In vitro efficacies of solubility-improved mebendazole derivatives against Echinococcus multilocularis.

Recently, we introduced an epoxy group to mebendazole by a reaction with epichlorohydrin and obtained two isoforms, mebendazole C1 (M-C1) and mebendazole C2 (M-C2). The in vitro effects of mebendazole derivatives at different concentrations on Echinococcus multilocularis protoscoleces and metacestodes as well as cytotoxicity in rat hepatoma (RH) cells were examined. The results demonstrated that the solubility of the two derivatives was greatly improved compared to mebendazole. The mortality of protoscoleces in vitro reached to 70-80% after 7 days of exposure to mebendazole or M-C2, and M-C2 showed higher parasiticidal effects than mebendazole (P > 0.05). The parasiticidal effect of M-C1 was low, even at a concentration of 30 µm. The percentage of damaged metacestodes that were treated with mebendazole and M-C2 in vitro at different concentrations were similar, and M-C1 exhibited insignificant effects on metacestodes. Significant morphological changes on protoscoleces and metacestodes were observed after treatment with mebendazole and M-C2. In addition, the introduction of an epoxy group to mebendazole also reduced its cytotoxicity in RH cells. Our results demonstrate that the introduction of an epoxy group not only improved the solubility of mebendazole, but also increased its parasiticidal effects on E. multilocularis and reduced its cytotoxicity in RH cells.

Biotransformation of flubendazole and fenbendazole and their effects in the ribwort plantain (Plantago lanceolata).

Although veterinary anthelmintics represent an important source of environmental pollution, the fate of anthelmintics and their effects in plants has not yet been studied sufficiently. The aim of our work was to identify metabolic pathways of the two benzimidazole anthelmintics fenbendazole (FBZ) and flubendazole (FLU) in the ribwort plantain (Plantago lanceolata L.). Plants cultivated as in vitro regenerants were used for this purpose. The effects of anthelmintics and their biotransformation products on plant oxidative stress parameters were also studied. The obtained results showed that the enzymatic system of the ribwort plantain was able to uptake FLU and FBZ, translocate them in leaves and transform them into several metabolites, particularly glycosides. Overall, 12 FLU and 22 FBZ metabolites were identified in the root, leaf base and leaf top of the plant. Concerning the effects of FLU and FBZ, both anthelmintics in the ribwort plantain cells caused significant increase of proline concentration (up to twice), a well-known stress marker, and significant decrease of superoxide dismutase activity (by 50%). In addition, the activities of four other antioxidant enzymes were significantly changed after either FLU or FBZ exposition. This could indicate a certain risk of oxidative damage in plants influenced by anthelmintics, particularly when they are under other stress conditions.

Flubendazole induces mitotic catastrophe and apoptosis in melanoma cells.

Flubendazole (FLU) is a widely used anthelmintic drug belonging to benzimidazole group. Recently, several studies have been published demonstrating its potential to inhibit growth of various tumor cells including those derived from colorectal cancer, breast cancer or leukemia via several mechanisms. In the present study we have investigated cytotoxic effects of FLU on malignant melanoma using A-375, BOWES and RPMI-7951 cell lines representing diverse melanoma molecular types. In all three cell lines, FLU inhibited cell growth and proliferation and disrupted microtubule structure and function which was accompanied by dramatic changes in cellular morphology. In addition, FLU-treated cells accumulated at the G2/M phase of cell cycle and displayed the features of mitotic catastrophe characterized by formation of giant cells with multiple nuclei, abnormal spindles and subsequent apoptotic demise. Although this endpoint was observed in all treated melanoma lines, our analyses showed different activated biochemical signaling in particular cells, thus suggesting a promising treatment potential of FLU in malignant melanoma warranting its further testing.

Mixture toxicity of flubendazole and fenbendazole to Daphnia magna.

Nowadays, residual amounts of many pharmaceuticals can be found in various environmental compartments including surface and ground waters, soils and sediments as well as biota. Even though they undergo degradability, their environmental discharge is relatively continuous, thus they may be regarded as quasi-persistent contaminants, and are also frequently regarded as emerging organic pollutants. Benzimidazoles, especially flubendazole (FLU) and fenbendazole (FEN), represent two anthelmintic drugs belonging to this group. Although their presence in environmental matrices has been reported, there is relatively little data concerning their (eco)toxicological impact. Furthermore, no data is available on their mixture toxicity. FLU and FEN have been found to have a strong impact on an environmentally important non-target organism - Daphnia magna. Moreover, these compounds are usually present in the environment as a part of pharmaceutical mixtures. Therefore, there is a need to evaluate their mixture toxicity, which was the main aim of this study. Single substance toxicity tests were carried out in parallel with mixture studies of FLU and FEN, with the application of two well established concepts of Concentration Addition (CA) and Independent Action (IA). As a result, both models (CA and IA) were found to underestimate the toxicity of mixtures, however CA yielded more accurate predictions.

