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Anthelmintic Resistance
... Resistance is present when the frequency of individuals in a population that can tolerate doses of a compound is greater than in a normal population of the species (Prichard et al. 1980). Resistance has had serious health, welfare, and economic consequences for many animal industries (Conder and Campbell 1995, Sangster and Dobson 2002, Wolstenholme et al. 2004, for example, costing Australian sheep farmers US$250 million/year (Besier et al. 1996). Specifically, nematode resistance to benzimidazoles, including fenbendazole, has occurred in many species (e.g., sheep and cattle), is geographically widespread (Sangster 1999), and can develop in 3-5 years of use (Drudge et al. 1964, Kumar andYadav 1994). ...
... There are several reasons why T. tenuis might develop resistance. First, a low and frequent dose of anthelmintic (Hudson 1992) can promote resistance (e.g., Barton 1983) by providing frequent opportunities for selection, and if drug efficacy is high, only worms carrying resistant alleles can survive and reproduce (Sangster and Dobson 2002). Second, persistent drugs (e.g., fenbendazole) select more strongly for resistance than short-acting drugs (Dobson et al. 1996). ...
... Second, persistent drugs (e.g., fenbendazole) select more strongly for resistance than short-acting drugs (Dobson et al. 1996). Third, resistance is most common in direct life-cycle parasites with short generation times (e.g., T. tenuis; Sangster and Dobson 2002). Fourth, red grouse have little if any acquired immunity against T. tenuis Moss 1989, Hudson 1992), and selection pressure for resistance could therefore be high compared with a host with a strong immune response because immunity selects parasites irrespective of drug resistance (Sangster 2001). ...
ABSTRACT The parasitic nematode Trichostrongylus tenuis has a detrimental effect on red grouse (Lagopus lagopus scoticus) at the individual and population levels. Treatment using grit coated with the anthelmintic fenbendazole hydrochloride reduces parasite infection and increases grouse density. However, a frequent and low dose of anthelmintic increases selection pressure for parasite resistance, a serious practical and economic problem. We used an egg hatch assay to test resistance of T. tenuis from 12 moors in northern England, which differed in grit treatment intensity. The anthelmintic concentration that prevented 50% and 95% of T. tenuis eggs from hatching (ED50 and ED95, respectively) did not differ among moors and were not related to treatment. We suggest annual monitoring and responsible anthelmintic use to prevent resistance so that medicated grit continues to enhance red grouse management.
... Much of the research investigating the mechanism of action for these drugs has been stimulated by the increasing incidence of anthelmintic resistance, an occurrence stimulated by the heavy reliance on these drugs as a means of managing control of nematode parasites (Geary et al., 1999a;Sangster & Dobson, 2002;Sangster & Gill, 1999). Anthelmintic resistance has now spread to numerous parasitic helminth phyla, affecting humans and animals, and involving all the major chemical groups of anthelmintics (Geary et al., 1999a;Sangster & Dobson, 2002;Sangster & Gill, 1999;Waller, 1997). ...
... Much of the research investigating the mechanism of action for these drugs has been stimulated by the increasing incidence of anthelmintic resistance, an occurrence stimulated by the heavy reliance on these drugs as a means of managing control of nematode parasites (Geary et al., 1999a;Sangster & Dobson, 2002;Sangster & Gill, 1999). Anthelmintic resistance has now spread to numerous parasitic helminth phyla, affecting humans and animals, and involving all the major chemical groups of anthelmintics (Geary et al., 1999a;Sangster & Dobson, 2002;Sangster & Gill, 1999;Waller, 1997). ...
p>Anthelmintic drugs are used as a control measure for parasitic nematode infections. However, heavy reliance on these compounds has led to the development of parasite resistance and a consequent need to develop new anthelmintics that function via novel biological pathways. In this project, seven potential anthelmintics were examined for activity in the free-living nematode C. elegans. Of the seven compounds, those belonging to the cyclooctadepsipeptide group were shown to inhibit C. elegans pharyngeal pumping. The most potent of the compounds tested was emodepside, which has previously been shown to act as a potent broad-spectrum anthelmintic. The major aim of this project was to define and characterise the mechanism of action of emodepside using the free-living nematode C. elegans. Emodepside potently inhibited the locomotion and pharyngeal pumping of adult and larval stage 4 (L4) C. elegans. The L4 pharynx demonstrated reduced emodepside sensitivity in the presence, but not absence, of the intact cuticle, suggesting that the L4 cuticle reduces drug access. Continuous exposure of C. elegans to emodepside from egg to adult resulted in a slowing of worm development, possibly due to the paralysis of locomotion and pharyngeal pumping, which are important for food location and ingestion. Emodepside inhibited C. elegans egg laying behaviour, but not egg production or egg hatching. These results suggest that emodepside functions at the neuromuscular junction of the pharyngeal, vulval and body wall muscles to produce paralysis of pharyngeal pumping, egg laying and locomotion. Analysis of specific C. elegans gene mutants for their sensitivity to the inhibition of pharyngeal 5-HT response by emodepside strongly suggested that the anthelmintic functions primarily via the Ca2+- activated K+ channel SLO-1, with minor contributory roles performed by the G protein-coupled receptor LAT-1, and the G proteins Go;, and G(V The C. elegans slo-1 (js379) null demonstrated a high level of resistance to emodepside, which was abolished upon rescue of slo-1 expression in neurons. Rescue of slo- 1 expression in the pharyngeal muscles did not restore emodepside sensitivity, suggesting that neuronal SLO-1 is activated by emodepside to achieve paralysis of pharyngeal pumping. A gain-of-function mutant for SLO-1 that increases the Ca2+ sensitivity of the channel was found to be hypersensitive to emodepside, suggesting that emodepside manipulates the Ca2+ sensitivity of SLO-1 to facilitate its activity. To establish how emodepside is affecting SLO-1, the role of this channel in the pharynx was investigated. The s/o-7 Qs379) null possessed a longer mean pump duration and a disrupted pattern of pumping. Rescue of neuronal SLO-1 restored the wild type phenotype, suggesting that neuronal SLO-1 contributes to the control of pharyngeal pumping via the regulation of neurotransmitter release. Therefore, it is possible that emodepside may function by increasing the Ca2+ sensitivity of neuronal SLO-1 at the pharyngeal neuromuscular junction, resulting in an inhibition of neurotransmitter release and muscle paralysis. However, whole cell patch clamp recordings from pharyngeal muscle cells showed that emodepside inhibits SLO-1 currents rather than activating them. It is possible that the different intracellular environments afforded by pharyngeal neurons and muscle may result in the SLO-1 channel being modified differently in these two locations. This could produce a neuronal channel that is stimulated by emodepside and a muscle channel inhibited by the drug. Interestingly, the lat-1 (okl465) mutant was resistant to the effect of emodepside on pharyngeal pumping but not locomotion, suggesting that components of the molecular pathways involved in emodepside activity at the pharyngeal and body wall muscle neuromuscular junctions may be subtly different. The cyclooctadepsipeptides verticilide, PF1022-222 and PF1022-888 were also shown to inhibit C. elegans pharyngeal pumping. The slo-1 (js379) and lat-1 (okl465) null mutants demonstrated reduced sensitivity to all three compounds, suggesting that SLO-1 and LAT-1 are involved in the mechanism of action of these compounds as well as emodepside. SLO-1 and LAT-1 are not known to be involved in the mechanism of action of any other currently available anthelmintic, highlighting the novel functioning of the compounds tested in this project, and their potential as anthelmintics that could break resistance.</p
... Macrocyclic lactones, on the other hand, act on glutamate-gated chloride channels (GluCl) causing paralysis of the parasite neuromusculature, including the pharynx, thereby preventing the worm from feeding [8]. The target of benzimidazoles is, however, the tubulin within the parasite intestinal cells, which forms into microtubules that are necessary for nutrient acquisition [10]. Benzimidazoles bind to the β-tubulin component preventing it from forming microtubules within the intestinal cells of the helminth. ...
... Egg hatch test and other in vitro tests generate dose-response lines [10]. This allows the calculation of parameters, such as the concentration that kills 95% of eggs (the EC95), a single parameter used to compare isolates. ...
... Although the neurological changes associated with Chagas' disease are often neglected, there is evidence that T. cruzi is able to parasite and induce inflammatory lesions in structures of the peripheral nervous system (PNS) (Marin-Neto et al., 2007) and central nervous system (CNS) (Masocha and Kristensson, 2012;Pittella, 2013). The CNS involvement during the acute phase of Chagas' disease can lead to meningitis, seizures, restlessness, continuous crying, insomnia and transient coma (Sangster and Dobson, 2002;Storino et al., 2003). The consequences of chagasic meningoencephalitis that occur at the chronic phase consist of motor and sensory disorders, psychic alterations and cerebellar impairment (Sangster and Dobson, 2002). ...
... The CNS involvement during the acute phase of Chagas' disease can lead to meningitis, seizures, restlessness, continuous crying, insomnia and transient coma (Sangster and Dobson, 2002;Storino et al., 2003). The consequences of chagasic meningoencephalitis that occur at the chronic phase consist of motor and sensory disorders, psychic alterations and cerebellar impairment (Sangster and Dobson, 2002). In addition, electrophysiological changes were determined as a consequence of the deterioration of the cerebral cortical function in individuals with chronic Chagas' disease (Prost et al., 2000). ...
Currently, the types and distribution of the lesions induced in the central nervous system (CNS) by Trypanosoma cruzi remain unclear as the available evidence is based on fragmented data. Therefore, we developed a systematic review to analyse the main characteristics of the CNS lesions in non-human hosts infected. From a structured search on the PubMed/Medline and Scopus platforms, 32 studies were retrieved, subjected to data extraction and methodological bias analysis. Our results show that the most frequent alterations in the CNS are the presence of different forms of T. cruzi and intense lymphocytes infiltrates. The encephalon is the main target of T. cruzi , and inflammatory changes in the CNS are more frequent and severe in the acute phase of infection. The parasite's genotype and phenotype are associated with the tropism and severity of the CNS lesions. The methodological limitations found in the studies were divergences in inoculation pathways, under-reporting of animal age and weight, sample calculation strategies and histopathological characterization. Since the changes were dependent on the pathogenicity and virulence of the T. cruzi strains, the genotype and phenotype characterization of the parasite are extremely relevant to predict changes in the CNS and the neurological manifestations associated with Chagas’ disease.
