The response of adult and free-living stages of Necator americanus, in vitro, to anthelmintics

The in vitro response of adults (males and females) and free-living stages of Necator americanus one of the human hookworms, to a wide variety of 20 broad and narrow spectrum anthelmintics was tested. Almost all the broad spectrum anthelmintics influenced males, females and free-living stages at different levels and showed good activity with EC50 values varying from about 0.0002 and 0.0007 mg/l for pyrantel pamoate and tricofenol piperazine respectively to about 8.47 and 7.6 mg/l for morantel tartrate and amoscanate respectively. Certain drugs (emetine, praziquantel and suramin) exerted their effect either on male or female or free-living stages at 10.0 mg/l level. It is concluded that either sex or life-cycle stage alone may not be an effective criteria for screening of anthelmintics which have been employed at large; and females of nematodes (in particular those of N. americanus) should be taken into account for proposing EC50 as they were found to require relatively highest EC50 level in almost all the instances studied presently.     

A comparison of superoxide dismutase (SOD, EC:1.15.1.1) distribution in gastro-intestinal nematodes.

An examination of the distribution of superoxide dismutase (SOD) activities in whole worm homogenates from six gastro-intestinal nematodes, at different life-cycle stages, demonstrated that the third larval stages of each species contained notably more activity than the corresponding adults. Significant SOD activities were detected in culture fluids following in vitro maintenance of third and fourth larval stage and adult Trichostrongylus vitrinus and Teladorsagia circumcincta (formerly Ostertagia circumcincta). Marked interspecies variation was observed when SOD isoenzyme profiles of adult and third larval stage Nippostrongylus brasiliensis, Trichostrongylus vitrinus, Teladorsagia circumcincta and Haemonchus contortus were compared. In addition, SOD activity and isoenzyme polymorphism were greater in Nippostrongylus brasiliensis harvested from primed rats compared to parasites recovered from naive rats.     

Changes in the structure of Nippostrongylus brasiliensis intestinal cells during development from the free-living to the parasitic stages

The ultrastructure of Nippostrongylus brasiliensis intestinal cells was examined in free-living, feeding second-stage larvae, infective, nonfeeding third-stage larvae, and parasitic, feeding third-stage larvae. The intestinal cells of second-stage larvae were characterized by a well-developed microvillar border, large numbers of ribosomes, Golgi complexes, rough endoplasmic reticulum, and nuclei with prominent nucleoli. The intestinal cells of infective, third-stage larvae had very few microvilli and the cells were extremely narrow. Few ribosomes, Golgi complexes, and little rough endoplasmic reticulum were present. Nuclei did not contain nucleoli. When worms were introduced into an in vitro culture system, development of intestinal cells began. By 36 hr, microvilli were well differentiated and the cysoplasm contained numerous ribosomes and Golgi complexes, and rough endoplasmic reticulum, mitochondria, and nucleoli were prominent. These morphological changes were related to changes in the physiology of Nippostrongylus brasiliensis which occur during development from a free-living to parasitic form.     

Carboxypeptidase-like activity during development and aging of Nippostrongylus brasiliensis

An acid carboxypeptidase whose activity, based on several biochemical parameters, was determined to be similar to mammalian lysosomal carboxypeptidase was maximal at pH 5.0 during the early parasitic stages of development of the nematode Nippostrongylus brasiliensis. No such enzyme activity could be detected in the early free-living larval stages of this nematode. Carboxypeptidase activity declined as the age of the parasitic adult increased. Timing of maximum activity corresponds to the shift from free-living to parasitic stages of the life cycle.     

The effect of anaerobiosis on adenosine triphosphate levels in larval Nippostrongylus brasiliensis and Haemonchus contortus

The adenosine triphosphate (ATP) content of the pre-parasitic stages of Nippostrongylus brasiliensis, Haemonchus contortus (L1, L2 and L3) and the adults of the free-living nematode, Panagrellus redivivus, have been measured by bioluminescent photometry in aerated or near-anoxic conditions. The ATP content of the L1 and L2 stages of both parasitic species was unaltered by a lack of oxygen over a 90-min period. However, the L3 stage of both species and the adults of P. redivivus showed a significant fall in the level of ATP within 10 min of near-anoxia. This lower level of ATP was maintained during oxygen lack but the initial content was restored on return of the nematodes to aerobic conditions. The results suggest that measurement of ATP by bioluminescent photometry offers a readily measured and sensitive indicator of the capacity of a nematode to cope with transient changes in oxygen supply without undue metabolic stress.     

