3种苯并咪唑类药物对大熊猫西氏贝蛔虫的驱虫效果初步观察

西氏贝蛔虫Baylisascaris schroederi是圈养大熊猫Ailuropoda melanoleuca最常见、危害最严重的一种体内寄生虫,采用药物驱虫是目前控制圈养大熊猫蛔虫病的主要措施。为了筛选有效的驱虫药物,本研究观察了3种苯并咪唑类药物(阿苯达唑片剂、芬苯达唑膏剂和甲苯咪唑片剂)对大熊猫蛔虫的驱虫效果,统计了驱虫前后粪检蛔虫卵转阴率及排虫情况。结果表明,除芬苯达唑按5 mg·kg^-1体质量口服,每天1次,连续服用2 d,效果较差外,3种药物按10 mg·kg^-1体质量口服,每天1次,连续服用2 d,用药安全且有较好的驱虫效果。     

Speckle interferometry applied to pharmacodynamic studies: evaluation of parasite motility

The work reported here describes the application of the optical technique known as dynamic speckle interferometry to evaluate the motility of nematode parasites exposed to different anthelmintic drugs. This technique, a well proven tool for assessing the time evolution of different phenomena, is here successfully used to quantify parasite motility in pharmacodynamic assays. The characterization of the pharmacological properties of anthelmintic drugs is critical to optimize their use in parasite control. Besides, the evaluation of nematode motility is a relevant indicator of the pharmacodynamic effect of anthelmintic drugs. The application of this approach to study the motility of Haemonchus contortus (used as a model of nematode parasites) larvae exposed to different drugs is presented, showing its usefulness.Abbreviations ABZ albendazole - BZD benzimidazole - IVM ivermectin - LVM levamisole     

Understanding anthelmintic resistance: the need for genomics and genetics

Anthelmintic resistance is a major problem for the control of many parasitic nematode species and has become a major constraint to livestock production in many parts of the world. In spite of its increasing importance, there is still a poor understanding of the molecular and genetic basis of resistance. It is unclear which mutations contribute most to the resistance phenotype and how resistance alleles arise, are selected and spread in parasite populations. The main strategy used to identify mutations responsible for anthelmintic resistance has been to undertake experimental studies on candidate genes. These genes have been chosen predominantly on the basis of our knowledge of drug mode-of-action and the identification of mutations that can confer resistance in model organisms. The application of these approaches to the analysis of benzimidazole and ivermectin resistance is reviewed and the reasons for their relative success or failure are discussed. The inherent limitation of candidate gene studies is that they rely on very specific and narrow assumptions about the likely identity of resistance-associated genes. In contrast, forward genetic and functional genomic approaches do not make such assumptions, as illustrated by the successful application of these techniques in the study of insecticide resistance. Although there is an urgent need to apply these powerful approaches to anthelmintic resistance research, the basic methodologies and resources are still lacking. However, these are now being developed for the trichostrongylid nematode Haemonchus contortus and the current progress and research priorities in this area are discussed.     

New technologies for the control of human hookworm infection

Since the 1990s, the major approach to hookworm control has been morbidity reduction in school-aged children by periodic deworming with benzimidazoles. Now, efforts are underway to determine the feasibility of integrating deworming with control programs that target other neglected tropical diseases. However, the sustainability of benzimidazole deworming for hookworm is of concern because of the variable efficacy of mebendazole, high rates of post-treatment reinfection and possible development of drug resistance. This requires parallel efforts to develop new and complementary hookworm control tools, such as new anthelmintic drugs (e.g. tribendimidine) and a recombinant hookworm vaccine. It is hoped that, ultimately, anthelmintic vaccination will be linked to deworming as part of an expanded control package.     

