly in H. contortus, but also in Ostertagia and Trichostrongylus spp. These reports are based on individual farm, or isolate, and relate to only one parasite species. There has been no published survey in which ivermectin resistance has been detected in any of these species, let alone all three species, and therefore the results of this survey must be considered with great alarm. On the basis of precedent (Waller, 1994), clearly this situation will get worse unless effective counter measures are implemented without delay. The prevalence of resistance is not uniform over the survey area. The problem is clearly much greater in the province of Corrientes. This is closely correlated with the overall greater frequency of anthelmintic treatment farmers give to their flocks in this province where H. contortus is recognised as one of the most important disease problems. The problem is also particularly bad on those farms with large numbers of sheep. This may be attributed either to a generally higher stocking rate than on farms with small flocks, necessitating more frequent antheimintic treatment, or secondly, that these farmers are generally more affluent and thus spend more on anthelmintics. There is a tendency that resistance is also higher on farms which do not, or have no opportunity to, mix sheep with cattle. It is well known, certainly amongst veterinary parasitologists, that the nematode parasites of one host are, in general, unsuited to the other. Therefore, farmers are achieving what they consider to be satisfactory worm control of their sheep without having to resort as often to anthelmintic treatment. This survey demonstrates conclusively that anthelmintic resistance is a serious problem in northern Argentina and should be recognised as such by all parties, whether they be farmers, extension workers, research workers and the pharmaceutical industry. Although the problem is not as great as in other regions in South America surveyed concurrently, ameliorative measures need to be implemented with the same degree of urgency. It is hoped that all the above parties will work towards this goal co-operatively in the immediate future.
Acknowledgements This survey was financially supported by the Technical Cooperation Programme administered by the Food & Agricultural Organization of the United Nations.
References Anderson, N.. Martin, P.J. and Jarrett, R.G.. 1988. Mixtures of anthelmintics: a strategy against resistance. Aust. Vet. J., 65: 62-64.
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Anonymous, 1989. Antbelmintic resistance. Report of the Working Party for the Animal Health Committee of the Standing Committee on Agriculture, SCA Technical Report Series No. 28, Canberra. Kieran, P.J., 1994. Moxidectin against ivermectin - resistant nematodes - a global view. Aust. Vet. J., 71: 18-20. Prichard, R.K., 1990. Anthelmintic resistance in nematodes: extent, recent understanding and future directions for control and research. Int. J. Parasit., 20: 515-523. Romero, J., F_,spinosa, G. and Valera, A.R., 1992. Demostraci6n de resistencia al oxfendazole en Trichostrongylidos de ovinos de la zona deprimida del salado. Rev. Med. Vet., 73: 82-86. Rothwell, J.T. and Rolfe, P., 1994. Correspondence - Moxidectin against ivermectin-resistant nematodes - a global view. Aust. Vet. J., 71: 158. Shoop, W.L, 1993. lvermectin resistance. Parasitol. Today, 9:154-159. Waller, P.J., 1985. Resistance to anthelmintics and the implications for animal production. In: N. Anderson and P.J. Waller (Editors), Resistance in Nematodes to Antbelmintic Drugs. CSIRO Division of Animal Health, Glebe, Australia, pp. I - I I. Waller, P.J., 1994. The development of anthelmintic resistance in ruminant livestock. Acta Tropica, 56: 233-243.