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.     

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.     

Acaricidal activity of Palicourea marcgravii, a species from the Amazon forest, on cattle tick Rhipicephalus (Boophilus) microplus

Leaves of Palicourea marcgravii were extracted successively with hexane, ethyl acetate and ethanol in order to evaluate their acaricidal activity on larvae and adult stages of the cattle tick Rhipicephalus (Boophilus) microplus. The ethyl acetate extract showed the highest bioactivity of the tested extracts, which contained 0.12% monofluoroacetic acid. On engorged female, the ethyl acetate extract showed a lethal concentration 50% - LC(50)=30.08 mg ml(-1), inhibitory concentration 50% - IC(50)=5.79 mg ml(-1) and lethal time 50% - LT(50)=4.72 days; 100% reproduction was controlled at concentrations of 50 mg ml(-1) and on larvae the ethyl acetate extract showed a LC(50)=2.46 mg ml(-1). No alkaloids were detected in any of the extracts. This is the first report on the acaricidal activity of P. marcgravii extracts against R. microplus as well as the acaricidal properties of a plant species containing monofluoroacetic acid.     

Acaricidal activity of aqueous extract and synthesized silver nanoparticles from Manilkara zapota against Rhipicephalus (Boophilus) microplus

Traditional parasite control is primarily based on the use of chemical acaricides, which unfortunately have many negative side effects. The aim of the present study was to evaluate the effect of plant synthesized silver nanoparticles (AgNPs) using aqueous leaf extract of Manilkara zapota to control Rhipicephalus (Boophilus) microplus. The synthesized AgNPs were characterized by UV-vis spectrum, scanning electron microscopy (SEM), Fourier transform infrared and X-ray diffraction. The UV-vis spectrum of the aqueous medium containing silver nanostructures showed a peak at 421 nm corresponding to the surface plasmon resonance band of AgNPs. SEM supports the biosynthesis and characterization of AgNPs with spherical and oval in shape and size of 70-140 nm. Acaricidal activity of aqueous leaf extract of M. zapota and synthesized AgNPs were carried out against R. (B.) microplus and the results showed the LC(50) values of 16.72 and 3.44 mg/L; r(2)=0.856 and 0.783), respectively.     

Acaricidal activity of hydroethanolic formulations of thymol against Rhipicephalus sanguineus (Acari: Ixodidae) and Dermacentor nitens (Acari: Ixodidae) larvae

The aim of this study was to assess the acaricidal activity of hydroethanolic formulations of thymol at varying concentrations on Rhipicephalus sanguineus and Dermacentor nitens larvae. The larval packet test was used and the thymol concentrations tested were 2.5, 5.0, 10.0, 15.0 and 20.0 mg/ml. The control group was exposed only to water and ethanol (50/50%) and there were 10 repetitions for each treatment. The mortality was evaluated after 24 h. For the R. sanguineus larvae, the mortality rates were 47.5, 50.2, 96.7, 95.9 and 98.1%, while for D. nitens the rates were 14.1, 75.0, 90.2, 90.3 and 99.5%, at respective thymol concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/ml. The results indicate that the hydroethanolic formulations of thymol tested have acaricidal activity on R. sanguineus and D. nitens larvae exposed topically, causing mortality greater than 90% 24 h post-treatment starting at the concentration of 10 mg/ml.     

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.     

Acaricidal activity of Calea serrata (Asteraceae) on Boophilus microplus and Rhipicephalus sanguineus

Calea serrata (Asteraceae) is an endemic Southern Brazilian plant species used for religious and medicinal purposes. Previous study revealed the presence of chromenes, a class of natural compounds that possess insecticidal properties. This study reports the effect of the hexane extract from the aerial parts of this plant on egg hatchability, egg production and mortality rates of newly hatched larvae of the cattle tick Boophilus microplus. Larvae of Rhipicephalus sanguineus, the brown dog tick, received the same treatment. The extract was toxic to the eggs of B. microplus and to the larvae of both B. microplus and R. sanguineus.     

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.     

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.     

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.     

Efficacy of a single whole-body spray treatment of spinosad, against Boophilus microplus (Acari: Ixodidae) on cattle.

