Selection studies on anthelmintic resistant and susceptible populations of Trichostrongylus colubriformis of sheep

Waller P. J., Dobson R. J., Donald A. D., Griffiths D. A. and Smith E.F. 1985. Selection studies on anthelmintic resistant and susceptible populations of Trichostrongylus colubriformis of sheep. International Journal for Parasitology15: 669–676. A T. colubriformis population (BCK), formerly resistant to benzimidazole anthelmintics, but now highly resistant to levamisole after 6 years exposure to this drug alone in the field, was passed through 12 generations in the laboratory in three separate lines exposed either to selection with thiabendazole or levamisole, or to no selection. Another population (McM) not previously exposed to these anthelmintics was treated similarly in two lines, selected with thiabendazole or not selected.Selection with thiabendazole resulted in a return of benzimidazole resistance in the BCK line which occurred faster than in the McM line, but a similar level of resistance was reached in each by the twelfth generation. Resistance ratios in both selected lines compared with the unselected McM line were less than 20: 1, and only 1.5 times the recommended dose rate of thiabendazole was required to remove more than half of the resistant population. This suggests that a polygenic vigour tolerance rather than a specific resistance had been selected.In the case of levamisole resistance, the BCK population was found to contain two distinct subpopulations, one susceptible and the other highly resistant. Resistance ratios for the highly resistant subpopulation were greater than 4000: 1, implying a specific resistance controlled by a major gene. During the 12 generations of levamisole selection, the proportion of resistant phenotypes fluctuated about an average level of 70%, suggesting that susceptibility alleles were being maintained in the population through superior heterozygote fitness. This conclusion is supported by a significant decline in levamisole resistance in the absence of levamisole selection. Moreover, thiabendazole selection hastened the reversion to levamisole suceptibility.The results provide support for the reintroduction of a benzimidazole anthelmintic to control this helminth population, and for a slow rotation in the use of drugs with different modes of action.     

Ascaris suum: Differential effects of Avermectin B1a on the intact animal and neuromuscular strip preparations

Avermectin B_1a, a novel antiparasitic agent, paralyzes Ascaris suum without causing either flaccid paralysis or a hypercontraction. It reduces the lengthening of the acetylcholine-preconditioned A. suum muscle strip caused by γ-aminobutyric acid. It does not affect the contraction of the isolated muscle strip preparation caused by applying acetylcholine. However, preinjection with Avermectin B_1a does significantly reduce the shortening caused by acetylcholine injection without affecting the paralysis of an intact ascarid worm. These results suggest that Avermectin B_1a may act on the central nervous system of Ascaris sp. nematodes.     

Potential effects and relevant lead compounds of Vigna mungo (L.) Hepper seeds against bacterial infection,helminthiasis, thrombosis and neuropharmacological disorders

Multidrug-resistant bacterial infections, helminthiasis, thrombosis, anxiety and insomnia are some of the major global health concerns. Vigna mungo (L.) Hepper (VM) has been used traditionally to treat microbial infection, helminthic disorder, schizophrenia, memory loss, and blood circulatory problem. This research aims to discover antibacterial, anthelmintic, thrombolytic and neuropharmacological effects of the methanol extract of Vigna mungo seeds (MESVM), and also in-silico prediction of relevant lead compounds by molecular docking and ADME/T analysis. The crude extracts and subsequent fractions of MESVM were investigated for antibacterial activity by disc diffusion method, anthelmintic activity by paralysis and death test on earthworms, and thrombolytic activity by in vitro blood clot dissolution test. Open-field test and elevated plus maze test were performed for evaluating anxiolytic activity of the extracts. Using molecular docking, ligand poses of selected VM seeds’ phytoconstituents were predicted targeting tubulin, GlcN-6-P synthase, and human tissue plasminogen activator proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. In the antibacterial activity test, the MESVM at 10000 μg/mL concentration created highest and significant (P < 0.001) zone of inhibition against Staphylococcus aureus (15.42 mm) and Escherichia coli (12 mm) compared with tetracycline. The MESVM exhibited remarkable anthelmintic activity at 50 mg/mL concentration with 35.4 min paralysis time, 75.2 min death time and were closer to the durations of standard drug albendazole. No test extract showed anxiolytic activity. In thrombolytic activity test, all concentrations of MESVM produced clot lytic activity with high significance (P < 0.001) in comparison with the blank. In docking, 2′-hydroxygenistein, cyclokievitone hydrate, and aureol displayed maximum affinity to the target proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. This research revealed that the MESVM demonstrated potential anthelmintic, antibacterial and thrombolytic effects that confirmed the folkloric uses of VM and the found relevant lead compounds might be further optimized in future drug development.     

