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Visual deficits and retinotoxicity caused by the naturally occurring anthelmintics, Embelia ribes and Hagenia abyssinica.

The naturally occurring anthelmintics, Embelia ribes (Enkoko) and Hagenia abyssinica (Kosso), have been reported to possibly cause optic atrophy among the Ethiopian population. In this study we found retinal pathology and defects in visual behavior in chicks treated with Enkoko, Kosso, or embelin, a crystalline extract of E. ribes. The chicks were fed orally with a high dose of 0.25 g (5 g/kg) or a low dose of 0.025 g (0.5 g/kg) per day of Enkoko or Kosso, beginning on Day 2 of life. The high dose for Enkoko was administered for 1 or 5 days, while that for Kosso was administered for 1 or 9 days. For the low dose of both Enkoko and Kosso, the dosing regime was for a period of 1, 4, or 9 days. Embelin was administered at a dose of 0.001 g (0.02 g/kg) per day for 9 days. Control chicks were force fed an equivalent amount of chick feed. Treatment with Enkoko or Kosso significantly reduced the ability of chicks to detect a moving bead introduced into the peripheral field of vision. The degree of constriction of the visual field for detection was dependent upon the total amount of drug administered. Performance on a visual discrimination task, which required discrimination of feed grains from pebbles, was also impaired in chicks treated with total doses of 0.200 and 0.250 g of Enkoko or Kosso. Thus, the extent of deficit in visually guided tasks was found to be dose dependent. The visual deficits observed in Enkoko-treated chicks were mimicked by embelin, which suggests that embelin may be responsible for the visual defects. Anatomical evidence of degeneration of ganglion cells was found in retinae exposed to high doses of Enkoko (1.25 g) and Kosso (2.25 g). However, no retinal lesions were detected in chicks following treatment with cumulative doses of less than 0.25 g of Enkoko or Kosso. Similarly, retinal pathology was not observed in embelin-exposed retinae.

Administration, Oral↗

Comparative neuromuscular blocking actions of levamisole and pyrantel-type anthelmintics on rat and gastrointestinal nematode somatic muscle.

The basis for the comparative toxicity to parasitic nematodes and their mammalian hosts of the anthelmintics levamisole, pyrantel, and several related analogs on somatic nicotinic cholinergic transmission was examined. Measurements of muscle contractility and membrane potential were made using the isolated hemidiaphragm preparation of the rat and isolated axial muscle segments from the gastrointestinal nematode Haemonchus contortus. Pyrantel caused a dose- and time-dependent reduction of nerve-evoked twitches in the rat diaphragm. These effects were exacerbated by increasing the frequency of phrenic nerve stimulation from 0.5 to 50 Hz. Levamisole was less potent and the onset of its effects slower than pyrantel. Neither drug significantly affected twitches evoked from d-tubocurarine-blocked preparations following direct stimulation of the diaphragm. Twitch depression was reversed by washing, but not by application of physostigmine. In H. contortus, both drugs stimulated a spastic contraction and sustained paralysis in the concentration range of 1-10 microM, mimicking the action of nicotine. Neither nicotinic nor muscarinic antagonists blocked these responses. Moreover, neither nicotinic antagonists nor muscarinic agonists or antagonists had any independent effect on contractility of the parasite muscle segments. The blocking actions of levamisole and pyrantel on H. contortus axial muscle were associated with membrane depolarization at the muscle. In the rat-isolated hemidiaphragm, pyrantel, but not levamisole, depolarized end-plate regions of muscle fibers. d-Tubocurarine blocked the depolarizing action of pyrantel but not levamisole on rat-isolated hemidiaphragm. In axial muscle fibers of H. contortus, d-tubocurarine did not block the depolarizing actions of pyrantel, levamisole, or nicotine. 3-Bromo and 3-amino derivatives of levamisole were equipotent with and mimicked the actions of the parent compound on H. contortus axial muscle contractility. In the rat preparation, the 3-bromo derivative was more potent than levamisole or 3-amino-levamisole. 3-Amino-levamisole, but not 3-bromo-levamisole, depolarized muscle end-plate membrane in the rat diaphragm. Results of the present study are consistent with the following conclusions: (a) both levamisole and pyrantel block contractility of nematode axial muscle by causing sustained depolarization of the muscle membrane; (b) both drugs block neuromuscular transmission at the mammalian neuromuscular junction but their mechanisms appear to differ; (c) levamisole and pyrantel are more potent blockers of neuromuscular transmission in H. contortus than in the rat. These results suggest that potentially important pharmacological differences exist between nematode and mammalian somatic nicotinic receptors.