... Anthelmintics, which control helminth pests by removing them, are grouped according to their common chemistry and mode of action (Sangster & Dobson 2002;Vercruysse & Rew 2002). Currently, the avermectins (ivermectin, eprinomectin and doramectin) and the milbemycins (moxidectin), collectively known as macrocyclic lactones, are amongst the most effective anthelmintics on the market. ...
... The milbemycins, although structurally similar and with a similar range of biological activity to the avermectins, differ in substituents in a few of the side chains at the C-13 position and can basically be considered to be deglycosylated avermectins (Sangster & Dobson 2002;Steel 1993;Vercruysse & Rew 2002). Although they were discovered in 1973, before the discovery of ivermectin, they were originally developed for use in crop protection and have been used in veterinary practice from about 1986 only (McKellar & Benchaoui 1996;Takiguchi et al. 1980). ...
Avermectins and milbemycins are commonly used in agro-ecosystems for the control of parasites in domestic livestock. As integral members of agro-ecosystems with importance in maintaining pasture health through dung burial behaviour, dung beetles are an excellent nontarget bio-indicator taxon for examining potential detrimental effects of pesticide application. The current review focuses on the relative toxicity of four different anthelmintics (ivermectin, eprinomectin, doramectin and moxidectin) in dung residues using dung beetles as a bioindicator species. One of the implications of this review is that there could be an effect that extends to the entire natural assemblage of insects inhabiting and feeding on the dung of cattle treated with avermectin or milbemycin products. Over time, reduced reproductive rate would result in decreased dung beetle populations and ultimately, a decrease in the rate of dung degradation and dung burial.
... It is generally accepted that resistant genes spread through natural nematode populations, and AR evolves when selection pressure on these nematodes is high (Sangster and Dobson, 2002;Coles, 2005;Sutherland and Scott, 2010). AR evolves rapidly (Wolstenholme et al., 2004), and resistant genes persist in nematode populations for many generations (Palcy et al., 2010). ...
... This latter group should be identified according to suitable indicators ) and then treated. The animals unexposed to treatment are a source of nematodes not submitted to selection pressure and, thus, contribute to susceptible nematode population that can dilute AR alleles (Sangster and Dobson, 2002). This refugia concept has been widely accepted recently (van Wyk, 2001;Besier, 2012;Jackson et al., 2009). ...
The in vivo Faecal egg count reduction test was used for the evaluation of the benzimidazole, avermectin and moxidectin drug efficacies against gastrointestinal nematodes in ten flocks of sheep. The same samples were tested concurrently in vitro for benzimidazole resistance using the Egg hatch test, and for resistance to avermectins using the Microagar larval development test. The conformity of obtained results between in vivo and in vitro tests was recorded. When the selected methods were applied, anthelmintic resistance to benzimidazoles was detected at four (40%) farms while resistance to avermectins was evident at one (10%) farm. At one farm (10%) avermectin resistance was suspected. Moxidectin was effective at all surveyed farms. Teladorsagia was recognized as the only benzimidazole resistant genus in post-treatment coprocultures, whereas Haemonchus larvae were resistant to avermectins. This represents the first recorded occurrence of avermectin resistance in gastrointestinal nematodes of sheep in the Czech Republic. A linear mixed-effects model demonstrated that the majority of evaluated management practices have a significant effect on resistance to benzimidazoles. While application of preventive practices like quarantine and smart drenching maintains low levels of anthelmintic resistance, others like the dose-and-move strategy, administration of the same family of drugs over extended periods of time, and number of treatments per year are responsible for the increase of resistance to anthelmintics at evaluated farms. Only targeted selective treatments approaches had no effect on resistance status. This study proved that the development of anthelmintic resistance can be delayed if appropriate preventive management practises are followed.
... Resistance has been reported from virtually all major sheep-and goat-producing regions and in all major nematode species, in particular Haemonchus contortus, Teladorsagia (Ostertagia) circumcincta and Trichostrongylus spp. [8,9]. Although drench failure (i.e. ...
... Understanding the probable causal factors for resistance that are amenable to on-farm management will facilitate product longevity. For example, factors first implicated in resistance-development in the benzimidazole and imidothiazole anthelmintics were excessive frequency of use and under-dosing [8]: frequent use is likely to be of particular importance for H. contortus especially, and might involve under-dosing with sub-standard products [31]. Additional selection pressures include drug persistence (in some formulations) and environment-treatment interactions that favour resistant nematodes ('refugia' concept -survival of a proportion of worms that has escaped exposure to treatment and can dilute resistant parasites, resulting in a smaller increase in resistance levels) [13,32,33]. ...
The viability of small-ruminant enterprises is under threat in some locations because anthelmintic resistance continues to increase in prevalence and severity. Despite this, no new anthelmintic group has been developed for many years, and animal health companies seem unconvinced of the commercial justification for new compounds for the ruminant market. It is now argued, however, that market conditions are becoming favourable because of increases in multiple-drug resistance in nematodes of ruminants and other hosts and because few non-chemical options exist. A new anthelmintic is urgently needed and will find ready acceptance in an increasing market, and with better knowledge of resistance management, greater product longevity can be achieved.
... Regrettably, resistance has developed following use of these compounds (Sangster and Dobson, 2002) and is now recognized in parasites of both humans and domesticated animals. Resistance to cholinergic agonists may be produced by four general mechanisms : (1) changes in drug translocation (e.g. ...
... change in the downstream pathway to contraction). All of these mechanisms could play a role in resistance to levamisole and pyrantel and so it is anticipated that resistance to these drugs is polygenic (Sangster and Dobson, 2002). Fig. 2 shows the chemical structures of the cholinergic anthelmintics levamisole and pyrantel, together with a model for their mode of action. ...
Here we review molecular information related to resistance to the cholinergic anthelmintics in nematodes. The amount of molecular information available varies between the nematode species, with the best understood so far being C. elegans. More information is becoming available for some other parasitic species. The cholinergic anthelmintics act on nematode nicotinic acetylcholine receptors located on somatic muscle cells. Recent findings demonstrate the presence of multiple types of the nicotinic receptors in several nematodes and the numerous genes required to form these multimeric proteins. Not only are the receptors the product of several genes but they are subject to modulation by several other proteins. Mutations altering these modulatory proteins could alter sensitivity to the cholinergic anthelmitics and thus lead to resistance. We also discuss the possibility that resistance to the cholinergic anthelmintics is not necessarily the result of a single mutation but may well be polygenic in nature. Additionally, the mutations resulting in resistance may vary between different species or between resistant isolates of the same species. A list of candidate genes to examine for SNPs is presented.
... The rationale of regular deworming of horses kept on pasture is quite controversial. Since the 1960s, resistance to anthelmintics has been observed in horse parasitic nematodes (Kaplan & Nielsen, 2010), and the diversity and wide geographical distribution of Cyathostominae populations resistant to benzimidazoles or macrocyclic lactones (ivermectin), explain the recent widespread infections among horses subjected to regular deworming (Sangster & Dobson, 2002;Matthews, 2014). The benefits of controlling parasites in threatened species, to improve fitness and population performance, then, must be balanced with the individual ethical and ecosystem costs associated with control (Stringer & Linklater, 2014). ...
The first successful reintroduction programme for the Przewalski's horse in Russia was launched by FSFI «Orenburg Reserves» in 2015. A total of 36 Przewalski's horses were brought to the Pre-Urals Steppe site from semi-reserves in France and Hungary in 2015–2017. Parasite infections can significantly affect the survival of the wild horses during the process of adaptation. Investigation of the level of helminth infections in these reintroduced horses is the subject of their regular monitoring in the Pre-Urals Steppe. The studies were conducted on the territory of the Pre-Urals Steppe of 165.38 km2, in Orenburg State Nature Reserve in 2016–2019. The coprological examinations were conducted on a monthly basis using the McMaster method. The Baerman-Orlov larvoscopy technique was used to identify the larval stages of nematodes. Przewalski's horses were threatened with anthelmintic drugs every 3 to 12 months. All faecal samples collected in two or more months after deworming, contained eggs of nematodes of the family Strongylidae (Nematoda, Strongylida), the prevalence of infection was 100%. Single eggs of cestodes from the genus Anoplocephala (Cestoidea: Anoplocephalidae) were found in four samples out of 642. Larvoscopy revealed larvae of the nematodes from the genus Triodontophorus (subfamily Strongylinae) and larvae from the subfamily Cyathostominae. In addition, a small number of larvae of the II and III stages of the gastric botfly Gastrophilus spp. (family Gastrophilidae), were found in the faeces in some Przewalski's horses after deworming. Parascaris equorum was not registered in this study, although this parasite was found in domestic horses from the village nearest to the Pre-Urals Steppe. During the study period, there were no noticeable seasonal fluctuations in the number of helminth eggs; the level of EPG (eggs per gram of faeces) value gradually increased in all groups of horses after deworming. A high level of infection (> 500 EPG) was reached in 1–6 months after deworming; the maximal level was registered in September 2018 (2483.3 ± 718.5 EPG in one of the single-sex groups). The comparatively high EPG values did not affect the physical condition of Przewalski's horses in the Pre-Urals Steppe; no clinical signs of parasite infections were observed in our study. The level of strongylid infection in free-roaming breeding groups of Przewalski's horses was lower than that of animals kept in acclimatization enclosures. There was a noticeable trend towards a decrease in the average EPG level of the breeding groups. This may be due to two factors: 1) contamination of grass in the enclosures was higher because of higher density of horses; 2) the breeding groups transported in 2015 and 2016, had more time for the adaptation. Regular deworming is not recommended for relocated wild horses outside an adaptation period as it may influence their natural resistance to parasitic infections and poses a risk of the emergence of resistant helminth populations. Preventive deworming of the Przewalski's horses should be administered only within the first years after their relocation to new areas.
... Acquisition of new stock without quarantine and treatment has been identified as a major method of introducing resistant helminths parasite into stocks with minor or no incidence of resistance. Other measures of preserving chemical anthelmintic efficacy by livestock farmers include reduced frequency of treatment, accurate dosing (Sangster and Dobson, 2002), and selective treatment of infected animals. ...
