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Several compounds ( n = 13 single or combinations; most at therapeutic dosages) were evaluated between 1977 and 1992 in critical tests ( n = 91) against benzimidazole (BZ) resistant small strongyles (Population S) and several other species of internal parasites in Shetland ponies, mostly under 1 year old. The closed breeding herd, from which the test ponies were selected, had been treated every 8 weeks with cambendazole (CBZ) for 4 years (1974–1978) and oxibendazole (OBZ) for 14 years (1978–1992). Published field test data (1974–1992) on older ponies in the herd showed BZ resistance of small strongyles. Average efficacies in the present critical tests against small strongyles for OBZ ( n = 59 animals) were high in early years (95% or higher), but gradually declined to a low of 1% in 1991. Side-resistance of small strongyles was evident in critical tests ( n = 1–6/single drug or combination) for several other BZs and a pro-BZ; ivermectin and piperazine were highly active, but pyrantel pamoate exhibited weak activity. BZ resistance was evident for six small strongyle species ( Cyathostomum catinatum, Cyathostomum coronatum, Cylicocyclus nassatus, Cylicostephanus calicatus, Cylicostephanus goldi , and Cylicostephanus longibursatus ). Activity on bots, ascarids, large strongyles, and pinworms was essentially as expected, indicating no drug resistance.

The role of beta-tubulin genes in benzimidazole (BZ) resistance was investigated using one susceptible (S) and two resistant (Rt and Rc) strains of Haemonchus contortus. The Rt strain was isolated from the field on the basis of thiabendazole resistance. The Rc strain was derived from the S strain by treatment with cambendazole. cDNAs, derived from the S strain, encoding two isoforms of beta-tubulin (beta 12-16 and beta 8-9), alpha-tubulin and phosphofructokinase (Pfk) were used as probes for Southern hybridization analysis of genomic DNA digested by restriction enzymes. Genomic DNA was isolated from a pool of worms or single worms. The restriction-enzyme fragment length polymorphism (RFLP) differences among these strains depended on the enzyme and the probe used. When digested with Stu I or Hpa I, and probed under stringent conditions with beta 8-9 or beta 12-16, fewer fragments were seen in the Rt and Rc strains than in the S strain. Different hybridizing fragments were found in different individuals. The frequency of individuals bearing certain fragments hybridizing to beta 12-16 or beta 8-9 in the susceptible population was reduced significantly in the resistant populations. Some differences in RFLP between these strains were observed when probed with alpha-tubulin or Pfk, but the changes were not consistent with fragments being lost from the resistant strains as observed for beta-tubulin probes. These changes in RFLP pattern correlate with changes in the binding profiles of BZs and isoelectric isoform patterns reported previously for these strains. The data confirm that reduced heterogeneity within the population is associated with BZ resistance. Our results show that both the beta 8-9 and the beta 12-16 subfamilies of beta-tubulin are affected to a similar extent by this reduction in heterogeneity in a resistant population.

Benzimidazole resistant strains of Haemonchus contortus and Ostertagia spp were subjected to selection pressure over five laboratory generations with the recommended dose rates of either cambendazolc, oxfendazole or morantel. A change in response, with larger residual worm burdens remaining after treatment at the fifth generation, was shown for both cambendazole and oxfendazole against both species of nematode. No change in response against either species was shown for morantel. The results are discussed in terms of the problem associated with inefficient removal of the adult parasites after treatment.

Electronic measurements of the motility of third stage larvae of a susceptible and a levamisole/ benzimidazole-resistant strain of Haemonchus contortus were made after incubation in solutions of anthelmintics for 24 h. Results confirmed the resistance to benzimidazoles, but failed to show differences in response to levamisole. Visual observations of paralysis of first and third stage larvae in solutions of levamisole also failed to show significant differences between the two strains. The tests as conducted failed to demonstrate levamisole resistance, suggesting that nematode strains may vary in their mechanisms of resistance to levamisole.

The prevalence of benzimidazole-resistant small strongyles was determined in a survey, conducted on 14 thoroughbred studs, which compared the faecal egg counts of groups of horses before and after treatment with the recommended doses of cambendazole (20 mg kg −1 b.w.) or febantel (6 mg kg −1 b.w.). Benzimidazole-resistant cyathostomes were found on all farms examined. Pyrantel pamoate (19 mg kg −1 b.w.), oxibendazole (10 mg kg −1 b.w.) and ivermectin (0.2 mg kg −1 b.w.) reduced the strongyle egg counts on these studs by 97–100% at 2 weeks post-treatment. However, 6 weeks after dosing the reduction of the strongyle egg output had decreased to an average of 67.8% (8.7–97.1%) with pyrantel pamoate and 51.2% (0–95.8%) with oxibendazole, whereas ivermectin still suppressed the egg counts by 98.2% (95–100%).

