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Small Ruminant Research 76 (2008) 99103

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 sufciently 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 eld 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

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worms may occasionally take place. The more common these are, the faster AR develops. These mutations may be in the receptor sites where drugs work, or may constitute differences in enzymes or mechanisms modifying transport or metabolism of anthelmintics. Also, resistance will develop faster if genes for resistance are dominant than recessive. Finally, development of resistance depends upon whether resistant worms are as t, by means of life cycle completion, egg production, pasture survival and infectivity, or less t than susceptible ones (Coles, 2005). The rate of AR development depends on several factors. Within the most important of them, stands the frequency of treatments. 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., 1994). Underdosing is considered another important factor for development of AR, because it allows the survival of heterozygous resistant worms and therefore, contributes to selection of resistant strains (Egerton et al., 1988). In cases where sub-therapeutic doses were used in order to reduce costs, resistance was always promoted. Also, different bioavailability among animal species, as e.g., between sheep and goats, also results to an effectively lower dose. Goats require a dose rate 1.52 times higher than sheep, therefore, when goats are treated with the same dose as sheep, practically they are underdosed. This may explain the fact that AR occurs more frequently in goats than in sheep (Hennessy, 1994). Goats develop resistant strains which can be passed onto sheep, much easier when animals of these two species are kept together (Jackson, 1993). Cases have also been reported that resistant nematode strains can be introduced from another farm or even from another area by animal transportation. 3. Detection of resistance The great signicance of AR has led to development of a wide range of reliable and standardized detection tests for research and diagnostic purposes (Coles et al., 1992). Most of them however, have drawbacks concerning cost, reproducibility of ndings, applicability and interpretation of results (Varady and Corba, 1999). There are several in vivo tests suitable for all types of anthelmintics; others can be performed in vitro and measure the effect of anthelmintics on the development, growth or movement of nematodes (Jabbar et al., 2006). The test most commonly used to detect AR remains the Faecal Egg Count Reduction test (FECRT), which is suitable for all anthelmintics, including ones under-

going metabolism within the host, and can be easily applied in sheep (Coles et al., 2006). This test is considered to be reliable if more than 25% of the worms are resistant (Martin et al., 1989). Generally, 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 dened times thereafter, these depending on the specic anthelmintic tested. Obviously, an untreated control group of animals should also be included, in order to record any natural changes to egg counts, which may occur during the test period. Ideally, 10 animals with an epg count of >150 should be included per group (Coles et al., 2006). In these cases, it is important to weigh animals beforehand, in order to administer the correct dose of the anthelmintic drug being tested. Subsequently, faecal samples should be collected 810 d after treatment for benzimidazole-testing or 1417 d after treatment for macrocyclic lactone-testing; no nematode eggs should be found in faecal samples after these time periods. The modied McMaster technique used either on individual or on pooled samples, can be used (Coles et al., 1992). Use of larval cultures in pre- and post-treatment samples helps to determine specic nematode species involved, in order to identify resistance within a specic parasitic species. In this case, samples should not be stored at 4 C for more than 24 h, as this may affect hatchability of nematode eggs, particularly of those of Haemonchus contortus (McKenna, 1988). Also, it has to be noted that culture conditions may favour the development of one parasitic species over another and therefore, inclusion of correction factors (Webb et al., 1979; Presidente, 1985) should take place before interpretation of results. A small proportion of worms surviving treatment may indicate a resistance problem, which could further develop under drug pressure and thus, should be monitored. If reduction in faecal egg counts is over 95%, then the anthelmintic should be considered to be efcient and its use may be continued (Coles et al., 2006). The most reliable in vivo method to detect AR and conrm the results of FECRTs or to validate different in vitro assays is the Controlled Anthelmintic Efcacy test. In summary, animals are challenged with known susceptible and suspected resistant isolates of nematodes. Their worm burdens are compared after treatment, either when worms become adults or during different developmental stages. Despite the fact that this test is highly reliable, it requires a lot of labour and animals and hence, it is now seldom used (Presidente, 1985; Wood et al., 1995; Coles et al., 2006). Various laboratory tests have also been used to detect AR. The one most widely used is the Egg Hatch test

