The control of parasitic helminths in domestic animals relies largely on the use of anthelmintic drugs. Although anthelmintics are used in all domestic species, the largest market is undoubtedly the ruminant market, especially cattle, where millions of pounds are spent annually in an effort to reduce the effects of parasitism. The traditional method to control the gastrointestinal nematodes is the use of chemical dewormers. There are three major classes of anthelmintics that are widely used for the control of nematodes in small ruminants: Benzimidazoles, Macrocycliclacctones and Imidazothiazoles/ Tetrahydropyrimidines. However, the frequent use of these compounds has caused resistance to the anthelmintics in numerous species.
Factors responsible for failure of anthelmintics:
Inadequate integration between management strategies and chemotherapy.
Incorrect use of anthelmintic drugs.
Insufficient understanding of the relationship between pharmacological properties, and several host-related factors.
The time of parasite exposure to active drug concentrations determines the efficacy or persistence of activity.
Drug resistance is heritable and repeated dosing will therefore select for an increasing proportion of resistant individuals. The mechanisms involve either differences in drug metabolism within the parasite or mutations at the binding site of the drug. The prevalence and severity of anthelmintic resistance is increasing and leading to uncontrolled loss of production. Helminth resistance to anthelmintics has been most frequently recorded in sheep and goats (mainly Haemonchus spp and Trichostrongylus spp in tropical and subtropical regions and Teladorsagia in temperate areas) and horses (mainly the small strongyles), and initially involved the benzimidazole group of compounds. Over the last decade resistance to all three chemical classes of broad-spectrum anthelmintics, 1-BZ (benzimidazoles and probenzimidazoles), 2-LM (levamisole/morantel) and 3-AV (avermectins/milbemycins), and in some cases to the narrow-spectrum anthelmintics, such as closantel, has become more widespread in nematode parasites of small ruminants. Studies have shown minimal reversion to susceptibility in highly selected homozygous isolates following withdrawal of the selecting drug and, as a consequence, once resistant worms are present on a livestock enterprise they can be considered as permanent. Therefore, it is important to be able to detect the presence of emerging resistant isolates at an early stage. Unfortunately, the in vivo faecal egg count reduction test and the in vitro egg hatch assay, larval development assay and larval migration inhibition assay, used to detect the presence of resistant worms.
Detection of Anthelmintic Resistance
The presence of anthelmintic resistance can be detected in flocks in a number of ways.
In vivo methods
FECRT (Faecal egg count reduction test)
The FECRT provides an estimation of anthelmintic efficacy by comparing faecal egg counts of animals before and after treatment.
1. Randomly distribute or distribute based on egg counts.
2. Choose animals 3–6 months of age or if older with eggs counts >150 epg.
3. Use 10 animals per group if possible.
4. Rectal sample putting 3–5 g into individual pots.
5. Count using the McMaster technique as soon as possible after collection.
6. Individually weigh animals and give manufacturers recommend dose orally, from a syringe.
8. Take second rectal sample at the following time periods after treatment: Levamisole 3–7 days. Benzimidazole 8–10 days. Macrocyclic lactones 14–17 days.
Control efficacy test
When results from an FECRT undertaken 10 days post-treatment are not definitive, the efficacy test can be carried out to reach final conclusions. A complete parasitological necropsy should be performed in five animals selected at random from both groups used in the FECRT to determine the worm burden in each test group.
Critical anthelmintic test
It is based on the collection of faeces from animals for at least 4 days after treatment and count the number of expelled worms. A residual worm burden, if any, is then estimate by post slaughter examination of the GIT, and efficacy of the drug calculated. However, this test is not suitable for animal worms which undergo digestion during passing through the intestinal tract.
Controlled slaughter test
It is consider most reliable method for evaluating the anthelmintic activity and is recommended for dose determination and dose confirmation trials. Animals randomly allocated to medicated and non medicated group, and after a suitable period animals are slaughtered. The parasite remaining in the GIT or tissue is identified, counted and efficacy of the drug may be calculated as follows;
Percentage efficacy (%) = Xg controlled group - Xg treated group/ Xg controlled group
*Xg indicates the geometric mean number of parasites.
