Guideline for the Establishment of Efficacy and Management Data in Support of Applications for the Registration of Products to be Used in Control of the Cattle Tick (Boophilus Microplus)

1.2 Alternative in vitro procedures

It has been found that the susceptibility of larval cattle ticks, from various strains, to some of the recently developed macrocyclic lactones, including avermectins and milbemycins, can also be determined using a test method in which larvae of each strain are dipped in solutions of the test compound, in 0.02 % triton X‑100 in 1.0 % ethanol in demineralised water.  Again percentage mortality can be determined at various concentrations, LC50 values calculated, and resistance factors if present determined. Formulated material can be used in this test method.  Up to 10 ml of 1% formulated material could be required, again dependent on larvicidal activity of the active ingredient. Adult topical application, and/or injection procedures, have also been developed and are utilised at the CSIRO and DPI laboratories.  These methods enable investigators to assess potential resistance problems likely to affect compounds active against adult ticks, but with limited activity against other stages.

1.3 In vivo procedure

Chemicals with acaricidal activity derived from modes of action linked to interference with development of the cattle tick during the parasitic phase, eg growth regulators or hormone mimics, may not be amenable to resistance evaluation using in vitro procedures.  In these instances, an equivalent level of control, on animals infested with susceptible and nominated resistant strains, would demonstrate that the candidate chemical is not affected by established resistance mechanisms.  Such trials would need to be conducted as stall trials, according to the method set out in Appendix I.  A minimum of three treated animals per strain would be required in such an in vivo test, with three animals left untreated.  These untreated control animals should preferably be infested with the susceptible strain.

1.4 Mixtures

In cases where a potential new acaricide product is based on a mixture of two or more active ingredients, resistance factors should be determined for each of the individual components of the mixture.  This would not be necessary, however, where one of the chemicals is acting as a synergist in the combination and data supporting this effect is available.

The development of a product based on a mixture of two or more independently active ingredients could pose considerable problems, for their future long term use.  Skillful formulation may ensure the ratio of these individual components, of the mixture, is maintained over a prolonged period in a plunge dip.  Commonly however, in this situation the use concentration of these components is set lower than that normally recommended, when the chemicals are used alone.  Consequently selection of resistance mechanisms, in the tick population under treatment, could be considerably enhanced.  This would be particularly relevant during the residual decay period of the chemical on the beast, when protection against larval reinfestation is expected. Other problems, such as the potential for increased host toxicity when chemicals act in combination, should also be considered.  The development of such mixtures should therefore be treated with considerable caution. The relevant section in the "Interim Requirements for the Registration of Agricultural and Veterinary Chemical Products" 1993 and its succeeding registration documents, referred to earlier, should also be consulted in relation to this matter.

1.5 Recommended strains

In order to encompass the major resistance mechanisms currently present in the field, the susceptibility of strains listed in Table 1 to the potential new acaricide should be examined.

1.6 Correlations between in vitro and field tests

Results of in vitro resistance tests, such as LC50 values, should not be used to make direct comparisons of efficacy between various compounds against a particular strain, nor as a guide to likely field use concentrations for potential acaricides. Considerable research has been conducted demonstrating that such comparisons can be misleading.

TABLE 1: Nominated strains to be used in laboratory resistance tests with potential new acaricides.

Top

2. Efficacy Assessment

The traditional parameter for efficacy is derived from the control achieved during the period of three weeks, following treatment of cattle infested with all parasitic stages of the cattle tick (Wharton et. al. 1970).  Determination of this parameter has been based on a comparison made between the number of adult female cattle ticks engorging on treated versus untreated control animals.  These numbers have been determined either from daily collection of ticks, dropping from penned cattle (Roulston et. al. 1968), or by counting partly engorged "standard" ticks of a certain size, 4.5-8 mm in length, on one side of each animal.  Such counts have been shown to provide an accurate estimate of the number of ticks that will complete engorgement within 24 hours (Wharton and Utech 1970).

