Resistance In Nematode Parasites Research Articles

Anthelmintic resistance in trichostrongylid nematodes of sheep farms in Northwest Spain

A survey to determine the prevalence of anthelmintic resistance in nematode parasites of sheep against the three main families of anthelmintics (benzimidazoles, macrocyclic lactones and imidazothiazoles) was carried out from January 1999 to December 2003 involving 85 flocks in Northwest (NW) Spain. In the study on prevalence of resistance to benzimidazoles, faecal egg count reduction test (FECRT) showed there was resistance in 8 (12.7%) flocks, 1 (1.6%) was suspected, and 54 (85.7%) were susceptible. The results indicated that 9 flocks (34.61%) showed resistance, 1 (3.85%) showed suspected resistance, and 16 (61.54%) were susceptible to the imidazothiazoles. Against macrocyclic lactones, resistance was observed in 8 flocks (15.69%), 4 (7.84%) showed suspected resistance and 39 (76.47%) were susceptible. None of the flocks used in the study showed resistance to the three families of anthelmintics. Nevertheless, six were recorded as resistant or suspected of being resistant to two of these families of anthelmintics. Egg hatch assay (EHA) and FECRT were carried out jointly on 61 flocks, although EHA was done on a total of 83 farms. The results showed that 15 (18.07%) of the 83 flocks were resistant, with egg death 50 over 0.1 microg/ml thiabendazole, and 68 (81.93%) were susceptible to benzimidazoles. When the results between FECRT and EHA were compared, both techniques showed good correlation in field studies. Faecal cultures performed pre- and post-treatment indicated that Teladorsagia and Trichostrongylus were the main genera.

Anthelmintic resistance of nematode parasites of small ruminants in eastern Ethiopia: Exploitation of refugia to restore anthelmintic efficacy

Faecal egg count reduction tests (FECRT) were conducted in May 2003 to determine the efficacy of anthelmintics used for treatment against nematode parasites in separately managed sheep and goat flocks at Alemaya University in eastern Ethiopia. These tests revealed high levels of anthelmintic resistance to albendazole, tetramisole, the combination of these two drugs, and to ivermectin in the goat flock (predominantly infected by Haemonchus contortus and Trichostrongylus spp.), whereas all drugs were highly efficacious in the sheep flock. A second FECRT confirmed these observations. Following this, a new management system was implemented on the goat flock for a period of 9 months (January–September 2004) in an attempt to restore the anthelmintic efficacy. This involved a combination of measures: eliminating the existing parasite infections in the goats, exclusion from the traditional goat pastures, and introducing communal grazing of the goats with the university sheep flock and livestock owned by neighbouring small-holder farmers. A second series of FECRTs (Tests 3 and 4) conducted 7 months after this change in management, showed high levels of efficacy to all three drugs (albendazole, tetramisole and ivermectin) in the goat flock. This is the first field study to demonstrate that anthelmintic efficacy in the control of nematode parasites of small ruminants can be restored by exploiting refugia.

A survey on anthelmintic resistance in nematode parasites of sheep in the Slovak Republic

The prevalence of anthelmintic resistance on 27 sheep farms in Slovakia was investigated in 2003 and 2004 using the faecal egg count reduction test (FECRT) according to the WAAVP guidelines. Resistance to albendazole was detected on one farm (3.7%) and suspected on two farms (7.4%) out of 27 sheep flocks. Resistance to ivermectin was tested on 26 farms. On six (23.1%) farms, results indicated the presence of ivermectin resistance. Resistance to ivermectin was suspected on eight farms (30.8%). However, it is also possible that generic ivermectin anthelmintics used in survey have a lower efficacy against sheep nematodes.

Drug resistance in nematodes: a paper tiger or a real problem?

