Isolation of benzimidazole resistant strains of Ostertagia circumcincta from British sheep

Two strains of Ostertagia circumcincta were isolated from sheep in Great Britain; one (CVL strain) from a breeding flock maintained at the Central Veterinary Laboratory, the other (H2 strain) from a commercial flock in southern England. Their resistance to benzimidazole anthelmintics was assessed by means of in vitro egg hatch assays and slaughter trials. In vitro egg hatch assays gave calculated ED50 estimates of 0.799 micrograms thiabendazole/ml for the CVL strain and 0.794 micrograms thiabendazole/ml for the H2 strain, compared with ED50 estimates of 0.038 micrograms thiabendazole/m and 0.036 micrograms thiabendazole/ml for two known susceptible strains of O circumcincta. There was a 40.7, 28.4 and 66.9 per cent reduction in the group mean worm burdens of lambs infected with the CVL strain following treatment with thiabendazole, fenbendazole and oxfendazole, respectively, and 23.8, 0.0, 79.6, 52.7, 99.9 and 100 per cent reduction in the group mean worm burdens of lambs infected with the H2 strain following treatment with thiabendazole, fenbendazole, oxfendazole, albendazole, levamisole and ivermectin, respectively. Detailed field histories for both strains are given.     

The efficacy of netobimin against a benzimidazole susceptible and a resistant strain of Haemonchus contortus in sheep in The Netherlands

Netobimin was tested for efficacy against Haemonchus contortus using 7 groups of 5 parasite-free lambs of six months age. The lambs in group 1 and 2 were infected with 10,000 larvae of a benzimidazole susceptible strain and those in groups 3-7 with the same dose of a resistant strain. The following treatment scheme was applied 21 days after infection: lambs in groups 2 and 4-7.5 mg kg-1 netobimin, in group 5-20 mg kg-1 netobimin, in group 6-5 mg kg-1 oxfendazole and in group 7--3.8 mg kg-1 albendazole. The lambs in groups 1 and 3 remained untreated. All lambs were slaughtered 28 days after infection. Egg counts decreased in all lambs after treatment, but increased again in lambs in groups 4, 6 and 7. There was a slight increase in lambs in group 5, while those in group 2 remained negative. Post-mortem worm counts showed a reduction of 99.8 per cent in lambs in group 2 compared to those in group 1. In lambs in group 4-7 the reduction of worm counts was respectively 40.9, 89.5, 24.7 and 40.7 per cent compared to those in group 3. Egg development assays carried out 20 days after infection showed an average LD50 of 0.46 mg ml-1 thiabendazole for the resistant strain. After treatment (day 27) the LD50 was 0.53, 0.48, 0.58, 0.56 and 0.47 in lambs in the groups 3-7. It is concluded that netobimin and other (pro)-benzimidazoles should not be used in cases of benzimidazole resistance and that levamisole, pyrantel tartrate or ivermectin are preferable.     

Studies on an Ostertagia ostertagi strain suspected to be resistant to benzimidazoles.

Four calves were infected with a susceptible (laboratory) strain of Ostertagia ostertagi and four with a field strain suspected to be resistant to benzimidazoles. After 25 days two calves from each group were treated with 3.5 mg kg-1 fenbendazole. Egg output fell to zero in all treated calves. Treated calves did not harbour worms at slaughter 35 days after infection. Significant differences between the strains were shown for ED50 values for thiabendazole in the egg hatch assay, but not in the tubulin binding assay. It is concluded that benzimidazole resistance in the suspected strain cannot be confirmed.     

Evidence of multiple anthelmintic resistance in a strain of Teladorsagia circumcincta (Ostertagia circumcincta) isolated from goats in Scotland

The efficacy of ivermectin, fenbendazole and levamisole was determined in lambs individually infected with 5000 third stage larvae of Teladorsagia (Ostertagia) species isolated from a herd of goats with a history of benzimidazole resistance and where the efficacy of ivermectin had also suffered an apparent decline. Efficacies, calculated using geometric means, for groups where treatments were directed against adult worms were 90.3 per cent (ivermectin), 43.4 per cent (fenbendazole) and 100 per cent (levamisole). The efficacy of ivermectin against immature stages was 83.2 per cent. Ivermectin was highly effective (99.9 per cent) against a known susceptible ovine strain of Teladorsagia.     