... EC50 s were analyzed by twoway ANOVA followed by comparison with Bonferroni's test (P < 0.05). The resistance ratio (RR) was calculated with the following formula: RR = EC50 of the resistant isolate/EC50 of the susceptible isolate ( Sangster and Dobson, 2002). ...
This study aimed to evaluate the effects of Eucalyptus citriodora essential oil and citronellal on a Haemonchus contortus isolate susceptible to anthelmintics, the Inbred-susceptible Edinburgh (ISE) and a resistant Kokstad isolate. The oil was analyzed by gas chromatography (GC). The products were evaluated using an egg hatching test
(EHT), a larval development test (LDT) and an adult worm motility test (AWMT). The effective concentrations to inhibit 50% (EC50) of egg hatching and larval development and resistance ratios (RRs) were calculated. The results were analyzed by analysis of variance and Tukey’s or Bonferroni’s test. The major constituent of E. citriodora oil was citronellal (63.9%). According to EHT, EC50 values for the ISE isolate were 0.4 mg/ml and
0.3 mg/ml; for the Kokstad isolate, values were 0.5 mg/ml and 0.4 mg/ml for oil and citronellal, respectively. LDT EC50 values were 2.9 mg/ml and 2.3 mg/ml for the ISE isolate and 3.2 mg/ml and 2.4 mg/ml for the Kokstad isolate for oil and citronellal, respectively. In AWMT, 2 mg/ml of oil and citronellal completely inhibited
the motility of both the ISE and Kokstad isolates. RRs between the isolates ranged from 1 to 1.3. No significant differences were observed between the isolates or treatment comparisons (P > 0.05). Therefore, E. citriodora oil and citronellal both exhibited ovicidal and larvicidal effects and inhibited the motility of both H. contortus
isolates.
... EC50 s were analyzed by twoway ANOVA followed by comparison with Bonferroni's test (P < 0.05). The resistance ratio (RR) was calculated with the following formula: RR = EC50 of the resistant isolate/EC50 of the susceptible isolate (Sangster and Dobson, 2002). ...
This study aimed to evaluate the effects of Eucalyptus citriodora essential oil and citronellal on a Haemonchus contortus isolate susceptible to anthelmintics, the Inbred-susceptible Edinburgh (ISE) and a resistant Kokstad isolate. The oil was analyzed by gas chromatography (GC). The products were evaluated using an egg hatching test (EHT), a larval development test (LDT) and an adult worm motility test (AWMT). The effective concentrations to
inhibit 50% (EC50) of egg hatching and larval development and resistance ratios (RRs) were calculated. The results were analyzed by analysis of variance and Tukey’s or Bonferroni’s test. The major constituent of E. citriodora oil was citronellal (63.9%). According to EHT, EC50 values for the ISE isolate were 0.4 mg/ml and 0.3 mg/ml; for the Kokstad isolate, values were 0.5 mg/ml and 0.4 mg/ml for oil and citronellal, respectively. LDT EC50 values were 2.9 mg/ml and 2.3 mg/ml for the ISE isolate and 3.2 mg/ml and 2.4 mg/ml for the Kokstad isolate for oil and citronellal, respectively. In AWMT, 2 mg/ml of oil and citronellal completely inhibited the motility of both the ISE and Kokstad isolates. RRs between the isolates ranged from 1 to 1.3. No significant differences were observed between the isolates or treatment comparisons (P > 0.05). Therefore, E. citriodora oil and citronellal both exhibited ovicidal and larvicidal effects and inhibited the motility of both H. contortus isolates.
... Benzimidazole produce anthelminitic action through multiple targets such as inhibition of mitochondrial enzyme fumurate reductase, reduces glucose transport and also uncouples oxidative phosphorylation, but the primary anthelmintic activity is binding to β-tubulin leads to inhibition of microtubule polymerization [17,18]. The resistance development against benzimidazoles is associated with change in primary target β-tubulin. ...
Antibiotics are in wide use for the control of microbial infections and as growth promoters in food animals. The indiscriminate use of these antibiotics has given rise to the menace of resistance in bacteria against these antibiotics. In similar fashion, wide spread use of anthelmintics resulted in resistance among parasites which has become a challenge for the clinician. The anthelmintic resistance in parasites has wide implications in terms of animal health and their productivity. Resistance is said to be present when there is a large frequency of microbes within a population able to tolerate doses of a compound than in a normal population. The resistance against a particular antimicrobial agent is also heritable. Among the parasites, nematode infestations are of major economic concern, particularlyin domesticated animals throughout the world. Anthelmintics have been developed with objective to control the parasites and finally reduce the production loss. Unplanned and injudicious use of anthelmintics has resulted in the development of resistance. Anthelmintic resistance has been noted against all classes of anthelmntics. Parasites (nematodes) have developed resistance against most prescribed groups of anthelmintics like benzimidazole through changes in primary target β-tubulin,whereas,resistance against levamisole, morantel, and pyrantel occur through loss or changes in nicotinic-acetylcholine receptors (nAChR).The P-glycoprotein, a drug efflux protein, was over expressed in avermectin resistant nematodes. Main goal of clinician is to minimize the resistance against the anthelmintics in planned ways which include correct dose administration, cyclical changeover of anthelmintics, regular monitoring on development of resistance by in vivo as well in vitro tests, controlling of livestock density on particular pasture area and use of combination of anthelmintics.
... Synthetic anthelmintics are used to control helminth infections in livestock. However, all sheep and goat farming countries reported anthelmintic resistance (Jackson and Coop, 2000;Sangster and Dobson, 2002;Kaplan, 2004). The resistance to commercially available anthelmintic drugs and the entry of drug residues in the food chain have instigated the search for alternative drugs such as medicinal plants. ...
Parasitic diseases remain a major threat to livestock production around the world, particularly in India. Paramphistomosis caused by paramphistomes are one of the most common and economically important diseases of livestock. The high incidence of resistance to chemotherapeutics, toxicity and side effects has urged the necessity of finding alternative plant-based anthelmintics against helminth parasites. Therefore, the present investigation was aimed to assess the anthelmintic effect of the rind of Punica granatum Ethanol Extract (PgEE) against paramphistomes in infected sheep. Infected sheep were treated orally with 30 and 50 mg/ml concentrations of PgEE. Eggs Per Gram (EPG) count on faeces, haematological and biochemical parameters of sheep were investigated. In PgEE-treated sheep, the egg count reduced significantly in the faeces and the reduction was proportional to dosage and duration after treatment. The maximum reduction (97.95 %) was observed on day 21 post-treatment with 50 mg/ml concentration of PgEE. In infected sheep, the haemoglobin and protein content were below standard physiological values. Improvement of haematobiochemical profile was observed in sheep after treatment with PgEE.
... When first introduced, all these drugs were highly efficacious, but frequent and widespread use and misuse has resulted in the emergence of resistant parasite populations, such that AR is now a major global problem, especially (but not exclusively) in parasites of small ruminants [27], and is the greatest threat to the sustainable control of helminthoses in the short to medium term. Anthelmintic inefficacy and resistance has also become apparent in cattle, reported initially in the southern hemisphere [28][29][30], but more recently also within Europe [31,32]. The problem of AR is compounded by the fact that many of these parasite populations are resistant to more than one class of anthelmintic [33,34] and that the prevalence of resistance is increasing inexorably in many regions [35]. ...
Infections with parasitic helminths (nematodes and trematodes) represent a significant economic and welfare burden to the global ruminant livestock industry. The increasing prevalence of anthelmintic resistance means that current control programmes are costly and unsustainable in the long term. Recent changes in the epidemiology, seasonality and geographic distribution of helminth infections have been attributed to climate change. However, other changes in environment (e.g., land use) and in livestock farming, such as intensification and altered management practices, will also have an impact on helminth infections. Sustainable control of helminth infections in a changing world requires detailed knowledge of these interactions. In particular, there is a need to devise new, sustainable strategies for the effective control of ruminant helminthoses in the face of global change. In this paper, we consider the impact of helminth infections in grazing ruminants, taking a European perspective, and identify scientific and applied priorities to mitigate these impacts. These include the development and deployment of efficient, high-throughput diagnostic tests to support targeted intervention, modelling of geographic and seasonal trends in infection, more thorough economic data and analysis of the impact of helminth infections and greater translation and involvement of end-users in devising and disseminating best practices. Complex changes in helminth epidemiology will require innovative solutions. By developing and using new technologies and models, the use of anthelmintics can be optimised to limit the development and spread of drug resistance and to reduce the overall economic impact of helminth infections. This will be essential to the continued productivity and profitability of livestock farming in Europe and its contribution to regional and global food security.
... Thus it can be concluded that region of BZ resistance is same in phylogenetically-distant organisms viz., fungi to nematodes. There is also genetic evidence that resistance is outcome of single, sex linked, gene in T. columbriformis and O. dentatum, but in H. contortus multiple genes could be involved (Sangster et al., 2002). Our results correspond to an earlier report (Tiwari et al., 2007). ...
Benzimidazole is a synthetic anthelmintic against which nematode resistance especially in Haemonchus contortus, is emerging at a alarming speed. The mechanism of benzimidazole resistance appears to involve mutations in the gene encoding β-tubulin isotype 1 (β-tubulin-1). The present study was carried out to find out the variation existing in β-tubulin-1 which is directly involved with drug binding capacity involving microtubules
polymerization. DNA of adult nematode H. contortus was extracted, amplified and sequenced. Out of 50 worms investigated, 37 showed benzimidazole susceptible gene while 13 were resistant indicating single nucleotide mutation at amino acid 200 TTC/TAC. In addition, 12 worms showed several regions of consistent difference indicating single nucleotide polymorphism (SNPs) at various positions in coding region. It has been concluded that resistant alleles conferring anthelmintic resistance is prevalent in the local population of H. contortus of north-east Punjab, Pakistan
... Currently, a large number of veterinary parasites are exhibiting resistance (Pomroy, 2006). Even after a drug has been withdrawn from the market, there is no evidence of resumption of its previous anthelmintic activity (Sangster & Dobson, 2002). Sangster et al. (1999) and Xu et al. (1998) identified a P-gp homologue in parasites that expelled ivermectin, thus reducing drug activity and favoring the development of resistance to macrocyclic lactones. ...