A research agenda for helminth diseases of humans: intervention for control and elimination

Recognising the burden helminth infections impose on human populations, and particularly the poor, major intervention programmes have been launched to control onchocerciasis, lymphatic filariasis, soil-transmitted helminthiases, schistosomiasis, and cysticercosis. The Disease Reference Group on Helminth Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), was given the mandate to review helminthiases research and identify research priorities and gaps. A summary of current helminth control initiatives is presented and available tools are described. Most of these programmes are highly dependent on mass drug administration (MDA) of anthelmintic drugs (donated or available at low cost) and require annual or biannual treatment of large numbers of at-risk populations, over prolonged periods of time. The continuation of prolonged MDA with a limited number of anthelmintics greatly increases the probability that drug resistance will develop, which would raise serious problems for continuation of control and the achievement of elimination. Most initiatives have focussed on a single type of helminth infection, but recognition of co-endemicity and polyparasitism is leading to more integration of control. An understanding of the implications of control integration for implementation, treatment coverage, combination of pharmaceuticals, and monitoring is needed. To achieve the goals of morbidity reduction or elimination of infection, novel tools need to be developed, including more efficacious drugs, vaccines, and/or antivectorial agents, new diagnostics for infection and assessment of drug efficacy, and markers for possible anthelmintic resistance. In addition, there is a need for the development of new formulations of some existing anthelmintics (e.g., paediatric formulations). To achieve ultimate elimination of helminth parasites, treatments for the above mentioned helminthiases, and for taeniasis and food-borne trematodiases, will need to be integrated with monitoring, education, sanitation, access to health services, and where appropriate, vector control or reduction of the parasite reservoir in alternative hosts. Based on an analysis of current knowledge gaps and identification of priorities, a research and development agenda for intervention tools considered necessary for control and elimination of human helminthiases is presented, and the challenges to be confronted are discussed.     

Review of methodology for the determination of benzimidazole residues in biological matrices

Benzimidazoles are anthelmintic agents widely used in the treatment of parasitic infections in a range of species and as fungicidal agents in the control of spoilage of crops during storage and transport. In this paper, the more important benzimidazoles are introduced and their pharmacological effects and physiochemical properties discussed. The metabolism of these drugs is described relating to the occurrence and persistence of residues in biological matrices, providing information for selection of suitable matrices and target residues for testing. Methods for determination of benzimidazoles are reviewed for a range of biological matrices. The importance of selecting suitable extraction and clean-up procedures is discussed, along with the difficulties encountered in adapting single residue methods to multi-residue methods. The importance of suitable detection systems for determination of benzimidazoles, namely, screening, HPLC, GC and confirmatory methods is described in detail. The future for benzimidazole residue analysis is discussed, focusing on selection of appropriate residues for screening methods and protocols for confirmation of benzimidazole residues.     

Production losses and control of helminths in ruminants of temperate regions

In this review of research on production losses and control principles in ruminant helminthiasis, the discussion primarily refers to nematode infections under conditions in Europe.Among the loss-producing effects of nematodes much interest has recently been centered on the possible reduction in milk yield of cows brought about by Ostertagia ostertagi. Another important factor of new interest is the inappetence associated with trichostrongyle infections. Furthermore, an example is given of hidden losses in association with the adoption of less profitable production systems to avoid parasitism.It is imperative that scientists and advisors are aware of newer management practices which engender risks of loss-producing infections. High stocking rates and the spreading of slurry onto pastures are examples of procedures which may increase the hazards of parasitism. To implement practices of routine control it is important to examine the regularity with which the herbage infectivity patterns occur. Principles of controlling nematode infections by systems of grazing management and by strategic anthelmintic treatments are discussed and illustrated with examples. Finally, problems of integrating control in farm enterprises are discussed.     

A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility

Background

Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods.

Methodology/Principal Findings

Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC₅₀ values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus.

Conclusions/Significance

Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.     

