Anthelmintic resistance

The regular use of anthelmintics can lead to anthelmintic resistance. It is especially likely to occur if anthelmintics are the only form of control. Resistance against anthelmintics in Australia was first described in the late 1960’ s and has become a growing problem since then. Other

countries are also affected and anthelmintic resistance has become an increasingly serious worldwide problem in the sheep industry (JACKSON et al., 2000; KAPLAN, 2004) as well as in cattle and horses (KELLY et al., 1981; DONALD, 1983; KAPLAN, 2002; MCKELLAR et al., 2004).

In the most important sheep nematodes, Haemonchus, Trichostrongylus and Telodorsagia, resistance has been found widely in Australia (LOVE, 2002). The appearance of resistance followed the sequence in which the broad-spectrum anthelmintics were released to the market for commercial use. So, the first resistance were reported for phenothiazine, then for the benzimidazoles, imidazothiazoles, followed by macrocyclic lactones (WALLER, 1985). Some populations of H. contortus resistant to salicylanilides and organophosphate compounds have also been reported (SANGSTER, 1999).

1.1.6.1. Types of resistance

The following terms, frequently used in studies on anthelmintic resistance, were defined by Prichard (PRICHARD et al., 1980).

- Resistance: is present, when there is a greater frequency of individuals within a population able to tolerate doses of a compound than in a normal population of the same species. Resistance is a heritable character.

- Side-resistance: exists, where the resistance to a compound is the result of selection by another compound with a similar mode of action.

- Multiple-resistance: occurs, when the same nematodes are resistant to two or more classes of anthelmintics, either as a result of selection by each group independently, or as a result of cross-resistance.

- Reversion: is a decrease in the frequency of resistant individuals in a population following removal of a selecting agent.

1.1.6.2. Selection for resistance

Resistance is inherited. Initially, resistant alleles are rare in a population of worms. The treatment with an anthelmintic usually removes the large majority of worms, but a small number of survivors (drug selected worms) can remain. These survivors, who carry resistant genes, contribute to the genetic pool in subsequent generations. If they reproduce and the progeny survive, the level of resistance in the population will increase.

Continued anthelmintic treatment provides further selection and under environmental conditions appropriate for worm survival, subsequent generations of worms will inherit more and more resistance alleles leading to a more resistant population (MARTIN, 1985). This will reach a point where treatment failure occurs.

When selected worms are the only survivors and there are few worms in untreated refugia (refugia such as pasture during dry weather), resistance develops quickly. The following definition of refugia was given by J. van Wyk (VAN WYK, 2001): proportion of the population not affected by selection, e.g. free-living stages, worms in untreated animals.

Increased resistance is sometimes referred to as an increase in the frequency of resistant genes.

These genes may code for drug target sites or for proteins responsible for drug removal.

1.1.6.3. Detection of resistance

In order to detect resistance, several tests have been developed. They can generally be grouped in two categories – in vivo tests and in vitro tests.

In vivo tests include controlled tests involving treatment and slaughter of infected sheep. They are expensive and require the use of many animals. This is also time consuming, taking a minimum of a month to complete.

A more common alternative is the Faecal Egg Count Reduction Test (FECRT), which is simple and easy to perform. It relies on the principle, that faecal egg counts reflect adult worm numbers.

It involves the measurement of faecal egg counts before and after the treatment and the changes in egg counts are calculated. These tests have a poor sensitivity and fail to detect low levels of resistance (below 25% resistant alleles) and also provide a poor quantitative estimate of resistance (MARTIN et al., 1989). Another disadvantage is, that some drugs, like ivermectin, temporarily suppress egg laying (LE JAMBRE et al., 1995) so resistant worms appear to be susceptible in routine testing.

Compared with the in vivo tests, the in vitro tests are less expensive, often more reproducible, easy to perform and less time consuming (LACEY et al., 1990). Generally they require a single sampling from the farm. In general they involve incubating one of the free-living stages of the parasite in a range of drug concentration, then taking some measure of vitality which is then used to generate a dose response value.

The first in vitro test, the Egg Hatch Assay (LE JAMBRE, 1976), was used for BZ’ s and levamisole, but is unsuitable for avermectins and closantel. The drugs inhibit hatching of the eggs and therefore further development.

The Larval Development Assay (LDA) is the most common system. Lacey (LACEY et al., 1990) developed a LDA, which is available commercially as the DrenchRite® assay. Drugs inhibit development from eggs to L3. These tests are described in more detail in section 1.4.2.1. and 1.4.2.2.

L3 motility assays are useful for detecting BZ and ML resistance. The larvae are incubated in the presence of drugs and then motility is measured by electronic detectors (FOLZ et al., 1987), migration through a sieve (SANGSTER et al., 1988) or by observation (GILL et al., 1991).

Potentially, the most sensitive methods for detection of resistance are genetic tests. The genetic basis of resistance must be known in order to develop test systems. In case of the BZ’ s, the major mechanisms of resistance are known and a PCR-based test has been developed (SILVESTRE et al., 2000; HUMBERT et al., 2001).

BZ resistant and susceptible strains differ on the molecular level by a single nucleotide change in the codon for amino acid 200 of a -tubulin gene: a switch from TTC (phenylalanine) to TAC (tyrosine) (KWA et al., 1994).

There are a range of recommendations on how to control and prevent anthelmintic resistance.

Strategic worm control programs incorporating grazing management have been developed and research in the field of anthelmintic resistance is still going on. Understanding the mechanism of resistance on a pharmacological level is one of the most important targets of the current research and is the subject of this thesis – better understanding could lead to better diagnosis and treatment.