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).     

Utility of a Haemonchus contortus/jird (Meriones unguiculatus) model for studying resistance to levamisole

Trichostrongylid nematodes of sheep commonly are identified as exhibiting resistance to levamisole. In vitro assays have been developed to study levamisole resistance for Haemonchus contortus, but no in vivo model has been identified for this species. To determine the utility of a H. contortus/jird (Meriones unguiculatus) model for examining levamisole resistance, immunosuppressed jirds were inoculated with approximately 1,000 exsheathed infective larvae of H. contortus (resistant or susceptible to levamisole), treated per os on day 10 postinoculation (PI) with levamisole hydrochloride or analogs of the drug, and killed on day 13 PI. Stomachs were removed, opened longitudinally, incubated in distilled water at 37 C for 5 hr, fixed in formaldehyde solution, and stored for subsequent microscopic examination. Doses of levamisole and its analogs, which elicited percentage clearances of greater than or equal to 93.5 for the susceptible strain, cleared less than or equal to 68.9% of the resistant worms. These data are consistent with activities for the drugs against wild-type and levamisole-resistant strains of Caenorhabditis elegans. Thus, the H. contortus/jird model provides a useful in vivo tool to study resistance to levamisole and possibly other anthelmintics.     

Haemonchus contortus: characterization of a glutamate binding site in unselected and ivermectin-selected larvae and adults.

A specific ivermectin-sensitive, glutamate binding site has been identified in the parasitic nematode Haemonchus contortus. Glutamate binding in H. contortus was saturable and occurred in a single class of high-affinity binding sites which appeared to have pharmacological properties different from those of mammalian glutamate receptors. Adult and larval forms of H. contortus had dramatically different glutamate binding kinetics, the larvae showing nearly up to 200-fold higher Bmax values and up to 9-fold increases in Kd values compared to adults. Treatment of adult H. contortus with the anthelmintic, ivermectin, decreased the Bmax value for glutamate binding in the susceptible strain but not in the resistant parasites. Furthermore, selection for ivermectin resistance was associated with a significant increase in Bmax for glutamate binding in adults and a similarly significant increase in glutamate binding affinity in larvae. These results suggest that the H. contortus glutamate binding site identified in this study may be involved in the phenomenon of ivermectin resistance.     

Resistance to ivermectin by Haemonchus contortus in goats and calves.

Efficacy of ivermectin on susceptible or resistant populations of the parasitic nematode Haemonchus contortus was determined in cattle and goats held in a barn. Goats were each infected with 3000 infective, ivermectin-susceptible or -resistant H. contortus larvae on day 0 and reinfected with 2000 infective larvae on day 24. Goats were treated orally with 600 micrograms kg-1 ivermectin on day 31. No significant differences were detected in blood packed cell volume (PCV) or total protein (TP), prepatent period, or epg among the four groups of goats that were each infected with one of four parasite strains (one susceptible, three resistant). There were no differences among the four parasite strains in the numbers of infective larvae that developed to the third larval stage from fecal cultures or in the viability of cultured infective larvae when held in the laboratory at 27 +/- 1 degrees C for 14 weeks. After treatment with ivermectin, there were significant differences among the parasite strains in PCV, TP, and epg. Total worm counts were reduced by 94 to 97% with three times the recommended dose. Immature and adult Skrjabinema ovis were also present in two treated goats. In a second test, one goat infected once with 10,000 infective larvae of a resistant strain of H. contortus and then treated with nine doses of ivermectin, increasing from 500 to 2000 micrograms kg-1 over a period of 133 days, had 35 adult worms at necropsy. In a third test, three calves were readily infected with an ivermectin-resistant strain of H. contortus from goats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The activity of drug-metabolizing enzymes and the biotransformation of selected anthelmintics in the model tapeworm Hymenolepis diminuta