Rabbits infected with the ruminant parasites Trichostrongylus axei and T. colubriformis were administered eight drugs (phenothiazine, thiabendazole, haloxon, cambendazole, mebendazole, levamisole, fenbendazole, and oxibendazole) at dose rates known to be highly effective as anthelmintics in ruminants. All but phenothiazine and thiabendazole were highly active against adult T. colubriformis; mebendazole, levamisole, and fenbendazole were highly active against T. axei. When the dose rates of thiabendazole, haloxon, cambendazole, and oxibendazole were doubled, all but haloxon were highly active against T. axei adults. Efficacy against 3 and 10-day old worms was generally poor; oxibendazole showed best overall activity.

Fumarate reductase activity in a thiabendazole-resistant strain of Haemonchus contortus was found to be significantly lower than that from a susceptible strain. However, the fumarate reductase activity in a mebendazole-resistant strain did not differ from that in the susceptible strain, even though it was cross-resistant to thiabendazole. Published reports of fumarate reductase activity in strains of H. contortus susceptible or resistant to benzimidazoles were reassessed. A second, unrelated Australian thiabendazole-resistant strain also proved to have significantly diminished fumarate reductase activity, whereas two American strains, one resistant to thiabendazole and one to cambendazole, possess fumarate reductase activities indistinguishable from corresponding susceptible strains. It therefore appears that the phenomenon of benzimidazole resistance cannot be generally correlated with diminished fumarate reductase activity, although in the specific case of the Australian thiabendazole-resistant strains it may be a contributory factor.

The binding and inhibitory properties of 11 benzimidazoles for bovine brain tubulin were investigated. The effects of the benzimidazoles on the initial rates of microtubule polymerization were determined by a turbidimetric assay. The median inhibitory concentrations (I50) for nocodazole, oxibendazole, parbendazole, mebendazole and fenbendazole ranged from 1.97 . 10(-6) to 6.32 . 10(-6) M. Benomyl, cambendazole and carbendazim had I50 values from 5.83 . 10(-5) to 9.01 .10(-5) M. Thiabendazole had an I50 value of 5.49 . 10(-4) M. Inhibitor constants (Ki) were determined by the colchicine binding assay. Oxibendazole, fenbendazole, and cambendazole had Ki values of 3.20 . 10(-5), 1.73 . 10(-5) and 1.10 . 10(-4) M, respectively. Oxibendazole and fenbendazole were competitive inhibitors of colchicine. In contrast, cambendazole was a noncompetitive inhibitor of colchicine. The ability of these benzimidazoles to inhibit microtubule polymerization and the mode of action for the anthelmintic benzimidazoles is discussed.

Comley John C. W. and Wright Spdenis J. 1981. Succinate dehydrogenase and fumarate reductase activity in Aspiculuris tetraptera and Ascaris suum and the effect of the anthelmintics cambendazole, thiabendazole, and levamisole. International Journal for Parasitology11: 79–84. Succinate dehydrogenase and fumarate reductase activities from a particulate fraction of A. tetraptera and a soluble extract of A. suum have been determined using spectrophotometric methods. Fumarate reductase activity in A. suum could only be detected anaerobically. Succinate dehydrogenase activity from A. suum was partially characterized and shown to exist in several multimolecular forms (isoenzymes). The in vitro effect of the anthelmintics cambendazole, thiabendazole and levamisole on succinate dehydrogenase and fumarate reductase activity from the above nematodes are described. Significant inhibition of fumarate reductase activity of both nematodes was only achieved using 5 mM levamisole and 1 mM thiabendazole. After in vivo anthelmintic treatment of A. tetraptera only thiabendazole significantly inhibited fumarate reductase. It is suggested that the succinate dehydro-ogenase-fumarate reductase complex in these nematodes is unlikely to be the primary site chemotherapeutic attack for any of the anthelmintics tested.

