Multiple mutations in the para-sodium channel gene are associated with pyrethroid resistance in Rhipicephalus microplus from the United States and Mexico.

BACKGROUND: Acaricide resistant Rhipicephalus microplus populations have become a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically associated with mutations in domains I, II, III, and IV of voltage-gated sodium channel genes. In R. microplus, known resistance mutations include a domain II change (C190A) in populations from Australia, Africa, and South America and a domain III mutation (T2134A) that only occurs in Mexico and the U.S. METHODS: We investigated pyrethroid resistance in cattle fever ticks from Texas and Mexico by estimating resistance levels in field-collected ticks using larval packet discriminating dose (DD) assays and identifying single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that associated with resistance. We then developed qPCR assays for three SNPs and screened a larger set of 1,488 R. microplus ticks, representing 77 field collections and four laboratory strains, for SNP frequency. RESULTS: We detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. We expected to identify the domain III SNP (T2134A) at a high frequency; however, we only found it in three U.S. collections. A much more common SNP in the U.S. (detected in 19 of 21 field collections) was the C190A domain II mutation, which has never before been reported from North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexico field collections. The T170C transition mutation has previously been associated with extreme levels of resistance (super-knockdown resistance) in insects. We found a significant correlation (r = 0.81) between the proportion of individuals in field collections that carried any two resistance SNPs and the percent survivorship of F1 larvae from these collections in DD assays. This relationship is accurately predicted by a simple linear regression model (R2 = 0.6635). CONCLUSIONS: These findings demonstrate that multiple mutations in the para-sodium channel gene independently associate with pyrethroid resistance in R. microplus ticks, which is likely a consequence of human-induced selection.

Widespread movement of invasive cattle fever ticks (Rhipicephalus microplus) in southern Texas leads to shared local infestations on cattle and deer.

BACKGROUND: Rhipicephalus (Boophilus) microplus is a highly-invasive tick that transmits the cattle parasites (Babesia bovis and B. bigemina) that cause cattle fever. R. microplus and Babesia are endemic in Mexico and ticks persist in the United States inside a narrow tick eradication quarantine area (TEQA) along the Rio Grande. This containment area is threatened by unregulated movements of illegal cattle and wildlife like white-tailed deer (WTD; Odocoileus virginianus). METHODS: Using 11 microsatellite loci we genotyped 1,247 R. microplus from 63 Texas collections, including outbreak infestations from outside the TEQA. We used population genetic analyses to test hypotheses about ecological persistence, tick movement, and impacts of the eradication program in southern Texas. We tested acaricide resistance with larval packet tests (LPTs) on 47 collections. RESULTS: LPTs revealed acaricide resistance in 15/47 collections (32%); 11 were outside the TEQA and three were resistant to multiple acaricides. Some collections highly resistant to permethrin were found on cattle and WTD. Analysis of genetic differentiation over time at seven properties revealed local gene pools with very low levels of differentiation (FST 0.00-0.05), indicating persistence over timespans of up to 29 months. However, in one neighborhood differentiation varied greatly over a 12-month period (FST 0.03-0.13), suggesting recurring immigration from distinct sources as another persistence mechanism. Ticks collected from cattle and WTD at the same location are not differentiated (FST = 0), implicating ticks from WTD as a source of ticks on cattle (and vice versa) and emphasizing the importance of WTD to tick control strategies. We identified four major genetic groups (K = 4) using Bayesian population assignment, suggesting multiple introductions to Texas. CONCLUSIONS: Two dispersal mechanisms give rise to new tick infestations: 1) frequent short-distance dispersal from the TEQA; and 2) rare long-distance, human-mediated dispersal from populations outside our study area, probably Mexico. The threat of cattle fever tick transport into Texas is increased by acaricide resistance and the ability of R. microplus to utilize WTD as an alternate host. Population genetic analyses may provide a powerful tool for tracking invasions in other parts of the world where these ticks are established.

Invasive potential of cattle fever ticks in the southern United States.