Nematospiroides dubius: response of the late fourth-stage larva to anthelmintics in vitro

Approximately 60% of fourth-stage larvae of Nematospiroides dubius recovered from mice 6 days after infection, developed to the young adult stage when cultured over a 7-day period in a complex medium in vitro. Larvae at the late fourth stage of development were found to be highly susceptible to most broad spectrum anthelmintics under in vitro conditions, the benzimidazole, imidazothiazole, pyrimidine, isothiocyanate and macrocyclic lactone compounds all being active at very low concentrations. Narrow spectrum anthelmintics active only against ascarids, pinworms, filariae, cestodes or trematodes had little or no effect on these larvae. Ineffective also were those chlorinated hydrocarbon, substituted phenol and salicylanilide compounds known to affect Haemonchus but not trichostrongylid worms in general. It is concluded that in vitro assays employing larvae of N. dubius are useful for the stringent screening of compounds for broad spectrum antitrichostrongyle activity. Used in conjunction with in vivo screens employing N. dubius in mice, they also afford means for detecting intrinsic activity against the parasite in a system free from any complicating host pharmacokinetics.     

The use of zeolites as slow release anthelmintic carriers

This work examines the ability of commercial zeolite Y to act as a slow release agent for a number of anthelmintic drugs. Administration to rats, dosed with Nippostrongylus brasiliensis, of pyrantel and/or fenbendazole and pigs, dosed with Ascaris and Oesophagostomum, of dichlorvos (DDVP) loaded onto zeolite Y was more successful in killing adult worms than administration of the pure drug alone. The zeolite Y was used as supplied for initial studies and then later dealuminated for further studies. The drug loadings were monitored by thermal analysis and the loaded zeolites were used in several field trials. The results indicate that zeolite Y is a suitable vehicle for the slow release of some anthelmintics. The slow release of drug from the zeolite matrix improved its efficacy.     

Selection of different genotype larvae and adult worms for anthelmintic resistance by persistent and short-acting avermectin/milbemycins

To understand the factors that influence selection for anthelmintic resistance, it is necessary to examine the impact of drug treatment, particularly persistent drugs, on all phases of the worm life cycle. The efficacy of various avermectin/milbemycin anthelmintics was determined against resident worms, incoming larvae (L3) and development of eggs in faecal culture. Homozygote-resistant and maternal and paternal F1-heterozygote genotypes of Haemonchus contortus were used to infect sheep before or after treatment with ivermectin (IVM) oral, IVM capsule, moxidectin (MOX) oral or MOX injectable. Total worm count and quantitative larval culture were used to determine efficacy against parasitic and free-living stages, respectively. Selection for resistance by IVM capsules occurred at the adult and L3 stages because of poor efficacy against these stages for all resistant genotypes. However, the selective advantage of these surviving worms was reduced due to the low development of their eggs to L3 in faecal culture. For MOX, selection for resistance predominantly occurred after treatment because of high efficacy against resident adult worms of all resistant genotypes but poor efficacy against resistant L3 ingested after drug administration. The results indicated no evidence of sex-linked inheritance for IVM resistance. Mean IVM efficacies against homozygous and heterozygous resistant adult worms were not different, and IVM capsule efficacy against incoming L3 was approximately 70% for all resistant genotypes, consistent with a dominant trait. MOX was highly effective against adults of all resistant genotypes and approximately 76% effective against incoming L3 regardless of resistance genotype, also consistent with a dominant trait. These results will enable the impact of persistent drugs on worm control and anthelmintic resistance to be estimated. The results indicate that IVM capsules should not be used in populations where avermectin/milbemycin resistance is present.     

Effects of cholinergic drugs on longitudinal contraction in levamisole-susceptible and -resistant Haemonchus contortus.

