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.     

Benzimidazole resistance of sheep nematodes in Norway confirmed through controlled efficacy test

ABSTRACT: BACKGROUND: Resistance against benzimidazoles (BZ) has recently been detected in Norwegian sheep flocks through a large scale prevalence survey based on the faecal egg count reduction test (FECRT). The use of this test in combination with bulk larval culture only gives an indication of which gastrointestinal nematodes genera that are involved and these results have to be confirmed by a controlled efficacy test (CET) to get accurate information about resistant nematodes populations at species level. A CET was therefore performed with larvae from two flocks where BZ resistance was previously detected through FECRT. RESULTS: The latter test confirmed the previous results in both flocks. In flock A, the BZ resistant nematode population consisted solely of Haemonchus contortus, whereas H. contortus and Teladorsagia circumcincta comprised the resistant worm population in flock B. CONCLUSIONS: Some discrepancies that have been recorded between FECRT and CET results regarding time for post-treatment coproscopical examination and a temporary suppression of faecal egg excretion are discussed.     

Adaptation of gastrointestinal nematode parasites to host genotype: single locus simulation models

Background

Breeding livestock for improved resistance to disease is an increasingly important selection goal. However, the risk of pathogens adapting to livestock bred for improved disease resistance is difficult to quantify. Here, we explore the possibility of gastrointestinal worms adapting to sheep bred for low faecal worm egg count using computer simulation. Our model assumes sheep and worm genotypes interact at a single locus, such that the effect of an A allele in sheep is dependent on worm genotype, and the B allele in worms is favourable for parasitizing the A allele sheep but may increase mortality on pasture. We describe the requirements for adaptation and test if worm adaptation (1) is slowed by non-genetic features of worm infections and (2) can occur with little observable change in faecal worm egg count.

Results

Adaptation in worms was found to be primarily influenced by overall worm fitness, viz. the balance between the advantage of the B allele during the parasitic stage in sheep and its disadvantage on pasture. Genetic variation at the interacting locus in worms could be from de novo or segregating mutations, but de novo mutations are rare and segregating mutations are likely constrained to have (near) neutral effects on worm fitness. Most other aspects of the worm infection we modelled did not affect the outcomes. However, the host-controlled mechanism to reduce faecal worm egg count by lowering worm fecundity reduced the selection pressure on worms to adapt compared to other mechanisms, such as increasing worm mortality. Temporal changes in worm egg count were unreliable for detecting adaptation, despite the steady environment assumed in the simulations.

Conclusions

Adaptation of worms to sheep selected for low faecal worm egg count requires an allele segregating in worms that is favourable in animals with improved resistance but less favourable in other animals. Obtaining alleles with this specific property seems unlikely. With support from experimental data, we conclude that selection for low faecal worm egg count should be stable over a short time frame (e.g. 20 years). We are further exploring model outcomes with multiple loci and comparing outcomes to other control strategies.     

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.     

Experimental infection of pigs with three dose levels of Trichuris suis

The objective of the study was to follow the course of Trichuris suis infection in pigs given infective eggs at low (400 eggs), medium (4,000 eggs) and high inoculation dose (40,000 eggs), respectively. Interestingly, despite a 100-fold difference in dose level no significant difference was found in either blood parameters, total faecal egg excretion, fecundity or worm burdens at necropsy 12 weeks post inoculation. The highest and lowest median faecal egg output was found in the medium and high dose group, respectively. With increasing dose level, worm size, establishment and prevalence of T. suis positive pigs decreased while worms were dislocated aborally. In addition there was a highly significant correlation between female worm burden and faecal egg excretion.     

Rhythmic behaviour of intestinal helminths in rodents

Intestinal helminths exhibit two types of circadian rhythms, either involving the migration of adults worms ( Hymenolepis and Syphacia ) within the gut or periodic release of eggs by female worms ( Aspiculuris and Heligmosomoides ) to the exterior of the host to maximize transmission efficiency. By modifying the feeding regimes of rodents, there is clear evidence that these rhythmic processes are directly linked with the digestive and defaecating activities of the host with neurohormonal control mechanisms likely to be involved at least in the migratory behaviour of adult worms. Furthermore, in Heligmosomoides polygyrus rhythmic egg output appears to be dependent upon parasite age and the population density of the host and parasite. A brief reference is also made to studies on long-term rhythms of egg output by intestinal nematodes and their effect on patterns of host faecal production.     