The purpose of this review is to illustrate where drug resistance in parasitic nematodes has become a major problem. The mechanisms underlying anthelmintic resistance, the possible reasons for the development of anthelmintic resistance, and recommendations to minimize the further development of anthelmintic resistance in humans will be addressed. Resistance has developed to all drugs of the few anthelmintic classes currently available. Drug resistance has become a major threat to sheep production in Australia, New Zealand, Great Britain and South Africa. In addition, the treatment of nematode infections in horses has become insufficient because of anthelmintic resistance, whereas resistance in cattle has recently been found in New Zealand and southern America. In contrast, anthelmintic resistance to human helminths has only been reported anecdotally. The rate of development of anthelmintic resistance is determined by the resistance gene frequency and by selection pressure, as illustrated by the concept of 'worms in refugia' in ovine nematode infections. Although anthelmintic resistance has become a major constraint in livestock production of small ruminants, drug resistance is currently not a major issue in the treatment of human nematode infections. However, if recent community treatment programmes are pursued injudiciously, the inevitable evolutionary consequence of anthelmintic resistance in humans might emerge sooner rather than later.

Evolution of high-level, multiple anthelmintic resistance on a sheep farm in Malaysia.

Anthelmintic resistance in nematode parasites of sheep and goats on a government farm in north Malaysia was monitored over a 3-year period (1997-2000). The faecal egg count reduction test (FECRT) was conducted on young sheep at the beginning and end of this period. Changes in management, designed to reduce the selection pressure for the development of anthelmintic resistance, were also implemented during this time. By far the most important parasite problem was Haemonchus contortus. In 1997, this nematode was found to be resistant to levamisole, with suspected resistance to closantel and moxidectin. However, when the FECRT was repeated 3 years later, its resistance status had become much more severe, with resistance to benzimidazole, levamisole and ivermectin, and suspected resistance to moxidectin. This rapid evolution to multiple anthelmintic resistance is a major concern that needs to be arrested. There is an urgent need to evaluate other control strategies that incorporate livestock management, the 'smart' use of drugs and non-chemotherapeutic approaches, such as biological control agents.

Anthelmintic resistance in nematodes of horses.

Suppressive anthelmintic treatment strategies originally designed to control Strongylus vulgaris in horses were extremely successful in reducing morbidity and mortality from parasitic disease. Unfortunately, this strategy has inadvertently resulted in the selection of drug-resistant cyathostomes (Cyathostominea), which are now considered the principal parasitic pathogens of horses. Resistance in the cyathostomes to benzimidazole drugs is highly prevalent throughout the world, and resistance to pyrantel appears to be increasingly common. However, there are still no reports of ivermectin resistance in nematode parasites of horses despite 20 years of use. It is unknown why resistance to ivermectin has not yet emerged, but considering that ivermectin is the single most commonly used anthelmintic in horses most parasitologists agree that resistance is inevitable. The fecal egg count reduction test is considered the gold standard for clinical diagnosis of anthelmintic resistance in horses, but diagnosis is complicated by lack of an accepted standard for the performance of this test or for the analysis and interpretation of data. Presently there is very little data available on the molecular mechanisms of anthelmintic resistance in cyathostomes; beta-tubulin gene is the only anthelmintic-resistance associated gene that has been cloned. The increasingly high prevalence of anthelmintic-resistant cyathostomes must be taken into account when designing worm control programs for horses. Strategies to decelerate further selection for drug resistance thereby extending the lifetime of currently effective anthelmintics should be implemented whenever possible. Considering the nature of the equine industry in which horses often graze shared pastures with horses from diverse locations, transmission and widespread dispersal of resistant parasites is virtually assured. A proactive approach to this problem centered on understanding the molecular basis of anthelmintic resistance in cyathostomes is required if we are to expect chemical control of nematodes in horses to remain a viable element of parasite control in the future.

Triclabendazole-resistant Fasciola hepatica: beta-tubulin and response to in vitro treatment with triclabendazole.

