Test for acaricide resistance

  • To tailor your tick control program (which acaricide and application method to use) it is important to determine your property's acaricide resistance profile.
  • Testing is most frequently conducted in a laboratory.
  • Check the requirements of your local laboratory but generally, send the lab 30 – 60 live, fully fed, adult female ticks (~10 mm long) in a small cardboard box with air holes. Ideally collect the ticks from multiple animals.
  • Collect ticks prior to treatment or a minimum of 49 days post treatment.
  • Laboratory bioassays for tick resistance measure the proportion of ticks that survive a concentration of acaricide that is expected to kill almost all susceptible ticks.
  • Laboratory reporting of bioassay results generally takes around 8 weeks from the time of collection of the ticks.
  • Genetic testing can rapidly detect some types of acaricide resistance (SP and Amitraz) but they are limited to only detecting genetic mutations that have been identified. These tests are not used for routine diagnostic purposes as resistant ticks, with different mutations, may produce negative test results.

Why test for acaricide resistance?

Wherever ticks are a potential problem for cattle production the application of acaricides is one of the most important components of a control program. In order to make sensible selections from a range of possible strategies for controlling ticks, often the most important question is which acaricide should be applied? The selection of acaricide then influences the frequency and method of application. Although resistance to commercially available acaricides is common, each property will have its own profile of acaricide resistance and in designing a cost effective control program it is important to know which acaricides can be expected to be useful, and which are unlikely to work.

Samples required for testing

Specific requirements for collecting and storing ticks for acaricide resistance testing in cattle ticks should be obtained from the laboratory that will do the testing. However, most commonly the preferred sample is adult female ticks that are fully fed and approximately 10 mm in length. 30 to 60 fully engorged ticks are required, depending on the type of test to be done and the number of acaricides to be tested. Where possible, ticks should be collected from several cattle, not just one animal. If possible collect ticks prior to treatment or a minimum of 49 days post treatment. For transport, ticks should be put into small non-crushable boxes, preferably made of cardboard, with a few small holes allowing air to circulate. Only a small amount of freshly cut, green grass is needed to provide moisture. More problems occur with samples that are too wet, rather than too dry, provided that transport times and temperatures are not excessive. Where large numbers of ticks are submitted for bioassay testing, the cardboard containers should be placed in a cool box with a cooler brick (wrapped in paper towels) and transported to the laboratory by the fastest route. Do not submit ticks in airtight containers, plastic bags or glass tubes. Do not place ticks in cotton wool. Do not expose ticks or containers to excessive heat/sunlight or chemicals. It is important to contact and discuss specific requirements with the laboratory that will do the testing.

Types of test

Approaches for testing ticks for acaricide resistance include ‘Bioassays’ conducted on ticks of specified life-cycle stages in the laboratory to determine the proportion of ticks killed by varying concentrations of acaricide. For a limited number of acaricides it is possible to determine the proportion of a sample of ticks that carry a ‘specific DNA mutation’ that confers resistance through genetic testing.

Bioassays

Bioassays are the most common means of diagnosing resistance. Two tests are currently available for resistance testing in Australia; the Adult Immersion Test (AIT) is used for fluazuron resistance testing, and the Larval Packet Test (LPT) for organophosphate, synthetic pyrethroid, macrocyclic lactone and amitraz resistance detection. For all testing, fully fed, adult females are obtained as stated above.

The Adult Immersion Test (AIT) requires  30 – 60 fully engorged ticks for testing but send a larger sample if you can collect them. This allows for a minimum of 10 ticks each for two testing concentrations plus a control. For AIT testing, ticks are either collected prior to treatment, or a minimum of 49 days post treatment. The ticks must reach the laboratory in good condition, and before they have begun to lay eggs. In the AIT, engorged adult females are exposed to acaricide by immersion in solutions of different concentrations for a set amount of time. The adults are then left to lay eggs which are incubated until they hatch. The percentage of larvae that hatch from the submitted sample, compared to a laboratory control strain, provides the resistance percentage or resistance profile.

A larval packet test (LPT) determines the acaricide resistance status of the offspring of the submitted ticks. Adult females are incubated for generally two weeks until they lay eggs, the eggs are then incubated for around four weeks allowing the larvae to hatch and harden. Resistance testing is performed on the larvae. Each female produces some 2,000-3,000 larvae, so it is possible to test hundreds of larvae at a time, and to have replicated bioassays for each of several acaricides and at several concentrations. Bioassays quantify the proportions of a sample that survive or die at any given concentration of each acaricide. Resistance level is usually expressed as the proportion of survivors at a concentration that is expected to kill almost all susceptible ticks. These bioassays are time consuming and require specialist laboratory facilities for culturing and handling ticks. Careful technique is essential and the assays take around 8 weeks from the time of collection of samples until the delivery of results.

Pen trials

Pen trials for resistance testing are quite complicated because they require a large number of larvae and specialised facilities. For this reason they are mainly used for research on resistant strains, and in the development of new acaricides.

Detection of DNA mutations that confer resistance

It is now possible to extract the DNA from ticks and determine whether they carry a mutation that makes them resistant to synthetic pyrethroids (SPs) and amitraz. Although such genetic testing uses standard molecular genetic techniques, genetic testing for acaricide resistance has not become routine. The main reason for this is that genetic testing can only reveal the status of a tick or a sample of ticks in relation to the specific mutations that have already been discovered. For example, the target site of SPs has been well characterised and three mutations have been discovered that confer resistance to SPs. Most known SP-resistant populations will have one or more of these mutations, but resistant populations may exist that don’t carry any of these mutations. It is quite possible that as-yet unknown mutations exist in the same gene, but in a place not examined using the current genetic tests. For this reason, a positive result (i.e. a resistance-conferring mutation is found to be present) confirms that there is resistance, but a negative result does not confirm that resistance is absent, it just indicates that the known mutations are absent. There is also the possibility that different mechanisms are responsible for resistance in a population, e.g. detoxification. The main advantage of genetic tests for resistance is that they are quick (only require a day or two) and they are quantitative (it is possible to state exactly the proportion of the population that carries a mutation).