Despite advancements in pharmacogenetics in human medicine, the incorporation of pharmacogenetics into veterinary medicine is still in its early stages of development. To date, efforts to understand the pharmacologic impact of genetic variation in veterinary species have largely focused on genes encoding for the membrane transporter, P-glycoprotein (P-gp). The emphasis on the role of P-gp is largely because of safety concerns associated with the use of some macrocyclic lactones in dogs. Because of the body of information available on this topic, we use P-gp as a platform for understanding the importance of population diversity in veterinary medicine. The impact of P-gp on drug pharmacokinetics and pharmacodynamics is considered, along with endogenous and exogenous factors that can modulate P-gp activity. The review includes discussion of how population diversity in P-gp activity can lead to susceptibility to certain diseases or alter patient response to environmental stress or pharmaceutical intervention. In addition, phenotypic diversity also needs to be considered, as demonstrated by the impact of P-gp up-regulation and drug resistance. The aim of this review was to set the stage for further exploration into the impact of genetic and phenotypic variability on drug pharmacokinetics, disease propensity, product formulation and drug response in both companion and food-producing animals.
... However, the mild temperature and environmental humidity are also optimum for survival of many important livestock parasites. The use of chemical anthelmintics is the most common option to reduce the adverse effects caused by nematode parasites (Pedreira et al., 2006), but their usefulness is increasingly constrained by the emergence of drug resistance to several chemical groups (Sangster and Dobson, 2002). ...
A coprological survey to analyze the presence of flock resistance to benzimidazoles (BZ) and macrocyclic lactones (ML) was performed in sheep under field conditions. Fecal samples were collected from 2,625 sheep in 72 commercial farms from Galicia (NW Spain). The in vitro (FECRT, fecal egg count reduction test) and in vivo (EHA, egg hatch assay, and LFIA, larval feeding inhibition assay) tests were used to assess the efficacy of these anthelmintics. Coprocultures were also developed to obtain knowledge on the main genera of trichostrongylid nematoda prior to, and after, the administration of the anthelmintics. By using the FECRT, BZ resistance was observed in 13 (18%) flocks, whereas ML resistance was only detected in 2 (3%) farms. The number of resistant flocks to BZ was 21 (29%) by using the EHA and 7 (10%) by means of the LFIA. None of the flocks used in this study showed simultaneous resistance to both employed anthelmintics. The results from the in vitro and in vivo tests revealed that 92% of the flocks FECRT resistant to BZ were also resistant with the EHA. The LFIA confirmed all the farms resistant to ML by using the in vivo test. After the administration of BZ, nematode larvae belonging to Teladorsagia circumcincta (32.2%), Trichostrongylus spp. (29%), Nematodirus spp. (6.5%), and Chabertia ovis (3.2%) were identified. In the flocks receiving ML, only T. circumcincta was identified (57%). We recommend the use of in vitro tests because they are more efficient. As the use of macrocyclic lactones is increasing in this region, further investigation is needed for detecting resistance to the anthelmintic family compounds by the LFIA.
The aim of this study was to characterize the anthelmintic resistance (AR) of a sheep gastrointestinal nematode population, named Caucaia, from northeastern Brazil. Phenotypic tests performed were: egg hatch (EHT), larval development (LDT) and fecal egg count reduction (FECRT). Benzimidazoles (BZs) genotypic evaluation was by frequency of single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A, and for levamisole (LEV), by frequency of resistance alleles of Hco-acr-8 gene. The primers were designed specifically for Haemonchus contortus. Effective concentrations 50% (EC50) for BZs (EHT), and for macrocyclic lactones (MLs) and LEV (LDT) were 1.02 µg/mL, 1.81 ng/mL and 0.04 µg/mL, respectively. Resistance ratios for MLs and LEV were 0.91 and 3.07, respectively. FECRT efficacies of BZs, MLs, monepantel (MPTL) and LEV were 52.4; 87.0; 94.5 and 99.6%, respectively. qPCR for BZs demonstrated resistance allele frequencies of 0%, 26.24% and 69.08% for SNPs E198A, F200Y and F167Y, respectively. For LEV, 54.37% of resistance alleles were found. There was agreement between EHT, FECRT and qPCR for BZs, and agreement between LDT and qPCR for LEV. Thus, based on higher sensitivity of qPCR, and phenotypic evaluation, the Caucaia population was considered resistant to BZs, MLs, LEV and suspect for MPTL.
Two methodologies were tested to isolate pure Trichostrongylus colubriformis strains from naturally infected sheep. Also, the in vitro susceptibility status to commercial anthelmintic (AH) drugs was determined in these strains. These methods were performed in a tropical region of Mexico where naturally infected sheep and goats host Haemonchus contortus, T. colubriformis and Oesophagostomum columbianum. For the first strain, a group of 6 naturally infected lambs from the “Paraiso” farm were treated with closantel (subcutaneous (SC), 10 mg/kg). On day 10 post-treatment, the eggs per gram (EPG) of faeces were determined with the McMaster technique. The faeces from the two lambs with the highest EPG were used for coprocultures (4 days, 28 °C). The L3 larvae were recovered and identified as T. colubriformis (69%) and O. columbianum (31%). The latter was removed by 10-day refrigeration (4–5 °C) followed by sieving (25 μm), resulting in 100% T. colubriformis (PARAISO strain). The second strain was isolated using repeated doses of levamisole (LEV, SC 7.5 mg/kg) in an 8-year-old sheep. The sheep had 1700 EPG before the LEV treatments and maintained 1300 EPG after both LEV treatments (day 10). The coproculture (4 days, 28 °C) after the second treatment contained 100% T. colubriformis (FMVZ-UADY strain). The in vitro AH susceptibility was determined using the egg hatch test for benzimidazole (BZ), and the larval migration inhibition test for ivermectin (IVM) and LEV. The PARAISO strain was BZ- and LEV-susceptible, and IVM-resistant. Meanwhile, the FMVZ-UADY strain was BZ- and IVM-susceptible, and LEV-resistant. The conditions where these two protocols could be used in other parts of the world were discussed.
Anthelmintic resistance means that developing genetically transmitted lack of susceptibility to an anthelmintic which is previously known to be susceptible to a parasite population. Anthelmintic resistance has an increasing importance in recent years. The anthelmintic resistance which is developed especially due to use of unconscious anthelmintics also brings with it economic problems. Investigations have shown that resistance has developed to anthelmintics in a short period of time after launch to the market and even in some countries, several sheep and goat farms have been closed due to anthelmintic resistance. For this reason, especially in livestock breeding; The development of resistance should not be overlooked while planning of treatment and control programs and choosing anthelmintics. In this review, resistance mechanisms which is developed to anthelmentic drugs, resistance detection methods and anthelmentic resistance status of livestock in Turkey were evaluated.
In this chapter, we consider the similarities and differences between management of ecto‐ and endoparasites. We discuss the general approaches of prevention and control of ecto‐ and endoparasites (historic and current chemotherapies, environmental management/host management), while considering the different challenges faced relating to lifecycle, host distribution, genetics, and selection pressure.
Fifty years after anthelmintic resistance in livestock parasites was first reported, the prevalence of resistance has increased globally, and is of increasing significance in animal industries. It is now timely to reflect on what we have learnt, how research has unfolded, and what we hope to learn in the future. This Opinion paper examines ten important research events that were pivotal in resistance research. The moments include the discovery, description, and diagnosis of parasite resistance, as well as important physiological and genetic findings, and the development of online tools to help manage resistance. Despite our efforts, resistance remains the greatest challenge in parasite control. The future directions for research, including people and funding, are discussed.
This study explored the variation in susceptibility to acetone:water plant extracts between infective larvae (L3) of ten Haemonchus contortus isolates from different geographical origin. The L3 of 10 different isolates were exposed either to the acetone:water extract of a temperate plant (Onobrychis viciifolia) or a tropical plant (Acacia pennatula) and were evaluated with the larval exsheathment inhibition assay (LEIA). The L3 of each isolate were incubated with different concentrations of each extract (0, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200 μg/mL of phosphate buffered saline (PBS)). After incubation, the exsheathment process of L3 was induced using a solution with sodium hypochlorite (2%) and sodium chloride (16.5%). The proportion of exsheathed L3 was determined for each concentration at 0, 20, 40 and 60 min. Effective concentrations 50% (EC50) and the corresponding 95% confidence intervals (95% CI) were calculated for every isolate with both extracts. Moreover, a resistance ratio (RR) was calculated for each extract to compare isolates, using the most susceptible isolate as the respective reference for each extract. To determine the role of polyphenols on the reported effect, a second set of incubations was made for each isolate and each extract, using the extracts at a concentration of 1200 μg/mL PBS with or without polyvinylpolypyrrolidone (PVPP), a polyphenol blocking agent, and controls without extract. The ten different H. contortus isolates showed variation in susceptibility for each of the 2 extracts tested (P < 0.05). The EC50 values for A. pennatula extract ranged from 36 to 501 μg/mL (RR: 2.11–13.68). Meanwhile, the EC50 values for O. viciifolia extract ranged from 128 to 1003 μg/mL (RR: 1.25–7.82). The use of PVPP revealed that polyphenols were responsible for the anthelmintic activity recorded for both extracts. However, tested H. contortus isolates suggested that susceptibility to one polyphenol-rich extract did not determine the susceptibility to the other polyphenol rich extract. The latter result indicated that the different H. contortus isolates varied in their susceptibility to the polyphenols present in each extract evaluated.
In this report, beta-tubulin gene polymorphism was investigated in Haemonchus contortus populations isolated from sheep flocks in different regions of Khouzestan province, southwestern Iran. The samples were chosen on the basis of regional variation in benzimidazole treatment background of sheep flocks. Our objective was to study the relationship between treatment background and beta-tubulin gene polymorphism of H. contortus isolated from the selected regions. H. contortus isolated from Shooshtar revealed reduction in beta-tubulin gene polymorphism restriction fragment length polymorphism (RFLP) in respect to other isolates indicating that benzimidazole-resistant isolates of H. contortus developed in some sheep flock of Shooshtar region.
