Accumulation of acaricide resistance mechanisms in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) populations from New Caledonia Island

Rhipicephalus (Boophilus) microplus has been pesticide-controlled for several decades in the pacific island of New Caledonia. Since 1996, pesticide-control has been based on either deltamethrin (Butox) or amitraz (Taktic) in herds harbouring deltamethrin-resistant ticks. In this island, the first R. microplus deltamethrin- and amitraz-resistances were detected in 1992 and 2003, respectively. Using LPT bioassays, we have undertaken to update data regarding the geographical distribution and the physiological diversity likely to be involved in these resistances. We confirmed that after 17 years of intensive use of deltamethrin, several resistances of moderate levels (<30-fold) have evolved and/or diffused in any part of the island. We also evidenced that amitraz-resistant phenotypes have recently evolved in diverse western tick populations, although none has reached fixation in any tick population yet. According to synergists bioassays, the physiological changes involved in amitraz-resistance in New Caledonia would involve target modification and detoxifying P450 cytochrom oxydase(s). It may also involve detoxifying esterase(s) although this later point will need confirmation on samples bearing higher frequency of resistant phenotypes. Results are discussed with regard to the local evolutionary dynamics of resistance.     

Comparison of the reproductive biology between acaricide-resistant and acaricide-susceptible Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The reproductive fitness of Rhipicephalus (Boophilus) microplus (Canestrini) strains resistant to organophosphate (OP), pyrethroid (P), or formamidine (F) acaricides was compared to an acaricide-susceptible (SUS) strain to determine whether the acquisition of resistance affected reproductive fitness in the resistant strains. The SUS strain females had a 3.0 days preoviposition period, a 12.1 days oviposition period, a 22.5 days egg incubation period, a mean of 3670 eggs per female, and a mean percentage egg hatch of 78.1%, which were all remarkably similar to these same parameters reported for this species throughout the world. The reproductive biology of the P-resistant strain (PYR) and the F-resistant strain (FOR) were, for the most part, similar to those of the SUS strain. In the few instances where statistical differences did occur there was little evidence that the variation had any biological basis that could be attributed to a reduction in fitness related to resistance to P or F acaricides. Although the comparison of reproductive parameters of the OP-resistant strain (OPR) and the SUS strain identified statistical differences between the mean egg incubation and oviposition periods, the magnitude of the differences was not sufficient to conclude that the OPR strain was biologically less fit than the SUS strain. However, the OPR strain produced 30% fewer eggs (2562 eggs per female) than the SUS strain (3670 eggs per female) indicating the acquisition of resistance placed the OPR at a selective disadvantage relative to the SUS strain. This coupled with a lower, though non-significant, egg hatch was used to predict there would be a reduction of at least 34.1% in larval numbers available to potentially re-infest subsequent cattle than were available from the SUS strain. These data may aid the development of management strategies that can be used to control OP-resistant ticks.     

In vitro acaricidal effect of tannin-rich plants against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The objectives of this study were to evaluate the in vitro acaricidal effects of lyophilized extracts of four tannin rich plants (Acacia pennatula, Piscidia piscipula, Leucaena leucocephala and Lysiloma latisiliquum) against diverse stages of Rhipicephalus (Boophilus) microplus, and to asses whether tannins were involved in the acaricidal effect using polyethylene glycol (PEG) to block tannins. Larval immersion (LIT) and adult immersion (AIT) tests were used to evaluate the acaricidal effect of each of the lyophilized extracts against larval and adult stages of R. microplus respectively. Larvae and adult ticks were exposed to increasing concentrations of each plant extract (0, 1200, 2400, 4800, 9600 and 19,200 μg ml(-1)) for 10 min. Larval mortality was recorded at 48 h post-incubation. Adult mortality was recorded daily over 14 days, at which point their reproductive efficiency was evaluated. PEG was added to the extracts to verify whether tannins were involved in the acaricidal effect. The effect on egg laying inhibition and larval mortality was analyzed using the GLM procedure in SAS. A Kruskal-Wallis test was used to assess the effect of PEG on LIT results. Calculation of the lethal concentration 50 (LC50) was performed using a probit analysis. All extracts reduced the viability of R. microplus larval stages (P<0.001), and viability was restored with the addition of PEG suggesting an important role of tannins in the acaricidal effect (P<0.001). The LC50 values of L. latisiliquum and P. piscipula plant extracts were 6.402 and 2.466 μg ml(-1). None of the tannin-rich plant extracts affected adult mortality (P>0.05). Lysiloma latisiliquum extract inhibited egg hatching of R. microplus (P<0.01). Tannin-rich plant extracts from A. pennatula, P. piscipula, L. leucocephala and L. latisiliquum showed potential acaricidal activity. Further in vivo studies are needed to confirm this finding.     