A molecular diagnostic tool to replace larval culture in conventional faecal egg count reduction testing in sheep

The accurate diagnosis of parasitic nematode infections in livestock (including sheep and goats) is central to their effective control and the detection of the anthelmintic resistance. Traditionally, the faecal egg count reduction test (FECRT), combined with the technique of larval culture (LC), has been used widely to assess drug-susceptibility/resistance in strongylid nematodes. However, this approach suffers from a lack of specificity, sensitivity and reliability, and is time-consuming and costly to conduct. Here, we critically assessed a specific PCR assay to support FECRT, in a well-controlled experiment on sheep with naturally acquired strongylid infections known to be resistant to benzimidazoles. We showed that the PCR results were in close agreement with those of total worm count (TWC), but not of LC. Importantly, albendazole resistance detected by PCR-coupled FECRT was unequivocally linked to Teladorsagia circumcincta and, to lesser extent, Trichostrongylus colubriformis, a result that was not achievable by LC. The key findings from this study demonstrate that our PCR-coupled FECRT approach has major merit for supporting anthelmintic resistance in nematode populations. The findings also show clearly that our PCR assay can be used as an alternative to LC, and is more time-efficient and less laborious, which has important practical implications for the effective management and control strongylid nematodes of sheep.     

In vitro and in vivo studies on the bioactivity of a ginger (Zingiber officinale) extract towards adult schistosomes and their egg production

The bioactivity of an ethyl acetate extract of ginger (Zingiber officinale) towards Schistosoma mansoni adult pairs, both cultured in vitro and in vivo in laboratory mice, was investigated by monitoring worm mortality and fecundity. In vitro, a concentration of 200 mg l(-1) of extract killed almost all worms within 24 h. Male worms seemed more susceptible than female under these conditions. Cumulative egg output of surviving worm pairs in vitro was considerably reduced when exposed to the extract. For example, after 4 days of exposure to 50 mg l(-1), cumulative egg output was only 0.38 eggs per worm pair compared with 36.35 for untreated worms. In vivo efficacy of the extract was tested by oral and subcutaneous delivery of 150 mg kg(-1) followed by assessment of worm survival and fecundity. Neither delivery route produced any significant reduction in worm numbers compared with untreated controls. Worm fecundity was assessed in vivo by cumulative egg counts per liver at 55 days post infection with mice treated subcutaneously. Such infections showed egg levels in the liver of about 2000 eggs per worm pair in 55 days, in both treated and control mice, with no significant difference between the two groups. To ensure that density-dependent effects did not confound this analysis, a separate experiment demonstrated no such influence on egg output per worm pair, at intensities between 1 and 23 worms per mouse.     

Cattle nematodes resistant to macrocyclic lactones: Comparative effects of P-glycoprotein modulation on the efficacy and disposition kinetics of ivermectin and moxidectin

The role of the drug efflux pump, known as P-glycoprotein, in the pharmacokinetic disposition (host) and resistance mechanisms (target parasites) of the macrocyclic lactone (ML) antiparasitic compounds has been demonstrated. To achieve a deeper comprehension on the relationship between their pharmacokinetic and pharmacodynamic behaviors, the aim of the current work was to assess the comparative effect of loperamide, a well-established P-glycoprotein modulator, on the ivermectin and moxidectin disposition kinetics and efficacy against resistant nematodes in cattle. Fifty (50) Aberdeen Angus male calves were divided into five (5) experimental groups. Group A remained as an untreated control. Animals in the other experimental Groups received ivermectin (Group B) and moxidectin (Group C) (200 μg/kg, subcutaneuosly) given alone or co-administered with loperamide (0.4 mg/kg, three times every 24 h) (Groups D and E). Blood samples were collected over 30 days post-treatment and drug plasma concentrations were measured by HPLC with fluorescence detection. Estimation of the anthelmintic efficacy for the different drug treatments was performed by the faecal egg count reduction test (FECRT). Nematode larvae were identified by pooled faecal cultures for each experimental group. Cooperia spp. and Ostertagia spp. were the largely predominant nematode larvae in pre-treatment cultures. A low nematodicidal efficacy (measured by the FECRT) was observed for both ivermectin (23%) and moxidectin (69%) in cattle, which agrees with a high degree of resistance to both molecules. Cooperia spp. was the most abundant nematode species recovered after the different drug treatments. The egg output reduction values increased from 23% to 50% (ivermectin) and from 69% to 87% (moxidectin) following their co-administration with loperamide. Enhanced systemic concentrations and an altered disposition of both ML in cattle, which correlates with a tendency to increased anthelmintic efficacy, were observed in the presence of loperamide. Overall, the in vivo modulation of P-glycoprotein activity modified the kinetic behavior and improved the efficacy of the ML against resistant nematodes in cattle. The work provides further evidence on the high degree of resistance to ML in cattle nematodes and, shows for the first time under field conditions, that modulation of P-glycoprotein may be a valid pharmacological approach to improve the activity and extend the lifespan of these antiparasitic molecules.     