The drug-metabolizing enzymes of some helminths can deactivate anthelmintics and therefore partially protect helminths against these drugs' toxic effect. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (albendazole, flubendazole, mebendazole) in the rat tapeworm Hymenolepis diminuta, a species often used as a model tapeworm. In vitro and ex vivo experiments were performed. Metabolites of the anthelmintics were detected and identified by HPLC with spectrofluorometric or mass-spectrometric detection. The enzymes of H. diminuta are able to reduce the carbonyl group of flubendazole, mebendazole and several other xenobiotics. Although the activity of a number of oxidation enzymes was determined, no oxidative metabolites of albendazole were detected. Regarding conjugation enzymes, a high activity of glutathione S-transferase was observed. A methyl derivative of reduced flubendazole was the only conjugation metabolite identified in ex vivo incubations of H. diminuta with anthelmintics. The results revealed that H. diminuta metabolized flubendazole and mebendazole, but not albendazole. The biotransformation pathways found in H. diminuta differ from those described in Moniezia expanza and suggest the interspecies differences in drug metabolism not only among classes of helminths, but even among tapeworms.     

Genetic Variability of the β-Tubulin Genes in Benzimidazole-Susceptible and -Resistant Strains of Haemonchus Contortus

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

A spectrum in the susceptibility of leishmanial strains to intracellular killing by murine macrophages

The susceptibility of 26 strains and clones of Leishmania to in vitro killing by lymphokine (LK)-activated macrophages was determined. A spectrum in the susceptibility of Leishmania to macrophage killing was observed. Some leishmanias were completely resistant to killing, including some but not all of the L. mexicana strains studied. This resistance was expressed in amastigotes and stationary growth-phase promastigotes, but not in logarithmic promastigotes. In contrast, some L. braziliensis parasites failed to survive within either activated or nonactivated macrophages. Between these two extremes were strains that survived within nonactivated macrophages, but were readily killed within activated macrophages. These included L. donovani, L. major, and some L. mexicana strains. Finally, one L. mexicana strain (WR357) was found to be susceptible to killing at high LK concentrations, but was relatively resistant at lower LK concentrations or at cutaneous temperatures. The observed differences in susceptibility to macrophage-mediated microbicidal activity may explain, in part, the variable pathogenesis of leishmanial infections.     

Localization of p-glycoprotein mRNA in the tissues of Haemonchus contortus adult worms and its relative abundance in drug-selected and susceptible strains

Membrane transporter P-glycoproteins (Pgps) are present in a number of nematode species, including Haemonchus contortus. Allelic variation in some Pgp genes has been found to be associated with resistance to the anthelmintic ivermectin (IVM), although functional verification of a role for Pgps in IVM resistance has yet to be demonstrated. By in situ hybridization, the distribution of Pgp mRNA was visualized in transverse cryosections of adult H. contortus, using a digoxigenin-labeled cDNA probe encoding the ATP-binding region of an H. contortus Pgp. The probe sequence targeted a conserved ATP-binding region of Pgp-A (97.9% identity). It also shared 49.7-71.1% identity with 11 other Pgp sequences previously identified in H. contortus and may hybridize these sequences to give an overall measure of the total P-glycoprotein mRNA. Staining was predominately localized along the intestinal tract of the worms, with the most intense staining localized in the pharynx and anterior intestine. In the mid- and posterior intestinal regions, staining was restricted to the luminal side of the intestine. Some staining was also associated with the vas deferens and the lateral hypodermal chords anterior to the nerve ring. Using densitometry, the levels of Pgp mRNA in the pharynges of unselected and IVM- and moxidectin (MOX)-selected strains of male and female H. contortus were compared. No differences were detected between the levels of expression of Pgp in the susceptible strain versus the IVM- or MOX-selected strains. Evidence in the literature suggests that not all Pgp homologues are linked to chemical resistance phenotypes. It is thus possible that expression of I of the H. contortus Pgps is altered in IVM-resistant strains but that this phenomenon was undetectable in our experiments.     