Resistance in Fasciola hepatica to triclabendazole ('Fasinex') has emerged in several countries. Benzimidazole resistance in parasitic nematodes has been linked to a single amino acid substitution (phenylalanine to tyrosine) at position 200 on the beta-tubulin molecule. Sequencing of beta-tubulin cDNAs from triclabendazole-susceptible and triclabendazole-resistant flukes revealed no amino acid differences between their respective primary amino acid sequences. In order to investigate the mechanism of triclabendazole resistance, triclabendazole-susceptible and triclabendazole-resistant flukes were incubated in vitro with triclabendazole sulphoxide (50 microg/ml). Scanning and transmission electron microscopy revealed extensive damage to the tegument of triclabendazole-susceptible F. hepatica, whereas triclabendazole-resistant flukes showed only localized and relatively minor disruption of the tegument covering the spines. Immunocytochemical studies, using an anti-tubulin antibody, showed that tubulin organization was disrupted in the tegument of triclabendazole-susceptible flukes. No such disruption was evident in triclabendazole-resistant F. hepatica. The significance of these findings is discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.

Molecular aspects of sexual development and reproduction in nematodes and schistosomes

In contrast to the free-living nematode Caenorhabditis elegans, surprisingly little is known about the molecular aspects of reproduction in parasitic helminths. Investigations into such aspects would provide an improved understanding of the fundamentals of sexual differentiation, development, maturation and behaviour, as well as sex-specific genes and their expression. Such knowledge could lead to new means of parasite control by interfering with or disrupting one or more of these processes, which is particularly important given the emerging problems with genetic resistance in parasitic nematodes against anthelmintic drugs. This chapter brings together some relevant information on the sexual biology of C. elegans, summarizes studies of gender-specific expression in selected parasitic helminths of socio-economic significance, describes advanced molecular techniques for the analysis of gender-specific genes, and indicates the prospects for genomic research on reproductive processes and the implications thereof for controlling parasitic helminths.

Ivermectin-resistant Ostertagia circumcincta from sheep in the lower North Island and their susceptibility to other macrocyclic lactone anthelmintics

Aim: To confirm the ivermectin resistance status of a strain of Ostertagia circumcincta which was isolated from a sheep farm in the lower North Island of New Zealand and to assess the susceptibility of this strain to other macrocycliclactone anthelmintics. Methods: Twenty-five lambs housed indoors were each infected with 12,000 L3 larvae of the above parasite strain. Approximately 3 weeks after infection the lambs were allocated to 1 of 4 treatment groups (3 groups of 6, and 1 group of 7 lambs), one of which remained untreated while the others were drenched orally with ivermectin, moxidectin or abamectin at 0.2 mg/kg liveweight. Faecal egg counts (FECs) before and after treatment, and post-mortem worm burdens 10 days after treatment were examined to assess efficacies of each anthelmintic. Results: Treatment with ivermectin reduced the mean FEC by only 18% and the mean worm burden by only 42%, whereas moxidectin and abamectin reduced FECs by > 92% and worm burdens by >95%. Conclusion: These results, together with a similar case described recently from the South Island, confirm the emergence of ivermectin resistance in nematode parasites of sheep in New Zealand. The superior efficacy of moxidectin and abamectin in this case indicates that, following the emergence of resistance to ivermectin, some short-term practical use may still be made of these other anthelmintics. However, their continued use will undoubtedly result in increased levels of resistance and eventual therapeutic failure of these products also.

Haemonchus contortus: Selection at a Glutamate-Gated Chloride Channel Gene in Ivermectin- and Moxidectin-Selected Strains

