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Small Ruminant Research 76 (2008) 99–103

Anthelmintic resistance in sheep nematodes
E. Papadopoulos
School of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Macedonia, Greece Available online 19 February 2008

Abstract Many parasitic nematodes of veterinary importance have genetic features that favour development of anthelmintic resistance, this becoming a major worldwide constrain in small ruminant production. The development of anthelmintic resistance poses a large threat to future production and welfare of grazing sheep. Development of variable degrees of resistance among different species of gastrointestinal nematodes has been reported for all the major groups of anthelmintic drugs. Reliable detection of resistance is important, in order to design appropriate strategies for controlling and delaying the development of resistance. Maintaining parasites in refugia and not exposed to anthelmintics, seems to be a key point, because the susceptible genes are preserved. Targeted selective treatments attract the interest of scientists towards this direction. None of the non-chemical methods for parasite control, i.e. nutrition, vaccines, parasite resistant breeds, is sufficiently effective without anthelmintic support and thus do not offer a practical option. However, most of them reduce reliance on the use of chemicals and are environmental friendly. Extensive research is required to manage resistance and field evaluation of any control suggestion. © 2008 Elsevier B.V. All rights reserved.
Keywords: Sheep; Health; Welfare; Parasitic disease; Nematodes; Flock; Planning; Health management; Preventive medicine; Anthelmintic resistance; Benzimidazoles; Macrocyclic lactones

1. Introduction Many parasitic nematodes of veterinary importance have genetic features that favour the development of anthelmintic resistance (AR). They possess the genetic potential to respond successfully to chemical attack and the means to assure dissemination of their resistant genes through host movement (Kaplan, 2004). A large number of papers have been published on AR to report any available information concerning the species of nematodes involved, drugs, geographical spread, detection methods, possible reasons for development, control and recommendations (Coles et al., 1992, 2006; Coles, 2005; Jabbar et al., 2006).

2. Development of anthelmintic resistance It is expected that during anthelmintic treatments, a small number of worms survive, these being the most resistant proportion of the population. These worms contaminate the pasture with a majority of resistant larvae for subsequent generations, leading gradually to the selection pressure of AR. This selection rate depends on the percentage contribution to the next generation between nematodes surviving treatment and other ones not exposed to it (in refugia). Any action increasing the percentage contribution that survivors of treatment make to the next generation, will contribute to development of resistance, whilst any action increasing the prevalence of the untreated population will slow down its development. The gene frequency for resistance within untreated populations plays an important role to determine how fast AR develops. Even in nature, amongst the genetic diversity of nematode population, random mutations in unselected

This paper is part of the special issue entitled “Current issues in Sheep Health and Welfare” guest edited by George C. Fthenakis. E-mail address: eliaspap@vet.auth.gr. 0921-4488/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2007.12.012