Animals↗

Mutagenicity of antiamebic and anthelmintic drugs in the Salmonella typhimurium microsomal test system.

4 amebicides (chloroquine diphosphate, diiodohydroxyquin, iodochlorohydroxyquin and dehydroemetine) and 6 anthelmintics (bephenium hydroxynaphthoate, 4-hexylresorcinol, mebendazole, niclosamide, pyrantel pamoate and pyrvinium pamoate) were tested for mutagenicity in the Salmonella typhimurium microsomal test system. Frameshift mutations were induced by dehydroemetine and niclosamide following activation by microsomal enzymes, while pyrvinium pamoate induced both frameshift and base-pair substitution mutations with or without metabolic activation. The urine of mice treated with dehydroemetine or pyrvinium pamoate showed no mutagenic activity. However, urine obtained from mice treated with niclosamide was mutagenic in strains TA98 and TA1538. The fluctuation assay showed chloroquine diphosphate to be mutagenic in TA1537, a strain which detects frameshift mutations.

Amebicides↗

Genotoxicity of amebicide and anthelmintic drugs in Escherichia coli pol A+/pol A-.

The amebicides dehydroemetine and chloroquine diphosphate and the anthelmintic pyrvinium pamoate, previously reported to be mutagenic in Salmonella typhimurium (Cortinas de Nava et al., 1983), were clearly shown to be genotoxic in the Escherichia coli pol A+/pol A- assay. Two other antiparasitic drugs, diiodohydroxyquin and 4-hexylresorcinol, were also found to be genotoxic in E. coli, while iodochlorhydroxyquin preferentially inhibited the pol A+ strain. From the 3 alternative testing methods employed, the liquid suspension succeeded in detecting 5 antiparasitic drugs as genotoxic; the microsuspension identified 2, and the disc diffusion method only 1. However, the metabolic activation system could only be coupled successfully and in a reproducible way to the microsuspension assay.

Amebicides↗

A comparison of the genetic activity of pyrvinium pamoate with that of several other anthelmintic drugs in Saccharomyces cerevisiae.

Several anthelmintic drugs that are used routinely in oxyuriasis therapy were analyzed for genotoxicity in a diploid mitotic recombination and gene conversion assay (strain D5 of Saccharomyces cerevisiae), and in a haploid yeast reversion assay (strain XV185-14C). Piperazine citrate, piperazine adipate, mebendazole and thiabendazole did not appear to be genotoxic in either yeast strain. Pyrvinium pamoate induced the reversion of the missense, nonsense and frameshift alleles in strain XV185-14C, whereas pyrantel pamoate induced only the reversion of the frameshift allele. Pyrvinium pamoate was recombinogenic in strain D5, and there is an indication that pyrantel pamoate, at the lowest dose that was tested, might induce gene conversion or aneuploidy.

Anthelmintics↗

Energy metabolism in Cotugnia digonopora and the effect of anthelmintics.

Incorporation of 32Pi into organic phosphate by mitochondria of Cotugnia digonopora was supported maximally by malate. Fumarate and succinate induced lower but significant production of ATP. Pyruvate, alpha-ketoglutarate and oxalacetate proved to be poor substrates and citrate and isocitrate had no effect. A net phosphorylation of approximately 2 mol of ADP was observed for each mol of CO2 liberated from malate or succinate. In contrast, with pyruvate, in spite of a high rate of decarboxylation, the production of ATP was extremely low. 2,4-Dinitrophenol inhibited phosphorylation. All anthelmintics examined interfered with the mitochondrial phosphorylation of ADP, with maximum inhibition by salicylanilide compounds. The anticestodal activity of the latter group of compounds, niclosamide for example, may, therefore, be attributed to their ability to inhibit mitochondrial phosphorylation.

Animals↗

Purification of cytosolic glutathione transferases from Schistocephalus solidus (plerocercoid): interaction with anthelmintics and products of lipid peroxidation.