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(EHT) for detection of resistance to benzimidazoles, which is based on the nematode ovicidal activity of these anthelmintics (Taylor et al., 2002; Coles et al., 2006). It is particularly important to use fresh (within 3 h of collection) eggs, since sensitivity to thiabendazole decreases with embryogenesis (hence, the test would yield a false negative result). Alternatively, eggs can be stored anaerobically for up to 7 d after sample collection (Hunt and Taylor, 1989). By using discriminating doses and then choosing one preventing hatching of 99% of susceptible eggs (i.e., eggs hatching at this concentration are resistant), rather than calculating the LD50, the sensitivity of the test can be signicantly increased. Discriminating doses have been established using susceptible isolates of certain nematodes; however, further evaluation is required to conrm these values, so that the percentage of hatched eggs diagnostic of low levels of resistance can be agreed. The percentage of eggs hatching in the discriminating dose corresponds to the percentage of benzimidazole-resistant eggs in the sample (Coles et al., 2006). The advantage of this test is the requirement for only one faecal sample. However, some reports (Dorny et al., 1994) have indicated a poor correlation between the results of FECRT and EHT. Also, Coles et al. (2006) reported failure of several European laboratories to obtain the same answer by using precisely the same population of H. contortus worms, likely as a consequence of using different water supply or of applying different sample dilutions. The above two tests, FECRT and EHT, are widely used to detect AR. 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., 1989). This means that FECRT and EHT are able to detect AR when it may be too late to interfere, 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., 1995). Several tests have been developed to detect AR using nematode larvae; these include the microagar larval development test, the larval motility test, the larval paralysis test, etc. The larval development test is more laborious and time consuming than the EHT, but allows detection of AR to the major broad-spectrum anthelmintic groups, including macrocyclic lactones (Jabbar et al., 2006). Two versions of this test have been described; the rst is liquid-based and the other agarbased. Unlike the EHT, in this test the age of eggs used is not important. Nematode eggs or rst stage larvae are exposed to different concentrations of anthelmintics, incorporated either in a small test tube/Petri dish containing nutrient medium or into agar wells in a microtiter

plate. The effect of the drugs on subsequent development to third stage larvae at the end of the test is measured. Additionally, the 3rd stage larvae can be speciated, in order to identify nematode species present (control wells) and those surviving the anthelmintic. Here again, discriminating doses can be used to reduce the numbers of drug concentrations required and to increase the sensitivity of the test (Coles et al., 2006; Jabbar et al., 2006). This test is considered to be more sensitive than FECRT and EHT, apparently detecting AR when 10% of the worm population carries resistant genes (Dobson et al., 1996). Tests using adult worms have been also developed, like the adult development test, the adult migration inhibition test, but there has been little progress in this area due to the complexity of the cultural techniques required or to the requirements for sacricing animals in order to collect adult worms. Also, the colorimetric biochemical assays that have been developed, mainly to compare non-specic esterases and acetylcholinesterases of benzimidazole-resistant and susceptible trichostrongylid nematode strains, have not been used widely. They suffer from limitations like necessity for control parasite strains to compare color change, requirement for a very stable ambient temperature in order to maintain activity of enzymes and therefore false changes in color, etc. (Sutherland and Lee, 1989). Finally, recent research has focused to developing sensitive molecular based tests. PCR is able to detect 1% of resistant individuals within a susceptible worm population (Roos et al., 1995). Investigation of the molecular basis of AR, up to now, has been mainly conned to benzimidazole-resistance. The molecular mechanisms for the rest of the anthelmintic groups are currently little understood and still under investigation. 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- -tubulin gene (Kwa et al., 1994). 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. Since the same mutation is responsible for benzimidazole-resistance in many parasitic nematodes, this method may be used to investigate the frequency of alleles bearing it in a wide range of nematodes (Jabbar et al., 2006). On the other hand, genotyping a single larva or worm is very laborious and relatively expensive, therefore molecular tests they must be developed for RT-PCR or pyrosequencing in order to be of practical use in the eld. RT-PCR requires the use of very expensive equipment, but saving of time may