Blackhall, W. J., Pouliot, J.-F., Prichard, R. K., and Beech, R. N. 1998.Haemonchus contortus: Selection at a glutamate-gated chloride channel gene in ivermectin- and moxidectin-selected strains.Experimental Parasitology90, 42–48. Anthelmintic resistance in nematode parasites of livestock is a serious problem worldwide. Ivermectin, an avermectin, and moxidectin, a milbemycin, are potent endectocides commonly used to control these parasites. The proposed mode of action of avermectins and possibly the milbemycins involves the binding of the drug to the α-subunit of a glutamate-gated chloride channel, which opens or potentiates gating of the channel and leads to the hyperpolarization of the target neuromuscular cell. Glutamate gates the channel by binding to the β-subunit. We have cloned a fragment of a putative α-subunit gene fromHaemonchus contortus.The sequence of the beta subunit is available from GenBank. Genetic variability of this fragment was analysed by single-strand conformation polymorphism in five strains ofH. contortus: two strains passaged without drug selection, two strains selected with ivermectin, and one strain selected with moxidectin. One allele of the putative α-subunit gene appeared to be associated with resistance to the drugs, increasing in frequency in the three drug-selected strains relative to the unselected strains. Another allele appeared to be associated with susceptibility, decreasing in frequency in the three drug-selected strains relative to the unselected strains. A similar analysis of the β-subunit gene showed no significant differences in allele frequencies between the unselected and drug-selected strains. Our findings suggest a correlation between changes in allele frequencies of the putative α-subunit gene and resistance to ivermectin and moxidectin.

Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog

Resistance to ivermectin and related drugs is an increasing problem for parasite control. The mechanism of ivermectin resistance in nematode parasites is currently unknown. Some P-glycoproteins and multidrug resistance proteins have been found to act as membrane transporters which pump drugs from the cell. A disruption of the mdr1a gene, which encodes a P-glycoprotein in mice, results in hypersensitivity to ivermectin. Genes encoding members of the P-glycoprotein family are known to exist in nematodes but the involvement of P-glycoprotein in nematode ivermectin-resistance has not been described. Our data suggest that a P-glycoprotein may play a role in ivermectin resistance in the sheep nematode parasite Haemonchus contortus. A full length P-glycoprotein cDNA from H. contortus has been cloned and sequenced. Analysis of the sequence showed 61–65% homology to other P-glycoprotein/multidrug resistant protein sequences, such as mice, human and Caenorhabditis elegans. Expression of P-glycoprotein mRNA was higher in ivermectin-selected than unselected strains of H. contortus. An alteration in the restriction pattern was also found for the genomic locus of P-glycoprotein derived from ivermectin-selected strains of H. contortus compared with unselected strains. P-glycoprotein gene structure and/or its transcription are altered in ivermectin-selected H. contortus. The multidrug resistance reversing agent, verapamil, increased the efficacy of ivermectin and moxidectin against a moxidectin-selected strain of this nematode in jirds ( Meriones unguiculatus). These data indicate that a P-glycoprotein may be involved in resistance to ivermectin and other macrocyclic lactones in H. contortus.

Anthelmintic resistance in nematode parasites from goats in Spain

In a flock of cashmere goats sited in Asturias (NW, Spain) diminished efficacy was observed following treatment with netobimin and consequently a study to demonstrate the existence of anthelmintic resistance and the species of nematode involved was conducted. Results from faecal egg count reduction tests before and after treatment with netobimin or Ivermectin showed efficacies of 89.4 (81.8–94.8) and 99.7% (93.9–99.9%), respectively. Teladorsagia circumcincta was found to be the dominant species involved in netobimin resistance. Effective dose (ED 50) values in the egg hatch assay of 0.22 μg thiabendazole ml −1 confirmed the existence of benzimidazole carbamate resistant nematodes. To the best of our knowledge, this is the first report on the existence of anthelmintic resistant nematodes from ruminants in Spain.

A survey of anthelmintic resistance on ten sheep farms in Mashonaland East Province, Zimbabwe.

