Genetic variability of the beta-tubulin genes in benzimidazole-susceptible and -resistant strains of Haemonchus contortus.

Benzimidazole anthelmintics are the most common chemotherapeutic agents used to remove intestinal helminths from farm animals. The development of drug resistance within helminth populations is wide-spread and can render these drugs essentially useless. The mechanism of benzimidazole resistance appears to be common to many species ranging from fungi to nematodes and involves alterations in the genes encoding beta-tubulin. During the selection process resulting in resistance, there must be quantitative changes in the population gene pool. Knowledge of these changes would indicate the mechanisms underlying the spread of resistance in the population, which in turn could be used to design more effective drug administration strategies. To this end we have identified allelic variation at two beta-tubulin genes in Haemonchus contortus using restriction map analysis of individual adults. Extremely high levels of variation were identified at both loci within a susceptible strain. In two independently derived benzimidazole resistant strains, allele frequencies at both loci were significantly different from the susceptible strain but not from each other. The same alleles at both loci, in both resistant strains, were favored by selection with benzimidazoles, suggesting that both loci are involved in determining benzimidazole resistance. These data confirm that changes in allele frequency, rather than novel genetic rearrangements induced by exposure to the drug, explain the changes associated with benzimidazole resistance. These results also show that any DNA based test for the development of benzimidazole resistance must take into account the frequency of alleles present in the population and not simply test for the presence or absence of specific allelic types.

Observations on the fumarate reductase system in Haemonchus contortus and their relevance to anthelmintic resistance and to strain variations of energy metabolism.

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.

Haemonchus contortus: the role of two beta-tubulin gene subfamilies in the resistance to benzimidazole anthelmintics.

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.

Control of cambendazole-resistant small strongyles (Population S) with oxibendazole in a pony band: an 8 year field test (1984-1992).

Studies in a band of ponies harboring Population S benzimidazole-resistant small strongyles were initiated in 1974 and have continued for 18 years. Treatment (bimonthly) was with cambendazole for the first 4 years and with oxibendazole (OBZ) for the next 14 years. Data on the first 10 years have been published. The present investigation includes the last 8 years (4 October 1984-11 September 1992), which are the seventh through fourteenth years, of treatment with OBZ. Pre- and posttreatment mean counts of strongyle eggs (epg) and larvae (lpg) per gram of feces were determined biweekly during the current study to monitor the efficacy of OBZ. The average annual percent reductions of epg counts effected by OBZ treatments were 51%, 53%, 38%, 38%, 39%, 28%, 40%, and 19% for the seventh through fourteenth years, respectively. Similar levels of reductions were observed for lpg counts. Although OBZ was initially highly effective on this population of small strongyles, epg and lpg counts gradually declined, but have remained more or less constant since the fifth year of research. However, reductions of the counts were the lowest for the last year of the study.

Motility response of levamisole/benzimidazole-resistant Haemonchus contortus larvae.

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.

A comparison of the effect of albendazole, cambendazole, and thiabendazole on the larval development of three hymenolepidid cestodes.

Flour beetles (Tribolium confusum) parasitized either by Hymenolepis diminuta, H. nana, or H. microstoma were fed continuously on flour mixed either with thiabendazole, cambendazole, or albendazole (drug concentration was always 10%) from day 1 (24 hr) to day 10 postinfection when the experiments were terminated. All drugs markedly inhibited the development of H. diminuta and H. nana. Populations of these species recovered from beetles fed anthelmintics were composed mostly of under-developed forms, many of which still retained the size and appearance of newly hatched oncospheres, whereas all the parasites recovered from the control beetles (fed only flour) reached full development. Parasites inhibited by cambendazole and albendazole recovered and reached full development within 9 days after treatment was terminated. Also, results were obtained which implied that some parasites were able to continue their development at a reduced rate in the presence of the drugs. Hymenolepis microstoma differed from the other species in its response to the drugs. Albendazole and thiabendazole had no effect on its development and it was only slightly inhibited by cambendazole. Larvae recovered from beetles fed the latter drug had all developed beyond the oncosphere stage but 3 to 5% of them repeatedly failed to reach full development. The drugs varied in their effects on the flour beetles. An average of 63% and 33% of those fed thiabendazole and cambendazole, respectively, died before the 10th day of infection. Albendazole, on the other hand, had no effect on beetle survival.

Biochemical effects of thiabendazole and cambendazole on Hymenolepis diminuta (Cestoda) in vivo.