Safety of Mebendazole Use During Lactation: A Case Series Report.

INTRODUCTION: Mebendazole is an effective drug widely used in the treatment of parasitic infections. Although theoretically considered as safe during lactation, no studies have evaluated its potential adverse effects in infants of breastfeeding mothers. OBJECTIVES: We aimed to evaluate the safety of mebendazole in infants of lactating women treated with the drug. METHODS: Women referred for consultation regarding mebendazole use were invited to participate in the study. Overall 45 lactating women treated with various protocols of mebendazole were recruited in this case series study. RESULTS: Regardless of the treatment protocol used (single or repeated doses) mebendazole was well tolerated and was not associated with any adverse effects in infants of lactating mothers. There was mild GI irritability in two treated women. CONCLUSION: This study provides first evidence in humans as to the safety of mebendazole in breastfeeding.

Acute Toxicity and Environmental Risks of Five Veterinary Pharmaceuticals for Aquatic Macroinvertebrates.

Due to the high use of antibiotics and antiparasitics for the treatment of livestock, there is concern about the potential impacts of the release of these compounds into freshwater ecosystems. In this context, the present study quantified the acute toxicity of two antibiotics (sulfadiazine and sulfadimidine), and three antiparasitic agents (flubendazole, fenbendazole, ivermectin) for nine freshwater invertebrate species. These experiments revealed a low degree of toxicity for the sulfonamide antibiotics, with limited implications in the survival of all test species at the highest test concentrations (50 and 100 mg/L). In contrast, all three antiparasitic agents indicated on the basis of their acute toxicity risks for the aquatic environment. Moreover, chronic toxicity data from the literature for antiparasitics, including effects on reproduction in daphnids, support the concern about the integrity of aquatic ecosystems posed by releases of these compounds. Thus, these pharmaceuticals warrant further careful consideration by environmental risk managers.

Genotoxicity of flubendazole and its metabolites in vitro and the impact of a new formulation on in vivo aneugenicity.

The anti-parasitic benzimidazole flubendazole has been used for many years to treat intestinal infections in humans and animals. Previous genotoxicity studies have shown that the compound is not a bacterial mutagen and a bone marrow micronucleus test, using a formulation that limited systemic absorption, was negative. The purpose of this study is to explore the genotoxicity of flubendazole and its main metabolites in in vitro micronucleus studies and to test a new oral formulation that improves systemic absorption in an in vivo micronucleus test. The isolated metabolites were also screened using the Ames test for bacterial mutagenicity. It was found that flubendazole, like other chemically related benzimidazoles used in anti-parasitic therapies, is a potent aneugen in vitro The hydrolysed metabolite of flubendazole is negative in these tests, but the reduced metabolite (R- and S-forms) shows both aneugenic and clastogenic activity. However, in vitro micronucleus tests of flubendazole in the presence of rat liver S9 gave almost identical signals for aneugenicity as they did in the absence of S9, suggesting that any clastogenicity from the reduced metabolite is not sufficient to change the overall profile. Like flubendazole itself, both metabolites are negative in the Ames test. Analysis of dose-response curves from the in vitro tests, using recently developed point of departure approaches, demonstrate that the aneugenic potency of flubendazole is very similar to related anti-parasitic benzimidazoles, including albendazole, which is used in mass drug administration programmes to combat endemic filarial diseases. The in vivo micronucleus test of the new formulation of flubendazole also showed evidence of induced aneugenicity. Analysis of the in vivo data allowed a reference dose for aneugenicity to be established which can be compared with therapeutic exposures of flubendazole when this has been established. Analysis of the plasma from the animals used in the in vivo micronucleus test showed that there is increased exposure to flubendazole compared with previously tested formulations, as well as significant formation of the non-genotoxic hydrolysed metabolite of flubendazole and small levels of the reduced metabolite. In conclusion, this study shows that flubendazole is a potent aneugen in vitro with similar potency to chemically related benzimidazoles currently used as anti-parasitic therapies. The reduced metabolite also has aneugenic properties as well as clastogenic properties. Treatment with a new formulation of flubendazole that allows increased systemic exposure, compared with previously used formulations, also results in detectable aneugenicity in vivo. Based on the lack of carcinogenicity of this class of benzimidazoles and the intended short-term dosing, it is unlikely that flubendazole treatment will pose a carcinogenic risk to patients.