A survey to detect anthelmintic resistance in nematode parasites of sheep was conducted on 10 randomly-distributed farms in the Chivhu District, Mashonaland East Province, Zimbabwe. Before the survey, a questionnaire was circulated to the farmers concerning nematode parasite control. Results showed that parasite control using anthelmintic treatment was the only method practised and that the benzimidazoles were the most frequently used anthelmintic drugs. The faecal egg count reduction test was used to detect resistance. The anthelmintic groups tested were benzimidazoles, levamisole and ivermectin. Resistance to benzimidazoles was detected on 6 of 10 farms and levamisole resistance on 2 of 3 farms. Ivermectin resistance was not observed on the farms surveyed. Post-treatment larval cultures indicated that Haemonchus contortus survived administration of fenbendazole, albendazole, oxfendazole and levamisole. A Cooperia sp. strain resistant to albendazole was detected and this is the first report in Zimbabwe of a resistant parasite in this genus.

Anthelmintic resistance in nematode parasites of sheep in Denmark

The occurrence of anthelmintic resistance on sixteen sheep farms in Denmark was investigated using the faecal egg count reduction test (FECRT), the egg hatch assay (EHA) for thiabendazole (TBZ) and a microlarval development assay (LDA) for TBZ, levamisole (LEV) and ivermectin (IVM). At least two anthelmintics with different modes of action were tested on each farm in the FECRT, depending on the number of sheep available. Less than 95% faecal egg count reduction (FECR) were recorded 10–14 days after treatment with both LEV and IVM on two farms, LEV on two farms, benzimidazoles (BZs) on two farms and IVM on one farm. This indicated resistance to the respective anthelmintics according to the definitions of the World Association for the Advancement of Veterinary Parasitology. Trichostrongylus and Ostertagia spp. were the predominant nematode species in both pre-treatment and post-treatment faecal cultures. The LD50 values for TBZ in the EHA for isolates from the two farms where BZ resistance was detected in the FECRT were 0.52 and 1.5 μ M TBZ, indicating resistance. The LD50 values for TBZ for these isolates in the LDA were 0.09 and 1.1 μ M. The LD50 values in the LDA for isolates declared resistant to LEV ranged from 0.14 to 1.1 μ M LEV. For IVM, the two resistant isolates had LD50 values of 23 and 24 n M IVM in the LDA. There were gross disagreements in the declaration of resistance between methods of calculating %FECR based on the arithmetic mean and those that use geometric mean eggs per gram (EPG) of faeces. Similarly, inclusion of pre-treatment EPG or control group EPG in the calculation of %FECR influenced declaration of resistance.

A survey of anthelmintic resistance in nematode parasites of goats in Denmark

The occurrence of anthelmintic resistance in 15 goat herds in Denmark was investigated using the faecal egg count reduction test (FECRT), egg hatch assay (EHA) for thiabendazole (TBZ) and a microlarval development assay (LDA) for TBZ, levamisole (LEV) and ivermectin (IVM). Resistance to both benzimidazole (BZ) and levamisole (LEV) anthelmintics was detected on six farms, and to both BZs and IVM on one farm. Resistance to BZs was also detected on two farms and to LEV on two farms. Ivermectin resistance was present on one farm. Trichostrongylus and Ostertagia spp. were the predominant nematode species in both pre-treatment and post-treatment faecal cultures. In all nine instances where BZ resistance was detected in the FECRT, the LD50 values for TBZ in the EHA were higher than 0.5 μM TBZ (0.1 μg TBZ/ml), indicating resistance. LD50 values for TBZ in the LDA were also higher than 0.5 μM where resistance to BZs was detected in the FECRT, except two farms where LD50 values of 0.21 and 0.29 μM TBZ were recorded. LD50 values in the LDA for isolates declared resistant to LEV ranged from 0.78 to 5.62 μM LEV. For IVM, the two resistant isolates had LD50 values of 25 and 68 nM IVM. There were disagreements in the declaration of resistance between methods of calculating faecal egg count reduction percentage (FECR%) based on the arithmetic mean and those where geometric mean EPG is used. Similarly, inclusion of pre-treatment EPG or control group EPG in the calculation of FECR% influenced declaration of resistance.