Glutathione (GSH) transferase isoenzymes have been partially resolved from the cytosol of Schistocephalus solidus (plerocercoid) by GSH affinity chromatography and chromatofocusing at pH 7-5. The presence of isomeric forms was also suggested by analytical isoelectric focusing and high-performance liquid chromatography (HPLC). Gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that GSH transferase forms were dimers with a subunit size of approximately 24 kDa. The major GSH transferase form in S. solidus (plerocercoid) showed greater biochemical relationship to the Mu family of mammalian GSH transferase compared to the mammalian Alpha or Pi families. The major subunit purified by GSH affinity chromatography and reversed-phase HPLC also showed high N-terminal homology with the Mu family. A minor GSH transferase form appeared more biochemically related to the Alpha family with respect to substrate specificity and inhibitor sensitivity. The major GSH transferase was inhibited by haematin-related compounds, bile acids and a number of anthelmintics including members of the benzimidazole and phenol-based class of compounds. The major GSH transferase had conjugating activity with members of the trans, trans-2,4-alkadienal and trans-2-alkenal series, secondary products of lipid peroxidation.

Amino Acid Sequence↗

Specific interaction of benzimidazole anthelmintics with tubulin: high-affinity binding and benzimidazole resistance in Haemonchus contortus.

The specific (high-affinity) binding of tritiated benzimidazole [(3H]BZ) anthelmintics-mebendazole [(3H]MBZ) and oxibendazole [(3H]OBZ) to, and the specific displacement (inhibition) of these radioligands by unlabelled BZs (oxibendazole, mebendazole, oxfendazole, albendazole, fenbendazole and thiabendazole) from crude tubulin extracts prepared from thiabendazole (TBZ)-susceptible (S) or TBZ-resistant (R) strains of adult Haemonchus contortus, have been examined. The most striking difference between R and S was that the drug specifically bound at infinite ligand concentration (Bmax), was markedly reduced for the R strain, with no apparent change in association constant (Ka). Thus, resistance was associated with a loss of high-affinity receptors. TBZ-resistance was not associated with a change in low-affinity binding. There was a greater loss of high affinity receptors for [3H]OBZ than for [3H]MBZ. Using the displacement data. BZs were ranked according to their Ka and IC50 (concentration of BZ inhibiting 50% of radioligand binding) values. The Ka and IC50 values and the rank order of the BZs were approximately independent of the radioligand displaced or source (S or R) of the tubulin extracts used. The results are consistent with tubulin binding being the primary mechanism of action for all of these BZs.

Animals↗

In vitro activity of various anthelmintic compounds against Haemonchus contortus larvae.

Twenty-five known anthelmintic compounds were evaluated in vitro against the highly motile exsheathed non-feeding third-stage of Haemonchus contortus larvae. Activity was based on lack of motility or death of larvae after 24 h of chemical exposure. Six compounds (avermectins, closantel, levamisole, morantel, phenylhydrazone and ticarbodine) were active at a concentration of 100 micrograms cm-3 or less. The most active compounds were avermectins and levamisole. When higher in vitro concentrations were used, ten compounds (bephenium, coumaphos, dichlorovos, disophenol, hygromycin b, methyridine, parbendazole, phenothiazine, pyrantel and thiabendazole) exhibited activity. Nine compounds were found to be inactive; among these were the new benzimidazoles, i.e., albendazole, fenbendazole, mebendazole and oxibendazole. Because of the inactivity of the new benzimidazoles, this in vitro system is unsuitable as a routine screening tool. Also, the system appears to favor drugs that act quickly through percuticular entry. In an initial group of 5280 untested compounds, 254 (4.8%) exhibited in vitro activity at 100 micrograms cm-3 against the non-feeding larvae stage. The exogenous and in vitro cultivation techniques required for collecting, cleaning and exsheathing the larvae are described.

Animals↗

The efficacy of benzimidazole anthelmintics against late fourth stage larvae of Trichostrongylus colubriformis in gerbils and Nippostrongylus brasiliensis in rats.

The efficacy of eight benzimidazole anthelmintics has been examined against Trichostrongylus colubriformis in gerbils and Nippostrongylus brasiliensis in rats, when the parasites were entering into the fourth and final moult. The dose--response slopes obtained from the data for both host--parasite systems did not deviate significantly from a non-parallel model, and were used for relative potency determinations. The T. colubriformis assay ranked the benzimidazoles in order of potency as follows: albendazole, oxfendazole, fenbendazole, cambendazole, mebendazole, oxibendazole, parbendazole and thiabendazole. This compares favourably with the expected relative efficacies against trichostrongyles in sheep. N. brasiliensis was found to be far less useful in this respect. All compounds tested, with the exception of thiabendazole, were highly effective against both parasites. The T. colubriformis/gerbil assay could be a very useful tool for preliminary in vivo evaluation and possibly in the early selective optimisation of chemical series.