Papadopoulos / Small Ruminant Research 76 (2008) 99–103 worms may occasionally take place. it is now seldom used (Presidente. when goats are treated with the same dose as sheep. therefore. as this may affect hatchability of nematode eggs. and can be easily applied in sheep (Coles et al. 1994). 2006). If reduction in faecal egg counts is over 95%.. Use of larval cultures in pre. it has to be noted that culture conditions may favour the development of one parasitic species over another and therefore. much easier when animals of these two species are kept together (Jackson. Their worm burdens are compared after treatment. Obviously. 1985) should take place before interpretation of results. particularly of those of Haemonchus contortus (McKenna. 1989). There are several in vivo tests suitable for all types of anthelmintics. these depending on the specific anthelmintic tested. Presidente. faecal samples should be collected 8–10 d after treatment for benzimidazole-testing or 14–17 d after treatment for macrocyclic lactone-testing. it requires a lot of labour and animals and hence.. Cases have also been reported that resistant nematode strains can be introduced from another farm or even from another area by animal transportation. 1999). resistance was always promoted. an untreated control group of animals should also be included. 1985. 1995. Generally. as e.. development of resistance depends upon whether resistant worms are as fit. In summary. 3. Most of them however. Finally. by means of life cycle completion. Also. 1992).. In cases where sub-therapeutic doses were used in order to reduce costs. between sheep and goats. 2006). can be used (Coles et al. or may constitute differences in enzymes or mechanisms modifying transport or metabolism of anthelmintics. Wood et al. In this case. practically they are underdosed. samples should not be stored at 4 ◦ C for more than 24 h. In these cases. 1988).. resistance will develop faster if genes for resistance are dominant than recessive. Various laboratory tests have also been used to detect AR.. in order to administer the correct dose of the anthelmintic drug being tested. A small proportion of worms surviving treatment may indicate a resistance problem. egg production. Goats develop resistant strains which can be passed onto sheep. This test is considered to be reliable if more than 25% of the worms are resistant (Martin et al. Despite the fact that this test is highly reliable. or less fit than susceptible ones (Coles. stands the frequency of treatments. animals are challenged with known susceptible and suspected resistant isolates of nematodes.. growth or movement of nematodes (Jabbar et al.... 2006). The rate of AR development depends on several factors. 1979. reproducibility of findings. It has been documented that AR develops more rapidly in cases where animals would be treated regularly and particularly when the same group of anthelmintic would be used (Dorny et al. also results to an effectively lower dose. in order to record any natural changes to egg counts. then the anthelmintic should be considered to be efficient and its use may be continued (Coles et al. Subsequently. others can be performed in vitro and measure the effect of anthelmintics on the development. Coles et al. in order to identify resistance within a specific parasitic species. Detection of resistance The great significance of AR has led to development of a wide range of reliable and standardized detection tests for research and diagnostic purposes (Coles et al. including ones under- going metabolism within the host. 2005). These mutations may be in the receptor sites where drugs work. which is suitable for all anthelmintics. applicability and interpretation of results (Varady and Corba. 1994). no nematode eggs should be found in faecal samples after these time periods. Ideally. The modified McMaster technique used either on individual or on pooled samples. 2006).and post-treatment samples helps to determine specific nematode species involved. which could further develop under drug pressure and thus. Goats require a dose rate 1. 10 animals with an epg count of >150 should be included per group (Coles et al. have drawbacks concerning cost. The more common these are. 1992). either when worms become adults or during different developmental stages.5–2 times higher than sheep.g. The most reliable in vivo method to detect AR and confirm the results of FECRTs or to validate different in vitro assays is the Controlled Anthelmintic Efficacy test.. pasture survival and infectivity. 2006). which may occur during the test period. Also.100 E. 1988). different bioavailability among animal species. Also. The test most commonly used to detect AR remains the Faecal Egg Count Reduction test (FECRT). This may explain the fact that AR occurs more frequently in goats than in sheep (Hennessy. inclusion of correction factors (Webb et al. because it allows the survival of heterozygous resistant worms and therefore. it is important to weigh animals beforehand. Within the most important of them. Underdosing is considered another important factor for development of AR. contributes to selection of resistant strains (Egerton et al. should be monitored.. The one most widely used is the Egg Hatch test . the faster AR develops. 1993).. it compares the egg count before and after treatment with an anthelmintic: nematode eggs are counted in faecal samples at the time of treatment and at defined times thereafter.