The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America: Paraguay

This survey to detect anthelmintic resistance in nematode parasites of sheep was conducted on 11 farms in the Occidental and 26 farms in the Oriental Regions of Paraguay using the faecal egg count reduction test (FECRT). The anthelmintic groups tested were the benzimidazoles, levimasole and avermectins (both oral and injectable). Overall the levels of resistance were 73%, 68%, 73% and 47%, respectively. Levels of resistance were similar for all three important nematode genera, viz. Haemonchus contortus, Ostertagia and Trichostrongylus. This survey clearly indicates that a large, and ever increasing, proportion of sheep farmers are rapidly approaching the time when they will have exhausted all chemotherapeutic options to control parasites. Unless they face having to abandon their sheep farming operations, radical changes will need to be implemented with urgency.

The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America: General overview

South America has a large population of small ruminants. Current estimates are approximately 100 million sheep and 23 million goats. A large percentage of these flocks are raised in the humid tropics/sub-tropics. Nematode parasitism is singly the most important disease of these animals and typically farmers resort to frequent anthelmintic treatment in attempts to control this problem. Because of this reliance on drugs, price consideration is an important determinant in a farmer's choice of anthelmintic. In some circumstances, this has led to unethical practices of drug adulteration and substitution, or the offering to farmers of cheap, sub-standard genetic products. The prevalence of anthelmintic resistance has not been investigated in any widespread sense in South America, although some of the first reports of resistance were made in southern Brazil almost 30 years ago. The following series of papers outline surveys conducted in the humid tropics/sub-tropics of southern Latin America to assess the significance of resistance to the broad-spectrum anthelmintic groups in nematode parasites of sheep flocks.

The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America: Brazil

This survey was conducted in the southern Brazilian state of Rio Grande do Sul and involved 182 farms located in 26 countries. In addition to the three major broad-spectrum anthelmintic groups (viz. benzimidazole, levamisole and ivermectin) the combination benzimidazole and leyamisole and the H. contortus specific anthelmintic, closantel, were tested by the faecal egg count reduction method for the prevalence of anthelmintic resistance. Resistance was found to be 90%, 84%, 13%, 73% and 20%, respectively. This is a crisis situation. Immediate, drastic action needs to be implemented, otherwise the sheep industry in this region (approx. 10 million head) will soon face a lack of any effective anthelmintics with the inevitable consequences of major restructuring or abandonment.

The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America: Uruguay

This survey was conducted on 252 farms randomly distributed over all the sheep raising areas in Uruguay. The study involved farms with more than 600 sheep, which represented 80% of the total sheep population of the country. Three anthelmintic groups were assessed, namely, benzimidazoles, levamisole and avermectins. Overall, the results showed 80% of sheep flocks had benzimidazole resistance, 71% had resistance to levamisole, and 1.2% of flocks showed resistance to avermectins. Approximately 28% of farms had resistance to one anthelmintic group, 64% to two anthelmintic groups, and 1% resistance to all three groups. Only 7.5% of farms had no detectable levels of anthelmintic resistance. More than 80% of farms had Trichostrongylus populations resistant to both benzimidazoles and levamisole. Resistance was recorded in all three anthelmintic groups for Haemonchus and resistance also occurred to benzimidazoles and levamisole in Ostertagia.

The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America: Argentina

Sixty-five sheep farms in the northern provinces of Buenos Aires, Entre Rios, Corrientes, Cordoba and Sante Fe were used in this survey on anthelmintic resistance. Anthelmintic groups tested were the benzimidazoles, levamisole, the combination levamisole + benzimidazole product and the avermectins. The overall level of resistance was 46% of properties, with resistance to the individual drug groups being 40%, 22%, 11% and 6%, respectively. On a large proportion of farms the resistance recorded was to two, or more, anthelmintic groups. Haemonchus contortus, Ostertagia spp. and Trichostrongylus spp. were the most abundant parasite species recorded. Resistance was greatest in the province of Corrientes where the frequency of treatment is generally high due to the endemic nature of H. contortus. Management practices were also important with resistance greatest on farms where frequent drenching is carried out and on farms carrying only sleep.