Errata

Summary

Netobimin was tested for efficacy against Haemonchus contortus using 7 groups of 5 parasite‐free lambs of six month age. The lambs in group 1 and 2 were infected with 10,000 larvae of a benzimidazole susceptible strain and those in groups 3–7 with the same dose of a resistant strain. The following treatment scheme was applied 21 days after infection: lambs in groups 2 and 4 ‐ 7.5 mg kg^‐1 netobimin, in group 5 ‐ 20 mg kg^‐1 netobimin, in group 6 ‐ 5 mg kg^‐1 oxfendazole and in group 7 ‐ 3.8 mg kg^‐1 albendazole. The lambs in groups 1 and 3 remained untreated. All lambs were slaughtered 28 days after infection. Egg counts decreased in all lambs after treatment, but increased again in lambs in groups 4, 6 and 7. There was a slight increase in lambs in group 5, while those in group 2 remained negative. Post‐mortem worm counts showed a reduction of 99.8 per cent in lambs in group 2 compared to those in group 1. In lambs in group 4–7 the reduction of worm counts was respectively 40.9, 89.5, 24.7 and 40.7 per cent compared to those in group 3. Egg development assays carried out 20 days after infection showed an average LD₅₀ of 0.46 mg ml^‐1 thiabendazole for the resistant strain. After treatment (day 27) the LD₅₀ was 0.53, 0.48, 0.58, 0.56 and 0.47 in lambs in the groups 3–7. It is concluded that netobimin and other (pro) ‐benzimidazoles should not be used in cases of benzimidazole resistance and that levamisole, pyrantel tartrate or ivermectin are preferable.     

The influence of a heavy infection with sensitive and resistant strains of Ostertagia circumcincta and with Trichostrongylus colubriformis on the pharmacokinetics of febantel in lambs

Plasma concentrations of febantel and its major metabolites, fenbendazole, oxfen-dazole and fenbendazole sulphone, were determined after oral administration of 7.5 mg/kg febantel in lambs before and 28 days after infection with 100 000 L3 larvae of a benzimidazole (BZ)- sensitive or BZ-resistant strain of Ostertagia circumcincta or with 75 000 L3 larvae of a BZ- sensitive Trichostrongylus colubriformis strain. The febantel concentrations were always low, and in only a few samples were higher than the limit of detection. A mean decrease in the area under the curve ( AUC ) for the three metabolites of 10.2%, 16.4% and 4.9% in lambs infected, respectively, with BZ-sensitive O. circumcincta , BZ-resistant O. circumcincta and T. colubriformis was observed. The C _max for all the metabolites was higher in the BZ-sensitive O. circumcincta group than in the naive sheep, while the T _max occurred earlier. The C _max and the T _max values for all the metabolites were lower in the BZ-resistant O. circumcincta group than in their own naive controls. In the T. colubriformis group the C_max values of the metabolites were lower and the T_max occurred much later.     

Characteristics of a flubendazole resistant isolate of Oesophagostomum dentatum from Germany.

Two controlled tests were performed to investigate the benzimidazole resistance of a nodular worm isolate "GIBZ" from a pig breeding farm in Germany. In Trial I, groups of five pigs, artificially infected with Oesophagostomum larvae isolated from that farm were treated with flubendazole at a single dose of 5 mg kg(-1) bodyweight (BW) or remained untreated. In Trial II, three groups of three pigs each infected with larvae after a further laboratory passage of this isolate were treated with flubendazole either at a single dose of 5 mg kg(-1) BW or at a divided dose of 1.5 mg kg(-1) BW daily for 5 consecutive days, or with fenbendazole at a single dose of 5 mg kg(-1) BW, the fourth infected group remained untreated. The respective doses of anthelmintics were mixed with a small amount of feed and administered to individual pigs in both trials. Fecal egg counts before and after treatment and post-mortem worm burdens 7 days after (last) treatment were examined to assess the anthelmintic efficacies. Only infections with Oesophagostomum dentatum were found in both trials. In Trial I, the mean worm count reduction by flubendazole was 30% as compared to the untreated controls. In Trial II, flubendazole administered at a single or divided dose reduced the mean worm burden by 0 and 85%, respectively, whereas fenbendazole was 100% effective. These results establish resistance to flubendazole in the isolate "GIBZ" of O. dentatum. The failure to reveal side resistance to fenbendazole may be explained by that the currently recommended dose rate of this compound is supra-optimal for porcine nodular worms.     