Anthelmintic resistance of parasites in small ruminants, cattle and horses is increasing worldwide as a consequence of the over usage of the currently available products. In Belgium, Cooperia oncophora is the most common cattle nematode in which resistance, especially against macrocyclic lactones, occurs. Once resistance has been diagnosed, a change to another drug with a different mode of action is advised. However, effective anthelmintics will be hardly available in the near future. Therefore, it is important that farmers and veterinarians find a balance between achieving good parasite control and the sustainability of their control strategies. In this way, anthelmintic resistance may be delayed, and the effectiveness of anthelmintic drugs may be prolonged. This requires sensitive detection tools. With a sensitive detection technique, anthelmintic resistance can be diagnosed in a very early stage. Hence, the spread of resistance alleles in the parasite population may be prevented. In this review, different diagnostic assays for the detection of anthelmintic resistance are discussed, an overview is given of the current status of anthelmintic resistance in Belgian cattle, and measures are suggested to avoid or delay the development of anthelmintic resistance.
Anthelmintic drugs remain the principal means of intervention for therapy and prophylaxis of nematode parasitic diseases in humans and animals. Other than improvements in sanitation, there are no effective alternatives to chemical control of parasitic nematodes. However, resistance to anthelmintics has become a major problem in veterinary medicine, threatens both agricultural production and animal welfare, and there is increasing concern that drug resistance could arise in nematode parasites in humans.
An in vivo study in goats evaluated the effect of superimposing a single artificial infection with a benzimidazole (BZ)-susceptible Haemonchus contortus isolate upon established H. contortus populations of known BZ resistance by measuring the phenotypic BZ resistance of eggs collected from faeces before and after re-infection. Two H. contortus isolates, one benzimidazole resistant (BZR) and the other susceptible (BZS), were used to infect worm-free goats. Eight goats were initially infected with 2000 third-stage larvae (L3). In each case the inoculum contained a pre-determined proportion of the two isolates: 100% BZS (one goat), 75% BZS/25% BZR (two goats), 50% BZS/50% BZR (two goats), 25%BZS/75% BZR (two goats) and, finally, 100% BZR (one goat). The phenotypic BZ susceptibility of the H. contortus population formed in each goat after the first infection was determined on days 30 and 36 post-infection using an egg-hatch assay (EHA) that estimated the concentration of thiabendazole (TBZ) required for 95% inhibition of larval hatching (EC95) with a 95% confidence interval (95% CI). On day 49 post-infection, goats were re-infected with 2000 L3 of the BZS isolate alone. A second set of EHA bioassays was performed 28 days and 34 days after re-infection. The first infection protocol produced three populations classified as BZS (EC95 0.055-0.065 μg TBZ/ml) while four were categorized as BZR (EC95 0.122-0.344 μg TBZ/ml). The status of one other population could not be determined. After re-infection with BZS L3, the number of susceptible populations increased to six (EC95 0.043-0.074 μg TBZ/ml) while the remaining two were deemed resistant (EC95 0.114-119 μg TBZ/ml). Re-infection with BZS L3 thereby reduced the resistance status of most H. contortus populations.
Triclabendazole (TCBZ) is the drug of choice for Fasciola hepatica control and reports of F. hepatica resistant to this drug from a wide range of geographic regions are very concerning. This study investigated the presence of TCBZ resistance in F. hepatica in naturally infected Australian beef and dairy cattle herds and evaluated methods of measuring the levels of resistance. Faecal egg count and coproantigen reduction tests (FECRT and CRT, respectively) were conducted on 6 South-eastern Australian beef properties and one dairy property where treatment failure by triclabendazole (TCBZ) was suspected. The CRT was conducted on an additional beef property. On each property 15 animals were treated with an oral preparation of TCBZ at the recommended dose and 15 animals remained as untreated controls. Fluke eggs in faeces were counted and coproantigen levels were measured before treatment and 21 days after treatment and in the untreated control animals. These data were evaluated using three different methods to calculate % reductions compared with controls. Resistance (<90% reduction) was detected on the dairy property using both FEC and CRT, and on 3/6 beef properties using FECRT and 4/7 beef properties using CRT. Using the FECRT, reductions of 6.1–14.1% were observed in dairy cattle and 25.9–65.5% in beef cattle. Using the CRT, reductions of 0.4–7.6% were observed in dairy cattle and 27.0–69.5% in beef cattle. Live flukes were recovered at slaughter following TCBZ treatment of 6 cattle from 3 of the beef properties, confirming the TCBZ resistance status of F. hepatica in these cattle. This is the first report of F. hepatica resistant to TCBZ in cattle in Australia and the results suggest that resistance is widespread in the South-eastern region. The CRT is shown to be a robust alternative to the FECRT for evaluation of TCBZ resistance in F. hepatica in cattle.
Drug resistance has become an increasingly serious and widespread problem worldwide, impacting across a broad phylogenetic
range of important medical and veterinary parasite species. These include parasites from the groups of the Apicomplexa, the
fl agellates and the helminths. Accurate measurement of resistance is vital for several reasons. In addition to making decisions
on effective drug use for parasite control, resistance assays (sometimes called sensitivity assays) are also used to monitor
prevalence, severity and dissemination of resistance and to evaluate the impact of control regimes on resistant parasites.
In some cases they are used to assist the choice of drugs in clinical situations. A review of the assays currently available
and knowledge of the information they provide gives a vital signpost of our current situation across a variety of parasite
species and also as a guide in the design of future assays for resistances yet to develop.
The development of drug resistance in Eimeria is common because of extensive use of anticoccidial drugs for the control of avian coccidiosis. The significance of chemotherapy is evident from the fact that, in spite of advancement in the field of immunological, biotechnological and genetic methods, prophylactic chemotherapy with anticoccidial drugs is still widely used for the control of coccidiosis. In such situations, new drugs should be available to replace the older ones against which resistance has developed, however it takes a long time to develop any new compounds. It is therefore currently necessary to develop strategies to minimise the emergence of resistance in Eimeria strains and to prolong the effect of available anticoccidial drugs. This paper summarises the resistance status of Eimeria species in different parts of the world and reviews different types of resistance, mechanism of resistance development, factors involved in the development and spread of resistance, management of resistant strains and strategies to preserve the efficacy of the available anticoccidial drugs. Use of vaccines, synthetic and botanical anticoccidials and educating farmers about recommended coccidiosis control practices are discussed in this review, along with the integration of currently available options for the management of drug resistance and, ultimately, the control of coccidiosis.
To report the level of anthelmintic resistance on 13 commercial cattle properties in south-west Victoria, Australia.
Between 2006 and 2009 worm egg count reduction tests were conducted on calves on the 13 properties. Samples were collected 10-14 days post anthelmintic treatment and worm egg counts and larval differentiation tests were conducted. Resistance was defined if there was less than 95% reduction (lower confidence limit <90%) in the faecal worm egg count for the particular genus.
The percentage of properties with anthelmintic resistance in at least one species was 54% for benzimidazole (BZ), 100% for levamisole (LEV) and for ivermectin (IVM) it was 100% for the half-dose (0.1 mg/kg) and 62% for the full dose (0.2 mg/kg). A substantial frequency of resistance was detected in Ostertagia ostertagi to BZ (5/11), LEV (3/3) and IVM (5/11), in Trichostrongylus spp. to BZ (4/7) and in Cooperia spp. to IVM (6/11). No resistance to LEV was detected in Trichostrongylus or Cooperia spp. Suspected IVM-resistant Trichostrongylus spp. and BZ-resistant Cooperia spp. were only detected on one property each.
This is the first Australian report of macrocyclic lactone-resistant O. ostertagi in the refereed literature. The frequency of resistance in O. ostertagi to BZ, LEV and IVM and in Trichostrongylus spp. to BZ in the present study appears higher than levels detected in the 2004-05 New Zealand survey, whereas the resistance frequency in Cooperia spp. to IVM and BZ was less.
The increasing prevalence of anthelmintic (drench) resistance in gastrointestinal parasite populations is decreasing the profitability of the Australian sheep industry. Refugia management can delay its development by not exposing a proportion of the worm population to chemical control. A dynamic-optimisation model is used to assess the economic value of refugia for management of the worm species Teladorsagia circumcincta and macrocyclic lactone drenches in an application to Western Australian sheep flocks. A low rate of refugia (2 per cent) is most profitable under standard circumstances because it slows the development of resistance, but also reduces the proportion of the flock not exposed to chemical control. Frequent drench application should remain the primary method of control. However, its efficacy should be preserved through refugia management, rather than greatly reducing treatment frequency. Copyright 2009 The Authors. Journal compilation 2009 Australian Agricultural and Resource Economics Society Inc. and Blackwell Publishing Asia Pty Ltd.
Parasitism in New World camelids (NWC), which is associated with both ecto- and endoparasites, is a major health concern throughout the world. Clinical disease has been noted as causing severe economic losses; subclinical issues have yet to be addressed. Despite the advances made in the knowledge and understanding of parasites of NWC, old parasites continue to plague producers, and new issues have arisen. This article updates information on the major ecto- and endoparasites of NWC, including diagnostic techniques and issues relative to anthelmintic resistance in nematodes.
Resistance to antiparasitic drugs is all too common in parasites of veterinary interest. The fact that resistance has arisen in so many different species of parasite and hosts against so many different drugs and in so many geographic areas suggests that the resistances may have common features. Such features may be useful in generating ideas for resistance management. Although describing the nature and presence of resistance remains an important objective, there is now a pressing need to develop improved methods of detection of resistance and to devise schemes for integrated parasite management (IPM). Multidisciplinary teams of researchers and extension workers are exploring new ways to deal with the problem of resistance.
Chemotherapy is central to the control of many parasite infections of both medical and veterinary importance. However, control has been compromised by the emergence of drug resistance in several important parasite species. Such parasites cover a broad phylogenetic range and include protozoa, helminths and arthropods. In order to achieve effective parasite control in the future, the recognition and diagnosis of resistance will be crucial. This demand for early, accurate diagnosis of resistance to specific drugs in different parasite species can potentially be met by modern molecular techniques. This paper summarises the resistance status of a range of important parasites and reviews the available molecular techniques for resistance diagnosis. Opportunities for applying successes in some species to other species where resistance is less well understood are explored. The practical application of molecular techniques and the impact of the technology on improving parasite control are discussed.