Risk Factors for High Endoparasitic Burden and the Efficiency of a Single Anthelmintic Treatment of Danish Horses

A questionnaire survey regarding endoparasite control practices in Danish horse herds was carried out in 1995. The participating veterinarians and herd owners were sampled using convenience and purposive sampling. In the analysis of risk factors for development of a high endoparasitic burden (>200 eggs per gram faeces) 903 horses were sampled and the analysis of the efficiency of a single anthelmintic treatment was based on 605 horses. The following factors had a significant effect on the endoparasitic burden: herd type, age of the horses, use of pasture rotation, anthelmintic treatment of horses visiting the herd, use of an adviser in the planning of endoparasite control and advice regarding pasture rotation. An interaction between pasture rotation and advice regarding pasture rotation was found, but due to high collinearity this was not reported. The factors influencing significantly on the reduction of the faecal egg count after a single anthelmintic treatment were the type of herd, the age of the horse, the drug used, and the anthelmintic-resistance-status of the herd. A negative effect of permanent pastures was observed. If pasture hygiene was performed on the advice of the veterinarian, the effect of a single anthelmintic treatment was less compared to a single anthelmintic treatment without any advice. An interaction between the treatment group and the resistance-status of the herd was found. Additional factors, normally accounted for, when endoparasites and anthelmintic resistance is discussed, were investigated, but not found significant in this study.     

Population dynamics of Trichostrongylus colubriformis in sheep: computer model to simulate grazing systems and the evolution of anthelmintic resistance

A computer model was developed to simulate Trichostrongylus colubriformis populations, their level of resistance to the common anthelmintics, host mortalities and acquired immunity. Predictions were based on sheep management practices such as lambing, weaning, sheep/paddock rotation, anthelmintic treatment, the use of controlled release devices (capsules) for anthelmintic delivery and daily meteorological records to determine the development and survival of infective larvae (L3) on pasture. Evolution of drug resistance was determined by a simple genetic system which allowed for up to three genes, each with two alleles, to give a maximum of 27 genotypes associated with one drug or three genotypes for each of three drugs. The model was validated against egg counts, L3 counts on pasture and host mortalities observed in a grazing trial, however, aspects of the model such as the development of drug resistance and use of the model in a variety of climatic zones have yet to be tested against field observations. The model was used to examine the impact of grazing management and capsule use on anthelmintic resistance and sheep production over 20 years using historical weather data. Predictions indicated that grazing management can play a dominant role in parasite control and that capsule use will reduce sheep mortalities and production losses, and in some circumstances will not cause a substantial increase in anthelmintic resistance for up to 5 years.     

Prevalence of anthelmintic resistance in gastrointestinal nematodes of dairy goats under extensive management conditions in southwestern France

The occurrence of benzimidazole (BZ) and levamisole resistance was investigated in 18 randomly selected dairy goat herds located in southwestern France and characterized by extensive management. On each of the 18 farms, 45 adult goats were randomly allocated into three groups of 15 animals each: an untreated control group, a group that was orally administered fenbendazole (10 mg kg(-1) body weight) and a group that received orally a levamisole drench (12 mg kg(-1) body weight). Individual faecal egg counts and pooled larval cultures were done 10 days after anthelmintic treatment. Naive lambs were infected with larvae obtained from control and fenbendazole treated groups and were necropsied 35 days after infection for worm recovery. Faecal egg count reductions (FERC) were calculated for fenbendazole and levamisole and, when less than 95 per 100, were considered as indicative of anthelmintic resistance. An in vitro egg hatch test (EHT) was conducted with thiabendazole on eggs isolated from pooled faeces of fenbendazole treated goats in nine farms. Faecal egg count reductions indicated the occurrence of benzimidazole resistance in 15 out of 18 farms. Among these farms, nine had EHT values above 0.1 microg thiabendazole ml(-1) confirming the benzimidazole resistance status. Levamisole resistance was detected in two farms through FECR. Based on necropsy results, the prevalence of benzimidazole resistance was higher in Trichostrongylus colubriformis, medium in Haemonchus contortus and lower in Teladorsagia circumcincta. In nine farms the benzimidazole resistance was monospecific whereas multispecific resistance was found in the six remaining farms. A negative relationship was found between FECR for fenbendazole and the average number of anthelmintic treatments given per year on the farm. Despite extensive management including a low number of treatments, the prevalence of benzimidazole resistance was very high suggesting that the repeated and sometimes exclusive use of benzimidazole drugs, even at low frequency, is probably the main cause in developing nematode resistance in dairy goat herds. The importance of other factors such as under-dosing or buying animals already carrying resistant nematodes are discussed.     