An investigation of the chemotherapeutic and biochemical effects of two benzimidazole anthelmintics, thiabendazole (TBZ) and cambendazole (CBZ), on Hymenolepis diminuta in experimentally infected rats is reported. Thiabendazole was active against H. diminuta at a relatively high dosage. A single oral dose of TBZ at 250 mg/kg body weight on day 15 of infection eliminated 100% of the tapeworms as determined at necropsy 5 days after treatment. The chemotherapeutic actions of TBZ on H. diminuta were accompanied by marked changes in worm weight and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of TBZ 24 hr earlier were significantly smaller and contained much less glycogen (as a percent of the wet weight) than worms from unmedicated controls. Protein concentrations increased in TBZ-treated worms and at a rate sufficient to offset the decline in glycogen concentration. Glycogen/protein ratios in TBZ-treated worms were significantly lower than the corresponding control values. Cambendazole proved to be five times more potent than TBZ against H. diminuta and produced the same basic changes in worm weight and chemical composition within 18 hr of treatment of the host. Administration of a single oral dose of TBZ or CBZ to the host produced in H. diminuta another change, the onset of which coincided with, or preceded, the gross alterations in worm weight and chemical composition. That change, observed in in vitro studies carried out 14 hr after treatment, revealed that tapeworms from drug-treated rats absorbed and metabolized much smaller quantities of exogenous glucose than did the controls, and the ability of the worm to accumulate glucose against a concentration difference was significantly depressed.

Glucose metabolism of Haemonchus contortus adults: effects of thiabendazole on susceptible versus resistant strain.

Analyses of the in vitro glucose metabolism of Haemonchus contortus adults have confirmed a complex series of end-products: ethanol, n-propanol, propionate, acetate, and CO2 as the major end-products of catabolism. No difference in end-product accumulation was seen between the cambendazole sensitive (BPL) and cambendazole resistant (CR) strains. Thiabendazole (5 mM) in vitro depressed ethanol, propanol, acetate, and propionate accumulation by approximately 42% in the BPL strain. However, in the resistant strain (CR), these end-product accumulations increased by 50% when the worms were exposed to drug in vitro and 80% when exposed in vivo. Resistance manifested by the CR strain appeared to be associated with the ability to increase its carbon flow in the presence of thiabendazole.

The Hymenolepis diminuta-golden hamster (Mesocricetus auratus) model for the evaluation of gastrointestinal anticestode activity.

A novel laboratory anticestode assay was developed using Hymenolepis diminuta in the hamster. The commercial anticestode compounds, praziquantel, bunamidine, and niclosamide were active against patent infections of Hymenolepis diminuta in golden hamsters (Mesocricetus auratus) when given orally at 3.125, 100, and 200 mg/kg, respectively. The gastrointestinal nematode anthelmintics, cambendazole and mebendazole, were active at 50 mg/kg. Rafoxanide (fasciolicide) was active at 25 mg/kg, the lowest level tested. The coccidiostat, nicarbazin, was active at experimental levels (800 mg/kg and up). The anthelmintic-ectoparasiticide (endectocide), ivermectin, was inactive against the tapeworm at 0.5 mg/kg, as expected.

Colorimetric assay for the detection of benzimidazole resistance in trichostrongyles.

A modified version of the aphid tile-test, which is used to detect insecticide resistance in single adult aphids that are resistant or susceptible to organophosphate or carbamate insecticides, was used to compare the levels of non-specific esterases in strains of the trichostrongyle nematodes Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis which were known to be resistant or susceptible to benzimidazole (BZ) anthelmintics. This colorimetric assay has shown that there is significantly more non-specific esterase in the infective-stage larvae of BZ-resistant strains than in susceptible strains and this may prove to be of use in the detection of resistance to benzimidazole anthelmintics.

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.

The dose response of several benzimidazole anthelmintics against resistant strains of Haemonchus contortus and Trichostrongylus colubriformis selected with thiabendazole.

Dose response lines for eight benzimidazole anthelmintics and thiophanate were determined, using standardised strains of thiabendazole selected and resistant Haemonchus contortus and Trichostrongylus colubriformis. Against H contortus, thiophanate, thiabendazole, parbendazole and oxibendazole were inactive. Mebendazole was inactive at dose rates of 6.26 and 12.5 mg/kg, although significant activity occurred at 25 mg/kg. Fenbendazole, cambendazole, oxfendazole and albendazole demonstrated significant activity at dose rates equal to or greater than the recommended therapeutic level. Thiophanate was inactive against resistant T colubriformis. The remaining compounds only showed significant activity when used at dose rates in excess of the recommended therapeutic level. These results show that a side resistance exists among the benzimidazole anthelmintics and suggests that changes in dose response lines could be expected to occur if resistant strains are selected with benzimidazoles other than thiabendazole.