Sequence variation in the Trichuris trichiura beta-tubulin locus: implications for the development of benzimidazole resistance

Benzimidazole resistance has evolved in a variety of organisms and typically results from mutations in the beta-tubulin locus at specific amino acid sites. Despite widespread treatment of human intestinal nematodes with benzimidazole drugs, there have been no unambiguous reports of resistance. However, since beta-tubulin mutations conferring resistance are generally recessive, frequencies of resistance alleles less than 30% would be difficult to detect on the basis of drug treatment failures. Here we investigate sequence variation in a 1079 bp segment of the beta-tubulin locus in the human whipworm Trichuris trichiura from 72 individual nematodes from seven countries. We did not observe any alleles with amino acid mutations indicative of resistance, and of 40 point mutations there were only four non-synonymous mutations all of which were singletons. Estimated effective population sizes are an order of magnitude lower than those from another nematode species in which benzimidazole resistance has developed (Haemonchus contortus). Both the lower diversity and reduced population sizes suggest that benzimidazole resistance is likely to evolve less rapidly in Trichuris than in trichostrongyle parasites of livestock. We observed moderate levels of population subdivision (Phi(ST)=0.26) comparable with that previously observed in Ascaris lumbricoides, and identical alleles were frequently found in parasites from different continents, suggestive of recent admixture. A particularly interesting feature of the data is the high nucleotide diversities observed in nematodes from the Caribbean. This genetic complexity may be a direct result of extensive admixture and complex history of human populations in this region of the world. These data should encourage (but not make complacent) those involved in large-scale benzimidazole treatment of human intestinal nematodes.     

Enhancing a search for traditional medicinal plants with anthelmintic action by using wild type and stress reporter Caenorhabditis elegans strains as screening tools

Traditional healers in Sarawak, Malaysia, use plants such as Picria fel-terrae, Linariantha bicolor and Lansium domesticum to treat gastrointestinal infections. This study aimed to test whether their nematocidal activities could be confirmed in vitro using highly standardised Caenorhabditis elegans models. We applied eight different ethanol solubilised plant extracts and two commercial anthelmintic drugs to larval and adult stages of C. elegans in vitro. Seven C. elegans strains were evaluated, one wild type and six strains with GFP-tagged stress response pathways to help characterise and compare the pathways affected by plant extracts. Our in vitro screen confirmed that both of the commercial anthelmintic drugs and five of the eight traditionally used plant extracts had significant nematocidal activity against both larval and adult C. elegans. The most effective extracts were from P. fel-terrae. The plant extracts triggered different stress response pathways from the commercial anthelmintic drugs. This study showed that using traditional knowledge of plant medicinal properties in combination with a C. elegans in vitro screen provided a rapid and economical test with a high hit rate compared with the random screening of plants for nematocidal activities. The use of transgenic C. elegans strains may allow this approach to be refined further to investigate the mode of action of active extracts.     