The efficacy of a single whole-body spray of spinosad, a naturally derived control agent, applied at three concentrations was evaluated against cattle infested three separate times prior to treatment and at four weekly intervals following treatment with Boophilus microplus (Canestrini). At 0.0167% active ingredient (AI) both tick numbers (1894 ticks per calf) and index of fecundity (IF) of females (258.3) were no different than that of the control group. However, spinosad treatment at both 0.05 and 0.15% AI resulted in fewer ticks per calf (600 and 935, respectively) with lower IF values for females (43.4 and 38.4, respectively). The percent control of ticks on the animals at the time of treatment (acute efficacy) was dramatically lower at 0.0167% AI (21.4%) than at 0.05 (86.3%) and 0.15% AI (87.9%). Spinosad treatments appeared to be more effective against immature stages (nymphs and larvae) than against adult ticks that were on the animals at the time of treatment. The mean weight of females that survived to repletion was similar (322-348 mg) in all groups. By contrast, the mean weight of egg masses produced by females was highest in the control group (155 mg), whereas each increase in spinosad concentration resulted in a substantial decrease in egg mass weight, with the 0.15% AI group averaging only 73 mg. The hatch rate of eggs derived from females ranged from 93.4% in control females down to 53.9% hatch for females treated at 0.15% AI spinosad. The residual efficacy of spinosad at 0.0167% AI was poor even at 1 week following treatment, resulting in 101 ticks per calf and a level of control of only 66.4%. At 0.05% AI, protection against successful reinfestation was high at 1-week post-treatment where only five ticks per calf reached repletion, and control of the IF of these females was 99.3%. The 0.15% AI treatment provided almost complete protection against reinfestation for 2 weeks following treatment (< or =5 ticks per calf), and control of the IF of these ticks was >99.9%. Thus, the use of spinosad at US ports-of-entry would be unacceptable because of the critical necessity of achieving 100% control with a single treatment to prevent the reintroduction of ticks. However, it is likely ticks could be eradicated using spinosad in tick infested areas of the US if repeated (systematic) treatments were applied to cattle maintained on the premises.     

Acaricidal activity of limonene, limonene oxide and beta-amino alcohol derivatives on Rhipicephalus (Boophilus) microplus

Limonene, limonene oxide and eight beta-amino alcohol derivatives obtained by synthesis were investigated for the effect on egg hatchability and mortality rates of newly hatched larvae of the cattle tick Rhipicephalus (Boophilus) microplus. At the doses between 10mug/ml and 2.5mug/ml all the compounds were highly lethal to the larvae and some of them showed activity at lower concentrations. The effect on the eggs hatchability was observed in all the treatments.     

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.     

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.     

Effect of moxidectin against natural infestation of the cattle tick Boophilus microplus (Acarina: Ixodidae) in the Mexican tropics

The study was divided in to two trials and carried out in a ranch in eastern Yucatan state, Mexico. In the first trial, two groups of 15 BostaurusxBosindicus heifers, 6-12 month of age and naturally infested with Boophilus microplus ticks were used. Heifers in Group 1 were treated with a 1% injectable formulation of moxidectin at the dose of 0.20mg/kg body weight by subcutaneous injection. The other group remained as untreated controls. Number of immature and engorging female ticks were assessed on days 0, 7, 14, 21, 28 and 35 post-treatment (PT). The efficacy of moxidectin on adult ticks from day 7 to 28 PT was greater than 95%. The efficacy decreased to 74.9% by day 35. In the second trial, animals in Group 1 were treated with the moxidectin product as before, while cattle in Group 2 were treated according to the routine procedure for the control of ticks on that property (125 g/l amitraz as a dip). Treatment of all cattle was repeated four times at intervals of 28 days. The efficacy of the experimental moxidectin treatment was similar to that of the routine amitraz treatment, i.e., greater than 99%.     