Treatment of rats infected with Nippostrongylus brasiliensis with a single, oral therapeutic dose of the anthelmintic benzimidazole carbamates oxfendazole or mebendazole resulted, 24 hr later, in a marked reduction (60–90%) in the secretion of a low molecular weight acetylcholinesterase from the parasites when they were incubated in vitro . This effect coincided with the expulsion of parasites from the host as a result of the therapy. When parasites were incubated in vitro with 0.1 mM oxfendazole, mebendazole, flubendazole, parbendazole, cambendazole or thiabendazole a similar effect was observed; with oxfendazole and mebendazole the effect was apparent within 1 hr and lasted for at least 4hr after removal to fresh, drug-free medium. Whether treated in the host or in vitro the reduction in secretion was balanced by an equivalent rise in acetylcholinesterase activity within the parasites. It is suggested that the inhibition of protein secretion may be a specific manifestation of a general effect of these compounds on microtubule function.

Cambendazole (CBZ), fenbendazole (FBZ), oxfendazole (OFZ) and thiabendazole (TBZ) all inhibited the fumarate stimulated oxidation of NADH in Haemonchus contortus mitochondria. These observations plus the phenomenon of cross resistance to benzimidazoles suggested that the different benzimidazole anthelmintics affect parasitic helminths in a similar manner. The variation in efficiency and spectrum of activity may therefore be due to differences in their pharmacokinetic behaviour. To test this hypothesis, the magnitude and duration of concentrations of TBZ, FBZ and OFZ in plasma and other body compartments after administration were compared with their effectiveness against parasites. The effects of similar doses against benzimidazole-resistant. Trichostrongylus colubriformis and H. contortus were found to correlate with the period high plasma concentrations were maintained. Further evidence of this relationship was obtained by infusion or multiple drenching of TBZ to cattle harbouring arrested Ostertagia ostertagi larvae, so as to maintain high circulating concentrations for an extended period. This resulted in the removal of 90% of the arrested larvae, whereas TBZ as normally administered is considered quite ineffective against these larvae. These observations suggest that the spectrum and effectiveness of benzimidazoles may be improved by extending the period during which parasites are exposed to toxic concentrations.

Effects of cambendazole and mebendazole on the respiratory metabolism of the anterior portion of Moniezia expansa were investigated in vitro . Anaerobically and in the presence of glucose, both drugs inhibited glucose uptake and increased glycogen utilisation. They reduced succinate production, by inhibiting fumarate reductase and (in the case of cambendazole) phosphoenolypyruvate carboxykinase activities, and increased lactate production. The additional lactate formed was accumulated in the worms. The drugs diminished ATP synthesis and/or turnover of adenine nucleotides. Aerobically, the drugs exerted similar effects on glucose uptake, glycogen utilisation and adenine nucleotides but the formation of end products was unaffected. Hexokinase and phosphofructokinase activities were inhibited by the drugs in vitro , but were not inhibited in extracts of parasites preincubated with the drugs.

SUMMARY The anthelmintic action of cambendazole against 10-day-old Hyostrongylus rubidis and Oesophagostomum spp. larvae was evaluated in a controlled test using artificially-infected pigs. Twenty-two pigs divided into four groups were orally infected with 3000 H. rubidis and 38,000 Oesophagostomum spp. larvae. Three groups of pigs were given cambendazole orally at dose rates of 15, 20 and 25 mg/kg bodyweight respectively 10 days after infection. The fourth group remained untreated as controls. Two control “indicator” pigs were killed on the day of treatment to determine the number and location of the larvae. Anthelmintic efficiency was estimated by comparing faecal egg counts and numbers of worms in the stomach and large intestine at post-mortem examination 46 days after infection. At the dose rates given cambendazole had little or no effect against immature 10-day-old Hyostrongylus larvae found both in the stomach wall and lumen, or against 10-day-old Oesophagostomum spp. larvae, the majority of which were found to be in the fourth stage in the large intestinal contents and in the third and fourth stages in the large intestinal wall.

Systematic dose- and time-response trials were first conducted to determine a treatment regimen with cambdendazole (CBZ, 2-[4′-thiazolyl]-5- iso propoxycarbonylaminobenzimidazole) that would be therapeutically effective against adult Trichinella spiralis in experimentally-infected mice. CBZ proved to be highly effective against adult Trichinella in a 3-day treatment course during the enterai phase of experimental trichinellosis. When treatment began 72 h after the mice were inoculated with parasites, the number of adult worms recovered from the host intestine was greatly reduced by twice-daily oral doses of CBZ at 25 mg kg −1 body wt. The efficacy of the divided daily oral dosage treatment regimen that was effective against adult T. spiralis during the enteral phase of infection was then tested during the parenteral phases of trichinellosis. Gavage administration of CBZ at 25 mg kg −1 bis m die for 3 consecutive days during the invasive (days 14–16) and encystment (days 28–30) phases of infection significantly reduced (29 and 90 %, respectively) the number of larvae subsequently recovered from the host musculature on day 44 postinoculation.