The difference in efficacy of ivermectin oral, moxidectin oral and moxidectin injectable formulations against an ivermectin-resistant strain of Trichostrongylus colubriformis in sheep

AIM: To evaluate the efficacy of ivermectin oral, moxidectin oral and moxidectin injectable formulations against an ivermectin-resistant strain of Trichostrongylus colubriformis in sheep. METHODS: Twenty-four mixed breed lambs were infected with 15,000 infective third-stage larvae of an ivermectin-resistant strain of T. colubriformis which had originally been isolated from a goat farm in Northland in 1997. Twenty-six days post infection, the lambs were divided into 3 treatment groups and a control group (n=6 lambs/group). Treatment consisted of either ivermectin oral formulation (0.2 mg/kg), moxidectin oral formulation (0.2 mg/kg), or moxidectin injectable formulation (0.2 mg/kg). Faecal egg counts (FECs) were determined at 0, 3, 5, 7 and 10 days after treatment. All animals were necropsied 12 days after treatment and worm counts were performed. Larval development assays were conducted 24 days post infection. A further 3 lambs were infected with 15,000 infective third-stage larvae of a fully susceptible strain of T. colubriformis for comparative purposes in the larval development assay. The efficacy of the moxidectin injectable formulation was also confirmed in these 3 lambs. RESULTS: The FEC reduction test at day 10 after treatment revealed 62%, 100% and 0% reductions in arithmetic-mean FECs for ivermectin oral, moxidectin oral and moxidectin injectable groups, respectively. The ivermectin oral, moxidectin oral and moxidectin injectable formulations achieved 62%, 98% and 4% reductions in arithmetic-mean worm burdens, respectively. Larval development assays showed resistance ratios for ivermectin of 4:1, avermectin B2 of 2.7:1, ivermectin aglycone of 37:1, moxidectin of 1.4:1, thiabendazole of 14.6:1 and levamisole of 1.8:1. CONCLUSIONS: The moxidectin oral formulation provided a high degree of control against ivermectin-resistant T. colubriformis whereas the moxidectin injectable formulation had very low efficacy. Ivermectin aglycone was the analogue of choice for diagnosis of ivermectin resistance in T. colubriformis in the larval development assay.     

Field observations on the control of ovine parasitic gastroenteritis in south-east England

Three worm control strategies, on a commercial farm in south east England, were monitored parasitologically for a grazing season. Three peaks of larval infection in June, late August and late October/early November occurred on paddocks grazed by dosed ewes and lambs on a new ley (preventive strategy). Lambs dosed and moved on to a hay aftermath in July (evasive strategy), had a mean egg count of 2090 by late August, attributable to infection with Haemonchus contortus. Three weekly drenching of lambs (suppressive strategy), suppressed faecal egg counts until September when the treatment interval was extended due to other on-farm commitments. In addition to these observations, autoinfection, with Haemonchus contortus, was seen to occur in lambs grazing summer turnips. An in vitro egg hatch assay, and critical efficacy trial, confirmed the presence of a benzimidazole resistant strain of Haemonchus contortus on this farm. The implications of these findings are discussed.     