A novel force transducer was used to measure the effects of cholinergic agonists on longitudinal contraction in Haemonchus contortus. Drugs were applied to whole worms or injected via a cannula in the pseudocoelomic cavity. A number of agonists, including nicotine and the anthelmintics m-aminolevamisole, levamisole and morantel, caused contractions in whole worms. Four- to 25-fold increases in concentration of the active compounds were required to cause contractions in each of two levamisole-resistant strains of H. contortus. Of the other compounds tested, bephenium had equivalent activity against susceptible and resistant strains. Anticholinesterase compounds caused contractions after a slight delay in susceptible, but not resistant worms. Numerous cholinergic agonists and other compounds did not cause contraction when applied to whole worms. One of these, acetylcholine, caused contractions in cannulated worms. Compared with the susceptible strain, five- to six-fold higher concentrations of acetylcholine were required to cause equivalent contractions in the resistant strains. Levamisole resistance in adult H. contortus is likely to be due to a change in the characteristics of the cholinergic receptor(s).     

Nippostrongylus brasiliensis and Ascaridia galli: mitochondrial respiration in free-living and parasitic stages

Aerobic respiratory pathways have been delineated and respiratory efficiency has been assessed in mitochondria isolated from embryonated eggs, infective larvae, and adult Nippostrongylus brasiliensis and Ascaridia galli. Mitochondrial respiration in free-living stages of N. brasiliensis is mediated mainly by a mammalian-like antimycin A- and cyanide-sensitive pathway; specific respiratory activity is high and oxidative phosphorylation efficient. In mitochondria of adult N. brasiliensis, antimycin A- and cyanide-sensitive respiration is decreased relative to respiration though an alternative pathway, and specific respiratory activity and mitochondrial efficiency are lower. Respiration in mitochondria from embryonated eggs and tissues of adult A. galli is comparable, and apparently mediated by an antimycin A- and cyanide-insensitive alternative respiratory pathway; no evidence for the presence of a mammalian-like respiratory pathway in embryonated eggs of A. galli was found. The results of this study are compared to mitochondrial respiration in eggs, larvae, and adult body wall muscle of Ascaris suum.     

Haemonchus contortus: in vitro drug screening assays with the adult life stage

Motility and feeding assays were assessed as in vitro systems for screening of novel compounds for anthelmintic activity against adult Haemonchus contortus. The study aimed to develop an assay with the parasitic adult stage of this species that could be used in conjunction with, or as an alternative to, the free-living larval stage screens commonly used for drug discovery with many parasitic nematode species. The feeding assay showed limitations due to the apparent continuation of a significant degree of feeding in worms showing greatly reduced motility in the presence of some drugs. Hence, it appeared most likely that the feeding assay would underestimate the toxicity of these drugs. The motility assay was able to detect toxicity of known anthelmintics, including the 'slow-acting' benzimidazoles. A small-scale screening exercise used the motility assay to detect toxicity towards adult parasites in 10 compounds out of a group of 200 chemicals (selected due to known toxic effects in larval development assays). The motility assay appeared suitable for drug screening against adult H. contortus. The use of the adult stage for drug screening in this way ensures that the drug is toxic towards the parasite life stage to be targeted in vivo. A lack of activity in subsequent in vivo trials could, therefore, be most likely attributable to host pharmacokinetic factors rather than an intrinsic lack of activity of the drug towards the adult parasite.     

Biochemical characterisation of the enzyme responsible for "activated L-serine sulphydrase" activity in nematodes.