-tubulin gene (Kwa et al. 1995). 1989). 1989). however. the 3rd stage larvae can be speciated. the adult migration inhibition test. Also. Alternatively. The molecular mechanisms for the rest of the anthelmintic groups are currently little understood and still under investigation. but there has been little progress in this area due to the complexity of the cultural techniques required or to the requirements for sacrificing animals in order to collect adult worms.. apparently detecting AR when 10% of the worm population carries resistant genes (Dobson et al. rather than calculating the LD50. eggs can be stored anaerobically for up to 7 d after sample collection (Hunt and Taylor. This test is considered to be more sensitive than FECRT and EHT. They suffer from limitations like necessity for control parasite strains to compare color change. recent research has focused to developing sensitive molecular based tests. Unlike the EHT. Tests using adult worms have been also developed. Additionally. FECRT and EHT. likely as a consequence of using different water supply or of applying different sample dilutions.. The larval development test is more laborious and time consuming than the EHT. PCR is able to detect 1% of resistant individuals within a susceptible worm population (Roos et al. The effect of the drugs on subsequent development to third stage larvae at the end of the test is measured. Also.E. 2006). Finally. discriminating doses can be used to reduce the numbers of drug concentrations required and to increase the sensitivity of the test (Coles et al. given that reversion to susceptibility is easily possible only when the resistant genes are present in less than 5% of the worm population (Roos et al. since sensitivity to thiabendazole decreases with embryogenesis (hence. Coles et al.. The percentage of eggs hatching in the discriminating dose corresponds to the percentage of benzimidazole-resistant eggs in the sample (Coles et al. further evaluation is required to confirm these values. Discriminating doses have been established using susceptible isolates of certain nematodes.. Since the same mutation is responsible for benzimidazole-resistance in many parasitic nematodes. 2006). 1994) have indicated a poor correlation between the results of FECRT and EHT. so that the percentage of hatched eggs diagnostic of low levels of resistance can be agreed. (2006) reported failure of several European laboratories to obtain the same answer by using precisely the same population of H. eggs hatching at this concentration are resistant). which is based on the nematode ovicidal activity of these anthelmintics (Taylor et al.. 1994).. genotyping a single larva or worm is very laborious and relatively expensive. some reports (Dorny et al. Nematode eggs or first stage larvae are exposed to different concentrations of anthelmintics. but saving of time may . mainly to compare non-specific esterases and acetylcholinesterases of benzimidazole-resistant and susceptible trichostrongylid nematode strains. has been mainly confined to benzimidazole-resistance. the sensitivity of the test can be significantly increased. Two versions of this test have been described. This means that FECRT and EHT are able to detect AR when it may be too late to interfere. the colorimetric biochemical assays that have been developed. The limitation of both tests is that they are able to detect AR only when at least 25% of the worm population contains resistant genes (Martin et al. 2006).e.. 1996). these include the microagar larval development test. incorporated either in a small test tube/Petri dish containing nutrient medium or into agar wells in a microtiter plate. 2002. like the adult development test. 1989). the test would yield a false negative result). (Sutherland and Lee. etc. 2006. therefore molecular tests they must be developed for RT-PCR or pyrosequencing in order to be of practical use in the field. The advantage of this test is the requirement for only one faecal sample. are widely used to detect AR. Several tests have been developed to detect AR using nematode larvae. the larval paralysis test. contortus worms. Here again. However. RT-PCR requires the use of very expensive equipment.. 2006). The most common molecular mechanism for benzimidazole-resistance in trichostrongyles of small ruminants involves a phenylalanine to tyrosine mutation at position 200 of the isotype 1. the larval motility test. Tests based on the use of a single point mutation to detect AR suffer from the potential problem that resistance might have resulted from multiple mutations. including macrocyclic lactones (Jabbar et al. Coles et al. Jabbar et al. Investigation of the molecular basis of AR. By using discriminating doses and then choosing one preventing hatching of 99% of susceptible eggs (i. It is particularly important to use fresh (within 3 h of collection) eggs.. 1995). On the other hand. requirement for a very stable ambient temperature in order to maintain activity of enzymes and therefore false changes in color. The above two tests. 2006). but allows detection of AR to the major broad-spectrum anthelmintic groups. Papadopoulos / Small Ruminant Research 76 (2008) 99–103 101 (EHT) for detection of resistance to benzimidazoles. in this test the age of eggs used is not important... up to now. in order to identify nematode species present (control wells) and those surviving the anthelmintic. the first is liquid-based and the other agarbased. have not been used widely.... etc. this method may be used to investigate the frequency of alleles bearing it in a wide range of nematodes (Jabbar et al.