Animals↗

The anthelmintic efficacy of netobimin against experimental infections of Fasciola hepatica in sheep.

Netobimin (coded SCH 32481, Schering Corporation), a new broad-spectrum anthelmintic having both fasciolicidal and nematocidal properties was evaluated for efficacy against mature Fasciola hepatica infections in sheep. The trial was conducted with 30 cross-bred spring lambs, each experimentally infected with 250 F. hepatica metacercariae. A single treatment of netobimin was administered at 17 weeks post-infection (PI) by oral drench at 7.5 or 20 mg kg-1 body weight while 10 animals remained as untreated controls. At necropsy (either 1 or 2 weeks post-treatment), the mean number of adult flukes recovered from the control, 7.5 and 20 mg kg-1 groups were 94.7, 35.9 and 8.8, respectively. The resulting efficacies were 62% (P less than or equal to 0.05) and 90.7% (P less than or equal to 0.01), respectively. No clinical signs of fascioliasis were noted in any sheep during the trial. No signs of toxicosis nor any adverse reactions to the drug were observed.

Animals↗

Anthelmintic efficacy of netobimin against naturally acquired gastrointestinal nematodes in yearling heifers.

The efficacy of netobimin (Coded SCH 32481, Schering Corporation) in removing naturally acquired gastrointestinal nematode infections was evaluated in 10 treated and 10 untreated (control) yearling beef heifers. The anthelmintic was administered as an oral drench at a dose level of 7.5 mg kg-1 body weight. Fecal egg per gram (EPG) counts were reduced with netobimin by 98% (P less than or equal to 0.01) at both 1 and 2 weeks post-treatment (PT). The compound was highly effective in removing Oesophagostomum radiatum (100% at P less than or equal to 0.01), Cooperia spp. (97.66% at P less than or equal to 0.01) and Nematodirus helvetianus (100%, although not significant), but was ineffective against immature Ostertagia ostertagi (3.19%) and only moderately effective against adult forms (66.14% at P less than or equal to 0.05). The low efficacy against adult O. ostertagi was partially attributed to the maturation of fourth-stage larvae, not removed by the 7.5 mg kg-1 treatment, during the 14-day treatment-slaughter interval. Efficacy against Trichuris spp. could not be evaluated due to low levels and unequal numbers of worms recovered in the groups. The overall efficacy against adult species, excluding Trichuris spp., was 67.40% (P less than or equal to 0.05). No adverse reactions or signs of toxicosis were observed in heifers treated with netobimin.

Administration, Oral↗

The anthelmintic efficacy of netobimin against naturally acquired gastrointestinal nematodes in sheep.

The broad-spectrum anthelmintic efficacy of netobimin (SCH 32481, Schering Corporation) was evaluated using 30 cross-bred spring lambs with naturally acquired infections of gastrointestinal nematodes. Three groups of 10 animals each were allotted into either control (given a tap water drench as a placebo) or 7.5 and 20 mg kg-1 dosage groups (given the netobimin as an oral drench). Seven to fourteen days post-treatment, animals were necropsied and nematodes recovered by standard techniques. Examination of fecal samples taken on dates of necropsy showed median egg production was reduced in treated animals (61.98% with 7.5 mg kg-1 and 100% with 20 mg kg-1). The compound was highly effective in removal of adult nematodes representing a number of genera and species of trichostrongyloids at the 7.5 and 20 mg kg-1 dose levels (shown, respectively, below). These included Ostertagia spp., with O. circumcincta, O. trifurcata, O. ostertagi and Teladorsagia davtiani (96.20%; 100%), Trichostrongylus spp., with T. axei, T. vitrinus and T. colubriformis (100%; 98.72%), Nematodirus spp., with N. spathiger, N. filicollis and N. battus (100% both levels) and Haemonchus contortus (100% both levels). High efficacies against other species of nematodes (at both dose levels) were not statistically significant (Cooperia spp., Chabertia ovina and Oesophagostomum venulosum). At 20 mg kg-1, netobimin significantly reduced populations of early and late fourth stage larvae of Ostertagia spp. by 100%. The overall efficacy (all life stages included) was 90.16% at 7.5 mg kg-1 and 98.77% at 20 mg kg-1 dose levels. No adverse reactions or signs of toxicosis were observed.

Administration, Oral↗

Economics of a dry-cow anthelmintic drenching programme for dairy cows in New Zealand. Part 2. Influence of management factors and other herd characteristics on the level of response.