Acaricidal properties of extracts from the aerial parts of Hypericum polyanthemum on the cattle tick Boophilus microplus

Laboratory tests were carried out on larvae and adults of the cattle tick Boophilus microplus to determine the toxicity of n-hexane and crude methanolic extracts of Hypericum polyanthemum (Guttiferae) using the larval immersion test (LIT) and adult immersion test (AIT). In the AIT, the effectiveness of treatment against engorged females was assessed by measuring egg production: the n-hexane extract was found to have a small effect on the egg laying at the highest concentration (19.2% of egg-laying inhibition) whilst the crude methanolic extract did not affect the egg production. For the LIT the n-hexane extracts were highly toxic to the larvae at all the concentrations (100% of mortality). The crude methanolic extract was also toxic to the larvae at higher concentrations killing 100, 96.7, 84.7 and 52.7% at the concentrations of 50, 25, 12.5, and 6.25 mg/ml, respectively, 48 h after the immersion of the acarus.     

A new in vitro test to evaluate the resistance level against acaricides of the cattle tick, Rhipicephalus (Boophilus) microplus

In this article we present a new bioassay to assess the resistance status of ticks to acaricides. The Larval Tarsal Test (LTT) is a sensitive, highly time-effective in vitro test. It allows the investigation of a large number of compounds and doses on the cattle tick Rhipicephalus (Boophilus) microplus in a short period of time. The ability of the LTT to assess the lethal concentration at 50% mortality (LC(50)) and resistance ratios (RRs) of a susceptible and a resistant R. microplus strain was compared with the FAO-recommended Larval Packet Test (LPT). Representative compounds of the carbamate, organophosphate (OP), synthetic pyrethroid (SP), formamidine (FOR), macrocyclic lactone and pyrazole classes were used for this comparison. The resistance status against OP, SP and FOR of the resistant R. microplus strain was confirmed in vivo. The LTT resulted in resistance ratios comparable to those obtained with the LPT. However, the lethal concentrations were up to 150-fold lower in the LTT than in the LPT. The advantage of the LTT is to simplify the methodology by avoiding the handling of larvae and using multi-well plates. The LTT is therefore a suitable test for the assessment of the level of resistance of R. microplus and is very promising to evaluate the resistance profile of field strains. Additionally, the LTT is also suitable to test other ixodid species.     

Moxidectin pour-on for control of natural populations of the cattle tick Boophilus microplus (Acarina: Ixodidae)

Fifty Bos taurus x Bos indicus heifers naturally infested with Boophilus microplus ticks were divided into two groups of 25 heifers each. Individuals of one group were treated with moxidectin 0.5% pour-on at a dosage of 500 microg of moxidectin/kg body weight and heifers from the other group remained as untreated controls. An efficacy higher than 95% was found on days 7-21 after treatment by using female ticks 4.5-8.0 mm long as the main infestation parameter. A lower, but significant efficacy (p < 0.05) was also found on days 1 (32.3% efficacy) and 27 (73.4% efficacy) post-treatment. Significantly (p < 0.05) lower numbers of immature ticks were also observed on heifers of the treated group from days 7 through 27 after treatment. A lower engorgement weight of female ticks from treated heifers was found on days 1 and 21 after treatment. Treatment also affected reproductive performance (oviposition, egg hatch and number of eggs laid) of female ticks collected on Day 1.     

Survival of larvae of Boophilus annulatus, Boophilus microplus, and Boophilus hybrids (Acari: Ixodidae) in different temperature and humidity regimes in the laboratory.

The survival period for larvae of Boophilus annulatus (Say), Boophilus microplus (Canestrini) and hybridized Boophilus ticks was determined by exposure to various combinations of temperature (20, 25, 30 and 35°C) and relative humidity (32, 63, 75, 84 and 97% RH) in the laboratory. Results indicated that within a given temperature and RH regime, there was no difference (P> 0.05) in larval survival among the three species tested, indicating that these ticks respond similarly over a wide range of temperature and RH combinations. Larval survival in all three species was longest (P < 0.05) at 20°C and either 84 or 97% RH. With each increase in temperature at the 84 and 97% RH treatment levels, there was a corresponding significant (P < 0.05) decrease in larval survival. When the temperature reached 35°C at all humidities or when the RH was 63% or less at all temperatures, the mean larval survival period was 43 days or less in all cases and little difference (P> 0.05) was observed among the treatment regimes included. Results suggest that at a RH of 75% and more, the temperature is the determining factor in larval survival, whereas at a RH of 63% and less the RH is the determining factor in larval survival, regardless of temperature.