Larval development test for detection of anthelmintic resistant nematodes.

The growth, using freshly cultured Escherichia coli with ampicillin or heat-treated lyophilised E coli as a food source, of the larvae of the mouse nematode Nematospiroides dubius and the infectivity of resulting third stage larvae were determined. Concentrations of E coli between 0.5 and 1 mg dry weight ml-1 permitted optimal larval development for both N dubius and Trichostrongylus colubriformis. Development of larvae of susceptible and cambendazole-resistant strains of Haemonchus contortus in thiabendazole solutions showed clear differences between the strains and the larval development test was more sensitive than the egg hatch test. The test also detected a levamisole resistant strain of H contortus, although the degree of resistance could not be adequately measured. It is concluded that the test can be run with any anthelmintic to which resistance is suspected.

Inheritance of cambendazole resistance in Haemonchus contortus.

Fourth-molt larvae (M4) of cambendazole-resistant (CBZ-R) and cambendazole-susceptible (BPL) strains of Haemonchus contortus were recovered from donor lambs and inoculated orally into source lambs to effect mating in 2nd lamb. Eggs produced by these parasitic matings were collected, cultured, and inoculated into lambs to compare the anthelmintic activity of cambendazole (CBZ), given at a dose rate of 20 mg/kg, against the F1 adults with its activity against the 2 parenteral strains (CBZ-R and PBL). In 2 replications, CBZ was 94% and 99% efficacious against the progeny of the cross matings and 91% and 95% against the BPL strain; CBZ was only 39% and 42% efficacious against the CBZ-R strain. In 1 test of F2 progeny of the reciprocal matings, CBZ was 81.2% efficacious against the F2 adults, whereas CBZ was 97.5% efficacious against the BPL strain and 37% against the CBZ-R strain. The results indicated that heredity of resistance to CBZ in H contortus is not sex-linked and is probably a result of a heterozygous recessive allele. The infective larvae of the CBZ-R groups in these 3 trials were from the 20th and 24th passages through lambs without exposure to any anthelmintics, yet the anthelmintic activity of CBZ ranged from 37% to 42%, indicating that there had been no reversion to susceptibility.

Resistance of population-B equine strongyles to thiabendazole, oxfendazole, and phenothiazine (1981 to 1987).

Critical tests were completed on foals (n = 15) naturally infected with benzimidazole (BZ)-resistant population-B strongyles during the period between 1981 and 1987. Thiabendazole at a dosage of 44 mg/kg was tested in 8 foals, oxfendazole at 10 mg/kg was tested in 4 foals, and phenothiazine at 55 mg/kg, cambendazole at 20 mg/kg, and fenbendazole at 5 mg/kg were tested in 1 foal each. Efficacies of thiabendazole, cambendazole, and fenbendazole against the 5 species of BZ-resistant small strongyles (Cyathostomum catinatum, Cyathostomum coronatum, Cylicocylus nassatus, Cylicostephanus goldi, and Cylicostephanus longibursatus) were partial. Removals in the aggregate averaged 48%, 52%, and 68%, respectively, indicating continued resistance of the 5 species of small strongyles to thiabendazole, cambendazole, and fenbendazole. In contrast, oxfendazole was efficacious, removing an aggregate average of 95% of the 5 BZ-resistant species of small strongyles in the 4 foals. This apparent reversion to susceptibility to oxfendazole may be attributable to paucity of reference data (1 test foal) done in 1976. All 5 species of the BZ-resistant small strongyles had cross resistance to phenothiazine (38% aggregate removal), whereas 9 of the BZ-susceptible species were effectively (99%) removed. This suggests a similar mode of action between phenothiaznie and the BZ compounds.

Cambendazole for strongyle control in a pony band: selection of a drug-resistant population of small strongyles and teratologic implications.