Concluding remarks Sustainable control strategies of nematode resistance to anthelmintics require an integrated approach. Kyriazakis and Houdijk. Modeling of anthelmintic use has shown that the administration of combination drenches should reduce the chances of the development of AR (Barnes et al. Waller. In practice. developed in South Africa. but usually these breeds are less productive. Papadopoulos / Small Ruminant Research 76 (2008) 99–103 make up for the additional cost of the reagents and costs of the machine. has been widely recognized (Van Wyk.. 2005. Parasitol. Barger. Borgsteede.. 13–18. 2005). The FAMACHA system. Today 11. Practical implementation of holistic internal parasite management in sheep. none of the nonchemical methods for parasite control is sufficiently effective without anthelmintic support. 78. the cost increases significantly and safety is questioned. as well as chemoprophylaxis to minimize the pressure for parasite adaptation (Jabbar et al.J. 99–108. G. still has to be fully evaluated. No system currently exists on deciding which animals infected with other non-blood sucking nematodes can be left untreated. Research on AR should be a priority area. 1995). Worms in refugia come from larvae on pasture.C. 2006). There have been some encouraging results from the use of nematophagous fungal spores to reduce pasture contamination.J... Coles. Geerts. G. 2005). Though this approach was highly successful.. 2006... The great impact of refugia. P. The introduction of susceptible worms (genes) may also contribute towards this direction.. which offers another alternative for biological control of nematodes. quality nutrition reduces the effect of parasitism. and caused by H. 1995. Bath. In the past almost all methods of parasite control were based on the frequent use of anthelmintics. I. Control of anthelmintic resistance The enormous problem of AR is easily appreciated. 2005). F. faecal egg counts or other parameters for selective treatments will have to be further studied. World Association for the .C. this being especially true in systems where grass is grown as a rotational crop between other crops. relies on examining the animal’s conjuctiva. Coles. Sissay et al. including a macrocyclic lactone (Coles.. Small Rumin Res. 62. 2006. any management system markedly reducing larvae on pasture and being used in conjunction with an anthelmintic. In any case. that sensitive molecular tests will be limited for research use and for designing management strategies to slow down the development of resistance (Coles et al. but its economic basis has been questioned (Coles. keeping nematodes unexposed to anthelmintics. S. in order to ensure that resistant worms do not come along with them. R. 2006).. Body condition score.. Unfortunately. but animals have to receive an adequate daily dose of these (Larsen. 2006).R.H.F. T. furthermore. as indicated by a color score chart (Van Wyk and Bath. 2000). 2006. 5. the use of plants containing condensed tannins or other active compounds. A good policy could be to isolate and treat them with a combination of anthelmintics. References Barnes.M. Dobson. 2002.A. 2004). Protein supplementation can increase the rate of acquisition of immunity and resilience against gastrointestinal nematodes (Coop and Kyriazakis. subsequently treating only sheep with signs of anaemia. Van Wyk et al... but up to now nothing really effective against nematodes is commercially available. It is recommended to quarantine animals entering the farm. Bauer.. will select for AR. Taylor. reevaluation and adjustment of control strategies. 4. Effort has been put through molecular biology to develop vaccines.102 E. These worms will provide the next generation of parasites. Vet. in combination with the fact that no flow of new classes of anthelmintics took place. 2005. 56–63. Res. enabling constant monitoring. benzimidazole resistance among sheep nematodes is so common in many parts of the world. Breeding sheep able to tolerate nematodes offers less requirements for use of chemicals. Bath. Among the alternative strategies to parasite control. Worm control and anthelmintic resistance: adventures with a model. However. Klei. C. 2001. G. 2006). E.. The most dramatic reduction in pasture larval burdens comes with drought. contortus parasitism. Finally. 1992. effort should be put to avoid introducing resistant genes into a farm with new stock. Current research is now looking into estimating the proportion of animals and which of them should be left untreated. Sci. the use of mixtures has been applied when AR is already present and therefore their full benefit is not well known. from untreated animals and from stages of nematodes inside animal hosts not susceptible to treatment (Coles. 2006. 1999.A. changes in nematode control and measures to delay the development of resistance must be taken (Kaplan. it proved to be shortsighted and unsustainable. von Samson-Himmelstjerna. According to Coles (2005). Anthelmintic resistance—looking to the future: a UK perspective. Waller. whereas the ones surviving treatment must contribute as little as possible to that next generation.. including environmental management and taking into account climate and parasites in different areas.. Coles. On the other hand. M.H. 2006).

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