The results of a series of trials conducted on 47 seasonal supply dairy farms in the southern North Island of New Zealand, which indicated a statistically significant overall production response of 2.24 kg milkfat/cow/lactation following a 2-treatment dry-cow anthelmintic drenching programme (Bisset et al., 1987), were subjected to further analyses in an attempt to identify any management factors or herd characteristics which may have influenced the levels of production response to treatment in individual herds. The management factors examined included calf drenching practices, types of grazing system, mean pre-calving condition of the herds, and 'nutritional status' of the herds over the milking season. The influence of herd quality, herd size and geographical location were also examined. Considered separately, only calf drenching practice had a significant influence on the level of herd response to treatment. Increases in milkfat production/cow due to the treatment programme were significantly greater (P less than 0.05, t-test) on farms where calves had received a minimal number of drench treatments (less than or equal to 2) between weaning and the end of March (mid-autumn) (+5.32 kg/cow/lactation, P less than 0.01) (= 108.2 l milk), than on farms where calves had received regular 3-4 weekly treatments over the same period (+0.42 kg/cow/lactation, N.S.) While none of the other factors, considered in isolation, appeared to significantly influence the production response of herds, observations on possible interactive effects suggested that the grazing system employed was probably of importance in so far as it determined the level of exposure of cows to calf-contaminated pasture. Thus, the greatest mean response to the treatment programme occurred in herds where cows were overwintered on areas grazed during the milking season by calves which had received less than or equal to 2 drench treatments from weaning until the end of March (+5.95 kg milkfat/cow/lactation, P less than 0.01).

Analysis of Variance↗

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of equine anthelmintics.

These guidelines have been designed to assist in the planning, operation and interpretation of studies which would serve to assess the efficacy of drugs against internal parasites of horses. Although the term anthelmintic is used in the title and text, these guidelines include studies on drug efficacy against larvae of horse bot flies, Gasterophilus spp, which are non-helminth parasites commonly occurring in the stomach of horses. The advantages, disadvantages and application of critical and controlled tests are presented. Information is also provided on selection of animals, housing, feed, dose titration, confirmatory and clinical trials, record keeping and necropsy procedures. These guidelines should assist both investigators and registration authorities in the evaluation of compounds using comparable and standard procedures with the minimum number of animals.

Animals↗

A field evaluation of anthelmintics in horses in Sweden.

A field evaluation of anthelmintics in 336 horses on 37 farms was conducted between February and May 1986 in Sweden. The herds, each comprising at least eight horses, had histories of grazing on permanent pastures and receiving regular treatments against parasites at least three times a year. Small strongyles were refractory to (pro)-benzimidazole drugs in all but one of 23 herds examined. There was an average reduction in egg output of approximately 60%, and approximately 30% of 205 horses examined were shedding less than 100 eggs g-1 7 days after treatment. There was great between-herd variation of both the faecal egg-count depression (6.4-90.4%) and drug efficacy (0.0-80.0%). The non-benzimidazole drugs under investigation were fully effective against mature small strongyles.

Animals↗

A new anthelmintic assay using rats infected with Trichostrongylus colubriformis.

A new anthelmintic assay is described which uses immunosuppressed (60 ppm hydrocortisone acetate in diet) rats infected with the nematode Trichostrongylus colubriformis. Immunosuppressed rats were infected with 1500 T. colubriformis larvae, treated either orally or subcutaneously on Day 14 post-infection and necropsied 4 days after treatment. The worm counts in immunosuppressed control animals averaged 775 worms per rat. A range of benzimidazoles, levamisole hydrochloride, morantel tartrate, 22,23-dihydroavermectin B1a and alpha-milbemycin have been evaluated in the assay. The ED95 values obtained indicate that rats infected with T. colubriformis provide a highly predictive model for assaying the activity of experimental drugs in vivo prior to studies in ruminants.

Animals↗

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance.

Methods have been described to assist in the detection of anthelmintic resistance in strongylid nematodes of ruminants, horses and pigs. Two tests are recommended, an in vivo test, the faecal egg count reduction test for use in infected animals, and an in vitro test, the egg hatch test for detection of benzimidazole resistance in nematodes that hatch shortly after embryonation. Anaerobic storage for submission of faecal samples from the field for use in the in vitro test is of value and the procedure is described. The tests should enable comparable data to be obtained in surveys in all parts of the world.

Animals↗