Toxicidad de agentes antiparasitarios, antimicrobianos e insecticidas sobre larvas del camarón salino Artemia franciscana (Crustacea: Artemiidae) / Toxicity of antiparasitic, antimicrobial agents and insecticides on larvae of brine shrimp Artemia franciscana (crustacea: artemiidae)

Artemia franciscana "camarón salino", es un crustáceo sensible a un amplio rango de compuestos químicos, de fácil manejo en el laboratorio, y con un cultivo relativamente sencillo y barato. El objetivo del presente trabajo fue evaluar la toxicidad de agentes antiparasitarios, antimicrobianos e insecticidas sobre A. franciscana para establecer la concentración prevista que no causa efectos (PNEC) sobre los organismos marinos y obtener los niveles guía para la protección de la vida acuática. Con los nauplios II de A. franciscana, dentro de las 24 h de eclosión, se procedió a realizar los bioensayos de toxicidad calculando la Concentración letal media (CL50) a 24 h y 48 h de exposición. Se observó la siguiente secuencia de mayor a menor toxicidad a 48 h de exposición para tres agentes antiparasitarios comerciales: Mebendazol >Albendazol >Metronidazol. Con relación al efecto tóxico letal de seis agentes antimicrobianos comerciales se vio la siguiente secuencia de mayor a menor toxicidad a 48 h de exposición: Triclosan >Clotrimazol >Itraconazol >Ketoconazol >Oxitetraciclina >Mimosa. El camarón salino mostró efectos de mortalidad por acción de cinco sustancias con propiedades insecticidas, encontrándose el siguiente orden de mayor a menor mortalidad a 48 h de exposición: Cipermetrina >Rotenona >Carbaryl >Canela >Malation. Las tres sustancias químicas calificadas como muy tóxicas y que presentaron los niveles guía más bajos para la protección de la vida acuática fueron Triclosan (0,72 ug·L-1), Cipermetrina (0,84 ug·L-1) y Clotrimazol (0,97 ug·L-1). Se observó que diez (71,42%) de las sustancias químicas mostraron fuerte actividad citotóxica (AU)

Artemia franciscana "brine shrimp", is sensitive to a wide range of chemical structures, and easy handling in the laboratory and with a relatively simple and inexpensive crustacean culture. The aim of this study was to evaluate the toxicity of parasiticides agent, antimicrobials agent and insecticides on A. franciscana to establish Predicted No-Effect Concentration (PNEC) on marine organisms and obtain guidance levels for the protection of aquatic life. With A. franciscana nauplii II, within 24 h of hatching, we proceeded to perform toxicity bioassays calculating the average lethal concentration (LC50) at 24 h and 48 h of exposure. The following sequence of high to low toxicity to 48 h of exposure to three commercial antiparasitic agents were observed: Mebendazole> Albendazole> Metronidazole. Regarding the lethal toxic effect of six commercial antimicrobial agents about A. franciscana, the following sequence of toxicity at 48 h of exposure was observed: Triclosan> Clotrimazole> Itraconazole> Ketoconazole> oxytetracycline> Mimosa. The brine shrimp mortality showed effects on five substances with insecticidal properties, meeting the following order from highest to lowest mortality at 48 h of exposure Cipermethrin >Rotenone >Carbaryl >Cinnamon >Malathion. The three chemicals were classified as very toxic and presented lower levels guidance for the protection of aquatic life were Triclosan (0,72 ug·L-1), Cipermetrina (0,84 ug·L-1) y Clotrimazol (0,97 ug·L-1). Ten of chemicals (71.42%) showed strong cytotoxic activity (AU)

Toxicity of anthelmintic drugs (fenbendazole and flubendazole) to aquatic organisms.

Flubendazole (FLU) and fenbendazole (FEN) belong to benzimidazoles-pharmaceuticals widely used in veterinary and human medicine for the treatment of intestinal parasites as well as for the treatment of systemic worm infections. In recent years, usage of these drugs increased, which resulted in a larger contamination of the environment and possible negative effects on biota. Hence, in our research, we investigated an aquatic ecotoxicity of these pharmaceuticals towards: marine bacteria (Vibrio fischeri), green algae (Scenedesmus vacuolatus), duckweed (Lemna minor) and crustacean (Daphnia magna). Ecotoxicity tests were combined with chemical analysis in order to investigate the actual exposure concentration of the compounds used in the experiment as well as to stability and adsorption studies. As a result, study evaluating sensitivity of different aquatic organisms to these compounds and new ecotoxicological data is presented. The strongest negative impact of FLU and FEN was observed to D. magna.