On-farm risk factors associated with ivermectin resistance on sheep farms in Western Australia were identified from data derived from a postal survey of 235 farmers who had conducted a fecal egg-count-reduction test in 1999, 2000, or both years. A response of 54% was achieved. We developed a logistic-regression model. Contributory main effects in the final model were selling 10% more sheep in 2000 than is the usual policy (OR = 4.00), farm purchased since 1975 (OR = 2.34), and number of winter flock anthelmintic treatments in the previous 5 years (OR = 1.04). A secondary logistic-regression model assessed risk factors for farms selling 10% more sheep than usual in 2000; these farmers appeared less committed to their sheep enterprises than other farmers. These results are discussed in relation to current hypotheses of anthelmintic resistance. This is the first time that the farmer's management of the flock has been implicated in the development of anthelmintic resistance.
At present, there is no effective alternative to chemical control of parasitic helminths where livestock are grazed intensively. Resistance to anthelmintics has become a major problem in veterinary medicine, and threatens both agricultural income and animal welfare. The molecular and biochemical basis of this resistance is not well understood. The lack of reliable biological and molecular tests means that we are not able to follow the emergence and spread of resistance alleles and clinical resistance as well as we need. This review summarizes some of the recent findings on resistance mechanisms, puts forward some recommendations for limiting its impact and suggests some priorities for research in this area.
A mail survey of 235 Western Australian sheep farmers who had performed faecal egg count reduction tests for anthelmintic resistance in 1999 or 2000 was conducted, with some telephone follow-up. A response of 56% was achieved. Resistance to ivermectin, a member of the macrocyclic lactone class of anthelmintics, had developed on 44% of the farms surveyed. We used time to occurrence of resistance to ascertain factors that contributed to extending the time ivermectin remained an effective drench on these farms (median time=10.5 years). This time was significantly longer when farmers implemented more worm control practices on their farms (P=0.003). We developed a multivariable survival model that contained the following main effects: reduced winter drenching frequency, 0-2 flock treatments in 5 years (hazard ratio (HR) 0.52); availability of alternative effective anthelmintic classes on the farm (HR 0.30); always using safe pastures (HR 0.23); and veterinarians as the primary source of worm control advice (HR 0.58). The relationship of these findings to the understanding of anthelmintic resistance is discussed.
The problem of anthelmintic resistance prevents efficient control of parasites of livestock and may soon compromise human parasite control. Research into the mechanisms of resistance and the quest for diagnostic tools to aid control has required research that focuses on field resistance. On the other hand, resistant worms, including those kept in the laboratory, provide useful tools for studying drug action, especially at neuromuscular targets in worms. While the needs and directions of these research aims overlap, this review concentrates on research on drug targets. In this context, resistance is a useful tool for site of action confirmation. For example, correlations between molecular expression studies and resistance assays conducted on whole worms can strengthen claims for sites of anthelmintic action. Model systems such as Caenorhabditis elegans have been very useful in understanding targets but give a limited picture as it is now clear that resistance mechanisms in this worm are different from those in parasites. Accordingly, research on parasites themselves must also be performed. Resistant isolates of the sheep nematode parasite Haemonchus contortus are the most widely used for this purpose as in vivo, in vitro, physiological and molecular studies can be performed with this species. Neuromuscular target sites for the anthelmintics levamisole and ivermectin are the best studied and have benefited most from the use of resistant worm isolates. Resistance to praziquantel and the newer chemical groups should provide new tools to explore targets in the future.
The gastrointestinal nematodes are considered to be the most important group of internal parasites. Gastrointestinal parasitism is a major constraint to animal health, productivity, and profitability in grazing livestock production systems. The clinical manifestations of infection may range from no apparent effect to death. Despite years of research, assessment of production losses in many regions of the United States and of the potential impact of gastrointestinal nematode control remains to be determined.
The nucleotide sequences of the mitochondrial DNA (mtDNA) molecules of two nematodes, Caenorhabditis elegans [13,794 nucleotide pairs (ntp)], and Ascaris suum (14,284 ntp) are presented and compared. Each molecule contains the genes for two ribosomal RNAs (s-rRNA and l-rRNA), 22 transfer RNAs (tRNAs) and 12 proteins, all of which are transcribed in the same direction. The protein genes are the same as 12 of the 13 protein genes found in other metazoan mtDNAs: Cyt b, cytochrome b; COI-III, cytochrome c oxidase subunits I-III; ATPase6, Fo ATPase subunit 6; ND1-6 and 4L, NADH dehydrogenase subunits 1-6 and 4L: a gene for ATPase subunit 8, common to other metazoan mtDNAs, has not been identified in nematode mtDNAs. The C. elegans and A. suum mtDNA molecules both include an apparently noncoding sequence that contains runs of AT dinucleotides, and direct and inverted repeats (the AT region: 466 and 886 ntp, respectively). A second, apparently noncoding sequence in the C. elegans and A. suum mtDNA molecules (109 and 117 ntp, respectively) includes a single, hairpin-forming structure. There are only 38 and 89 other intergenic nucleotides in the C. elegans and A. suum mtDNAs, and no introns. Gene arrangements are identical in the C. elegans and A. suum mtDNA molecules except that the AT regions have different relative locations. However, the arrangement of genes in the two nematode mtDNAs differs extensively from gene arrangements in all other sequenced metazoan mtDNAs. Unusual features regarding nematode mitochondrial tRNA genes and mitochondrial protein gene initiation codons, previously described by us, are reviewed. In the C. elegans and A. suum mt-genetic codes, AGA and AGG specify serine, TGA specifies tryptophan and ATA specifies methionine. From considerations of amino acid and nucleotide sequence similarities it appears likely that the C. elegans and A. suum ancestral lines diverged close to the time of divergence of the cow and human ancestral lines, about 80 million years ago.
Nine anthelmintic products in pharmacies and from agricultural merchants in Kenya were tested for pharmaceutical quality. The concentration of active drug was compared with the claim on the label, and the variability of several products was tested between batches and between bottles within the same batch. All the products purchased claimed to contain levamisole but its mean (sd) concentration varied from 0 to 118.0 (13.3) per cent of the claimed. The concentration of levamisole in different batches of the same product ranged from 0 to 85.4 per cent of that claimed. One product consisting in part of mebendazole was found to contain 73.2 (9.4) per cent of the claimed concentration of this active component and two products consisting in part of oxyclozanide were found to contain 106.0 (14.4) and 120.6 (6.1) per cent of the expected concentration of oxyclozanide.
P‐glycoproteins can cause resistance of mammalian tumor cells to chemotherapeutic drugs. They belong to an evolutionarily well‐conserved family of ATP binding membrane transporters. Four P‐glycoprotein gene homologs have been found in the nematode Caenorhabditis elegans; this report describes the functional analysis of two. We found that PGP‐3 is expressed in both the apical membrane of the excretory cell and in the apical membrane of intestinal cells, whereas PGP‐1 is expressed only in the apical membrane of the intestinal cells and the intestinal valve. By transposon‐mediated deletion mutagenesis we generated nematode strains with deleted P‐glycoprotein genes and found that the pgp‐3 deletion mutant, but not the pgp‐1 mutant, is sensitive to both colchicine and chloroquine. Our results suggest that soil nematodes have P‐glycoproteins to protect themselves against toxic compounds made by plants and microbes in the rhizosphere.
A technique is described in which thiabendazole resistance is assayed by hatching nematode eggs in solutions of thiabendazole. Eggs of Haemonchus contortus and Ostertagia circumcincta were hatched in a series of concentrations of thiabendazole in a 0.1% NaCl solution. Thiabendazole resistant strains hatched in higher concentrations of thiabendazole than non-resistant strains. The dose response thus obtained for a strain of worms can be tested statistically against strains which are known to be either resistant or non-resistant.
Le Jambre L. F. and Royal W. M. 1977. Genetics of vulvar morph types in Haemonchus contortus: Haemonchus contortus from the Northern Tablelands of New South Wales. International Journal for Parasitology7: 481–487. The inheritance of the vulvar morph types, smooth, knobbed, linguiform A-right, linguiform A-left and linguiform C-type was studied in Haemonchus contortus from the Northern Tablelands of New South Wales. The dominance hierarchy of these characters was found to be smooth > knobbed > linguiform. The linguiform phenotype was the most common in the wild type population. The modifications on the linguiform phenotype were found to be highly heritable with A-right responding most to selection. Realised heritabilities for these characters was A-right 0.37, A-left 0.15, and C-type 0.21. It appeared that the dominance of a phenotype increased following selection.
The molecular basis for the resistance of the sheep parasitic nematode Haemonchus contortus to the benzimidazole (BZ) group of anthelmintics was investigated. Three BZ-susceptible and three resistant populations from different geographical locations were characterized with respect to the egg-hatch assay with thiabendazole (TBZ), mebendazole (MBZ) binding tests and restriction fragment length polymorphism (RFLP) after Southern blotting. Cloned H. contortus alpha- and beta-tubulin genes were used as probes to analyze the RFLPs of genomic DNA prepared from mixtures of infectious larvae (L3) or adults. The susceptible populations showed, with both alpha- and beta-tubulin probes, 2 to 6 different fragments, depending on the restriction enzyme used. The three resistant populations showed as many fragments with the alpha-tubulin probe as the susceptible populations, but when probed with beta-tubulin only 1 or 2 fragments were visible, but always less than in the susceptible populations. An in vitro selection experiment was carried out using a susceptible population that was isolated in the laboratory before BZ came on the market. The results showed that after two selections with increasing amounts of TBZ, the population had become resistant, according to the egg-hatch assay values and MBZ binding assay. Using RFPL, the number of beta-tubulin probe reactive DNA fragments was reduced from 5 to 1. Analysis of the DNA of individual male adults of susceptible populations indicated a heterogeneity among the individual worms regarding the number of beta-tubulin probe reactive fragments (1 to 4) and frequency of the specific fragments. Usually, only one specific fragment (9 kb) was found in the resistant individuals. This 9-kb fragment was already present in some individuals in the susceptible population although it was in combination with other fragments. This would imply that genes conferring BZ resistance were present in H. contortus populations before BZ came on the market, and could explain the fast selection for BZ resistance in the field.