Use of the Larval Tarsal Test to determine acaricide resistance in Rhipicephalus (Boophilus) microplus Brazilian field populations

Acaricide resistance of the cattle tick Rhipicephalus (Boophilus) microplus is widespread in most of the countries where this parasite is present. Bioassays are used to diagnose the level and pattern of resistance in tick populations. In the present study, we describe a detailed protocol of the Larval Tarsal Test (LTT) using simplified equipment and data on the resistance of 17 tick field populations originating from 5 Brazilian states. Nine acaricidal compounds from 5 major classes were tested: organophosphates (OP), synthetic pyrethroids (SP), macrocyclic lactones (ML), phenylpyrazols (PYZ) and amidines. For comparison, four of the tick populations were also tested with the Larval Packet Test (LPT) with one compound per class. The most common resistances were to SP, amitraz and OP, with frequencies of 94%, 88% and 82%, respectively. Resistance to PYZ was also found to be widespread (65%), suggesting a rapid development of fipronil resistance in Brazil. One case of ML resistance and 2 cases of suspected ML resistance were identified with the LTT. The LTT led to higher resistance ratios to all compounds than the LPT, reflecting its high sensitivity to detect resistance. Finally, the LTT allowed testing a larger number of compounds and doses with reduced labour in comparison to the LPT and turned out to be a reliable bioassay to detect resistance in field populations.     

Applicability of in vitro bioassays for the diagnosis of ivermectin resistance in Rhipicephalus microplus (Acari: Ixodidae)

The applicability of laboratory bioassays to diagnose ivermectin (IVM) resistance in Rhipicephalus microplus was evaluated. Adult immersion tests (AITs), larval immersion tests (LITs) and larval packet tests (LPTs) were performed to characterise the effects of ivermectin toxicity on adults and larvae of a susceptible reference strain. The AIT was determined to be a reasonable assay but requires a large number of individuals to attain interpretable results. The LIT and LPT were validated with an IVM resistant strain, revealing resistance ratios (RRs) of 6.73 and 1.49, respectively. In a field survey, nine different populations of cattle tick from the states of S?o Paulo and Mato Grosso do Sul, Brazil, were analysed with the LIT. Populations without previous exposure to ivermectin exhibited RRs between 0.87 and 1.01. Populations previously exposed to IVM showed RRs between 1.83 and 4.62. The LIT was more effective at discriminating between resistant and susceptible populations than the LPT. The use of the LIT is recommended for the diagnosis of ivermectin resistance in R. microplus.     

Efficacy of amitraz applied as a dip against an amitraz-resistant strain of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) infested on cattle

Selection pressure with the acaricide, amitraz was applied to a strain of Rhipicephalus (Boophilus) microplus (Canestrini) in 13 out of 18 generations, resulting in a 28.7-fold increase in resistance, indicating a shift in the phenotypic composition of the ticks, from a heterogenous mixture of both susceptible and resistant ticks when selection began to a homogenous composition of predominantly resistant ticks when selection ended. Resistant larvae of the last selected generation were infested on cattle three separate times at 20, 13, and 6 d (7 d intervals) before being dipped in a total immersion vat charged at 0.02, 0.044, and 0.096% active ingredient (AI) amitraz. There was a dose related, though not always significant, response to amitraz whereby each increase in concentration produced increased adverse effects. The number of ticks and index of fecundity (IF) of females recovered from cattle treated at 0.02 and 0.044% AI were not different from that of untreated ticks. However, female weight and egg mass weight of females recovered from cattle treated at 0.044% were lower than untreated females or females treated at 0.02% AI. All measured parameters obtained from ticks recovered from cattle treated at 0.096% AI were significantly lower than the untreated control group, but tick numbers and IF were not different from the other treated groups. At 0.02% AI the level of control was significantly lower (34.6%) than treatment at 0.096% AI (81.2%), while the level of control at 0.044% AI (57.6%) was intermediate between the other treated groups. Therefore, none of the amitraz treatments provided the required 99% control necessary for use in the U.S. Cattle Fever Tick Eradication Program, and the estimated line derived from regression analysis showed that a concentration of ca. 0.2% AI amitraz would be needed to achieve 99% control of the resistant ticks.     