In vivo preliminary investigations of the effects of the benzimidazole anthelmintic drug flubendazole on rat embryos and fetuses.

Flubendazole, in a new formulation with high systemic bioavailability, has been proposed as a macrofilaricide against filarial diseases. To investigate embryotoxic activity, the new flubendazole formulation was administered orally to Sprague Dawley rats at 2, 3.46, 6.32mg/kg/day on gestation day (GD) 9.5 and 10.5. Embryos/fetuses were evaluated on GD 11.5, 12.5 or 20. At 6.32mg/kg/day (Cmax=0.801µg/mL after single administration), flubendazole initially induced an arrest of embryonic development followed by a generalized cell death that led to 100% embryolethality by GD 12.5. At 3.46mg/kg/day (Cmax=0.539µg/mL after single administration), flubendazole markedly reduced embryonic development by GD 12.5 without causing cell death. On GD 20, 80% of fetuses showed malformations. At 2mg/kg/day (Cmax=0.389µg/mL after single administration), it did not interfere with rat embryofetal development.

Effects of the benzimidazole anthelmintic drug flubendazole on rat embryos in vitro.

Filarial diseases affect millions of people in poverty-stricken areas. In 2011, an investigation of the potential of flubendazole as a safe, highly efficacious, and field-usable macrofilaricidal drug was begun by Drug for Neglected Diseases initiative. As part of the preclinical development program, whole embryo culture was used to investigate the potential embryotoxicity of flubendazole and its metabolites, reduced and hydrolyzed flubendazole. Albendazole was included as a comparator. Flubendazole and albendazole showed similar potency in affecting rat embryonic development in vitro, inducing retardation of growth and dysmorphogenic effects at concentrations ≥0.5 µg/mL. The head, optic and otic systems, branchial arches and posterior body portion were affected. Diffuse areas of cell death were seen in various embryonic districts. The No Observed Effect Level (NOEL) was 0.25 µg/mL for both drugs. Reduced and hydrolyzed flubendazole were less embryotoxic than the parent compound, with NOELs 4-fold and >40-fold higher than that of flubendazole, respectively.

Toxic effects of mebendazole at high dose on the haematology of red-legged pademelons (Thylogale stigmatica).

OBJECTIVE: To investigate the effects of mebendazole at high dose on the haematology of macropods. Experimental. PROCEDURE: Five red-legged pademelons (Thylogale stigmatica) were dosed orally with mebendazole at 50 mg/kg/d for 5 to 6 days. Two control pademelons were dosed with water. Regular blood samples were taken for haematology over 20 days. RESULTS: All four treated pademelons sampled at 5 days developed severe leucopenia and neutropenia, moderate lymphopenia, thrombocytopenia, eosinopenia and monocytopenia, as well as bone marrow aplasia within 5 to 11 days after the first mebendazole dose. Four pademelons died unexpectedly or became ill and were euthanased 5 to 11 days after the first dose while the other animal recovered after 5 days of illness. Necropsy revealed systemic infection with opportunistic enteric bacteria, non-suppurative inflammation in tissues, haemorrhage and ulceration of the gastrointestinal tract. CONCLUSIONS: Red-legged pademelons rapidly develop bone-marrow aplasia and subsequent septicaemia after administration of high doses of mebendazole. Mebendazole at high doses should not be used for macropods.

Assessing behavioral toxicity with Caenorhabditis elegans.

Behavior, even in simple metazoans, depends upon integrated processes at the subcellular, cellular, and organismal level, and thus is susceptible to disruption by a broad spectrum of chemicals. Locomotor behavior (movement) of the small free-living nematode Caenorhabditis elegans has proven to be useful in assessing toxicity. Recently reported observations suggest that behavioral change (reduced movement) occurs after 4 h of exposure to heavy metals, and that with abbreviated exposure, the concentration-response relationship for Pb (a known neurotoxic metal) differs from that for Cu. In this study, movement was evaluated after 4-h exposures for nine compounds from three chemical classes: organic pesticides, organic solvents, and heavy metals. Concentration-dependent reduction of movement was observed for all test compounds with the exception of mebendazole, for which test concentrations were limited by solubility. Within each chemical class, movement was more sensitive to the neurotoxic compounds than to substances not believed to be neurotoxic, as evidenced by behavioral effective concentration to reduce average worm movement to 50% of the control movement values (e.g., levamisole and chlorpyrifos < mebendazole, ethanol and acetone < dimethylsulfoxide, and Pb and Al < Cu). These observations are discussed as they relate to the use of acute behavioral tests in assessing general chemical toxicity, and the enhanced value of 4-h testing for the detection of neural toxicants.