Trends and challenges in the effective and sustainable control of Haemonchus contortus infection in sheep. Review

Haemonchosis, with its very wide distribution, has become a very important production constraint in sheep farms in tropical, subtropical and temperate regions worldwide. Various intrinsic and extrinsic factors determine the survival of Haemonchus contortus and hence the development of the disease in the animal. In general, control of gastrointestinal nematode infestation in sheep relies heavily on anthelmintic treatments. However, the indiscriminate use of these drugs has led to the widespread emergence of drug resistant strains of parasites, that has necessitated the development and use of various parasite control methods such as grazing management, biological agents and vaccines and the selection of resistant breeds of animals, with or without moderate use of anthelmintics. The ultimate goal of such control programs is to enhance productivity, while minimising risks regarding drug resistance and consumer and environmental concerns. This review attempts to highlight the different methods employed in the control of haemonchosis in sheep and the practical limitations associated with both control programs and the internal and external factors associated with the parasite and its microenvironment.     

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.     

The fumarate reductase reaction of Haemonchus contortus and the mode of action of some anthelmintics

Prichard R. K. 1973. The fumarate reductase reaction of Haemonchus contortus and the mode of action of some anthelmintics. International Journal for Parasitology3: 409–417. Fumarate reductase activities in both thiabendazole susceptible and tolerant strains of Haemonchus contortus have been determined using spectrophotometric and radioisotopic methods. The effects of the anthelmintics, thiabendazole, cambendazole, 1-tetramisole, morantel tartrate, mebendazole and disophenol on the fumarate reductase system of these strains were assayed. Thiabendazole inhibited the fumarate reductase system of the susceptible strain, but no effect could be observed in the tolerant strain with a concentration of thiabendazole of 2 × 10^?3 M. Cambendazole inhibited the system in both strains, but the thiabendazole tolerant strain was significantly less susceptible to cambendazole than the thiabendazole susceptible strain. 1-tetramisole, morantel tartrate and disophenol inhibited the fumarate reductase system in both strains. Mebendazole was not found to affect the fumarate reductase system in either strain. The significance of the fumarate reductase system and its associated phosphorylation in the metabolism of helminths and in the susceptibility of helminths to anthelmintics is discussed.     

Specificity of the sensitivity of inbred mouse strains TPS, WR and CBA/LacY to the action of chemical mutagens

The cytogenetic effect of thioTEPA, ethyleneimine, mitomycin C, cyclophosphamide and phtorafurum in bone marrow cells of mouse strains TPS, WR, CBA/LacY was studied. Mice of the TPS strains were most sensitive to the action of all mutagens. Mice of the WR strain were sensitive to thioTEPA and phtorafurum but relatively resistant to mutagens which require metabolic preactivation, i. e. mitomycin C and cyclophosphamide. It was suggested to carry out the study of the cytogenetic activity of chemical compounds using the panel of TPS, WR, 101/H, C57BL/6Y, CBA/LacY strains.     

捻转血矛线虫丙硫咪唑耐药相关lncRNA的转录组学测序与分析

分析捻转血矛线虫敏感株和耐药株中长链非编码RNA (Long non-coding RNA,lncRNA)的表达谱,探讨lncRNA与捻转血矛线虫丙硫咪唑耐药机制的关联性,为捻转血矛线虫耐药机理提供依据。文中对捻转血矛线虫敏感株和耐药株进行cDNA测序文库构建,使用Illumina HiSeq 4000平台进行双端测序,筛选出差异的lncRNA,基于顺式调控(cis)和反式调控(trans)对差异显著的lncRNA进行靶基因预测,并对靶基因进行过Gene Ontology (GO)功能注释和KEGG Pathway富集分析,利用FPKM法估计lncRNA和mRNA的表达水平。结果表明,敏感株和耐药株候选lncRNA分别为6 377和6 356个,两个文库中筛选出168个差异显著的lncRNA,其中在敏感株中表达上调有92个,表达下调76个。筛选得到的差异显著lncRNA候选靶基因416个,这些基因共注释到641条GO terms和92条信号通路;其中富集到耐药性相关的通路有药物代谢-其他酶、药物代谢-细胞色素P450、细胞色素P450对异生素的代谢等。综上表明,部分lncRNA介导的靶基因与捻转血矛线虫丙硫咪唑耐药性相关,lncRNA在捻转血矛线虫耐药性中具有潜在的重要的调节作用。探究了对于敏感虫株和耐药虫株中lncRNA的表达谱,发现了敏感虫株和耐药虫株中差异表达的lncRNA,有助于找出捻转血矛线虫如何抵抗丙硫咪唑的发生机制,为探讨捻转血矛线虫丙硫咪唑耐药机制提供科学的依据。     