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make up for the additional cost of the reagents and costs of the machine. Unfortunately, benzimidazole resistance among sheep nematodes is so common in many parts of the world, 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., 2006; von Samson-Himmelstjerna, 2006). 4. Control of anthelmintic resistance The enormous problem of AR is easily appreciated. In the past almost all methods of parasite control were based on the frequent use of anthelmintics. Though this approach was highly successful, it proved to be shortsighted and unsustainable; furthermore, in combination with the fact that no ow of new classes of anthelmintics took place, changes in nematode control and measures to delay the development of resistance must be taken (Kaplan, 2004). On the other hand, none of the nonchemical methods for parasite control is sufciently effective without anthelmintic support. The great impact of refugia, keeping nematodes unexposed to anthelmintics, has been widely recognized (Van Wyk, 2001; Coles, 2005; Sissay et al., 2006). These worms will provide the next generation of parasites, whereas the ones surviving treatment must contribute as little as possible to that next generation. Worms in refugia come from larvae on pasture, from untreated animals and from stages of nematodes inside animal hosts not susceptible to treatment (Coles, 2005). According to Coles (2005), any management system markedly reducing larvae on pasture and being used in conjunction with an anthelmintic, will select for AR. The most dramatic reduction in pasture larval burdens comes with drought, this being especially true in systems where grass is grown as a rotational crop between other crops. The introduction of susceptible worms (genes) may also contribute towards this direction. Current research is now looking into estimating the proportion of animals and which of them should be left untreated. The FAMACHA system, developed in South Africa, relies on examining the animals conjuctiva, subsequently treating only sheep with signs of anaemia, and caused by H. contortus parasitism, as indicated by a color score chart (Van Wyk and Bath, 2002; Bath, 2006; Van Wyk et al., 2006). No system currently exists on deciding which animals infected with other non-blood sucking nematodes can be left untreated. Body condition score, faecal egg counts or other parameters for selective treatments will have to be further studied. Modeling of anthelmintic use has shown that the administration of combination drenches should reduce

the chances of the development of AR (Barnes et al., 1995). However, the cost increases signicantly and safety is questioned. In practice, the use of mixtures has been applied when AR is already present and therefore their full benet is not well known. In any case, effort should be put to avoid introducing resistant genes into a farm with new stock. It is recommended to quarantine animals entering the farm, in order to ensure that resistant worms do not come along with them. A good policy could be to isolate and treat them with a combination of anthelmintics, including a macrocyclic lactone (Coles, 2005). Among the alternative strategies to parasite control, quality nutrition reduces the effect of parasitism. Protein supplementation can increase the rate of acquisition of immunity and resilience against gastrointestinal nematodes (Coop and Kyriazakis, 1999; Kyriazakis and Houdijk, 2006), but its economic basis has been questioned (Coles, 2005). There have been some encouraging results from the use of nematophagous fungal spores to reduce pasture contamination, but animals have to receive an adequate daily dose of these (Larsen, 2000). Effort has been put through molecular biology to develop vaccines, but up to now nothing really effective against nematodes is commercially available. Breeding sheep able to tolerate nematodes offers less requirements for use of chemicals, but usually these breeds are less productive. Finally, the use of plants containing condensed tannins or other active compounds, which offers another alternative for biological control of nematodes, still has to be fully evaluated. 5. Concluding remarks Sustainable control strategies of nematode resistance to anthelmintics require an integrated approach, including environmental management and taking into account climate and parasites in different areas, as well as chemoprophylaxis to minimize the pressure for parasite adaptation (Jabbar et al., 2006; Waller, 2006). Research on AR should be a priority area, enabling constant monitoring, reevaluation and adjustment of control strategies. References
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