Effect of oral dosing vehicles on the developmental toxicity of flubendazole in rats.

Flubendazole was suspended in deionized water or olive oil and administered by gavage once daily to pregnant rats on Days 8-15 of pregnancy to examine if the embryolethal and teratogenic doses were affected by the vehicles used. Flubendazole in olive oil caused a statistically significant increase in embryolethality at doses of 7.83 mg/kg per day and higher, with complete resorption in all dams at 31.33 mg/kg per day. When flubendazole was suspended in deionized water, a significant increase in embryolethality occurred only at a maternal dose of 125.32 mg/kg per day. The proportion of litters with anomalous fetuses was significantly increased at doses of 31.33 mg/kg per day and above when flubendazole was administered in deionized water, but increased at doses at four times lower when flubendazole was administered as in olive oil. Administered as a single dose in olive oil on any one of Days 6-12 of pregnancy, a flubendazole dose of 31.33 mg/kg caused significant increases in embryolethality and decreased fetal body weights on Days 7-9, with an 82.7% incidence of embryolethality on Day 8, with complete resorption in 5 of the 8 dams. The critical periods for teratogenic effects were between Days 8 and 11 of pregnancy, with Day 9 being the most critical. Fetuses with gross, skeletal, or internal anomalies were seen in dams given a single dose of as low as 7.83 mg/kg.

Albendazole, mebendazole and praziquantel. Review of non-clinical toxicity and pharmacokinetics.

The pharmacokinetics and toxicity of albendazole, mebendazole and praziquantel are extensively reviewed, drawing on original published work and reviews in the open scientific literature and on assessments by international agencies and official regulatory bodies in Europe and the USA. Information about human and veterinary medical uses and adverse reactions is evaluated. The totality of the non-clinical information available about these long-established drugs may not comply with current official guidelines for new medicines but reasons are given why the "deficiencies" are only apparent and the data gaps can be replaced by other results, largely obtained from the target species and the many years of clinical experience of safe use of these drugs in humans and animals.

Benzimidazole treatment of cystic echinococcosis.

Hydatidosis (cystic echinococcosis, CE) constitutes a serious public health problem worldwide. Total surgical removal of a hydatid cyst is still considered the gold standard treatment for CE. Percutaneous treatment (PAIR), using either hypertonic saline or alcohol as a larvacidal agent, appears to be an additional effective form of treatment. Benzimidazoles (albendazole, ABZ; mebendazole, MBZ), given either alone or combined with praziquantel (PZ) are currently used for the treatment of non-surgical cases and as a supplementary treatment prior and post-surgery. Combined chemotherapy was found to be more effective than either of the agents given alone. ABZ is easily absorbed and more effective than MBZ. ABZ (12-15 mg/kg/day) and MBZ (30-70 mg/kg/day) given for 14-20 days prior to surgery and continued for an additional 3-24 months in a cyclic monthly form were found effective against the disease. Either increased or decreased circulating antigen levels, which consequently cause changes in the humoral (IgG, IgG1, IgG4, IgE) immune responses, have a prognostic value in successfully treated CE cases. However, although the cellular immune response to echinococcal antigens decreased in improved or cured CE patients, it was not considered of practical use in determining treatment efficacy. In certain cases successful treatment was also followed by elevated eosinophilia and erythrocyte sedimentation rates. In the present article, the mechanism of drug activities as well as the development of resistance against the drugs available are further discussed.

An in vivo evaluation of induction of abnormal sperm morphology by some anthelmintic drugs in mice.

Using the murine sperm-head abnormality test, the mutagenicity of pyrantel pamoate, levamisole, albendazole, mebendazole and niridazole was evaluated. Pyrantel pamoate and niridazole induced increases in sperm-head abnormalities statistically significant over the negative controls at all the dose levels that were considered; the induction was dose-dependent indicating that both drugs might be mutagenic. Levamisole, albendazole, mebendazole and thiabendazole, all were unable to induce statistically significant increases in sperm-head abnormalities over the negative controls at all the dose levels tested; there was no correlation between dose level of administered drugs and incidence of abnormal sperms, indicating that the drugs might not be mutagenic.