Anthelmintic tolerance in free-living and facultative parasitic isolates of Halicephalobus (Panagrolaimidae)

SUMMARY Studies on anthelmintic resistance in equine parasites do not include facultative parasites. Halicephalobus gingivalis is a free-living bacterivorous nematode and a known facultative parasite of horses with a strong indication of some form of tolerance to common anthelmintic drugs. This research presents the results of an in vitro study on the anthelmintic tolerance of several isolates of Halicephalobus to thiabendazole and ivermectin using an adaptation of the Micro-Agar Larval Development Test hereby focusing on egg hatching and larval development. Panagrellus redivivus and Panagrolaimus superbus were included as a positive control. The results generally show that the anthelmintic tolerance of Halicephalobus to both thiabendazole and ivermectin was considerably higher than that of the closely related Panagrolaimidae and, compared to other studies, than that of obligatory equine parasites. Our results further reveal a remarkable trend of increasing tolerance from fully free-living isolates towards horse-associated isolates. In vitro anthelmintic testing with free-living and facultative parasitic nematodes offers the advantage of observing drug effect on the complete life cycle as opposed to obligatory parasites that can only be followed until the third larval stage. We therefore propose Halicephalobus gingivalis as an experimental tool to deepen our understanding of the biology of anthelmintic tolerance.     

Biological control of ruminant trichostrongylids by Arthrobotrys oligospora, a predacious fungus

Sheep and cattle are prey to many parasitic worms, including the trichostrongylid nematodes. Conventional control involves the use of anthelmintic drugs, but in this article Hadji Ahmad Hashmi and Roger Connan discuss the possible biological control of these nematodes by means of a 'living lasso', the predacious fungus Arthrobotrys.     

Drug transfer into target helminth parasites

The pharmacokinetics of an anthelmintic drug includes the time course of drug absorption, distribution, metabolism and elimination from the host and determines the concentration of the active drug that reaches the location of the parasite. However, the action of the anthelmintic also depends on the ability of the active drug to reach its specific receptor within the target parasite. Thus, drug entry and accumulation in target helminths are important issues when considering how best to achieve optimal efficacy. Passive drug transfer through the external helminth surface is the predominant entry mechanism for most widely used anthelmintics and is discussed in this article. Despite the structural differences between the external surface of nematodes (the cuticle) and the external surface of cestodes and trematodes (the tegument), the mechanism of drug entrance into both types of helminth depends on the lipophilicity of the anthelmintic and this is the major physicochemical determinant for the drug to reach a therapeutic concentration in the target parasite. Understanding the processes that regulate drug transfer into helminth parasites is an important aspect in improving the control of parasites in human and veterinary medicine.     

Changes in anthelmintic resistance status of Haemonchus contortus and Trichostrongylus colubriformis exposed to different anthelmintic selection pressures in grazing sheep

This experiment was designed to study, over a 5-year-period, the effect of different frequencies of treatment with three different anthelmintic groups, namely, benzimidazoles, levamisole and ivermectin, and different frequencies of alternation between them, on existing levels of anthelmintic resistance in the nematode parasites Haemonchus contortus and Trichostrongylus colubriformis of grazing sheep. No evidence of ivermectin resistance emerged, even in suppressively treated groups. Likewise, H. contortus failed to develop resistance to levamisole under a similar selection regimen. Thiabendazole was shown to select positively against levamisole resistance in T. colubriformis resulting in significantly greater susceptibility to this drug than for the natural reversion which occurred in the untreated control. There was no evidence that an anthelmintic treatment combined with a movement of sheep to pastures of low infectivity selected more rapidly for resistance than where the same number of treatments were given to set-stocked sheep. Rotation between anthelmintic groups at yearly intervals appeared to be more beneficial in delaying resistance than rotation of drugs with each treatment.     