The binding of tritiated benzimidazoles (BZs) — albendazole, parbendazole, oxibendazole, mebendazole, oxfendazole and fenbendazole — to crude tubulin extracts from BZ-susceptible and -resistant Haemonchus contortus has been examined. For all BZs, the binding was substantially lower in the resistant isolate. The extent of this reduction was dependent on the structure of the BZ, with mebendazole demonstrating superior binding to the resistant isolate that the other BZs. Enrichment of the crude tubulin extract using polylysine-linked agarose demonstrated that for both isolates more than 85% of the observed binding was to protein eluted in the tubulin-containing fraction. Based on biochemical kinetics, the change in tubulin associated with resistance is reduced capacity in resistant tubulin to bind BZ with little or no reduction in the association constant of the BZ-tubulin complex. Comparative egg hatch assay demonstrated a similar structural specificity with the resistance factor of mebendazole observed to be lower than that of albendazole, parbendazole, oxibendazole and thiabendazole. The results of both studies support the hypothesis that BZ resistance is due to a change in tubulin and that this change is dependent on the structure of the BZ.
Investigations into genetic mechanisms underlying anthelmintic resistance in parasitic nematodes have indicated that even at moderate levels of resistance, reversion to the initial level of susceptibility may be impossible. Alleles that confer the higher levels of susceptibility may be lost from the population in the early stages of selection. Therefore close monitoring of early steps in the selection for resistance is needed, as discussed here by Marleen Roos. Marcel Kwa and Warwick Grant.
This study was undertaken to determine the level of resistance against pyrantel citrate in strains of Oesophagostomum quadrispinulatum and Oesophagostomum dentatum which have previously been found resistant to this anthelmintic. Groups of pigs were artificially infected with batches of infective larvae which were previously found either susceptible or resistant to pyrantel citrate. After treatment with 1, 2 and 4 times the recommended dose (14 mg kg-1) of pyrantel citrate, the resistant O. quadrispinulatum population was reduced by 51.0, 76.2 and 86.1%, and O. dentatum by 41.2, 47.9 and 78.5%. The results indicated that O. dentatum was slightly more resistant (P less than 0.05) than O. quadrispinulatum to pyrantel citrate. Treatment of the susceptible worms with 1 and 2 times the recommended dose caused a reduction in worm numbers by 61.0 and 99.4%, respectively.
Mathematical modelling offers an aid to the understanding of parasitic diseases. In the past, computational limitations have meant that the role of many factors influencing the control of disease has had to be ignored or oversimplified. Using computers, these shortcomings can be overcome. Site-specific and environmental modelling offer new prospects for the management of disease. This paper illustrates the approach of computational veterinary parasitology with an application to anthelmintic and acaricidal resistance.
A field evaluation of anthelmintics in 336 horses on 37 farms was conducted between February and May 1986 in Sweden. The herds, each comprising at least eight horses, had histories of grazing on permanent pastures and receiving regular treatments against parasites at least three times a year. Small strongyles were refractory to (pro)-benzimidazole drugs in all but one of 23 herds examined. There was an average reduction in egg output of approximately 60%, and approximately 30% of 205 horses examined were shedding less than 100 eggs g-1 7 days after treatment. There was great between-herd variation of both the faecal egg-count depression (6.4-90.4%) and drug efficacy (0.0-80.0%). The non-benzimidazole drugs under investigation were fully effective against mature small strongyles.
This study was undertaken to determine whether anthelmintic-resistant Oesophagostomum spp. populations occur in Danish swine herds. A controlled field trial on selected sow herds suggested resistance to pyrantel citrate in a herd where faecal egg count depression in response to treatment was only 28.3%. This was confirmed by subsequent experimental infection of pigs where the suspected resistant Oesophagostomum isolate was compared with a susceptible worm isolate. After treatment with the recommended dose of the drug, worm burdens of the suspected isolate were only reduced by 42.6% (p greater than 0.05) in contrast to the susceptible isolate, which was reduced by 94.6% (p less than 0.01). Differential counts of the resistant isolate suggested that O. dentatum might be more resistant than O. quadrispinulatum. The resistant isolate originated from a herd, where the anthelmintic had been administered four times a year over a period of 7 years. The potential for development of anthelmintic resistance in Oesophagostomum spp. under management practices in our country is discussed in the light of the current views on predisposing factors.
Anthelmintic resistance has emerged as the most important problem confronting the successful control of nematode parasites of grazing animals. Although the significance of the problem varies between, and within, countries and farming enterprises, there is little likelihood that it will disappear of its own accord. On the contrary, it is reasonable to assume that it will increase if there is no change in traditional methods of parasite control. Although progress is being made in non-chemotherapeutic methods of control, these are unlikely to provide any practical alternatives in the short-term future. Nor can the pharmaceutical industry be expected to solve the problem because of the long period and the exceedingly high costs involved in bringing a completely new class of drug on to the market. The answer must lie in carefully husbanding the currently available anthelmintics, by providing farmers with programs which give good levels of parasite control and maintain high productivity in animals with fewer anthelmintic treatments. To be enthusiastically adopted by farmers, the programs require a commitment by both research and advisory workers. Such success can be achieved, as exemplified by the "Drenchplan" and "Wormkill" programs in Australia. It behoves workers in all countries which have a significant grazing livestock industry, not only those with an existing resistance problem, to consider how such schemes could be implemented.
Avermectin (AVM) inhibition of the development of the free-living stages of Haemonchus contortus has been quantified in an assay in which nematode eggs are placed on an agar matrix containing serial dilutions of a drug in the wells of a microtitre plate. Development is allowed to proceed for 6 days by which time larvae in control wells (no drug) have reached the infective, third (L3) stage. At high concentrations (> 30 nM) ivermectin (IVM) paralyses L1 larvae soon after hatching, however, much lower concentrations (approximately 1 nM) are sufficient to inhibit development to the L3 stage which suggests that effects of the drug other than those relating to gross motor activity are responsible for the latter effect. The larval stages of IVM-susceptible H. contortus isolates from both Australia and South Africa, including isolates known to be resistant to levamisole or rafoxanide and/or the benzimidazoles, were equally sensitive to inhibition by AVMs. In contrast, 6 isolates of H. contortus resistant to IVM in vivo showed a reduced sensitivity to AVM inhibition of development. The order of potency of a limited range of AVMs as inhibitors of larval development was consistent with in vivo efficacy. Resistance ratios for IVM-resistant isolates were dependent on AVM structure, with AVM B2 the most sensitive probe for IVM resistance in the isolates tested.
A strain of Haemonchus contortus (CAVR) isolated in Australia was found to be resistant to ivermectin (IVM) with 0.4 mg kg-1 of the anthelmintic failing to significantly reduce worm burdens. Resistance to IVM was sex-influenced in the CAVR strain with adult males showing a greater sensitivity to IVM. Cross resistance to moxidectin was evident with approximately 15% of the population surviving a dose of 0.1 mg kg-1. The free-living stages of the CAVR isolate had a reduced sensitivity to avermectin (AVM) inhibition of development and motility. Similar structure-activity patterns and resistance factors were obtained for a series of related AVMs as inhibitors of larval development and L3 motility in CAVR and White River II, an IVM-resistant H. contortus isolate from South Africa. Further, both isolates were found to be 3 times more sensitive to paraherquamide than a susceptible H. contortus isolate. This suggest that the same resistance mechanism is operating in both isolates. The CAVR strain is susceptible to the benzimidazoles, levamisole and closantel.
An absolute pre-requisite for a genetic response to a selective pressure is genetic variation within the population under selection. Helminth populations are clearly able to respond to selective pressures and must, therefore, be genetically heterogeneous. While not quite tautological, this is at best indirect evidence for the existence of genetic variation but there are few examples of well documented helminth phenotypic variation with a proven genetic basis. Isozyme analysis has provided more direct evidence for variation but attempts to link this variation to responses to selection or to identify the forces maintaining that variation have been largely unsuccessful. Thus there is a clear need for new techniques. The recent application of PCR and direct sequencing technology to the study of helminth genetics has allowed the genotypes of individual worms to be determined and the first direct measurements of allele frequencies to be made in this group of organisms. In addition, the application of genetic and molecular data from Caenorhabditis elegans is a potentially rich source of new markers. These techniques do not require that the genetic basis of the phenotype in question be known since a large number of loci can be examined and selection detected through changes in the frequency of anonymous linked marker loci. Phenotypes with complex genetic bases can, therefore, be analysed. I have applied these techniques to the study of anthelmintic resistance genetics and others have applied them to the genetics of inhibited development in Ostertagia. Other phenotypes that are of great interest are the potential for selection of resistance to vaccination and the use of genetically resistant hosts. The ease with which helminths have countered all classes of anthelminitics and the apparently high levels of polymorphism in helminth populations suggest that immunological control methods may also prove to be vulnerable to the adaptive capabilities of the parasite. Evidence from a mouse-helminth model system has already provided evidence that worms can meet the challenge.
The application of molecular techniques such as restriction length polymorphisms, mitochondrial DNA analysis and sequence tagged microsatellite sites to determine the amount of genetic variation and population structure of trichostrongylid parasites of domestic ruminants is examined. Information already provided by these techniques indicates that the parasite populations have a large amount of genetic variation and that gene flow between populations is greater than previously expected. The implications are that parasites can rapidly respond to selection by either anthelmintics or genetically resistant hosts. Advance warning of the response to selection can be obtained, even when the trait under selection is unknown, by measuring changes in the DNA polymorphisms of the parasite populations. Resistance to the benzimidazoles in the trichostrongylids now appears to be dependent on changes at two beta-tubulin loci. The first stage in the development of benzimidazole (BZ) resistance involves selection for an allele encoding a resistant form of isotype 1 beta-tubulin. A higher level of resistance is achieved with the selection of the gene encoding isotype 2 beta-tubulin. Techniques that maintain BZs in the host for extended periods of time may select for the higher level of resistance. A DNA assay to test this hypothesis is proposed.