Spindle poisons can induce polyploidy by mitotic slippage and micronucleate mononucleates in the cytokinesis-block assay.

The human in vitro cytokinesis-blocked micronucleus (MN) assay has been extensively used for detection of clastogenic and aneugenic agents. In this test binucleate cells are generally considered to be the main target cell population for assessing genotoxic effect and almost no attention is paid to the biological information contained in mono-nucleate cells. In this study we analysed the frequencies of micronucleate mononucleates in a control population and after in vitro exposure to clastogens or aneugens. A clear increase in MN in mononucleates was found only after exposure to aneugenic compounds. By means of fluorescence in situ hybridization using a chromosome 1-specific probe we further analysed the proportion of mononucleate cells with and without MN which were tetrasomic (tetraploid) and would have been induced during aneugen treatment by mitotic slippage. The data indicate that treatment with nocodazole induces tetrasomy for chromosome 1 (tetraploidy) and an increase in MN frequency in mononucleate diploid and tetraploid lymphocytes. The results thus confirm that some mononucleates pass mitosis without chromatid segregation to daughter nuclei. These data suggest that MN in mononucleates may be useful to distinguish clastogens from aneugens and increase the sensitivity of the test.

Indication for thresholds of chromosome non-disjunction versus chromosome lagging induced by spindle inhibitors in vitro in human lymphocytes.

Risk assessment from exposure to spindle inhibitors should take into account the possibility of threshold concentration-response curves for aneuploidy induction. We analysed concentration-dependent induction of chromosome non-disjunction by well known spindle poisons (colchicine, carbendazim, mebendazole and nocodazole) and a reference clastogen, methyl methanesulphonate (MMS) in vitro in human lymphocytes; and integrated these findings with earlier results of chromosome loss in micronuclei. Chromosome non-disjunction was estimated on cytokinesis-blocked lymphocytes after simultaneous fluorescent in situ hybridization labelling with two chromosome-specific centromeric probes (chromosomes 1 and 17). The frequencies of spontaneous non-disjunction showed important inter-individual variations and were surprisingly high (7.04-15.39%). Lower concentrations of aneugens did not induce a statistically significant increase of non-disjunction frequencies over the respective control levels, whereas higher concentrations clearly induced a concentration-dependent increase in the non-disjunction frequencies with the four aneugens tested. On the contrary, even at high concentrations, MMS induced a slight increase in the frequency of non-disjunction but without being statistically significant when compared with the control frequencies. We estimated the inflection points, the first statistically significant concentrations, the last non-statistically significant concentrations and the number of events from concentration-response curves of chromosome non-disjunction and chromosome loss. A threshold-type of concentration-response for non-disjunction is highly probable for colchicine and nocodazole. For carbendazim and mebendazole the inflection point fell above the first statistically significant concentrations. But since it is obvious from dose-response curves where the inflection point/threshold lies, it appears that the model might be picking up some irregularities (possibly due to experimental variability in the dose-response curve at concentrations greater than the threshold). For accurate estimation of the threshold, analysis of more concentrations or more cells might be needed. Our data strongly indicate that in cultured human lymphocytes chromosome non-disjunction is a major mechanism of aneuploidy induction by spindle inhibitors and since non-disjunction occurs at lower concentration than chromosome loss, the aneuploidy threshold should be estimated on the basis of non-disjunction rather than on micronuclei frequencies (chromosome loss).

Multiple anthelmintic resistance in Haemonchus contortus on a sheep farm in India.

Multiple resistance to benzimidazoles (fenbendazole, albendazole and mebendazole) in a strain of Haemonchus contortus in sheep was detected on a farm where fenbendazole resistance had already been identified. Following a faecal egg count reduction test, this was confirmed by both critical and controlled anthelmintic tests. Different groups of sheep infected naturally or given an experimental infection with the fenbendazole-resistant strain were treated with the recommended doses of various anthelmintics. Compared to the control group, percentage reductions in faecal egg counts of sheep treated with fenbendazole, albendazole, mebendazole, levamisole and morantel varied between 56% and 81% and worm counts between 71% and 86%. The results indicate the presence of multiple anthelmintic resistance in this strain of H. contortus on this farm. Sheep treated with ivermectin and closantel showed 100% reductions in faecal egg and worm counts, suggesting high efficacy of these drugs against the population of H. contortus on this farm.