ABSTRACT' BACKGROUND: For >100 years cattle production in the southern United States has been threatened by cattle fever. It is caused by an invasive parasite-vector complex that includes the protozoan hemoparasites Babesia bovis and B. bigemina, which are transmitted among domestic cattle via Rhipicephalus tick vectors of the subgenus Boophilus. In 1906 an eradication effort was started and by 1943 Boophilus ticks had been confined to a narrow tick eradication quarantine area (TEQA) along the Texas-Mexico border. However, a dramatic increase in tick infestations in areas outside the TEQA over the last decade suggests these tick vectors may be poised to re-invade the southern United States. We investigated historical and potential future distributions of climatic habitats of cattle fever ticks to assess the potential for a range expansion. METHODS: We built robust spatial predictions of habitat suitability for the vector species Rhipicephalus (Boophilus) microplus and R. (B.) annulatus across the southern United States for three time periods: 1906, present day (2012), and 2050. We used analysis of molecular variance (AMOVA) to identify persistent tick occurrences and analysis of bias in the climate proximate to these occurrences to identify key environmental parameters associated with the ecology of both species. We then used ecological niche modeling algorithms GARP and Maxent to construct models that related known occurrences of ticks in the TEQA during 2001-2011 with geospatial data layers that summarized important climate parameters at all three time periods. RESULTS: We identified persistent tick infestations and specific climate parameters that appear to be drivers of ecological niches of the two tick species. Spatial models projected onto climate data representative of climate in 1906 reproduced historical pre-eradication tick distributions. Present-day predictions, although constrained to areas near the TEQA, extrapolated well onto climate projections for 2050. CONCLUSIONS: Our models indicate the potential for range expansion of climate suitable for survival of R. microplus and R. annulatus in the southern United States by mid-century, which increases the risk of reintroduction of these ticks and cattle tick fever into major cattle producing areas.

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 LC(50) and the LC(99) 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.

Analysis of doramectin in the serum of repeatedly treated pastured cattle used to predict the probability of cattle fever ticks (Acari: Ixodidae) feeding to repletion.

Analysis of doramectin concentration in blood serum of pastured cattle injected repeatedly (12 treatments) at two different dosage rates and 28-day intervals throughout the year was used to predict the probability that cattle fever ticks could successfully feed to repletion during the interval between any two consecutive treatments. Treatment at ~270 µg/kg indicated that serum doramectin concentration dropped below the baseline concentration estimated for tick survival (8 ppb) in 7 of the 12 treatments. However, the longest period between any two treatments during which the doramectin concentration remained below the 8 ppb baseline level for successful tick feeding was 15 days, making it virtually impossible for any ticks to reach ovipositional status prior to a subsequent treatment. At a dosage rate of ~540 µg/kg, the concentration dropped below the baseline tick survival level (8 ppb) only once, following the initial treatment, and the duration during which the concentration remained below the baseline level prior to the subsequent treatment was only 6 days. Thus, at the high dosage rate results indicated, with absolute certainty, that no ticks could successfully feed to repletion between any two consecutive treatments. Based on the data obtained in the study it was concluded that analysis of doramectin concentration in serum of treated animals would be a reliable predictor for assessing the probability that ticks could successfully develop to repletion. More importantly, results demonstrated that the trial policy, instituted by the Cattle Fever Tick Eradication Program, of repeatedly treating cattle with doramectin injections at 25-28 day intervals for eliminating cattle fever ticks would produce little or no risk of any viable ticks developing to repletion and re-infesting the field between treatment applications.

Absence of Mycobacterium bovis in feral swine (Sus scrofa) from the southern Texas border region.

Free-ranging wildlife, such as feral swine (Sus scrofa), harbor a variety of diseases that are transmissible to livestock and could negatively impact agricultural production. Information is needed regarding the exposure and infection rates of Mycobacterium bovis and many other diseases and parasites in feral swine occurring in the Texas border region. Our main objective was to determine exposure rates and possible infection rates of M. bovis in feral swine by opportunistically sampling animals from the Texas border region. From June to September 2010, we obtained samples from 396 feral swine and tested 98 samples for M. bovis by histopathology and mycobacteriologic culture. We found no evidence of M. bovis infection. We believe that it is important to periodically and strategically sample feral swine for M. bovis in high-risk areas of the United States because they are capable of becoming reservoirs of the disease.

Efficacy and blood sera analysis of a long-acting formulation of moxidectin against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) on treated cattle.