棉蚜抗吡虫啉品系和敏感品系主要解毒酶活性比较

通过生物测定和生物化学方法比较了棉蚜 Aphis gossypii 对吡虫啉抗性（约为7倍）和敏感品系几种主要解毒酶的活性。结果表明：氧化胡椒基丁醚对两个品系均无明显增效作用。抗性品系中羧酸酯酶和谷胱甘肽S-转移酶的比活力均明显高于敏感品系,抗性品系中羧酸酯酶的K_m值也显著高于敏感品系,说明抗性品系羧酸酯酶与底物的亲和力明显高于敏感品系。上述结果证明羧酸酯酶和谷胱甘肽S-转移酶活力增强在棉蚜对吡虫啉的抗性中起重要作用。     

Uptake of thiabendazole and its effects on glucose uptake and carbohydrate levels in the thiabendazole-resistant and susceptible Trichostrongylus colubriformis

Sangster N. C. and Prichard R. K. 1984. Uptake of thiabenzadole and its effects on glucose uptake and carbohydrate levels in thiabendazole-resistant and susceptible Trichostrongylus colubriformis. International Journal for Parasitology14:121–126. Some possible mechanisms of thiabenzadole (TBZ) resistance have been investigated in Trichostrongylus colubriformis. Uptake of TBZ and 3-0-methyl glucose and changes in carbohydrate levels in a TBZ-resistant and a TBZ-susceptible strain of T. colubriformis were measured after in vitro incubation, in the absence or presence of TBZ. The rate of TBZ uptake in adult worms was greater in the resistant strain but the difference was not statistically significant. Final concentrations of TBZ in resistant worms were significantly greater than in the susceptible strain, but there was no difference in TBZ uptake by eggs of either strain. A mechanism of resistance based on decreased drug uptake by resistant parasites is thus unlikely. Resistant worms absorbed 3-0-methyl glucose faster than susceptible parasites, however, this rate was unchanged in either strain when the worms were exposed to TBZ and indicates that the drug does not affect glucose uptake. Levels of glucose, trehalose and glycogen fell during the incubation with the decrease being more pronounced in the resistant strain. When the worms were exposed to TBZ the same changes in carbohydrate levels occurred except for resistant worms maintained anaerobically where levels were higher, showing that TBZ does not affect the rate of carbohydrate utilization.     

Biochemical characterization of hydrolytic and oxidative enzymes in insecticide resistant and susceptible strains of the German cockroach (Dictyoptera: Blattellidae).

We have identified resistance mechanisms in the German cockroach, Blattella germanica (L.), for propoxur and chlorpyrifos in strains of cockroaches that display multiresistance to several organophosphate and carbamate insecticides. The resistance mechanisms involve the combined effects of increased oxidative and hydrolytic metabolism and both strains are resistant to chlorpyrifos and propoxur. Experiments designed to test for similarity in metabolic enzymes suggest that, although the mechanisms involve similar processes, the enzymes responsible for insecticide detoxification are different in the two strains. Both resistant strains exhibited enhanced activity toward alpha-naphtholic esters relative to a standard susceptible strain; however, analysis of the progeny from resistant X susceptible crosses suggests that this general esterase activity is inherited differently than propoxur or chlorpyrifos resistance. Hybrids of the propoxur-resistant strain displayed the highest activity of all cockroaches tested, in contrast to hybrids of the chlorpyrifos-resistant strain, which were similar to the susceptible strain. Native gel electrophoresis of cytosolic preparations provided further evidence for differences in the pattern of hydrolytic enzymes and inheritance of resistance in the two strains. Analysis of components of the cytochrome P450-dependent monooxygenase system and activities toward model substrates indicate that the two resistance mechanisms also involve different oxidative processes. The propoxur-resistant strain displayed significantly higher levels of total cytochrome P450, but no other components were correlated with resistance. In contrast with the chlopyrifos-resistant strain, which was similar to the susceptible strain in all parameters measured, activity toward model substrates was higher in the propoxur-resistant strain than in any of the other strains and hybrids tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Schistosoma mansoni in Susceptible and Resistant Snail Strains Biomphalaria tenagophila: In Vivo Tissue Response and In Vitro Hemocyte Interactions