Efficacy of ivermectin against benzimidazole-resistant Nematodirus spathiger in sheep

Eighteen helminth-free lambs were randomly allocated to six groups of three. Each lamb was dosed with 3300 infective larvae pooled from two isolates of Nematodirus spathiger known to be benzimidazole resistant. One lamb from each group was treated with oral ivermectin, one with oral oxfendazole and one left untreated 21 days after infection. All lambs were humanely killed 14 days later and small intestine worm counts performed. No Nematodirus were found in the ivermectin-treated lambs. Nematodirus numbers were reduced by 13% in oxfendazole-treated lambs relative to the control lambs.     

Anthelmintic evaluation of a thiabendazole-resistant strain of Ostertagia circumcincta recovered from sheep in England

A total of 35 worm-free lambs were infected with a strain of Ostertagia circumcincta isolated earlier from sheep in Cheshire, England, and found to be resistant to thiabendazole (TBZ). When patency was established the sheep were divided into groups of six, and dosed orally with either TBZ (44 mg kg-1, 88 mg kg-1), fenbendazole (FBZ; 5 mg kg-1) or levamisole (7.5 mg kg-1) or not treated. Three of the remaining five animals were dosed with FBZ at 10 mg kg-1. Egg hatch tests, post-dosing faecal egg counts and post-mortem worm counts confirmed resistance to TBZ, and a degree of side-resistance to FBZ was also revealed. Only levamisole gave the clearance expected of modern anthelmintics.     

The effect of single and divided dose administration on the efficacy of fenbendazole against adult stages of benzimidazole resistant sheep trichostrongylids.

Sheep infected with benzimidazole resistant strains of Haemonchus contortus and Trichostrongylus colubriformis were used to compare the anthelmintic efficacy of fenbendazole given as a single dose or administered in a divided dose regime over five days. Statistical analysis showed no significant difference between the two methods of administration for H contortus. On the other hand, divided dose fenbendazole was significantly less effective than single doses against adult T colubriformis at dose rates of 5 and 7.5 mg/kg. In the case of H contortus a highly significant correlation coefficient between post treatment egg counts and worm counts (r = 0.789) was obtained. This suggests that reduction in faecal egg output following drug treatment would provide a useful field indication of anthelmintic performance of fenbendazole (and possibly related compounds) against benzimidazole resistant strains of this parasite.     

Anthelmintic resistance in nematode parasites of sheep in Denmark with special emphasis on levamisole resistance in Ostertagia circumcincta.

This study was undertaken to elucidate the presence of anthelmintic resistance in nematode parasites of sheep in Denmark. Twenty two flocks of sheep were selected for Faecal Egg Count Reduction (FECR) tests, based on a prior history of either the same anthelmintic, or anthelmintic class having been used 3 times or more over the previous 5 years. Evidence of anthelmintic resistance was detected in 7 flocks. Two flocks showed FECR of 88% and 94% after treatment with thiabendazole, FECR of another 2 were 90% and 94% following treatment with fenbendazole. Three flocks showed FECR of 73%, 89% and 94%, respectively following the use of levamisole. Ostertagia circumcincta was isolated from 1 of the latter flocks and subjected to an in vivo controlled slaughter assay. Following treatment with levamisole at the recommended dose rate of 7.5 mg/kg, FECR was 44.5% and worm counts were reduced by 67.7%. These results were further substantiated by an in vitro egg hatch paralysis assay and by measuring pepsinogen levels in treated and non-treated lambs. This is the first instance of anthelmintic resistance in sheep nematodes in Scandinavia.     

Levels of benzimidazole resistance in Haemonchus contortus and Trichostrongylus colubriformis recorded from an egg hatch test procedure

A method of using commercially available benzimidazole anthelmintics in an egg hatch test, to record levels of resistance is described. The test shows that a side-resistance is present to all the benzimidazole anthelmintics tested against both Haemonchus contortus and Trichostrongylus colubriformis after selection with thiabendazole. Additional selection on the field resistant strain of H contortus with thiabendazole showed increased levels of resistance to all the compounds tested while similar selection of T colubriformis failed to change the level of resistance after 20 generations. The level of resistance recorded by the egg hatch test in vitro is greater than that recorded for adults in vivo for the CF strain of H contortus and T colubriformis. Problems associated with reading the test are mentioned as are the different levels for the threshold of activity.     