The therapeutic and persistent efficacy of a single subcutaneous injection of a long-acting formulation of moxidectin at a concentration of 1 mg/kg body weight was determined against Rhipicephalus (Boophilus) microplus (Canestrini), along with the concentration-time blood sera profile in treated cattle. The therapeutic efficacy against ticks of all parasitic stages on cattle at the time of treatment was >99.9%, and the mean tick number, index of fecundity, engorgement weight, and egg mass weight of ticks recovered from treated animals were all significantly lower than ticks from untreated animals. The index of fecundity, engorgement weight of females, and egg mass weight of ticks recovered from treated animals infested at weekly (7-d) intervals between 14 and 63 d posttreatment were significantly lower than for ticks on untreated animals, whereas the number of ticks per animal recovered from treated cattle remained lower than that of untreated cattle for up to 49 d posttreatment. The percentage control remained >99% at weekly intervals between 14 and 49 d posttreatment, which is the minimum level of efficacy considered acceptable for use in the United States Cattle Fever Tick Eradication Program. The serum concentration of moxidectin in treated cattle increased to 25.6 ppb (parts per billion) within 1 d after treatment, and peaked at 47.3 ppb at 8 d posttreatment. Moxidectin sera levels remained above the estimated 100% threshold level for elimination of feeding ticks (5-8 ppb) for 44-53 d after treatment. The label claim of 50 d of prevention against reinfestation for the long-acting moxidectin formulation used in the study was supported by the efficacy and sera concentration data obtained. Based on these results, cattle could be treated at 63-d intervals with minimal risk of viable ticks detaching from treated animals. This treatment interval would be 4.5-fold longer than the presently required treatment interval of 14 d, thus leading to approximately 75% reduction in gathering and handling costs of cattle incurred by producers.

Efficacy of spinosad against acaricide-resistant and -susceptible Rhipicephalus (Boophilus) microplus and acaricide-susceptible Amblyomma americanum and Dermacentor variabilis.

Various acaricide-resistant strains of Rhipicephalus (Boophilus) microplus, representative of the major resistance mechanisms found in Mexico and Brazil, were exposed to spinosad using the Food and Agriculture Organization of the United Nations larval packet test and adult immersion test (AIT). Larvae of all strains tested were found to be susceptible to spinosad. Conversely, spinosad did not show toxic activity toward engorged females used in the AIT. In vitro tests against larvae, nymphs, and adults of acaricide-susceptible Amblyomma americanum and Dermacentor variabilis revealed differences in activity that were dependent on tick species and parasitic life stage. Spinosad seemed to be a viable alternative to current acaricides available for tick control in the species tested. The larval packet test should be used for future monitoring of resistance, as the AIT did not provide useful information with this chemical. The potential benefit for the use of spinosad in integrated pest management or eradication programs is discussed.

Bm86 midgut protein sequence variation in South Texas cattle fever ticks.

BACKGROUND: Cattle fever ticks, Rhipicephalus (Boophilus) microplus and R. (B.) annulatus, vector bovine and equine babesiosis, and have significantly expanded beyond the permanent quarantine zone established in South Texas. Currently, there are no vaccines approved for use within the United States for controlling these vectors. Vaccines developed in Australia and Cuba based on the midgut antigen Bm86 have variable efficacy against cattle fever ticks. A possible explanation for this variation in vaccine efficacy is amino acid sequence divergence between the recombinant Bm86 vaccine component and native Bm86 expressed in ticks from different geographical regions of the world. RESULTS: There was 91.8% amino acid sequence identity in Bm86 among R. microplus and R. annulatus sequenced from South Texas infestations. When South Texas isolates were compared to the Australian Yeerongpilly and Cuban Camcord vaccine strains, there was 89.8% and 90.0% identity, respectively. Most of the sequence divergence was focused in one region of the protein, amino acids 206-298. Hydrophilicity profiles revealed that two short regions of Bm86 (amino acids 206-210 and 560-570) appear to be more hydrophilic in South Texas isolates compared to vaccine strains. Only one amino acid difference was found between South Texas and vaccine strains within two previously described B-cell epitopes. A total of 4 amino acid differences were observed within three peptides previously shown to induce protective immune responses in cattle. CONCLUSIONS: Sequence differences between South Texas isolates and Yeerongpilly and Camcord strains are spread throughout the entire Bm86 sequence, suggesting that geographic variation does exist. Differences within previously described B-cell epitopes between South Texas isolates and vaccine strains are minimal; however, short regions of hydrophilic amino acids found unique to South Texas isolates suggest that additional unique surface exposed peptides could be targeted.