An in vitro egg development assay was used to test the resistance of eggs from a benzimidazole susceptible and a resistant strain of Haemonchus contortus to four benzimidazoles. Storage of eggs contained in faecal samples at 4°C for four, 24 or 72 hours did not appear to impair the results from the in vitro egg development assay or alter the LD50. Storage for one week, however, proved to be lethal for the eggs. The average LD50 values for thiabendazole, albendazole, mebendazole and cambendazole were 0·06, 0·07, 0·96 and 2·47 ppm for the susceptible strain. For the resistant strain the values averaged 0·26, 0·24, 1·07 and 5·11 ppm.

Two trials were conducted to determine the effect of cambendazole against natural infections of the swine kidneyworm, Stephanurus dentatus, in sows. In the first trial, nine sows were given a single dose of 20 mg cambendazole/kg body weight, in the feed, and eight sows were not treated. In the second trial, five sows were given 40 mg cambendazole/kg body weight and four were untreated. Urine volume, S. dentatus eggs per milliliter of urine, egg hatchability, and larval survival to the third stage were determined pre and post treatment. Adult kidneyworms were counted at necropsy, 14 days after treatments. Only a temporary decline in egg hatchability was observed on the day after treatment in each trial, and cambendazole was not found to be effective against adults of S. dentatus.

Summary Horses with a history of frequent benzimidazole (BZD) treatment were used in a trial to assess the effectiveness of oxibendazole (OBZ). Initial investigations established that these animals were infected with small strongyles resistant to thiabendazole (TBZ), mebendazole (MBZ), cambendazole (CBZ), oxfendazole (OXF) and fenbendazole (FBZ). The herd was subsequently divided into four groups and received TBZ, CBZ, OBZ, or no treatment. OBZ effectly reduced fecal egg counts. Similar reductions were not observed with TBZ or CBZ.

Cambendazole, 5-isopropoxycarbonyl-amino-2-(4-thiazolyl)bcuzimidazole, was given at dosages of 11 to 44 mg./kg. to cattle with naturally acquired helminth infectious. A single oral dose of 22 mg./kg. was highly effective in removing mature Haemonchus, Ostertagia, Trichostrongylus, Cooperia, Strongyloides and Oesophagostomum, as well as various lumen dwelling immature trichostrongylids.

A new anthelmintic 5-isopropoxy- carbonylamino-2-(4-thiazolyl)benzimidazole, was highly effective against immature and mature helminths of sheep, when given orally at 20 mg./kg. Helminths removed by this dosage, or even lower dosages, included the genera Haemonchus, Ostertagia, Trichostrongylus, Cooperia, Ncmatodirus, Strongyloides and Chabertia.

Swine were exposed experimentally to Ascaris suum, and cambendazole was fed prophylactically at dietary concentrations of 0.01 and 0.03%. The concentration was effective in controlling a moderate lower infection but not in controlling a very severe infection. The higher drug level gave virtually complete protection against both the moderate and the very severe infection. Cambendazole was given as a single oral dose to pigs with patent experimental infections of Strongyloides ransomi. Dosages of 7.5 and 15 mg./kg. (the only dosages tested) were more than 99% effective in reducing the number of eggs in the faeces and the number of worms recovered from the intestines.

The lethal effect of some benzimidazoles on Taenia hydatigena infections in dogs have been compared. Fenbendazole, parbendazole and oxibendazole were more effective than cambendazole and thiabendazole, while coarsely ground mebendazole did not differ significantly from any of them.

The resistance of the eggs of benzimidazole resistant Haemonchus contortus and Trichostrongylus colubriformis is not stable and will decrease in the absence of drug challenge. Dosing animals harbouring resistant H contortus with either thiabendazole or mebendazole selects for nematodes producing thiabendazole resistant eggs. The eggs of a thiabendazole selected strain of H contortus were more resistant to thiabendazole, parbendazole and oxibendazole but less resistant to cambendazole and mebendazole.