Anthelmintic efficacy of Achillea millifolium against gastrointestinal nematodes of sheep: in vitro and in vivo studies

Achillea millifolium Linn., commonly called 'Pehl-ghasa', is used by farmers in traditional animal health care as a vermifuge. The objective of this study was to evaluate the anthelmintic efficacy of crude aqueous extracts and crude ethanolic extracts of entire A. millifolium against the gastrointestinal nematodes of sheep. The worm motility inhibition assay was used for in vitro studies and faecal egg count reduction assay was used for in vivo studies. In vitro studies revealed significant anthelmintic effects of aqueous extracts and ethanolic extracts on live Haemonchus contortus worms (P < 0.05) as evident from their paralysis and/or death at 8 h post exposure. Aqueous extracts of A. millifolium resulted in a mean worm motility inhibition of 94.44%, while ethanolic extracts resulted in mean worm motility inhibition of 88.88%. The mean mortality index of aqueous extracts was 0.95 while for ethanolic extracts it was 0.9. The lethal concentration 50 was 0.05 mg ml(-1) for aqueous extracts and 0.11 mg ml(-1) for ethanolic extracts. The in vivo anthelmintic activity of aqueous and ethanolic extracts of A. millifolium demonstrated a maximum (88.40%) nematode egg count reduction in sheep treated with aqueous extracts at 2 g kg(-1) body weight on day 15 after treatment. Ethanolic extracts resulted in a maximum of 76.53% reduction in faecal egg counts on day 15 after treatment with 2 g kg(-1) body weight. Thus, the aqueous extracts exhibited greater anthelmintic activity under both in vitro and in vivo conditions, and could be due to the presence of water-soluble active principle/s in A. millifolium. It is concluded that the entire plant of A. millifolium possesses significant anthelmintic activity and could be a potential alternative for treating cases of helminth infections in ruminants.     

Merino ewes bred for parasite resistance reduce larval contamination onto pasture during the peri-parturient period

The peri-parturient period is crucial for controlling worms as the acquired immunity of ewes is disrupted, resulting in an increase in faecal worm egg counts. Two hypotheses were tested in this experiment - that ewes bred for worm resistance would have lower faecal worm egg counts than unselected control ewes, during late pregnancy and lactation, under similar but separate grazing areas; and also that numbers of infective nematode larvae would be lower on pastures grazed by resistant ewes than pastures grazed by unselected control ewes. Faecal samples were collected from resistant and unselected ewes in late pregnancy and early lactation, during the winter rainfall season, and analysed for numbers of Trichostrongylus colubriformis and Teladorsagia circumcincta. Pasture samples were taken 1 week before and 7 weeks after lambing started and analysed for infective larvae. In all sheep, worm egg counts rose 2 weeks prior to lambing and continued into lactation. Worm egg counts were significantly lower in the resistant ewes from 1 week before lambing to 2 weeks after lambing. There were no differences in egg counts between single- and twin-bearing ewes in the resistant line. However, twin-bearing control ewes had significantly higher egg counts than single-bearing control ewes. Following lactation, plots grazed by resistant ewes had substantially less contamination with T. colubriformis larvae, but there were no differences in numbers of T. circumcincta larvae. Our results demonstrate that sheep bred for worm resistance has lower worm burdens during the peri-parturient phase and that lambs born to resistant ewes face a lower larval challenge during their introduction to grazing. In our environment, selection for low worm egg counts has produced sheep highly resistant to T. colubriformis, but has had less impact on resistance towards T. circumcincta.     