Efficacy trials should be based on the final formulation planned for use in longer term field trials, and in the ultimate commercial product.  Where significant changes are subsequently made to the formulation, eg in relation to isomer composition of the active ingredient, or modifications to allow alternative application methods, bioequivalence of the new formulation should be confirmed in either stall or field trials, as outlined below in Section 2.1 and 2.2.

2.1. Stall Test

This method, utilising penned cattle, is recommended for the establishment of parasitic phase efficacy data.  It has the advantage not only of ensuring that an accurate record is made of the number of ticks maturing on treated and untreated animals, but also a sample of these ticks is subsequently incubated and production of viable eggs assessed.  These measurements provide a more realistic and accurate control figure, than assessments based only on tick counts on the host.  The advantage is apparent with some synthetic pyrethroid chemicals, and subsequently developed systemically active compounds, where an ovicidal effect adds considerably to overall tick control.

Details of the method and basis for analysis of results, is presented in Appendix I. Strains to be used will depend on previous results of laboratory tests utilising nominated resistant strains.  In some cases, for example where laboratory data has indicated a potential problem with either Parkhurst or Ulam strains, the limited availability of such strains, and quarantine precautions in the field, may mean that a stall test becomes the only practical method of generating parasitic phase efficacy data.  A minimum of three (3) treated and three (3) control animals is required for each potential acaricide under test, or for each dose rate, where dose titrations are being carried out.  Where a single dose rate only is evaluated, data supporting the choice of this rate should be cited.

2.2. Field assessment of parasitic phase efficacy

Where access to pen facilities is not available, and where laboratory resistance tests have not indicated a need for efficacy to be determined using a particular strain, acceptable estimates of this parameter may be determined using cattle in the field, as an alternative to the stall test described above.  Details of animal requirements, the assessment methods used, and calculations for the analysis of results are given in Appendix II.  As mentioned above the potency of a potential acaricide may be underestimated in such a system relying only on tick counts, without subsequent confirmation of the viability of ticks.

2.3. Determination of protective period or residual efficacy

The concept of a prolonged protective period against larval re-infestation, with extended intervals between treatments, is a commercially attractive attribute for any tick control product.  However the manufacturer and end user of such a product should exercise considerable caution in the application of this approach in cattle tick management.

There is little doubt that over reliance on the so called protective period can prove to be an illusory benefit, which will shorten the useful life of an effective acaricide through selection for resistance.  Nevertheless, the development of an optimal use strategy for a new acaricide will be dependent to a large degree on establishing a safe interval between treatments, that will minimise a build up in tick numbers in the field.  Establishment of the interval will depend, in turn, on soundly based knowledge of the residual efficacy of the acaricide.  It is useful therefore to derive this information during trials undertaken to determine efficacy described above.

In such trials the accepted method is to continue to infest treated and control animals twice weekly, beginning on Day +2.  The trial is continued until re-infestation of treated cattle is established, as determined by a comparison of adult tick collections, or counts, between treated and control groups.  Obviously it is essential that cattle should be exposed to normal weather conditions, during experiments to establish a protective period.  Preferably this should be in a field situation.  However if strain quarantine necessitates the use of penned cattle, these animals should be held in uncovered open pens for the duration of trials conducted to establish this residual protection.

The time taken for the first viable adult female ticks to reappear on treated cattle, less twenty-one days, the average duration of the parasitic phase, provides a measure of the minimum protective period.  Alternatively, the protective period could be based on the time taken for daily tick counts, from treated cattle, to reach that of the untreated group.  Assessment based on this latter premise is not acceptable, or conducive to the development of effective tick management.  Reasonable estimates should be based on the period taken for adult female ticks, on treated cattle, to re-establish to a level representing 3% of the count of these ticks on control animals.  In practice it is advisable to continue counts beyond this period for a further week, to confirm the continuing upward trend of tick numbers on the treated cattle.