Immunoprotective potential of a Rhipicephalus (Boophilus) microplus metalloprotease

Ticks have serious impacts on animal and human health, causing significant economic losses in cattle breeding. Besides damage due to the hematophagous behavior, they transmit several pathogens. Low cost and environmental safety have made vaccines a promising alternative control method against tick infestation. Metalloproteases (MPs) have been shown to be essential for diverse biological functions in hematophagous organisms, inhibiting blood clotting, degrading extracellular matrix proteins, and inhibiting host tissue repair via anti-angiogenic activity. In this study, we analyzed the immunoprotective potential of a recombinant MP against Rhipicephalus (Boophilus) microplus infestation. First, a cDNA encoding R. microplus amino acids sequence with highly conserved regions of the metzincin (reprolysin) group of MP was identified (BrRm-MP4). After expression and purification, recombinant BrRm-MP4 was used as a vaccinal antigen against R. microplus infestation in cattle (Bos taurus taurus). All vaccinated bovines developed immune response to the antigen, resulting in increased antibody level throughout the immunization protocol. Immunization with rBrRm-MP4 reduced tick feeding success, decreasing the number of engorged females and their reproduction potential, representing a 60% overall protection. These results show that rBrRm-MP4 provides protection against tick infestation, placing it is a potential candidate for an anti-tick vaccine.     

First report of fipronil resistance in Rhipicephalus (Boophilus) microplus of Mexico

Five strains of Rhipicephalus (Boophilus) microplus collected from Tamaulipas Mexico were tested for resistance against several classes of acaricides commonly used. All were resistant to fipronil. Four of five were co-resistant to permethrin and coumaphos in addition to being resistant to fipronil. One strain, El Zamora was found multi-resistant to permethrin, coumaphos, fipronil, and amitraz. Selection with fipronil for 3 generations produced a resistance ratio of 8.3 and 9.4 at the LC₅₀ and the LC₉₉ estimates, respectively. Permethrin resistance in El Zamora was possibly linked to elevated esterase (CZEST9) and could be a contributing factor of resistance to fipronil. The implications of resistance for the control of the southern cattle tick in the future are discussed.     

Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay

Rhipicephalus (Boophilus) microplus is an important cattle pest in Uruguay, and the law regulates its control. It is resistant to organophosphates, synthetic pyrethroids and, as recently discovered, to fipronil. Resistance to macrocyclic lactones (MLs) and amitraz have not been documented; however, veterinarians and farmers have reported treatment failures. The objective of the present work was to study the susceptibility of cattle tick strains from different Uruguayan counties to ivermectin (IVM) and fipronil by using the Larval Immersion Test (LIT). The Mozo strain was used as the susceptible reference strain. From 2007 to 2009, twenty-eight tick populations were collected from different cattle farms with and without history of IVM or fipronil use. A probit analysis estimated dose-mortality regressions, lethal concentrations (LC), and confidence intervals. The resistance ratio (RR) was determined at the LC(50) and LC(90) estimates. To classify a tick population in relation to resistance, three categories based on a statistical analysis of LC and RR between field populations and Mozo strains were defined: susceptible (no differences), incipient resistance (differences and RR(50)<2) and resistant (differences and RR(50)≥2). Eighteen field populations were tested with IVM and five of them presented a RR(50) range between 1.35 and 1.98 and the LC(50/90), which is statistically different from the Mozo strain (incipient resistance). However, the RR(90) increases ≥2 in four of the populations, confirming that tick resistance to IVM is emergent. The low RR values obtained could be a result of a low frequency of treatments with IVM. Twenty-seven tick populations were tested with fipronil and six were diagnosed as resistant according to the LIT. Cross-resistance was not observed between fipronil and IVM on these tick populations. The current study presents different R. (B.) microplus populations with an incipient resistance to IVM, and indicates that the fipronil tick resistance is restricted to certain areas in Uruguay.     