The biochemical properties of the enzyme responsible for nematode "activated L-serine sulphydrase" activity (L-cysteine + R-SH----cysteine thioether + H2S) have been investigated using primarily the gastro-intestinal nematodes Nippostrongylus brasiliensis and Haemonchus contortus. The activated L-serine sulphydrase enzyme was found to be cytosolic in origin and exhibited maximal activity at pH 9.0. Enzyme activity was widely distributed amongst the major tissues of adult female Ascaris suum but was particularly abundant in longitudinal muscle. The enzyme appeared to have a rigid specificity for L-cysteine as the primary thiol substrate, but was capable of utilising a number of sulphur amino acids (and derivatives) and nonphysiological thiols as second substrates. The best second thiol substrates were nonphysiological, hydroxyl-containing thiols that showed some structural similarity to the standard second substrate, 2-mercaptoethanol. Kinetic analyses revealed that the enzyme operates by a sequential catalytic mechanism, and the absolute Michaelis constants were: KL-cysteine = 0.21 +/- 0.02 mM and K2-mercaptoethanol = 5.58 +/- 0.59 mM. The enzyme was relatively insensitive to inhibition by a variety of substrate analogues and known inhibitors of pyridoxal 5'-phosphate dependent enzymes, whilst plant phenols caused significant levels of inhibition. The most potent inhibitors discovered were the anthelmintics bithionol, dichlorophene and hexachlorophene. Further characterisation revealed that hexachlorophene was a parabolic competitive inhibitor of the activated L-serine sulphydrase enzyme.     

Morphological differentiation and function of the coelomocytes in the parasitic stages of Nippostrongylus brasiliensis

Female and male worms of Nippostrongylus brasiliensis exhibited sexual dimorphism based on the number of coelomocytes present. A surprising multiplicity of diverse morphological types of coelomocytes developed in both female and male worms during the parasitic cycle. Cytoplasmic processes began to appear on the surface membrane of coelomocytes in the late third-stage larvae (L3s) in the lungs, and they increased greatly in type, size, and morphology during the fourth and fifth stages. These structures were characterized primarily as complex filopodia, pseudopodia, and cytoplasmic pearls, which resulted in the formation of highly pleomorphic cells. Pearls, starting as small protuberances, progressively increased in size and number with larval growth and development. In the adult worms, a novel process of autocannibalism was initiated in many of the very large coelomocytes. The pearls grew enormously in size at the expense of the cytoplasm, forming a peripheral garland in 1 plane surrounding a residual, small, flat, cytoplasmic core containing the nucleus. The underlying "strategy" was to increase the surface-to-volume ratio of these huge cells to overcome the restriction imposed by eutely; the coelomocytes do not undergo cell division. This morphological innovation makes possible a more efficient uptake of nutrients and exocytosis of waste matter. Vesicles (presumably lysosomes) in the coelomocytes of the infective L3 store an extraordinarily high concentration of vitamin B12 (cobalamin, Cbl). At present, the only physiological function that can be assigned to coelomocytes of N. brasiliensis is the uptake, concentration, and storage of Cbl in the free-living stages, with the subsequent release of the molecule from the vesicles in the early phase of parasitism. Thus, stored Cbl in the infective L3 is made available for biochemical processes during the critical period of larval growth and differentiation initiated in the lung. A model of a hypothetical coelomocyte is presented relative to the processing and use of Cbl. Based on many criteria, it is possible that functional differences exist between different morphological types of coelomocytes in the parasitic stages of N. brasiliensis and that future studies will have to address this matter.     

Concurrent infections with the ruminant nematodes Haemonchus contortus and Trichostrongylus colubriformis in jirds, Meriones unguiculatus, and use of this model for anthelmintic studies

Haemonchus contortus- and Trichostrongylus colubriformis-infected jirds (Meriones unguiculatus) are useful for anthelmintic studies. With concurrent infections of these parasites established in the jird, questions of not only anthelmintic activity, but to some extent spectrum, could be assessed in a single model system. This report outlines a model using immunosuppressed (0.02% hydrocortisone in feed) jirds concurrently infected with H. contortus and T. colubriformis. Immunosuppressed jirds were inoculated with approximately 1,000 exsheathed infective larvae of each species, treated per os on day 10 postinoculation (PI), and killed on day 13 PI. Stomachs and small intestines were removed, opened longitudinally, incubated in distilled water at 37 C for 5 hr, fixed in formaldehyde solution, and stored for subsequent examination. Contents of both organs were examined using a stereomicroscope (15-45 x). Various standard anthelmintics were evaluated in the model; modern broad-spectrum ruminant anthelmintics (benzimidazoles, febantel, ivermectin, levamisole hydrochloride, and milbemycin D) are active uniformly and in most cases at doses comparable to those required for efficacy against these parasites in ruminants. This model, using worms of 2 genera living in distinct sites, allows preliminary evaluation of anthelmintic activity and spectrum for experimental compounds in a single cost- and resource-efficient experiment.     