Schistosomiasis is a parasitic disease that is highly prevalent, especially in developing countries. Biomphalaria tenagophila is an important invertebrate host of Schistosoma mansoni in Brazil, with some strains (e.g. Cabo Frio) being highly susceptible to the parasite, whereas others (e.g. Taim) are completely resistant to infection. Therefore, B. tenagophila is an important research model for studying immune defense mechanisms against S. mansoni. The internal defense system (IDS) of the snail comprises hemocytes and hemolymph factors acting together to recognize self from non-self molecular patterns to eliminate the threat of infection. We performed experiments to understand the cellular defenses related to the resistance and/or susceptibility of B. tenagophila to S. mansoni. During the early stages of infection, fibrous host cells of both snail strains were arranged as a thin layer surrounding the sporocysts. However, at later stages of infection, the cellular reactions in resistant snails were increasingly more intense, with thicker layers surrounding the parasites, in contrast to susceptible strains. All parasites were damaged or destroyed inside resistant snails after 10 h of infection. By contrast, parasites inside susceptible snails appeared to be morphologically healthy. We also performed experiments using isolated hemocytes from the two strains interacting with sporocysts. Hemocyte attachment started as early as 1 h after initial infection in both strains, but the killing of sporocysts was exclusive to hemocytes from the resistant strain and was time course dependent. The resistant strain was able to kill all sporocysts. In conclusion, our study revealed important aspects of the initial process of infection related to immune defense responses of strains of B. tenagophila that were resistant to S. mansoni compared with strains that were susceptible. Such information is relevant for the survival or death of the parasites and so is important in the development of control measures against this parasite.     

Haemonchus contortus and Trichostrongylus colubriformis did not adapt to long-term exposure to sheep that were genetically resistant or susceptible to nematode infections

We tested the hypothesis that Haemonchus contortus and Trichostrongylus colubriformis would adapt to long-term exposure to sheep that were either genetically resistant or susceptible to H. contortus. Sheep genotypes were from lines with 10 years prior selection for low (resistant, R) or high (susceptible, S) faecal worm egg count (WEC) following H. contortus infection. Long-term exposure of H. contortus and T.colubriformis to R or S genotypes was achieved using serial passage for up to 30 nematode generations. Thus, we generated four nematode strains; one strain of each species solely exposed to R sheep and one strain of each species solely exposed to S sheep. Considerable host genotype differences in mean WEC during serial passage confirmed adequate nematode selection pressure for both H. contortus (R 4900 eggs per gram (epg), S 19,900 epg) and T. colubriformis (R 5300 epg, S 13,500 epg). Adaptation of nematode strain to host genotype was tested using seven cross-classified tests for H. contortus, and two cross-classified and one outbred genotype test for T. colubriformis. In the cross-classified design, where each strain infects groups of R, S or randomly bred control sheep, parasite adaptation would be indicated by a significant host genotype by nematode strain interaction for traits indicating parasite reproductive success; specifically WEC and, for H. contortus strains, packed cell volume. We found no significant evidence of parasite adaptation to host genotype (P > 0.05) for either the H. contortus or T. colubriformis strains. Therefore, we argue that nematodes will not adapt quickly to sheep bred for nematode resistance, where selection is based on low WEC, although selecting sheep using a subset of immune functions may increase adaptation risk. Our results support the hypothesis that nematode resistance is determined by many genes each with relatively small effect. In conclusion, selection of sheep for nematode resistance using WEC should be sustainable in the medium to long-term.