Evaluation of the Egg Hatch Assay and the Larval Migration Inhibition Assay to detect anthelmintic resistance in cattle parasitic nematodes on farms

Resistance to anthelmintic drugs, particularly to the widely used benzimidazoles (BZs) and macrocyclic lactones (MLs) is an increasing problem in cattle industries worldwide. Reliable methods for the assessment of anthelmintic efficacy in the field are required in order to react before resistance becomes an obvious problem on individual properties. The ability of the Egg Hatch Assay (EHA) and the Larval Migration Inhibition Assay (LMIA) to detect anthelmintic resistance under field conditions was evaluated on cattle farms in Northern Germany. As published previously Faecal Egg Count Reduction Test (FECRT) was performed using oral albendazole (Valbazen®) or injectable ivermectin (Ivomec®). Herein the FECRT results described earlier were compared with data from EHAs or LMIAs, respectively, performed with eggs from fresh faeces or larvae from faecal cultures of the tested animals before and after treatment. The obtained EC₅₀ values allowed the assessment of efficacy of albendazole and ivermectin on farm level. The results of the FECRTs and the results of both in vitro assays were comparable. In comparison to the FECRT the in vitro assays are less time, labour and cost intensive and are able to assess the susceptibility status of a worm population without treatment. Therefore both are beneficial alternatives for the reliable detection of reduced efficacy of these two drug classes on farms.     

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole in vitro

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC₅₀ value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity.     

Drug-Induced Exposure of Schistosoma mansoni Antigens SmCD59a and SmKK7

BackgroundSchistosomiasis is a serious health problem especially in developing countries and affects more than 243 million people. Only few anthelmintic drugs are available up to now. A major obstacle for drug treatment is the different developmental stages and the varying host compartments during worm development. Anthelmintic drugs have been tested mainly on adult schistosomes or freshly transformed cercariae. Knowledge concerning the larval stages is lacking.ConclusionThis study has revealed marked differences in anthelmintic drug effects against larvae. Drug treatment increases surface antigen presentation and renders larvae accessible to antibody attack.     

Anthelmintics as a risk factor in intestinal obstruction by Ascaris lumbricoides in children]

In a retrospective study the authors analyzed the clinical records of 199 children ages one month to 16 years hospitalized, with the diagnosis of intestinal ascariasis, in the Instituto Nacional de Pediatria of Mexico from 1984 to 1999. The purpose of the study was to evaluate the use of anthelmintics drugs as a risk factor of intestinal obstruction by A. lumbricoides. Two groups were made for the study: Group A (n = 66) of children who presented intestinal obstruction, Group B (n = 133) children with no complications. A comparative analysis of clinical data of both groups was made by means of chi square with Yates correction and a stratified analysis by means of chi square. Possible confusing elements were overcrowding, age and the use of antiparasitic drugs. The calculus of risk factors for intestinal obstruction by A. lumbricoides was done by means of contingency tables of 2 x 2 and odds ratio with an IC of 95%. The significant risk factors were included in a model of logistics regression with an impact variable consting in the presence or absence of intestinal obstruction in order to establish a multivariate model of predictive risk at level of significance of p < 0.05. Twenty-seven patients (40.90%) in group A (n = 66) were given anthelmintics medications prior to the intestinal obstruction: mebendazol, 14 (51-85%); two, albedazol (7.4%); eight, a non-specified anthelmintic (29.6%). In addition, an anthelmintic medication without a specified time of ingestion: two with mebendazol and one with piperazine (11.3%). In the case of mebendazol, the drug most frequently associated with intestinal obstruction, seven patients received it on the same day of the obstruction; five patients received it between one and seven days prior to the obstruction; two received it seven days prior to the complication. In the control group, only 7% had taken the anthelmintic one to seven days before the diagnosis of uncomplicated intestinal ascariasis diagnosis was made. With the step by step (Backward) logistic regression conditioned by the treatment variable with an anthelmintic, an X2 = 38.15 gl, p < 0.000 was obtained for which reason it was considered by A. lumbricoides. Of the probable risk factors analyzed in this study, the only one capable of influencing and predicting the presentation of intestinal obstruction by A. lumbricoides in children, was the prior anthelmintic treatment particularly with mebendazol.