Repeated treatment of populations of parasites with anthelmintics selects individuals that have innate or acquired resistance to the drugs. Ultimately treatment becomes ineffective because a large proportion of the parasites in the population are resistant.

It is now common for sheep to harbour at least one parasite species that is resistant to the major drench groups. It is becoming disturbingly common for worms to be resistant to anthelmintics in all of the major drench groups, including some combinations of unrelated drugs.

Drench resistance is common and often highly developed in goats. Only a limited range of anthelmintics are registered for use in goats. Due to their altered drug metabolism goats may require higher dose rates than recommended for sheep.

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Diagnosis

Diagnosis can be based on history and a failure to respond to treatment, with confirmatory testing such as an on-farm trial using a faecal egg count reduction test (FECRT). Because of limitations mainly with respect to the macrocyclic lactones, the in vitro larval development assay (LDA, DrenchRite^TM) is no longer available. The in vitro closantel resistance assay also is no longer available.

Faecal egg count reduction test (FECRT)

The faecal egg count reduction test (FECRT) requires considerable on-farm effort. FECRT can be performed using either:

1. A DrenchTest kit, or,
2. Several WormTest kits (one for each anthelmintic and one for a control group).

FECRT anthelmintics

Where nematode resistance is suspected, the trial should contain groups treated with a range of anthelmintics Traditionally 5 groups (benzimidazole (BZ), levamisole (LEV), BZ/LEV combination, ivermectin (IVM) and an untreated control group) were used. However, with recent extensive spread of resistance in these chemical groups, other drenches including naphthalophos (NAP, Rametin^TM), and its combinations with BZ and/or LEV, closantel (CLS), and other MLs such as abamectin, and moxidectin should be considered.

One-third dose CLS and, since 2002, half dose ivermectin have been recommended for the early detection of emerging resistance. Use of proprietary triple or quadruple combinations (for example Triton^TM , Hat-trick^TM and Q-drench^TM) may also be included. Groups with treatments given at greater than the recommended dose rates to fully evaluate the available anthelmintics may also be included. In practice, only double dose LEV or double dose LEV plus single BZ have been shown to be effective where the single dose is not effective. However, it is emphasized that the use of double dose rate or combinations of anthelmintics are not general recommendations. They are used to obtain a prompt answer to possibly a serious and immediate problem on specific farms, and now are infrequently used.

FECRT pre-trial check sampling (Day -10)

To avoid wasted effort, arrange for a pre-trial collection of faeces from the mob containing the trial sheep for worm egg counts. If this is not possible, faecal samples may be collected from the control group at day 0 to assess if egg counts are sufficient to continue the trial. Objective assessment of the efficacy of an anthelmintic is not reliable where the mean faecal worm egg count of the control group is less than 200 epg. Some authorities even suggest 300 epg. Subjective evaluation of the data may however be possible below this figure. A larval differentiation should be performed to see whether eggs from the worm species of interest are present at sufficiently high levels (>200 epg).

FECRT treatment (Day 0)

Selection of trial animals

Trial sheep should be preferably be young sheep at weaning, 3-6 months of age, of even body weight and bred on the property. Undrenched weaners are preferred, but if not possible they should NOT have received a drench during the previous 6 weeks (or 12 weeks if a persistent anthelmintic has been used) to avoid testing an already 'selected' worm population.

Number of trial animals

Each group should contain 15 sheep allowing that faeces may not be collected from all animals at the subsequent visit. A minimum of 10 sheep per group from which collections are made is necessary.

Randomisation of trial animals

Sheep should be allocated to treatment and control groups randomly. The best method of randomisation is based on pre-treatment egg counts; however, as these are usually not available, the sheep should be drafted into as many groups as there are anthelmintics to be tested plus one untreated control group. For example, if 4 drenches are to be tested, the first 5 sheep in the race are allocated at random to 5 groups; the next 5 are similarly allocated until each group contains 15 sheep. Each group should be identified with a different colour marking paint or coloured ear tag.

Anthelmintic treatment of trial animals

Anthelmintics should be administered accurately by either a calibrated syringe or a drench gun previously calibrated. In most situations, sheep should be orally dosed according to the weight of the heaviest animal in the group. If the mob is an even line of sheep the dose received on a mg/kg basis will not vary greatly.

FECRT sampling (Day 10-14)

Collection of faeces

All trial sheep should be sampled between 10 and 14 days after treatment. Faecal samples should be collected from the rectum of sheep within the first hour after yarding and before much handling has occurred. A minimum of 5 g (10 to 15 pellets) should be collected from each sheep. If there are treatment groups where a persistent anthelmintic has been used at the recommended dose rate, an additional post-treatment sampling of these groups, for example at day 28 or some other interval, depending on the expected persistence, may be performed. The reason for this is that, in the case of persistent anthelmintics, the first indication of resistance may be the appearance of worm eggs in the faeces sooner after treatment than otherwise would be expected.

Transportation of faeces

Samples in individually sealed containers filled as close to the top as possible to exclude air, should be transported to the laboratory as soon as possible. The samples should be kept cool in an esky with freezer bricks wrapped in newspaper to avoid freezing. Storage or transport of faeces at or below 4°C may affect the hatching of H. contortus eggs in a faecal culture.

FECRT laboratory testing

Larval differentiation

Pooled faecal cultures and larval differentiations should always be carried out on the control group and preferably each of the test groups. This will allow determination of the species composition and species resistance levels. This is important as an anthelmintic may remain useful although some species have developed resistance to it (eg levamisole may still be effective as a narrow spectrum against Haemonchus, but not as a broad spectrum against other species).

Calculation of FECRT results

The arithmetic mean ('average') on epg of samples collected 10-14 days after treatment, together with a 95% confidence interval, most accurately describes the range of in efficacy of an anthelmintic. The arithmetic mean is the most stringent test of a percentage reduction in faecal egg output and therefore is a more conservative measure of an anthelmintic's performance. Geometric means are sometimes requested by trial sponsors, but arithmetic calculations should always be conducted as well.

Each set of results will include the following information per group for each genus present (provided a larval differentiation has been conducted):

• Arithmetic mean ('Average')
• Range (Highest and lowest counts)
• Standard deviation
• Mean percent reduction in epg (i.e. = FECR)
• 95% confidence interval of FECR (Upper and lower limit of percent reduction for 95% confidence interval)

Interpretation of FECRT results

Resistance should be regarded as being present if:

1. the FECR is < 95%, and,
2. the lower 95% confidence limit of the % reduction in egg count is less than or equal to 90%.

An example of simple test groups without larval differentiation:

Comment: Interpret these results with caution due to low egg counts in the controls. However it appears that:

1. BZ resistance(*) is present.
2. Levamisole resistance (*) is present.
3. BZ + Levamisole resistance (*) is present.