Eprinomectin pour-on for control of Boophilus microplus (Canestrini) ticks (Acari: Ixodidae) on cattle

The efficacy of a commercial pour-on formulation of eprinomectin, a macrocyclic lactone, against experimental infestations of Boophilus microplus (Canestrini) ticks was evaluated in two trials involving 27 Bos taurus calves. The first trial was designed to evaluate the effects of a single treatment at a dose of 0.5 mg/kg of body weight against standard size B. microplus females (4.5-8.0 mm long). A significant reduction in tick numbers (P<0.05, Wilcoxon test) was observed between treated calves as compared to untreated ones from Day 3 (44% efficacy) after treatment to the end of the trial on Day 28 (96.9%), with a peak efficacy of 97.1% on Day 21. In the second trial the effect of eprinomectin on standard size tick numbers, engorgement weight and fertility of female ticks from calves with a single treatment dose of 1 mg/kg on Day 0 and calves treated twice at a dose of 0.5 mg/kg on Days 0 and 4 was evaluated. An efficacy >93% was obtained from Day 2 to Day 28 after treatment in calves treated twice at 0.5 mg/kg, and to the end of the trial (Day 35) in calves treated once with 1 mg/kg. The 1mg/kg treatment provided >98% residual efficacy for at least 7 days. During the first part of the second trial the efficacy of eprinomectin resulted from a dramatic adverse effect on engorgement weight and fertility of female ticks, with 100% control on Day 5 (dosage of 1 mg/kg) and on Days 6 and 7 (two doses of 0.5 mg/kg). Following Day 7, most of the effect was due to reduction in the number of standard size female ticks.     

In vitro and in vivo evaluation of deltamethrin and amitraz mixtures for the control of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in New Caledonia

Acaricide resistance is a major problem that hinders the control of the tropical cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), in many parts of the world where cattle production continues to suffer severe economic losses to tick infestation. Deltamethrin and amitraz have been used alone to control R. microplus in New Caledonia for the past decade, and tick populations have developed resistance to both acaricides. A study was conducted to evaluate the effectiveness of deltamethrin and amitraz mixtures, through in vitro laboratory bioassays and in vivo on-animal efficacy trials, for the control of resistant R. microplus on cattle at two dairy farms in New Caledonia. Results of laboratory bioassays using modified larval packet tests (LPT) revealed up to 16.59-fold resistance to deltamethrin, and up to 5.86-fold resistance to amitraz. Significant synergism was observed when amitraz was used as a synergist in deltamethrin bioassays. Amitraz significantly increased deltamethrin toxicity to tick larvae, while deltamethrin was much less effective on amitraz toxicity. Synergism of amitraz by deltamethrin only occurred when the deltamethrin concentration was relatively high. Results of on animal efficacy trials of deltamethrin and amitraz alone and mixtures of both at different concentrations revealed a similar pattern of synergism. Adding amitraz to a deltamethrin formulation led to dramatic increases of percent reduction of both immature and adult ticks. In contrast, adding deltamethrin to an amitraz formulation did not increase control efficacy. Results from this study may lead to the adoption of an acaricide mixture strategy for the control of pyrethroid-resistant R. microplus in New Caledonia and elsewhere.     

Structural and cytochemical changes in the salivary glands of the Rhipicephalus (Boophilus) microplus (CANESTRINI, 1887) (Acari: Ixodidae) tick female during feeding

This study describes the morphology of salivary glands of Rhipicephalus (Boophilus) microplus female ticks at beginning of feeding (24-48 h of attachment) and semi-engorged (4-5 days of attachment) to verify the degenerative characteristics of these organs and the secretory phase in which the process begins. At the beginning of feeding, secretion granules had been observed only in the cytoplasm of cells b, c1, c2, c4 (type II acinus) and d (type III acinus), as well as large nuclei with regular and preserved morphology. In the semi-engorged females the acini presented few normal cells, few partially preserved ones, and the remaining ones in several stages of degeneration, that is, with retraction and cytoplasmic vacuolization, and nuclei with chromatin in several stages of condensation, picnotic and/or in fragmentation. In type I acinus and in the excretory ducts of the studied glands, at both feeding stages, no degenerative characteristic was observed. In females of R. (B.) microplus, the salivary glands degenerate asynchronically and precociously when compared with those of others tick's species.     