Nippostrongylus brasiliensis infections in mice: the immunological basis of worm expulsion.

Nippostrongylus brasiliensis infections in mice were terminated more rapidly than in rats and immunologically induced damage occurred earlier. Like rats, mice expelled damaged worms more rapidly than normal worms. Recipients of cells from the spleen or mesenteric node of immune mice expelled their worms by day 8 of the infection. Recipients of cells alone or antiserum alone did not expel their worms by day 5 but mice given both cells and antiserum expelled their worms by this stage of the infection. Damaged worms were expelled more rapidly than normal worms from mice given immune cells, This work indicated that antibodies and cells collaborate to expel N. brasiliensis from mice as has been shown to occur in rats.     

Multiple resistance to anthelmintics by Haemonchus contortus and Trichostrongylus colubriformis in sheep in Brazil

The objective of this study was to determine the level of resistance of Haemonchus contortus and Trichostrongylus colubriformis in sheep to levamisole, albendazole, ivermectin, moxidectin, closantel and trichlorfon. The parasites were isolated from sheep naturally infected by gastrointestinal nematodes and were then kept in monospecifically-infected lambs for production of infective larvae (L3) of both species. Forty-two lambs, at three months of age, were simultaneously artificially infected with 4000 L3 of H. contortus and 4000 L3 of T. colubriformis. The animals were allocated into seven groups with six animals each that received one of the following treatments: Group 1--control, no treatment; Group 2--moxidectin (0.2mg/kg body weight (BW)); Group 3--closantel (10mg/kg BW); Group 4--trichlorfon (100mg/kg BW); Group 5--levamisole phosphate (4.7 mg/kg BW); Group 6--albendazole (5.0mg/kg BW); and Group 7--ivermectin (0.2mg/kg BW). Nematode fecal egg counts (FEC) were carried out on the day of treatment and again at 3, 7, 10 and 14 days post-treatment. On the same occasions, composite fecal cultures were prepared for each group for production of L3, which were identified into genus. The animals were sacrificed for worm counts at 14 days after treatment. The efficacy of each treatment was calculated from the arithmetic mean of the FEC or worm burden of the treated group, compared with the values of the control group. Only trichlorfon and moxidectin treatments resulted in a significant reduction of H. contortus recorded at necropsy (73% and 45% respectively). Moxidectin reduced T. colubriformis worm burdens by 82% and albendazole by 19%. All other anthelmintics resulted in no significant reduction in the numbers of worms found at necropsy. In conclusion, the isolates of H. contortus and T. colubriformis showed multiple resistance to all groups of anthelmintics tested. This is the first report, based on the controlled efficacy test, to show resistance of T. colubriformis to macrocyclic lactones in Brazil.     

Inhibition of Nb2 T-lymphoma cell growth by transforming growth factor-beta.

1. Mitochondria isolated from the gut-dwelling nematodes Nippostrongylus brasiliensis and Ascaridia galli (muscle and gut + reproductive tissue) were examined for cytochromes, and it was observed that N. brasiliensis and A. galli muscle tissue mitochondria contained a-, b- and c-type cytochromes, but their stoichiometries were quite different (1:2:1.9 and 1:11.4:13.6 respectively); A. galli gut + reproductive-tissue mitochondria, however, only contained b and c cytochromes, in a ratio of 1:0.8. 2. CO difference spectra showed the presence of CO-reacting b-type cytochrome(s) in all three types of mitochondria; the fast-reacting species comprised 30, 44 and 39% of the total in N. brasiliensis, A. galli muscle and A. galli gut + reproductive-tissue mitochondria respectively. 3. Cytochrome aa3 was observed in N. brasiliensis mitochondria and in those from A. galli muscle, but was below the level of detectability (less than 0.005 nmol/mg of protein) for A. galli gut + reproductive-tissue mitochondria. 4. Photochemical action spectra for the reversal of CO inhibition of the endogenous respiration of whole worms (at 24 microM- and 40 microM-O2 respectively for N. brasiliensis and A. galli) gave maxima at 598 and 542-543 nm, corresponding to the alpha- and beta-absorption maxima of cytochrome aa3, and at 567 nm (b-type cytochrome) for both worms. These results suggest that cytochrome aa3 is the major functional oxidase in N. brasiliensis, whereas the CO-reacting b-type cytochrome dominates in A. galli.     