In vitro methods
Egg Hatch Test
Benzimidazole anthelmintics prevent embryonation and hatching of nematode eggs. A number of egg hatch/embryonation assays have been developed for the detection of resistance to this group of anthelmintics.
The Egg hatch test methods
1. Add 1.89 ml water to each well in a 24 well plate. This should be deionised water with a neutral pH. Then add 10 ml of thiabendazole solution dissolved and diluted in DMSO to the water. Add DMSO to the control wells
2. To determine the degree of resistance use 0.05, 0.1, 0.2, 0.3 and 0.5 mg/ml thiabendazole. A single concentration of thiabendazole can be used, the discriminating dose.
3. Place 100 ml of fresh eggs (less than 3 h old or anaerobically stored) in each well. Since thiabendazole may bind to debris the eggs should be as clean as possible. Incubate at 250C for 48 h.
4. Add two drops of Lugol’s iodine to each well. Count at least 100 of the remaining eggs and hatched larvae.
Larval development tests (LDTs)
First stage larvae were cultured to third stage larvae in the presence of heat treated lyophilised Escherichia coli, as a food source, and the anthelmintic under test. Suitable controls were also run without the presence of anthelmintic. The test was able to show clear differences between benzimidazole resistant and susceptible strains of H. contortus and was also able to detect a levamisole resistant strain of H. contortus.
Larval paralysis and motility assay (Larval migration inhibition assay)
The test is used for levamisole and morantel resistance. This assay discriminates between
Resistant and susceptible strains of parasites, by estimating the proportion of third stage larvae in tonic paralysis after incubation with a range of levamisole and morantel drug concentrations. It is relatively easy to carry out, stocks of infective larvae are readily obtained and it is reported that there is a fairly good reproducibility of the test, any differences in repeatability being attributed to the age of larvae. However, the interpretation is complicated by the fact that if the anthelmintic is added to the egg suspension too early, the development has not proceeded far enough; if it is added too late the drug has no effect.
The mechanism of benzimidazole resistance appears to be associated with a reduced affinity of tubulin for the anthelmintic. Biochemical assays comparing non-specific esterases and acetylcholinesterases of benzimidazole resistant and susceptible trichostrongylid nematode strains have also been described. In these studies a simple colorimetric assay was developed in which samples were compared either by visual examination or through the use of a densitometer. Significantly greater esterase or acetylcholinesterase activity was found in the benzimidazole resistant strains.
Knowledge on the molecular mechanisms of anthelmintic resistance is mainly confined to the benzimidazoles. It is correlated with single nucleotide polymorphism on the β tubulin isotype 1 gene. Three mutation can be used as markers for the detection of resistance, namely at position 200 and 167 (both TTC to TAC) or at position 198 (GAA to GCA) by allele-specific PCRs. The molecular basis of resistance to other anthelmintic drugs remains largely unknown. The mechanism of levamisole resistance is thought to be associated with a reduction in the number of nicotinic acetylcholine receptors (nAChR) of nematodes or a change in their binding characteristics. The proposed mode of action of the avermectins and milbemycins involves the binding of the drug to α subunit of a glutamate-gated chloride channel that opens or potentiates gating and leads to hyperpolarisation of the target neuromuscular cell. Research on p-glycoproteins, which appear to act as drug transporters at the cell membrane, suggests that they may be involved in resistance to ivermectin.
Due to emergence of resistant strains of the parasite we need alternative control strategies, such as vaccines, breeding ‘worm-resistant’ breeds and pasture control using nematophagous fungi, dietary supplementation with rumen bypass protein or forages rich in condensed tannins. Much effort has been directed towards the development of a vaccine against H. contortus and a number of promising candidate vaccine antigens have been identified.
1, 2, 3, 4, 5, 8. Phd Scholar, 6. M.V.Sc Scholar, 7. Research Associate, Veterinary Parasitology Division, ICAR- IVRI, Izatnagar, Bareilly.
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