Cambendazole (CBZ) treatments (20 mg/kg) given at 8-week intervals were used for parasite control in a breeding band of ponies (n = 33 to 43) during the period July 1974 to August 1978. Pre- and posttreatment worm egg counts on feces were used to evaluate efficacy of treatments after every 2nd treatment interval by monitoring changes in strongyle egg counts. Initially, effective reductions (expressed as 92% to 96% fewer worm eggs) were lessened to 70% by the 9th treatment (18 months) and to 28% by the 12th treatment, and thereafter, they fluctuated between 0% and 38%. Critical tests on animals culled during the 3rd year revealed that (i) large strongyles (Strongylus vulgaris and S edentatus) were efficaciously removed, (ii) 5 species of small strongyles (Cyathostomum catinatus, Cyathostomum coronatum, Cylicocyclus nassatus, Cylicostephanus longibursatus, and Cylicostephanus minutus) exhibited some degree of resistance, and (iii) 11 species of small strongyles from 6 genera, including the 3 already named, were highly susceptible. Cylicostephanus minutus, in this population exhibited benzimidazole resistance for the first time, whereas Cylicostephanus goldi, previously recognized as resistant in other populations (B and C) in Kentucky, was susceptible to CBZ. A 2-fold increase in the drug-dose rate did not increase efficacy against the CBZ-resistant species of small strongyles, but a 4-fold increase small stronglyles, efficaciously removed 93% of the resistant small strongyles. Foaling rates in the brood band ranged between 38% and 61% for the 5 seasons. Three deformed foals occurred--1 in 1976 and 2 in 1977--among the total of 83 births. Present data did not evidence a probable week of early gestation for possible insult of the fetus by CBZ treatment.

Detection of activity for various anthelmintics against in vitro-produced Cooperia punctata.

The in vitro-grown parasitic stages of Cooperia punctata were used to evaluate 28 compounds with different kinds and degrees of in vivo activity. Using presumptive and confirmatory tests, it was possible to establish a group order of in vitro potency that compared favorably with an order based on established in vivo use of these compounds. The procedure lends itself to evaluating activity against a given parasitic growth stage and gives a quantitative estimate (range) of the concentration that produces 50% nematode kill. The system was most successful in detecting compounds with in vivo activity for C punctata, followed in order by compounds active against Cooperia spp, other gastrointestinal nematodes of ruminants, and other nematodes of non-bovine hosts. The procedure showed some differentiation between activity against nematodes versus that against cestodes, trematodes, and arthropods. The system permits considerable flexibility in experimental design, thus making possible the acquisition of the particular information desired. In addition to establishing lethal effects on the nematode, the procedure detected compounds with nematode-anesthetizing effects. The results indicate this in vitro system can be used with some expediency as a preliminary screening method in the search for new anthelmintic compounds.

Relationship between the antitrichinellous effect of seven derivates of benzimidazolecarbamates and their chemical structure.

The efficacy of seven more recently developed anthelmintics of the benzimidazolecarbamate group, i. e., parbendazole, mebendazole, fenbendazole, oxibendazole, cambendazole, oxfendazole and albendazole, has been tested in a series of experiments on white mice artifically infected with Trichinella spiralis. Our results disclosed a relationship between their anthelmintic effect and their chemical structure. This finding might be of importance in a targeted synthesis of new, effective, derivates of benzimidazole, e. g., in the therapy of trichinellosis and in the choice of the most effective drug.

Reproductive function in stallions treated with cambendazole.

Twenty stallions were used to determine if the anthelmintic cambendazole affected seminal characteristics, spermatogenesis, concentration of testosterone in blood serum, or weights of the reproductive orgrans. With minor exceptions, significant differences were not found between values for 10 control and 10 stallions treated with a single oral dose (40 mg/kg body weight) of cambendazole. The treatment did not affect reproductive function and it was concluded that the drug should have no effect on the fertility of stallions.

The prevalence of anthelmintic resistance of horse strongyles in The Netherlands.

A survey to determine the prevalence of anthelmintic resistance of horse strongyles was carried out with 616 horses on 22 farms. The tested drugs were cambendazole, pyrantel pamaote and ivermectin. Based on egg count reduction tests the efficacy of cambendazole varied from 0% to 93% and of pyrantel from 93% to 100%. Ivermectin treatments were 100% effective on all farms. Larval cultures after cambendazole treatments revealed exclusively cyathostome larvae. After pyrantel treatments besides cyathostome larvae other types of larvae were also found. After ivermectin treatments only a few cyathostome larvae were observed. It can be concluded that benzimidazoleresistance of cyathostomes is very common and widespread in the Netherlands. This means that benzimidazoles and pro-benzimidazoles should be excluded for strategic use in horses.