Potential synergistic effect of Melia azedarach fruit extract and Beauveria bassiana in the control of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in cattle infestations

The use of a concentrate emulsion of Melia azedarach green fruits and a suspension of the fungus Beauveria bassiana was evaluated in the control of Rhipicephalus microplus on artificially infested cattle. The evaluation was conducted following the protocol established by the Brazilian Agriculture Ministry. Five groups of 4 or 5 animals were allocated to one of the following treatments: emulsion concentrate of M. azedarach at 0.25% (T AZED 0.25%), emulsion concentrate of M. azedarach at 0.5% (T AZED 0.5%), B. bassiana at 2.4 × 10(8) conidia (T BASS), association of the concentrate of M. azedarach at 0.25% with B. bassiana at 2.4 × 10(8) conidia (T AZED 0.25%+BASS), and control (untreated). The association of the two compounds provided better results than any one isolated treatment, indicating compatibility or perhaps a synergy between M. azedarach and B. bassiana. This treatment resulted in fewer engorged females (129 ± 70) than in the control group (233 ± 82), showing high performance against all developmental stages of the tick. Results revealed an apparent synergistic effect of M. azedarach and B. bassiana in the control of R. microplus that should be further investigated.     

Kinetics of energy source utilization in Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) embryonic development

The present work evaluates the kinetics of utilization of the main potential energy sources throughout the embryonic developmental stages of Boophilus microplus. The embryonic development of this arthropod is completed in 21 days. Cellularization of the blastoderm occurs on the 6th day and is rapidly followed by germ band extension and segmentation, whose first signs are visible on the 7th day. Cellularization is typically a maternal-driven process, carried out by molecular determinants deposited in the oocyte during oogenesis. On the other hand, segmentation is of zygotic nature, being the consequence of the synthesis of various components by the growing embryo. The enhancement in total B. microplus RNA was observed after cellularization, corroborating the replacement of maternal-driven processes by embryonic zygotic expression. An abrupt increase in oxygen consumption was observed from cellularization until the 8th day of development. The reduction in dry weight at the same period and the susceptibility of oxygen consumption to KCN suggest that the respiration process is activated during early embryonic development. A marked decrease in total lipid content occurred between the 5th and 7th days of development, suggesting this is the main energy source for cellularization. A major reduction in carbohydrate content occurred later, between the 7th and 9th days, and it could be assigned to the morphological segmentation of the embryo. Although the total amount of proteins remains unchanged from oviposition to hatching, a 15% reduction in vitellin (VT) content was observed before cellularization, up to the 4th day after egglaying. This observation was correlated to the synthesis of new proteins needed to support early embryo development. Additional 20% of VT was consumed thereafter, mainly at the end of embryogenesis, and in this case VT is probably used as energy source to the older embryo. Altogether, these data indicate different energy sources for maternal and zygotic driven processes.     

Development of amitraz resistance in field populations of Boophilus microplus (Acari: Ixodidae) undergoing typical amitraz exposure in the Mexican tropics

The purpose of this study was to determine the effect of amitraz selection pressure on the development of resistance in field populations of Boophilus microplus in the Mexican tropics. Three farms (FA(1), FA(2) and FA(3)) in Yucatan, Mexico, were selected in this study. Amitraz was applied as a whole body sprays to all cattle on each farm for tick control once monthly for 15 months. From each farm, 20-30 B. microplus engorged females were collected every 3 months. The modified larval immersion test was used to test susceptibility of B. microplus to amitraz. Larvae were exposed to serial dilutions of amitraz. Probit analysis was used to determine lethal dose at 50% mortality and associated 95% confidence limits. The resistance factors found in the three farms during the 15 months of amitraz selection pressure were FA(1) (1, 2, 4, 4 and 13), FA(2) (1, 6, 23, 21 and 22) and FA(3) (2, 13, 2, 6 and 6). It is concluded that amitraz selection pressure on field populations of B. microplus increased the resistance level in all populations studied in the Mexican tropics.     