The detection limits for estimates of infection intensity in schistosomiasis mansoni established by a study in non-human primates

In human schistosomiasis mansoni, it is impossible to directly determine worm burden and hence infection intensity, so surrogates must be used. Studies on non-human primates revealed a linear relationship between worm burden and three surrogates, faecal egg output, circulating anodic and circulating cathodic antigens. By regression, the thresholds of detection were determined as 40, 24 and 47 worms, respectively. These observations provide a quantitative basis for the contention that low intensity infections in humans are being missed. The significance for estimates of disease prevalence, evaluation of the effects of chemotherapy and the implementation of vaccine trials is emphasised.     

Evaluation of anthelmintic activity of Iris hookeriana against gastrointestinal nematodes of sheep

The objective of this study was to evaluate the anthelmintic efficacy of Iris hookeriana Linn. rhizome against gastrointestinal nematodes of sheep. A worm motility inhibition assay was used for in vitro study and a faecal egg count reduction assay was used for an in vivo study. The in vitro study revealed anthelmintic effects of crude aqueous extracts and crude ethanolic extracts on live Trichuris ovis worms (P < or = 0.05) as evident from their paralysis and/or death at 8 h after exposure. The aqueous extracts of I. hookeriana resulted in a mean worm motility inhibition of 54.0%, while ethanolic extracts resulted in a mean worm motility inhibition of 84.6%. The mean mortality index of aqueous extracts was 0.55, while for ethanolic extracts it was 0.85. The lethal concentration 50 for aqueous extracts was 0.45 mg ml- 1 and for ethanolic extracts it was 0.15 mg ml- 1. The in vivo anthelmintic activity of aqueous and ethanolic extracts of I. hookeriana in sheep naturally infected with mixed species of gastrointestinal nematodes demonstrated a maximum (45.62%) egg count reduction in sheep treated with ethanolic extracts at 2 g kg- 1 body weight on day 10 after treatment, closely followed by ethanolic extracts at 1 g kg- 1 body weight on day 10 after treatment (43.54% egg count reduction). The aqueous extracts resulted in a maximum of 31.53% reduction in faecal egg counts on day 10 after treatment with 1 g kg- 1 body weight. Thus ethanolic extracts exhibited greater anthelmintic activity under both in vitro and in vivo conditions; this could be due to the presence of alcohol-soluble active ingredients in I. hookeriana. From the present study it can be suggested that I. hookeriana rhizome exhibited significant anthelmintic activity against gastrointestinal nematodes of sheep and has the potential to contribute to the control of gastrointestinal nematode parasites of small ruminants.     

An evaluation of the efficacy of monensin against Fasciola hepatica in the albino rat

The in vivo efficacy of monensin against Fasciola hepatica was determined in the albino rat. The results were variable, with monensin generally showing greater activity against juvenile (two-week-old) than adult (12-week-old) flukes. Significant (p less than 0.005) reductions in worm burdens were obtained only following treatment of adult flukes with 2 X 10 mg/kg monensin (52.9% efficacy), and of juvenile flukes with 1 X 10 mg/kg (42.4% efficacy) and 2 X 10 mg/kg (56.23% efficacy). Monensin administered in the diet (200 ppm) had a negligible effect on juvenile and adult fluke burdens. Prophylactic treatment of rats with monensin (100 ppm) produced a 45.5% efficacy, but this was not statistically significant. At doses of 1 X 5 mg/kg and 2 X 2.5 mg/kg, monensin had little effect on egg output by F. hepatica. No clear relationship was established between egg output and worm burden, and so faecal egg counting was not a reliable indicator of fluke burdens in the rat.     

Class I antigens of the bovine major histocompatibility system are weakly associated with variation in faecal worm egg counts in naturally infected cattle

Three bulls selected for high faecal worm egg counts and three bulls selected for low faecal worm egg counts were mated to Africander-Hereford cross cows. Faecal worm egg counts were taken on four occasions from the 132 offspring. Also, each animal was typed for 32 class I antigens of the bovine major histocompatibility system (BoLA). Least squares analysis of variance showed that line, sex and some of the antigens were associated with differences in worm egg output in the faeces. After adjusting for the effects of line and sex, cattle with antigen W9 had about twice as many worm eggs in their faeces as cattle without W9; cattle with antigen CA45 had about half the concentration of faecal worm eggs as cattle without CA45. However, the antigen associations were of borderline significance at the 5% level and more work in additional populations is necessary to confirm these associations.     