Membranes from both ivermectin-sensitive and -resistant Haemonchus contortus L3 larvae were examined for the presence of high affinity [3H]ivermectin binding sites. Both tissue preparations displayed high affinity drug binding sites (Kd = 0.13 nM). Receptor density (Bmax = 0.4 pmol/mg) was the same in both the sensitive and resistant nematodes suggesting that target site modification was not involved in the development of drug resistance in this particular strain of H. contortus. The H. contortus ivermectin binding site appeared to be similar to the well characterized Caenorhabditis elegans ivermectin binding site with respect to affinity for ivermectin and receptor density.
Rothwell J. T. and Sangster N. C. 1993. An in vitro assay utilising parasitic larval Haemonchus contortus to detect resistance to closantel and other anthelmintics. International Journal for Parasitology 23: 573-578. A new assay for detecting resistance to anthelmintics in vitro is described. The assay uses a simple culture system in which the ability of anthelmintics to kill or inhibit the migration of parasitic third and fourth stage Haemonchus contortus larvae through a 50 μm aperture mesh is assessed. The assay detects 2-10-fold resistance to closantel. Resistance to benzimidazoles, levamisole and ivermectin can also be detected.
Receptors for acetylcholine are present in nematodes. Studies using physiological and biochemical methods have revealed the existence of nicotinic acetylcholine receptors with a novel pharmacology. Caenorhabditis elegans provides a particularly suitable organism with which to investigate such receptors using molecular genetic approaches. Mutants resistant to the cholinergic agonist (and anthelmintic drug) levamisole have permitted the isolation of a number of genes, including structural subunits of the nicotinic acetylcholine receptor. The only known viable mutants of nicotinic receptors are those of Caenorhabditis elegans. This organism offers the prospect of studying the developmental and regulatory effects of the loss of a single component of the receptor. Using Caenorhabditis elegans it is possible to select interesting phenotypic mutations by in vivo mutagenesis before determining the causative lesion. Resistance genes other than those encoding structural subunits are of particular interest, as they will encode additional polypeptides closely associated with nicotinic receptor function. Such proteins are often difficult or impossible to identify using conventional biochemical approaches, whereas genetic selection should permit their identification.
P-Glycoprotein (P-gp) causes a multidrug resistance (MDR) phenotype in tumour cells. In some cancers, the expression of P-gp has been correlated with low clinical response to chemotherapy and survival of patients. Previous studies have shown that certain lipophilic drugs bind to P-gp and reverse the MDR phenotype of tumour cells. In this study, we extend that list of compounds and present evidence for the capacity of a potent and clinically safe anthelmintic, ivermectin (IVM), as an MDR-reversing drug. Using a highly drug-resistant human cell line, we compared IVM with other MDR-reversing agents and showed that IVM is 4- and 9-fold more potent than cyclosporin A and verapamil, respectively. The capacity of IVM to inhibit iodoaryl-azidoprazosin photolabeling of P-gp is consistent with direct binding to P-gp. Studies showed that [3H]IVM binding to membranes from resistant cells is specific and saturable with KD and Bmax values of 10.6 nM and 19.8 pmol/mg, respectively. However, while cyclosporin A or vinblastine inhibited [3H]IVM binding to membranes from drug-resistant but not drug-sensitive cells, neither verapamil nor colchicine had any effect. Furthermore, both IVM and cyclosporin A and, to a lesser extent, verapamil also inhibited [3H]vinblastine binding to membranes from drug-resistant cells. Drug transport studies showed that [3H]IVM is a substrate for the P-gp drug efflux pump. However, it was transported less efficiently by P-gp than [3H]vinblastine. Moreover, only cyclosporin A was effective in potentiating the accumulation of [3H]IVM in drug-resistant cells. Taken together, the high efficiency of MDR reversal by IVM combined with its low toxicity are consistent with the properties of an ideal MDR-reversing agent.
Three fecal egg count reduction assays (FECR) and one critical trial were performed to determine the efficacy of pyrantel pamoate (PP) at 6.6 mg base kg-1 on a well managed stud farm in Louisiana where a loss of efficacy was suspected. Efficacy of PP based on FECR varied from 25% in mares to 83% in yearlings. Second treatments with PP 2 weeks following an initial treatment failed to reduce eggs per gram (EPG). A critical trial was performed to determine the cyathostome species resistant to PP. Three strongyle-naive ponies which acquired infections on the farm were used for this purpose. Following treatment with PP at the recommended dose, 11 species of cyathostomes remained in the intestine of the tracer ponies. Reduced efficacies (62%-88%) were noted for seven species. Resistance to oxibendazole (OBZ), which was > 90% effective on this farm in 1982, was also evaluated by FECR and found to exist. The results of one experiment indicate that dual resistance of parasites to PP and OBZ also exists.
Caenorhabditis elegans is a rhabditid nematode. What relevance does this have for the interpretation of the complete genome sequence, and how will it affect the exploitation of the sequence for scientific and social ends? Nematodes are only distantly related to humans and other animal groups; will this limit the universality of the C. elegans story? Many nematodes are parasites; can knowledge of the C. elegans sequence aid in the prevention and treatment of disease?
Six in vitro methods for the detection of anthelmintic resistance were compared using benzimidazole/levamisole-resistant Haemonchus contortus and benzimidazole/levamisole/ivermectin-resistant Ostertagia circumcincta as well as susceptible strains of both parasite species. The degree of resistance to thiabendazole and levamisole was compared by (1) an egg hatch assay, (2) an egg hatch paralysis assay, (3) a larval development assay, (4) a larval paralysis assay (5) a larval paralysis assay with physostigmine and (6) larval micromotility assay. The degree of resistance for each assay was expressed as resistance factor--RF. For the detection of thiabendazole and levamisole resistance, the larval development test was observed as the most sensitive to measure quantitatively a degree of resistance between susceptible and resistant strains. For this test the RF for thiabendazole and levamisole was 14.3 and >32.5, respectively in H. contortus strains and 21.1 and 3.5 in strains of O. circumcincta. Egg hatch assay was also found to be sensitive and accurate in determining of resistance to benzimidazole. For measurement of levamisole resistance the egg hatch paralysis assay and larval paralysis assay were found to be able to distinguish between strains, but some disadvantages of these techniques make it unsuitable for field detection of levamisole resistance. Other in vitro assays as larval paralysis assay with physostigmine and larval micromotility assay were also investigated. Significant differences in paralysis of the larvae were observed using larval paralysis assay.
The potential for cytochrome P450 from Haemonchus contortus to operate in the oxygen-poor intestinal environment was investigated by examining the ability of the cytochrome to act in vitro as a peroxygenase in utilising cumene hydroperoxide for substrate oxidations not requiring molecular oxygen. Peroxygenase and NADPH-supported monooxygenase activities were measured in microsomes prepared from L3 and adult nematodes. Both cumene hydroperoxide- and NADPH-supported ethoxycoumarin O-deethylase and aldrin epoxidase activities were detected in larval microsomes. Adult microsomes showed low levels of cumene hydroperoxide-supported ethoxycoumarin O-deethylase, as well as NADPH- and cumene hydroperoxide-supported aldrin epoxidase activities. The use of inhibitors in ethoxycoumarin O-deethylase assays with larval microsomes indicated that the peroxygenase pathway does not proceed via ferrous cytochrome P450 (no inhibition by carbon monoxide), did not require molecular oxygen, and did not depend on electron flow from cytochrome P450 reductase. Larval activity was inhibited by typical cytochrome P450 inhibitors (piperonyl butoxide, SKF-525A, chloramphenicol, metyrapone, n-octylamine) and was unaffected by the peroxidase inhibitor salicylhydroxamic acid. In contrast, adult microsomal cumene hydroperoxide-supported ethoxycoumarin O-deethylase activity was significantly inhibited by both cytochrome P450 inhibitors and salicylhydroxamic acid. Adult microsomes also contained potassium ferrocyanide peroxidase activity utilising cumene hydroperoxide. This activity showed a similar pattern of inhibition by both cytochrome P450 and peroxidase inhibitors. Whilst the ability of larval H. contortus cytochrome P450 to act as a peroxygenase in vitro was demonstrated, the inhibition results with adult microsomes showing both cytochrome P450 and peroxidase activities require further investigation to clarify the nature of the adult microsomal cumene hydroperoxide-supported O-deethylase activity.
During the past decade, the prevalence of anthelmintic resistance in some economically important helminths of sheep, goats and horses has increased dramatically. In some regions of Australia, South America and South Africa, anthelmintic resistance has become a serious threat to the survival of the sheep industry. Mass treatment programmes and exclusive reliance on anthelmintics for worm control in livestock are amongst the most important reasons for the development of anthelmintic resistance. In this article, Stanny Geerts, Gerald Coles and Bruno Gryseels draw the attention to a number of errors that have occurred in the control of helminths in livestock and that should be avoided in the control of worms in humans.
Our knowledge of many aspects of the molecular biology of animal parasitic nematodes has rapidly expanded in recent years but the classical genetic analysis of this group of organisms has yet to emerge as a viable discipline. For example, it is not possible to routinely perform crosses between single males and females to examine the genetic basis of even simple phenotypes such as anthelmintic resistance. This has meant that the function of many cloned parasite genes can only be inferred from sequence comparison with genes from other organisms where the function is known, or by correlation of DNA polymorphisms linked to the gene with phenotypic differences between strains or individuals. In the absence of classical genetic techniques, a molecular solution is to transform a suitable host with the gene of interest, but what defines a suitable host? Here, Warwick Grant describes recent work that aims to provide such a host.
ExtractIn contrast to the situation in sheep, anthelmintic resistance in cattle nematodes in New Zealand appears to be relatively uncommon. A brief review in 1991 indicated that only six confirmed cases had been reported(l). All related to resistance to benzimidazole anthelmintics and involved infections of Cooperia and, to a lesser extent, Ostertugia and Trichostrongylus. Since then, however, a further 13 cases of benzimidazole resistance in cattle have been identified by faecal egg count reduction tests conducted on submissions to the Batchelar and Ruakura Animal Health Laboratories (Table I). In addition, another two cases have recently been detected by similar means by ethers . In the latter two instances, these not only involved resistance to benzimidazole drenches but to milbemycin/ avermectin type anthelmintics as well. Like those listed in Table I, the main parasite genus implicated on both of these occasions was Cooperia.