Artificial re-infestations following treatment may not be required, if a satisfactory field challenge of tick larvae is present for pastured animals.  However, this challenge would need to be verified from continuing satisfactory counts of engorged ticks on control animals after Day +21.

Top

3. Field Trials and Optimal Use Strategies

The use of acaricides in the field, in tick infested areas of Australia, covers a wide range of climatic and seasonal conditions.  These fluctuations will affect the performance of the product, as well as causing variations in the ecology and population dynamics of the pest.  Strain variation within the tick population further complicates management strategies.  Consequently all registration proposals for future acaricides need to be supported by convincing evidence, that these products will provide stable long term tick control in a variety of locations.

Some contemporary acaricides, or products with potential for use in tick control, may not provide the traditional >98% control, when assessed in efficacy tests as outlined in Section 2 above.  This may be due, in some instances, to differences in the mode of action or specific target of these chemicals, compared with the broad spectrum of toxicity associated with traditional acaricides.  These developments have increased the importance of longer term field trials, where the aim is to develop and demonstrate the potential of such methods in providing satisfactory, stable levels of tick control.  It is reasonable to expect that the results of such trials should be accorded particular significance, in the registration process.  Where <98% control is achieved, in parasitic phase efficacy tests, it is particularly important to demonstrate that the new approach can provide a tick management strategy equal to, or better than, that likely to be achieved with a traditional acaricide, under long term field use.  Variations in the method of application, and/or formulations, of acaricides currently providing >98% control when used in plunge dips, can result in significant reductions in efficacy.  Such variations could pose a considerable threat to the long term viability of the established method.  Hence it would be unwise to consider the development of a field treatment regime for these alternative formulations.

3.1       Sites and timing

In selecting suitable sites, and co-operators, it should be emphasized that treatments and observations should preferably be continued over two tick seasons, particularly where potential acaricides provide <98% tick control, in basic efficacy trials discussed in Section 2.  Best results in all field trials, will be achieved where a significant tick challenge is expected.  It is recommended that a minimum of two trial sites should be selected in each of the regions listed in Table 2.

3.2            Strategic treatment trials

A seasonal incidence of tick populations occurs, particularly in south-east Queensland where four distinct generations per year can be observed.  In order to maximize the efficacy of a treatment regime, and minimise the number of treatments required, recommendations have been developed to ensure that a control program is carried out at a time of year, when the tick population is most vulnerable.  In south-east Queensland this means that the program should be timed to control the "springrise" of overwintering larvae.  In tropical areas, where reproduction is virtually continuous, timing for the program is less critical.

TABLE 2: Recommended geographic areas and timing for field trials.

In accordance with these recommendations, at least one of the sites in each area should be selected to assess the performance of the product in a strategic treatment program, ie control achieved during a continuous period of eighteen weeks, beginning mid to late October in the south-east, September in central Queensland, and August or March in the tropics.  Tick populations on selected animals, minimum 20, should be monitored throughout this period, preferably with counts conducted immediately prior to each treatment.  It is obviously not reasonable to expect that untreated control animals should be retained on site, during this prolonged period.  However evidence should be provided, from local observations in the area, that a significant tick challenge existed for the trial animals during the selected period.  Treatment intervals should be planned on the basis of results obtained in previous parasitic and residual efficacy trials, as outlined in Section 2.

3.3            Confirmation of residual and parasitic efficacy

The second trial site in each area should be used to confirm previous detailed observations on parasitic and residual efficacy.  During the nominated period set out in Table 2, and at a time of significant tick challenge in the field, an efficacy assessment similar to that outlined in Section 2.2 and Appendix II, should be carried out, using a minimum of ten (10) animals.  Where the retention of untreated control animals is not feasible in the field situation, a comparison should be made between tick counts on animals treated with the experimental compound, and counts on a similar number treated with a registered acaricide, known to provide effective control at that site.  In addition to pre-treatment counts, at least three counts on trial cattle should be conducted on separate days during the 21 day period following treatment. Preferably these should be done to cover maturing ticks resulting from adult, nymph and larval infestations at the time of treatment, eg counts on Days 3, 10 and 19 would suffice as a minimum requirement.  Extension of the counting period beyond Day 21 should be used to verify protective period claims.