In vitro pharmacological evaluation and phenolic content of ten South African medicinal plants used as anthelmintics

Helminth infection is regarded as one of the neglected tropical diseases (NTDs). Although the disease is common in rural areas, information on the pharmacology of South African medicinal plants used against this disease is limited. We investigated the efficacy of ten South African medicinal plants against Caenorhabditis elegans. Because of the increased susceptibility of a host to microbial infections and other inflammatory responses associated with helminth infections, the antimicrobial and cyclooxygenase (COX) inhibitory activities of the plants were also investigated. Phenolics including flavonoids, condensed tannins and gallotannins have been linked to many pharmacological activities. Thus, the phenolic content of the plant extracts were quantitatively evaluated. In the three bioassays, organic solvent extracts from Cyathea dregei (roots and leaves), Felicia erigeroides (leaves and stems), Hypoxis colchicifolia (leaves) and Senna petersiana (leaves) exhibited noteworthy pharmacological activities while Acokanthera oppositifolia (leaves) had good COX inhibitory activity. The concentration of phenolics ranged from 56.7 to 1.7 mg GAE/g dry matter in Ocimum basilicum and Cotyledon orbiculata var. dactylopsis, respectively. Flavonoids, condensed tannin and gallotannin content also varied greatly among the plant extracts investigated.     

A novel approach for combining the use of in vitro and in vivo data to measure and detect emerging moxidectin resistance in gastrointestinal nematodes of goats

Ivermectin and moxidectin are closely related avermectin/milbemycin anthelmintics and available data suggest that side resistance occurs with these two drugs. However, moxidectin remains effective against many species of ivermectin-resistant worms due to its higher potency. The larval development assay (LDA) is routinely used to diagnose ivermectin resistance in Haemonchus contortus but laboratory diagnosis of moxidectin resistance is hampered by the lack of any validated in vitro tests. The objective of this study was to measure the relative susceptibility/resistance of H. contortus to moxidectin on goat farms in Georgia, and to validate the DrenchRite LDA for detecting resistance to moxidectin. Fecal egg count reduction tests (FECRT) were performed at five different moxidectin dose levels and DrenchRite LDAs were performed in duplicate on nine meat goat farms in Georgia, USA. To improve our ability to make inferences on the relative levels of resistance between farms, FECRT data were first analysed using a linear mixed model, and then Tukey's sequential trend test was used to evaluate the trend in response across dose levels. LDA data were analysed using log-dose logit-response and probit models. Using these statistical results, we were able to rank the nine farms from the least to the most resistant, and to develop a set of criteria for interpreting DrenchRite LDA results so that this assay can be used to diagnose both clinically apparent moxidectin resistance, as well as sub-clinical emerging resistance. These results suggest that our novel approach for examining these types of data provides a method for obtaining an increased amount of information, thus permitting a more sensitive detection of resistance. Based on results of the LDA, moxidectin-resistant farms had resistance ratios, compared with an ivermectin-sensitive farm, ranging from 32 to 128, and had resistance ratios of 6-24 compared with an ivermectin-resistant/moxidectin naive farm. Moxidectin resistance was diagnosed both in Haemonchus and Trichostrongylus on almost half of the farms tested, despite this drug only being used on these farms for 2-3 years.     

Evaluation of the in vitro activity of dermaseptin 01, a cationic antimicrobial peptide, against Schistosoma mansoni

Schistosomiasis is a neglected tropical disease that remains a considerable public health problem worldwide. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we examined the in vitro effects of dermaseptin 01 (DS 01), an antimicrobial peptide found in the skin secretion of frogs of the genus Phyllomedusa, on Schistosoma mansoni adult worms. DS 01 at a concentration of 100 μg/ml reduced the worm motor activity and caused the death of all worms within 48 h in RPMI 1640 medium. At the highest sublethal concentration of antimicrobial peptide (75 μg/ml), a 100% reduction in egg output of paired female worms was observed. Additionally, DS 01 induced morphological alterations on the tegument of S. mansoni, and a quantitative analysis carried out by confocal microscopy revealed extensive destruction of the tubercles in a dose-dependent manner over the concentration range of 50-200 μg/ml. It was the first time that an anthelmintic activity towards schistosomes has been reported for a dermaseptin.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.