Laboratory evaluation of verbutin as a synergist of acaricides against larvae of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Synergistic effects of verbutin, a member of aryl alkynyl derivatives, to three commonly used acaricides were evaluated with the modified Food and Agricultural Organization Larval Packet Test (FAO-LPT) against both susceptible and resistant strains of the southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae). These tick strains demonstrated various levels of resistance to coumaphos (2.5-8.2x), permethrin (57.9-711.7x), and amitraz (3.5-177.5x). Verbutin alone was more toxic to tick larvae than piperonyl butoxide (PBO), a conventional pesticide synergist. With synergism ratios (SRs) ranging from 1.5 to 6.0, verbutin was more potent than PBO (SR = 0.9-1.6) in synergizing coumaphos toxicity to resistant tick larvae. Similarly, verbutin (SR = 1.8-15) was also found to be more potent than PBO (SR = 0.9-2.5) in synergizing amitraz in resistant tick strains. Both verbutin and PBO significantly increased permethrin toxicity to larvae of all tick strains tested, and there was no significant difference between the two synergists. SRs ranged from 2.1 to 4.4 and from 2.1 to 3.6 for PBO and verbutin, respectively.

One Health approach to identify research needs in bovine and human babesioses: workshop report.

BACKGROUND: Babesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks. RESULTS: The involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein. CONCLUSIONS: The translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.

Therapeutic and persistent efficacy of a long-acting (LA) formulation of ivermectin against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and sera concentration through time in treated cattle.

The concentration-time profile, therapeutic, and persistent efficacy of a single subcutaneous injection of cattle with a long-acting (LA) formulation of ivermectin at a concentration of 630microg/kg of body weight were determined against Rhipicephalus (Boophilus) microplus. Ivermectin sera concentration in treated cattle increased to 13.0ppb within 1d after treatment, and peaked at 26.2ppb at 11d post-treatment. Ivermectin sera levels remained above the threshold level for control of feeding ticks (>or=8ppb) for 42.6d after treatment. Therapeutic efficacy of ticks on treated animals was >99.9%, and tick number, index of fecundity, engorgement weight, and egg mass weight of ticks from treated animals remained dramatically less than ticks from untreated animals. Tick number and reproductive capacity of ticks infested on treated animals at 14 and 28d post-treatment were less than for ticks on untreated animals, whereas engorgement weight and egg mass weight of treated ticks remained lower than that of untreated ticks 49d post-treatment. However, the level of control against ticks infested at 14d after treatment (99.9%) was the only post-treatment infestation interval that provided the required 99% control necessary for use in the U.S. tick eradication program. The 14d post-treatment infestation was also the only interval at which infested ticks were exposed to ivermectin levels above the threshold level of 8ppb for the entire parasitic development period. Cattle would have to be treated at intervals of no more than 31d apart to ensure that no viable ticks could reach repletion and detach from the host. Although this treatment interval is >2-fold longer than the present treatment requirement (14d), it is dramatically less than the label claim for the LA ivermectin formulation of 75d of prevention against re-infestation.

Effect of rainfall exposure immediately after a single dip treatment with coumaphos on the control of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) on infested cattle.