3.4       Strain identification

It is essential that samples of engorged adult female ticks should be collected from infested animals prior to the initial treatment at each site, and at regular intervals during the two season trial.  Resistance diagnosis on these samples is required to establish the composition of the initial strain present, and monitor resistance status of the tick population during the trial period.

3.5            Supplementary observations

The duration of these trials and the variety of locations involved, provide ideal opportunities to monitor stability of experimental chemicals intended for commercial use in dip vats.  Regular sampling, using the method outlined in Section 5, of at least one dip in each area subjected to heavy usage, should be used to develop an optimal management profile, for a formulation destined to be used in this way.  Stability and maintenance of mixture ratios, in multi‑component chemical systems, is critical where such mixtures are used, and will need to be verified during this time.

Records should also be maintained throughout the two tick seasons, of rainfall and any climatic changes which may significantly affect tick populations, or control methods.

Top

4. Supplementary Information

In addition to results of resistance tests and efficacy trials, and the development of optimal use recommendations, it is expected that submissions for registration of a chemical formulation as an acaricide should also include the following information:

4.1       Safety

Contra-indications in relation to animal use should be documented.  Information relating to adverse effects, causing discomfort to particular animal species, or where a particular formulation, or application method, has the potential to cause damage to hides, or animal products, should be recorded.

4.2       Rain fast tests

Acaricides are used under a wide range of climatic conditions.  The necessity to ensure effective tick control is achieved under these extremes, particularly where tick eradication or cattle movement is being undertaken, is important.  Acaricides used in dip vats, or as pour-ons, should be tested under artificial rainfall conditions following treatment, to demonstrate effective control in such circumstances.  Current recommendations are that animals should be subjected to the equivalent of 12.5 mm of rainfall over a 10 minute period, immediately following treatment.  Tick counts in this test on washed animals should be conducted as outlined in Appendix 2.  A minimum of five (5) treated animals should be used.

4.3       Use in cleansing tick infested cattle

Multiple treatments, commonly within a period of seven days, are used to ensure cattle from infested or quarantined areas can be moved to tick-free country.  Prior to movement these cattle are subjected to inspection to ensure no sign of tick life.  If it is intended to claim that a new product is suitable for this specific use, multiple treatments of infected cattle will need to be conducted, and efficacy assessments carried out, as described in Section 2.2.  However examinations to support this claim will need to include not only records of adult ticks collected, or counted, but also detailed observations as to the presence of other tick stages on the animals following treatment.

Top

5. Specific Requirements for Plunge Dip and Spray Race Use

Plunge dips and spray race vats provide a harsh environment which can adversely affect the stability, and long term efficacy, of any active chemical.  These application methods therefore require some particular observations, in order to ensure satisfactory performance and stability of an acaricide over an extended period.

5.1. Sampling and analytical techniques

The standard sampling procedure for dip vats has been to collect samples, minimum, 500 ml, of the vat contents at a depth of 60 cm at the "jump in" end of the dip.  Samples must be taken immediately behind the last animal treated, or used as a stirrer, and multiple samples should be collected as rapidly as possible.  The number of cattle used as stirrers prior to sampling, or the number of cattle treated since the last sample was taken, should be noted and the dip volume, at the time of sampling, must be recorded.  Details of methods developed for the chemical analysis of samples should be provided.