Population dynamics of Trichostrongylus colubriformis in sheep: mathematical model of worm fecundity

A mathematical model was constructed to predict the egg production of Trichostrongylus colubriformis worms as a function of worm age and host experience of infection. The model set egg production at zero until the worm was 14 days old, when a linear increase to maximum egg production levels occurred over 7 days. It was assumed that egg production remained at maximum levels until a threshold total worm burden was exceeded, when an exponential decline in egg production occurred. The rate of decline was assumed independent of worm age or worm burden. The estimated parameters (maximum egg production, threshold, lag and rate of decline) were optimized by fitting values predicted from the model to faecal egg counts observed in continuously infected sheep, giving R2 = 0.80. The model was validated against faecal egg counts obtained in two other continuous infection experiments, one performed at the same laboratory and the other in Britain.     

In vitro effects of artesunate on the survival of worm pairs and egg production of Schistosoma mansoni

The effect of artesunate (ART) on the survival time of adult worm pairs of Schistosoma mansoni and on their egg output during in vitro culture was assessed. ART significantly decreased the survival time of both paired male and female worms at concentrations of 5, 10, 20 and 40 mg l- 1 during in vitro cultivation. An inhibitory effect of ART on the daily egg output of paired female worms during in vitro cultivation was also observed.     

Correlation between discharged worms and fecal egg counts in human clonorchiasis

Background

Stool examination by counting eggs per gram of feces (EPGs) is the best method to estimate worm burden of Clonorchis sinensis in infected humans. The present study investigated a correlation between EPGs and worm burden in human clonorchiasis.

Methods and Findings

A total of 60 residents, 50 egg-positive and 10 egg-negative, in Sancheong-gun, Korea, participated in this worm collection trial in 2006–2009. They were diagnosed by egg positivity in feces using the Kato-Katz method. After administration of praziquantel, they were purged with cathartics on the next day, and then discharged adult worms were collected from their feces. Their EPGs ranged from 0 to 65,544. Adult worms of C. sinensis were collected from 17 egg-positive cases, and the number of worms ranged from 1 to 114 in each individual. A positive correlation between EPGs and numbers of worms was demonstrated (r = 0.681, P<0.001). Worm recovery rates were 9.7% in cases of EPGs 1–1,000 and 73.7% in those of EPGs over 1,000. No worms were detected from egg-negative subjects. Maximum egg count per worm per day was roughly estimated 3,770 in a subject with EPGs 2,664 and 106 collected worms.

Conclusions

The numbers of the worms are significantly correlated with the egg counts in human clonorchiasis. It is estimated that at least 110 worms are infected in a human body with EPGs around 3,000, and egg productivity of a worm per day is around 4,000.     

Fecundity and egg output by Toxocara canis in the red fox, Vulpes vulpes

The role of the red fox Vulpes vulpes in the dissemination of eggs of Toxocara canis into the environment is considered with reference to female worm fecundity and egg output in the faeces of infected foxes collected from four localities in southern England. A significant positive correlation was found between female worm size and the number of eggs in the uterus but there was no significant relationship between T. canis worm numbers and egg output in fox faeces. Reliable estimates of worm burdens in foxes could not, therefore, be determined from faecal egg counts alone. The highest mean egg output of 2145.0 epg recorded from adult foxes indicated that fox cubs are not necessarily the main sources of environmental contamination with T. canis eggs. Saturated magnesium sulphate was found to be a more effective flotation solution than zinc sulphate and sodium chloride for recovering eggs from fox faecal samples.     

Loss of Hymenolepis diminuta from the rat

One, 5, 15 and 30 worm infections of Hymenolepis diminuta were established in juvenile or adult male (Hooded Rowett or Sprague-Dawley) rats. Worm numbers and weight, and egg output were determined from day 15 to day 85 post infection. Gradual worm loss occurred only from 15 and 30 worm infections. In 5, 15 and 30 worm infections worm weight decreased from day 19 to day 50 but no weight loss occurred in single worm infections. The size range of individual worms from a multiple infection of a single rat increased markedly following infection. Adult rats showed a greater worm loss and harboured smaller worms than juvenile rats. The data will fit either a competitive or an immunological model.