Effects of azasteroids on growth and development of the free-living stages of Nippostrongylus brasiliensis and Nematospiroides dubius

25-Azasteroids were evaluated for their effects on the growth and development of the free-living stages of Nippostrongylus brasiliensis and Nematospiroides dubius. Increasing the concentration of 25-azasteroids in axenic cultures of either species resulted in a decrease in the percentage and mean length of larvae that developed to the third stage. Morphologic abnormalities of inhibited larvae were similar to those shown by larvae cultured in sterol-deficient medium. Addition of cholesterol to the culture medium reversed the inhibitive effects of azasteroid. Azasteroid completely inhibited growth and development of N. brasiliensis when the only sterol present in the culture medium was sitosterol. These results suggest similar pathways of sterol metabolism and similar mechanisms of action by azasteroids in the nematodes and insects that have been studied.     

A novel anthelmintic model utilizing jirds, Meriones unguiculatus, infected with Haemonchus contortus

Currently, no in vivo laboratory model is available for evaluating anthelmintics against the important ruminant helminth Haemonchus contortus. This report outlines a novel anthelmintic assay utilizing immunosuppressed (0.02% hydrocortisone in feed) jirds, Meriones unguiculatus, infected with H. contortus. Immunosuppressed jirds were inoculated with approximately 1,000 exsheathed infective larvae of H. contortus, treated per os on day 10 postinoculation (PI), and necropsied on day 13 PI. Each stomach was removed, opened longitudinally, incubated in distilled water at 37 C for 5 hr, fixed in formaldehyde solution, and stored for subsequent examination. Stomach contents were examined using a stereomicroscope (15-45x). A variety of standard anthelmintics has been evaluated in the model; modern broad-spectrum ruminant anthelmintics (benzimidazoles, febantel, ivermectin, levamisole hydrochloride, and milbemycin D) are active uniformly and in most cases at doses (mg/kg) comparable to those required for efficacy against H. contortus in ruminants. This model provides an important new tool to assess preliminarily the activity of experimental drugs against H. contortus in vivo prior to studies in ruminants and also may provide a useful tool for studying host-parasite interactions for H. contortus.     

Activity of novel nicotinic anthelmintics in cut preparations of Caenorhabditis elegans

The free-living nematode Caenorhabditis elegans is a useful model for studying the pharmacology of anthelmintics. Currently approved anthelmintics have various mechanisms of action, including activity at nematode nicotinic acetylcholine receptors (nAChRs). Classical anthelmintic agonists of these receptors (nicotine, levamisole, pyrantel and bephenium) caused intact specimens of C. elegans to undergo contracted paralysis. The nAChR antagonist mecamylamine paralysed intact worms and blocked the actions of the agonists. The time to onset of effects of these drugs was enhanced when worms bisected between the mid- and anterior-portions were tested. The novel anthelmintic nAChR antagonist derquantel (2-desoxoparaherquamide, 2-DOPH) was weakly active in intact specimens of C. elegans at concentrations of 50 μM over several days. No antagonism of the nAChR agonists was observed with this drug in intact worms. However, derquantel had direct and marked effects on motility in cut worms and blocked the effects of nAChR agonists in this preparation. A representative of the new amino-acetonitrile derivative (AAD) class of nAChR agonists was not antagonised by derquantel in cut C. elegans, suggesting that these two anthelmintics may not demonstrate unfavourable drug-drug interactions at the receptor level if used to treat livestock infected with parasitic nematodes. The permeability properties of the C. elegans cuticle may be more restrictive than those of adult parasites, calling into question primary anthelmintic screening strategies that rely on this model organism.