5.2. Stirring

Various manual approaches, including pumps and scoops, have been developed for stirring plunge dips.  These should be considered when the use of stirrer cattle may result in unacceptable residues, eg where these cattle may receive two successive treatments on the one occasion.  Whatever method is used to ensure vats are homogeneously stirred, evidence must be presented, from results of analyses of dip samples, that the procedure adopted is adequate in both fresh and polluted vats in a range of locations.  Analyses of samples taken during one season of continuous use, and at the initial treatments in a subsequent season, should satisfy this requirement.

5.3. Management

Recommendations must be presented as to the charging and replenishment or "top up" rates for acaricides used in dip vats and spray races.  The replenishment rates should cover normal losses following cattle treatment.  Procedures to be adopted, when evaporation or flooding cause significant concentration, or dilution of vat fluid should also be included.  Such replenishment must allow for "stripping" of the active ingredient during treatment.  Adequate studies should be undertaken to ensure the stripping rate of clean and polluted vats can be determined accurately.  The stability of the active ingredient should also be established, preferably during two seasons of continuous use of at least two dip vats, in three different geographical areas.

5.4. Spray race requirements

Results demonstrating optimal charging, stripping and replenishment rates, as described above, should be recorded for recirculating spray races.  A minimum of two geographically separate spray race facilities should be used to generate the information required, with a total of at least 500 cattle to be treated at each site.  Recommended management procedures for recirculating spray races should demonstrate that, at each trial site, vat acaricide concentrations were maintained at least at the minimum effective concentration established in extended dip trials.  This should apply even when spray race reservoirs are reduced to minimum operating volumes.  In spray race trials duplicate samples for analysis should be taken at charging, after each 200 litre depletion of the vat contents, when contents have been reduced to minimum operating volume, and before and after each replenishment or reinforcement with formulated concentrate.

Top

6. Clearance for use on Other Host Animals

Where a product has been approved for use in Boophilus control on cattle, a field efficacy trial, as outlined in Section 2.2, should provide sufficient evidence for extension of this registration to other hosts.  However care should be taken to ensure that the chemical, or application method, does not produce adverse effects in these alternate hosts.

Top

Appendix I

Basic short term efficacy trials - methods and analysis of results using tick infested cattle held in pens.

Materials and Methods

Bos taurus steers approximately 18 months to two years old are selected for the trial.  Cattle are prepared for treatment over a period of twenty‑four days during which time each animal is artificially infested with 10,000 larvae, of a selected strain of the cattle tick, on ten separate occasions.  Cattle are allowed to graze during this infestation period in an open paddock, with supplementary feeding of lucerne hay as required.  Four days prior to treatment cattle are penned individually.  For the duration of this period, and after treatment, penned animals are fed approximately 3kg of cattle pellets per animal twice daily and are provided water ad lib.  Adult female ticks dropping from each animal, or treatment group, are collected daily, at approximately 0900 hours, in wire mesh baskets following washing of the concrete floors of individual pens.  A random sample of fifty adult ticks, or maximum number available where less than fifty are collected are weighed and incubated at 27°C and 85% R.H., for seven days, to determine weight of eggs produced.  Viability of eggs produced is rated visually.  Prior to treatment, on Day - 1, all animals are weighed and ranked according to tick burden assessed on the basis of ticks collected on the previous three days.  Animals are randomly allocated, from this ranking, to the treatment and control groups, on the basis of three animals per group, to ensure an even distribution of tick infestations between the groups.  On Day 0, animals are treated immediately after the daily collection of ticks is completed.  Treatment may be carried out by hand spraying, where only limited quantities of formulated material are available, or by whatever application method is planned for the final commercial product.  Daily collection of adult ticks dropping from each animal, or treatment group, and incubation of a sample of the ticks collected, is continued, as described above, until Day +22, in order to assess the efficacy of the treatments in controlling the parasitic stages, present on the cattle at the time of treatment.