Acaricidal properties of the essential oil and precocene II obtained from Calea serrata (Asteraceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Calea serrata Less. (Asteraceae), an endemic species of south Brazil known as "quebra-tudo", is used in Afro-Brazilian religious rituals and in folk medicine for treating liver disorders. Phytochemical studies of the n-hexane extract of this plant demonstrated the presence of precocene II, a benzopyran derivative known for its insecticidal activity. The aim of this work was to isolate this benzopyran and determine the chemical composition of the essential oil of C. serrata and further to evaluate the acaricidal activity of the essential oil and precocene II against the larvae of Rhipicephalus (Boophilus) microplus. The LC(99.9) and LC(50) values obtained with the oil, which presents precocene II and sesquiterpenes, were 3.94 μL/mL and 0.28 μL/mL, respectively. For precocene II this values were 4.25mg/mL and 1.78 mg/mL, respectively. The results indicate a synergistic interaction between the components of the oil and precocene II.     

Synthetic pyrethroid resistance in Rhipicephalus (Boophilus) microplus ticks from north-western Himalayas,India

In the present study, adult immersion test (AIT) was used for evaluation of resistance against synthetic pyrethroids (deltamethrin and cypermethrin) in Rhipicephalus (Boophilus) microplus ticks collected from nine districts of three agro-climatic zones of north-western Himalayan region of India. Resistance factors (RFs) were calculated between 0.94 to 50.71 for deltamethrin and 0.32 to 13.18 for cypermethrin. Resistance to deltamethrin was detected at level I in two, level II in four, level III and level IV in one isolate each while one isolate was susceptible. Against cypermethrin, resistance at levels I and II was detected in three isolates each while three isolates were found susceptible. The low altitude sub-tropical zone revealed higher density of resistant ticks where intensive animal husbandry practices were followed and the synthetic pyrethroid usage was common. Data generated on pyrethroid resistance status of ticks in north-western Himalayan region will provide new insights in acaricidal resistance particularly from remote areas of this region and will help in formulating suitable control measures.

     

Susceptibility to two pyrethroids in Boophilus microplus (Acari: Ixodidae) populations of northwest Argentina. Preliminary results

Cattle are treated 6-12 times yearly to control Boophilus microplus ticks in the east zone of the Argentinean infested region, while 1-4 treatments are applied for tick control in the west zone. In the 1970s resistance to organo-phosphate acaricides was found in the east zone, but not in the west zone. However, a shift to synthetic pyrethroids (SP) was made through all infested regions. Currently, indications of resistance to SP in the east zone, but not in the west zone, are provoking to a switch to formamidine acaricides. During 1998 a total of 147 B. microplus engorged females were collected from 20 beef cattle ranches from the west zone of the Argentinean infested region. Individual progenies of these ticks were tested ('larval packet test') with cypermethrin and deltamethrin, and their LC 50 and LC 90 were compared to those estimated for the Milagro susceptible strain. No evidence of resistance to these SP was found. Due to sampling restraints the results are presented as preliminary. Nevertheless, a shift away from use of SP for control of B. microplus in the west zone appears to be unjustified and should be independent of the resistance circumstances observed in the east zone of the Argentinean tick infested region.     

Possible risk factors on Queensland dairy farms for acaricide resistance in cattle tick (Boophilus microplus)

A case control study was carried out within a cross-sectional survey designed to investigate the management by Queensland dairy farmers of the cattle tick Boophilus microplus. Although 199 farmers were surveyed, data on acaricide resistance were only obtained from 66 farms. Multiple models were used to predict the probability of acaricide resistance associated with 30 putative risk factors. The region of the state in which the farm was located and the frequency of acaricide application were consistently associated with acaricide resistance. The risk of resistance to all synthetic pyrethroids (Parkhurst strain) was highest in Central Queensland and increased when more than five applications of acaricide were made in the previous year, when spray races were used and when buffalo fly treatments with a synthetic pyrethroid were applied frequently. The probability of resistance to amitraz (Ulam strain) was highest in Central Queensland, increased when more than five applications of acaricide were made in the previous year, and decreased on farms when a hand-spray apparatus was used to apply acaricides to cattle. The probability of resistance to flumethrin (Lamington strain) was highest in the Wide Bay-Burnett region.