Efficacy of a single dip treatment in coumaphos at 0.182% active ingredient was determined against all parasitic stages of Rhipicephalus (Boophilus) microplus (Canestrini) on infested cattle exposed to various levels of rainfall immediately after treatment. One group of calves remained untreated with no exposure to rainfall to serve as a negative control. A second group of cattle treated with coumaphos, but not exposed to rainfall, acted as a positive treated control. Three additional groups of coumaphos-treated cattle were exposed to 14.3, 28.6, and 42.9 mm of rainfall, respectively. In the coumaphos-treated group not exposed to a rainfall, overall mean control (99.2%) was greater and mean female engorgement weight (200 mg), egg mass weight (43 mg), and index of fecundity (IF; 2.90) were all less than any group exposed to rainfall. Although exposure to the lowest level of rainfall (14.3 mm) resulted in substantially greater control (83.7%) with lower mean egg mass weight (65 mg) and IF (62.26) than ticks exposed to 28.6 or 42.9 mm of rainfall, differences were seldom significant (P > 0.05). This suggested that higher levels of rainfall exposure adversely impacted coumaphos efficacy somewhat more than lower levels of rainfall exposure. Control remained >97% against larval ticks regardless of rainfall exposure level; however, against nymphs or adults, dramatic declines in control occurred as a result of exposure to any rainfall. Thus, the movement of coumaphos treated cattle exposed to any level of rainfall would pose a high risk of dispersing viable ticks into uninfested areas.

Differential response to diazinon and coumaphos in a strain of Boophilus microplus (Acari: Ixodidae) collected in Mexico.

Boophilus microplus, collected from Nuevo Leon, Mexico, were found to be highly resistant to diazinon but not highly resistant to coumaphos, suggesting that different mechanisms of resistance were present in these ticks than other Mexican organophosphate (OP)-resistant ticks reported previously. When exposed to coumaphos and piperonyl butoxide or triphenylphosphate, the LCso estimate was reduced by 3.5- and 6.3-fold, respectively, suggesting that mono-oxygenases and/or esterases were involved in resistance to coumaphos. Additionally, it was determined that this strain had an Acetylycholinesterase (AChe) that was insensitive to the active form of coumaphos, coroxon, taking at least 24 min longer to reach 50% reduction in AChE activity compared with the susceptible strain. When exposed to diazinon, none of the synergists tested significantly lowered the LC50. However, it was determined that it took six times longer to reach 60% inhibition of AChE in the resistant strain compared with the susceptible strain when exposed to the active form of diazinon, diazoxon. Insensitive AChE seems to be very common in OP-resistant B. microplus. The potential benefits for the development of a field-portable AChE inhibition assay kit are discussed.

Genetics and mechanisms of permethrin resistance in the Santa Luiza strain of Boophilus microplus (Acari: Ixodidae).

The Santa Luiza strain of the southern cattle tick, Boophilus microplus (Canestrini) (Acari: Ixodidae), is resistant to both permethrin and amitraz. A study was conducted at the USDA Cattle Fever Tick Research Laboratory in Texas to investigate the genetic basis of permethrin resistance with cross-mating experiments, and to determine the mechanisms of permethrin resistance through synergist bioassays and biochemical analysis of esterase profiles. The Muñoz strain, an acaricide-susceptible reference strain, was used as the susceptible parent and the Santa Luiza strain, originating in Brazil, was used as the resistant parent. The Food and Agriculture Organization larval packet test was used to measure the levels of susceptibility of larvae of the parental strains, F1, backcross, F2, and F3 generations to permethrin. Results of reciprocal crossing experiments suggested that permethrin resistance was inherited as an incomplete recessive trait. There was no significant maternal effect on larval progeny's susceptibility to permethrin in the F1 and subsequent generations. The values of the degree of dominance were estimated at -0.700 and -0.522 for the F1 larvae with resistant and susceptible female parents, respectively. Results of bioassays on larval progeny of the F1 backcrossed with the resistant parent strain and of the F2 generations suggested that one major gene was responsible for permethrin resistance in the Santa Luiza strain. Selection of F3 larvae with either permethrin or amitraz led to significantly increased resistance to both permethrin and amitraz, indicating a close linkage between genes responsible for permethrin and amitraz resistance. The possible involvement of metabolic enzymes in permethrin resistance in the Santa Luiza strain of B. microplus was dismissed by the lack of enhanced synergism by TPP or PBO, as observed in synergist bioassays, as well as by the lack of enhanced esterase activity in the Santa Luiza strain relative to the susceptible strain. The results of this study suggest that other mechanisms, including a possible new sodium channel mutation that is different from the one currently known, may be responsible for permethrin resistance in the Santa Luiza strain of B. microplus.

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.

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.