Benzimidazole resistance in equine cyathostomes in Slovakia

The present study included 19 stud farms, including 243 horses, that were investigated for the occurrence of anthelmintic resistant cyathostomes. The number of horses on the farms varied from nine to more than 100, and horses of all ages were included. A minimum of seven horses were used for faecal egg count reduction (FECR) tests. The anthelmintics included were: fenbendazole (paste formulation), ivermectin (paste formulation) and pyrantel (powder). Resistance to benzimidazoles was detected on 14 farms, with FECR values ranging from 65.1 to 86.3%. Larval cultures after fenbendazole treatment revealed exclusively cyathostome larvae. Ivermectin was tested on eight farms and proved to be effective on all. Pyrantel was tested on two farms and FECR test indicated high efficacy (92-97%). Egg hatch assay (EHA) results showed that mean concentrations of thiabendazole that inhibited hatching in 50% of the eggs (ED(50)) in resistant populations were over 0.1 microg ml(-1). The results of our study suggest widespread resistance to fenbendazole in equine cyathostomes in Slovakia, and possible strategies to delay anthelmintic resistance are discussed briefly.     

Influence of dietary protein on the immune responsiveness of sheep to Haemonchus contortus

Twenty helminth-free lambs were fed diets containing either 169 g crude protein (CP) kg-1 dry matter (DM) or 88 g CP kg-1 DM from the age of seven months. One month later five lambs from each dietary group were vaccinated against Haemonchus contortus by the oral administration of 10,000 irradiated larvae on two occasions, four weeks apart. Four weeks following the administration of the second dose of irradiated larvae both the vaccinated and unvaccinated lambs were exposed to an experimental infection of 10,000 non-irradiated H contortus larvae Faecal egg output and haematological changes were monitored throughout the study. The lambs were slaughtered 28 days after challenge when worm burdens were assessed. Vaccination was equally successful in inducing a strong resistance to the challenge infection regardless of dietary status. It was concluded that dietary protein does not influence the response to vaccination with irradiated H contortus larvae of lambs more than seven months old.     

Anthelmintic resistance in cattle nematode in the western Pampeana Region of Argentina

During the past three years there have been several reports of cattle anthelmintic resistance (AR) in Argentina. Twenty-five cattle herds in the west fattening area of the Pampeana Region were surveyed to assess the prevalence of AR and the species of nematode involved. The survey involved fattening and cow-calf systems with a mean of 6760 bovines. Resistance was determined in vivo by a faecal egg count reduction test (FECRT). Efficacies from the World Association for the Advancement of Veterinary Parasitology (WAAVP) formula were compared with those obtained from the Abbott formula. Sixty 6-12-month-old calves were selected from each property and divided into four groups, namely, IVM (ivermectin, 0.2mg/kg), BZD (fenbendazole, 5mg/kg or albendazole 7.5mg/kg), LVM (levamisole, 7.5mg/kg) and Control (untreated group). Nematode larvae were identified by four pooled faecal cultures for each group. Cooperia and Ostertagia were the predominant nematode larvae in pre-treatment cultures. The mean FECRT efficacies for IVM, BZD and LVM were 81.0+/-21%, 93.3+/-8% and 97.6+/-4%, respectively. AR was detected in 16 (64%) herds. IVM resistance was present in 15 herds and BZD resistance in eight herds (32%). Resistance to both ivermectin and fenbendazole was present in seven herds. LVM resistance was not detected. Cooperia was the predominant post-treatment nematode in all the herds with IVM resistance. In one herd with IVM resistance, Ostertagia counts were slightly increased over pretreatment counts. Ostertagia was the prevalent post-treatment genera in herds with BZD resistance. A high level of resistance to ivermectin was recorded in this fattening region of Argentina. Also several herds with resistance to benzimidazole were observed. These results show the importance of AR in Argentinian cattle production. High correlation between WAAVP and Abbott formulae was observed. However, under some uncertain diagnosis, both formulae could be applied as complementary to help result interpretations.     