Analysis of Results

Percentage efficacy of treatment on parasitic stages based on tick survival is calculated on a daily basis as follows:

Daily percentage tick survival =            Tick count treated group x 100

                                                                        ADEQ

where ADEQ = Number of ticks expected in treatment group if left untreated

            =            Total pretreatment counts treated group               Daily

                        Total pretreatment counts control group   x            control

                                                                                                            count

Percentage efficacy based on egg production is calculated as follows, on a daily basis:

A          Egg production from ticks surviving in treated group

            Wt of eggs produced from incubated tick sample x          No of ticks collected

                        No of ticks incubated

B.            Expected egg production from treatment group if left untreated

            Wt of eggs produced from control incubated tick sample             x          ADEQ

                        No of ticks incubated (usually 50)

Therefore Percentage efficacy= A/B x 100

Top

Appendix II

Basic short term efficacy trials - methods and analysis of results using tick infested cattle held under paddock conditions

Materials and Methods

Bos taurus steers approximately 18 months to two years old should be selected for the trial.  Where field infestations of tick borne larvae are low cattle may be prepared for the trial over a period of twenty-four days, during which time each animal will be artificially infested with 10,000 larvae, of an OP-resistant strain of the cattle tick, on ten separate occasions, Synthetic pyrethroid, or amidine resistant strains, should not be used for artificial infestations under field conditions, because of the danger of contributing to the spread of these problem strains in the field population.  Under conditions favouring heavy field populations of the cattle tick, naturally infested animals may be used as an alternative to artificial infestations.  Tick burdens on selected animals, in this case should exceed 20 standard 4.5-8 mm adult female ticks counted on one side of each animal on Day-l prior to treatment.  Infestations should indicate that this level will be maintained, on control animals, for 21 days following Day 0, treatment day.  In the four days prior to treatment counts of 4.5-8 mm ticks should be carried out on at least three occasions.  On Day-1 all animals should be weighed, if treatment dose is to be based on this criterion, and ranked according to tick burden.  Animals should be randomly allocated from this ranking to the treatment and control groups to ensure an even distribution between the groups.  A minimum of five (5) animals per group will be required for this trial procedure.  Following treatment, counts on one side of each animal should be continued.  As a minimum requirement these should be carried out on Days 1, 2, 5, 7, 9, 12, 14, 16, 19, 21, and 23.  Where protective period is to be assessed counts, and possibly artificial infestations, will need to be continued as discussed in Section 2.3.

Analysis of Results

Percentage efficacy of treatment on parasitic stages, based on tick survival, will be calculated on a side count basis as follows:

Daily Percentage tick survival            =            AD            x            100
                                                                   BC

where

A  =     Number of ticks counted on control animals prior to treatment.
B  =      Number of ticks counted on the control group on a particular day after treatment.
C  =      Number of ticks counted on a treated group prior to treatment.
D  =     Number of ticks counted on the treated group on the particular day after treatment

Percentage tick survival for the 1 - 21 day period can be calculated similarly.  Counts refer to standard 4.5 - 8 mm adult female ticks.

Top

References

Anon. (1971).  Recommended methods for the detection and measurement of resistance of agricultural pests to pesticides.  Tentative method for larvae of cattletick, Boophilus spp.  FAO Method No. 7. FAO Plant Protection Bulletin 19, p 15.

Roulston, W.J., Stone, B.F., Wilson, J.T. and White, L.1. (1968).  Chemical control of an organophosphorous and carbamate resistant strain of Boophilus microplus (Can.) from Queensland. Bull. ent. Res. 58, 379-392.

Wharton, R.H., Roulston, W.J., Utech, K.B.W. and Kerr, J.D. (1970).  Assessment of the efficiency of acaricides and their mode of application against the cattletick Boophilus microplus. Aust. J. of agr. Res. 21, 985-1000.

Wharton, R.H. and Utech. K.B.W. (1970).  The relation between engorgement and dropping of Boophilus microplus to the assessment of tick numbers on cattle. J. Aust. ent. Soc. 9.171-182.