Acaricide resistance and synergism between permethrin and amitraz against susceptible and resistant strains of Boophilus microplus (Acari: Ixodidae).

The control of the southern cattle tick, Boophilus microplus (Canestrini), in Mexico and many other countries relies on chemical acaricides. Boophilus microplus has developed resistance to all major classes of acaricides in recent years. To gain a better understanding of the resistance and to develop resistance management strategies that benefit both Mexican ranchers and USDA's cattle fever tick eradication program (CFTEP), the authors used larval bioassay techniques to determine levels of resistance to permethrin and amitraz and then evaluated synergism between these two acaricides in one susceptible laboratory tick strain and four resistant strains originating from Mexico and Brazil. To examine mechanisms of resistance to permethrin in these strains, the frequency of a mutated sodium channel gene was determined using a PCR assay. The tick strains from Mexico and Brazil demonstrated 49.4- to over 672.2-fold resistance to permethrin, and up to 94.5-fold resistance to amitraz. While the San Roman strain from Mexico was the most permethrin-resistant strain, the Santa Luiza strain from Brazil was the most amitraz-resistant strain. A significant correlation was found between the permethrin resistance ratio and the allelic frequency of the sodium channel mutation. Significant synergism between permethrin and amitraz was found when one acaricide was tested in the presence of another. Synergism ratios ranged from 1.5 to 54.9 when amitraz was tested as a synergist for permethrin. Similar synergism ratios were obtained when permethrin was tested as a synergist for amitraz. Permethrin caused virtually no mortality in the San Roman strain, even at the highest concentration (3294 microg cm(-2)). Adding amitraz (11.0 microg cm(-2)) to permethrin led to a dramatic increase in larval mortality, even at very low concentrations of permethrin.

Mutations in a putative octopamine receptor gene in amitraz-resistant cattle ticks.

The mode of action of amitraz is thought to be its toxic effects on a receptor for a neuromodulator, octopamine. Resistance could arise from modifications of this receptor so that it would not be affected by amitraz. A putative octopamine receptor cDNA was cloned and sequenced from a cattle tick in Australia. However, when the sequence was compared between Australian strains of amitraz-susceptible and resistant ticks, no differences were detected. We have sequenced this putative octopamine receptor gene in tick strains from America. The American ticks have a sequence almost identical to that of the Australian ticks with no deletions or additions in the open reading frame. In a Brazilian strain and a Mexican strain that are very resistant to amitraz, there are two nucleotide substitutions that result in amino acids different from all the susceptible strains. Discovery of these mutations only in amitraz-resistant ticks provides the first evidence for the possibility of an altered pesticide target site as a mechanism of amitraz resistance in ticks.

Efficacy of a single doramectin injection against adult female Boophilus microplus (Acari: Ixodidae) in the final stages of engorgement before detachment.

The efficacy of injectable doramectin applied at 200 microg/kg was evaluated against adult female Boophilus microplus (Canestrini) (Acari: Ixodidae) in the later stages of engorgement before detachment. Lethal levels of doramectin in the serum of treated cattle (9.7-36.6 ppb) were reached and sustained within 24 h after treatment. However, treatment at 19 or 20 d postinfestation allowed greater tick survival and reproductive capability (IF) than at 18 d postinfestation, indicating that a significant portion of the ticks were able to engorge and detach before obtaining a lethal dose of doramectin. Thus, treatment at 18 d after infestation provided significantly higher overall control (99.5%) than treatment at 19 or 20 d postinfestation (95.8 and 89.1%, respectively). Analysis of control on a daily basis demonstrated that treatment at 18 d postinfestation provided > 99% on each day of the evaluation. Conversely, treatment at 19 or 20 d postinfestation produced levels of control ranging from 22.6 to 85.6% during the first 2 d of female detachment, and > or = 99% control was not achieved until after the fourth day of female detachment, where it remained throughout the study. Therefore, application of injectable doramectin at < or = 18 d after tick infestation was the only treatment regime considered acceptable for use in the U.S. Boophilus Eradication Program. Treatment intervals > 18 d postinfestation could pose a substantial risk of dispersing viable ticks to tick-free areas outside the permanent quarantine zone that has been established along the Texas-Mexico border.