Prevalence of anthelmintic resistant nematodes in sheep in south-east England

The prevalence of anthelmintic resistant nematodes among 52 commercial flocks in south-east England was investigated by comparing the faecal egg counts of groups of lambs before, and seven days after, treatment with thiabendazole and levamisole. Evidence of thiabendazole resistance was found on seven farms. in each case Haemonchus contortus was the only species of nematode involved. In vitro egg hatch assays carried out for isolates from these farms gave ED50 estimates of 0.065 to 0.332 micrograms thiabendazole/ml compared with estimates of 0.027 to 0.031 micrograms thiabendazole/ml for a known susceptible strain of H contortus assayed at the same time. In a series of slaughter trials, there was a 17 to 85 per cent reduction, as compared with controls, in the mean worm burdens of groups of lambs infected with these isolates and killed seven days after treatment with thiabendazole, confirming their resistance to this anthelmintic.     

Resistance to benzimidazole anthelmintics in field strains of Ostertagia and Nematodirus in sheep

SUMMARY: Resistance to a range of benzimidazole anthelmintics was investigated in 2 strains of Ostertagia spp. One strain (SRBO) had been exposed to fenbendazole, oxfendazole and thiabendazole, the other strain (KR79) only to thiabendazole. Both strains showed a high degree of resistance to albendazole, fenbendazole, oxfendazole and thiabendazole, which had efficiencies of 11–38% and 0–60% against all developmental stages of SR80 and KR79, respectively. There was no significant reduction in the KR79 worm count by thiabendazole at 132 mg kg-1 given either as a single dose or divided into 12 equal portions, one administered every 6h. Nematodirus spp were also found to be resistant to all benzimidazoles tested. Levamisole at 7 mg kg^-1 and naphthalophos at 30 mg kg-1 had efficiences of 89 and 66%, respectively, against SR80 Ostertagia and 99 and 19% against Nematodirus, all but the last of these being significant reductions in worm burdens. The resistant SR80 Ostertagia occurred on a research station, but possibly originated from a property which 3 years earlier had supplied ewes to the station. The use of benzimidazole anthelmintics and subsequent grazing on worm-free pasture may have enhanced the level of resistance. A relationship was established between egg counts and adult worm counts 10 days post-treatment, which suggested that for Ostertagia the worm count could be predicted from the geometric mean egg count from about 10 animals. Thus, where an Ostertagia population is suspected of being resistant, an anthelmintic efficiency assay using pre-and post-treatment faecal egg counts should provide a satisfactory diagnostic procedure.     

Development of a protective immunity against Ostertagia leptospicularis in trickle-infected sheep and parallel changes of serum gastrin, pepsinogen and antibody levels

Nine lambs, approximately 9 months of age were allocated to three groups (A, B, C), with three animals in each. Sheep in Groups A and B were trickle-infected with doses of 1000 third-stage larvae (L3) of Ostertagia leptospicularis (five times per week) over periods of 7.5 and 10.5 weeks, respectively, and were subsequently treated with fenbendazole (7.5 mg/kg). Approximately 3 weeks after anthelmintic treatment, all sheep were challenged with a single dose of 100,000 L3, whereas sheep of Group C received the same dose as a primary infection. Sheep of Groups A and B were almost completely refractory against the challenge infection, as indicated by negative faecal egg counts and adult worm burdens. A relatively high infection level was present in the sheep of Group C. The results indicate that a comparatively short immunization period of 7.5 weeks is sufficient to protect lambs against subsequent larval challenge. During immunization, the pepsinogen-, gastrin- and IgA-responses were similar in the individual sheep. In contrast to parasite-specific IgG1 and IgG2 levels, IgA decreased rapidly after cessation of trickle infection and parallel anthelmintic treatment, and may therefore indicate current exposure to parasite antigen. After challenge, the majority of the immunized sheep exhibited immediate and short-term responses of pepsinogen, gastrin and IgA in the serum. The time course and the level of each of these responses were very similar in the individual sheep, suggesting that the release of pepsinogen, gastrin and IgA into the circulation was influenced by related mechanisms.