Comparison of 3 Tests to Detect Acaricide Resistance in Boophilus Decoloratus on Dairy Farms in the Eastern Cape Province, South Africa

a INTRODUCTION The resistance of Boophilus decoloratus to acaricides has been a problem on farms in the Eastern Cape Province of South Africa for more than 60 years 16. The occurrence of resistance in the closely related and more widely distributed Boophilus micro-plus has resulted in the development of a number of laboratory and field tests, entailing the use of either larvae or adult ticks, to detect this phenomenon 9. The present study describes the evaluation of 3 of these tests in detecting acaricide resis


INTRODUCTION
The resistance of Boophilus decoloratus to acaricides has been a problem on farms in the Eastern Cape Province of South Africa for more than 60 years 16 .The occurrence of resistance in the closely related and more widely distributed Boophilus microplus has resulted in the development of a number of laboratory and field tests, entailing the use of either larvae or adult ticks, to detect this phenomenon 9 .The present study describes the evaluation of 3 of these tests in detecting acaricide resis-tance in B. decoloratus.The tests used were the Shaw Larval Immersion Test (SLIT) for tick larvae, and the Reproductive Estimate Test (RET) and the Egg Laying Test (ELT) for adult ticks.
A slight modification of SLIT, first described by Shaw 7 , was used.In it the larvae were incubated for 72 h after treatment before the test was read 8 .In RET, engorged female ticks are exposed to acaricide and their subsequent production of larvae is used as a measure of resistance 3 .This test was originally used to evaluate the efficacy of new acaricides 3 , as well as in acaricide resistance testing 13 .ELT is based on a comparison of the number of eggs laid by treated and untreated engorged female ticks.These tests were applied to ticks collected from cattle on dairy farms that had reported the failure of tick control regimes.
SLIT: the test used was that originally described by Shaw 7 and later modified to increase the period of larval incubation after treatment to 72 h before the test was read 8 .Empirical studies have shown that a factor of resistance (FOR) of >100 for both amitraz and cypermethrin and >5 for chlorfenvinphos indicate resistance.FOR values between 50 and 100 for both amitraz and cypermethrin and values between 2.5 and 5 for chlorfenvinphos are regarded as indicating emerging resistance (R J Taylor, unpubl.data, 2001).The susceptible Botshabelo reference strain of B. decoloratus was used to calculate the FOR values and the results could be read after 60 days.
RET: engorged female B. decoloratus of uniform size and free of visible abnormalities were collected from cattle on the 3 farms.The ticks were washed in water, air-dried 11 , and grouped according to size.Groups of 10 ticks were weighed and randomly allocated to 2 or 4 replicates for each treatment and the control group.The treatment groups were immersed in the recommended concentrations of the acaricides and the control group was immersed in sterile water.The test and control groups were incubated at 27 °C and 80-90 % relative humidity (RH), to permit oviposition and egg hatching 3 .
The %RE for female ticks was calculated as follows: An RE value of >80 % indicates resistance, and we consider RE values between 50 and 80 % as indicative of emerging resistance.
ELT: the collection and incubation of engorged female B. decoloratus was similar to that described for RET, but the number of ticks that had laid eggs was assessed on the 7th day of incubation at 27 °C and 80-90 % RH (Kemp, pers.comm., 2001).

% Resistance =
No. of treated ticks laying eggs No. of untreated ticks laying eggs 100 × A value of >80 % indicates resistance, and between 50 and 80 % emerging resistance.

SLIT
The larval offspring of B. decoloratus females collected on the farms Brycedale, Sunny Grove and Welgevind showed resistance to 1 or more of the test acaricides (Table 1).At Brycedale, emerging resistance to amitraz was recorded, while at Sunny Grove and Welgevind, larvae were susceptible to this chemical.At both Brycedale and Welgevind, resistance to chlorfenvinphos was present, while at Sunny Grove emerging resistance was recorded.At Brycedale and Welgevind the ticks were susceptible to cypermethrin, but at Sunny Grove they were highly resistant.

RET
On Brycedale, B. decoloratus was resistant to amitraz and chlorfenvinphos, whereas on both Sunny Grove and Welgevind it was susceptible to both these acaricides (Table 2).

ELT
Female B. decoloratus on Brycedale were resistant to amitraz and chlorfenvinphos and showed emerging resistance to cypermethrin (Table 3).At Sunny Grove this tick was susceptible to amitraz and chlorfenvinphos, but displayed an emerging resistance to cypermethrin.At Welgevind, resistance only to cypermethrin was detected.

DISCUSSION
The results obtained with SLIT cannot be compared directly with those obtained with either RET or ELT as the first test is based on assessing the resistance of tick larvae to acaricides, whereas the last 2 are based on assessments engorged female ticks.The pattern of resistance recorded on the 3 farms confirms these differences in that results obtained with SLIT often differed from those obtained with RET and ELT (Table 4).
Organophosphate (OP) acaricides had not been used for the past 10 years on either Sunny Grove or Welgevind (Amaral,  pers.comm., 2001)  chlorfenvinphos on both farms more than 10 years after the last use of an OP acaricide it would appear that once OP resistance has become established in a tick population its reversion to susceptibility is either very slow or does not occur 12 .The OP resistance on these farms could have originally been induced by acaricides containing OPs other than chlorfenvinphos, as resistance to 1 member of a group of chemically related acaricides can result in a degree of resistance to other members of the same group or a closely related group 1,8,15 .Furthermore cross-resistance between chemically related acaricides has previously been documented for B. decoloratus within the region of the present study 2 .'Ektoban' (Bayer Animal Health), which is a mixture of cypermethrin and cymiazol, had been used for tick control for nearly 10 years on both Sunny Grove and Welgevind and the owners reported that it no longer controlled ticks.Resistance to cypermethrin was detected on Sunny Grove with SLIT and on Welgevind with ELT.The high burdens of B. decoloratus observed on the cattle at Brycedale were in agreement with the results obtained from the acaricide resistance tests conducted in the laboratory.
Each of the 3 tests has certain practical advantages.SLIT uses unfed larvae, which are more easily standardised than adult ticks, and the mortality of the larvae can be recorded easily 14 .The larvae are also treated identically, leading to statistically more credible results 4 .A disadvantage of this method, however, is that the exposure of larvae to an emulsion of a commercial acaricide for 10 minutes is not a satisfactory imitation of the field situation 4 .Both RET and ELT require that engorged female ticks be immersed in commercial acaricides at the recommended field concentration 3 .The advantage of these tests is that they can be interpreted earlier than SLIT, which requires 60 days, namely within 42 days for RET and within 7 days for ELT.A disadvantage is that sufficient fully engorged female ticks are not always readily available for the tests 6 .
Although the results of the 3 test methods could not be compared statistically, RET and ELT in most cases showed similar results and these often differed from those obtain by SLIT (Table 4).In previous studies a poor correlation between larval and adult test results has also been observed 5 and it has been stated that the Adult Immersion Test (AIT) reflected field conditions better than SLIT 10 .