Molecular detection of Rickettsia rhipicephali and other spotted fever group Rickettsia species in Amblyomma ticks infesting wild birds in the state of Minas Gerais, Brazil.

The current study evaluated parasitism of wild birds by ticks in a fragment of Atlantic Forest in the state of Minas Gerais, Brazil, and examined the ticks for rickettsial agents. Birds were captured during 2014 and 2015 and ticks were identified by sequencing fragments of the 16S and 12S ribosomal DNA. Among 260 birds representing 19 families and 52 species, a total of 69 (26.5%) were found to be infested by larvae (LL) and/or nymphs (NN) of Amblyomma longirostre (Koch, 1844) (45 LL, 4 NN), Amblyomma calcaratum Neumann, 1899 (9 LL, 15 NN), Amblyomma nodosum Neumann, 1899 (2 NN), Amblyomma parkeri Fonseca and Aragão, 1952 (21 LL), Amblyomma sp. haplotype Nazaré (77 LL), and Haemaphysalis leporispalustris (Packard, 1869) (17 LL, 1 NN). The use of PCR and sequencing of the rickettsial genes gltA, htrA, ompA and ompB, revealed the presence of "Candidatus Rickettsia amblyommii" in A. longirostre (13/49; 26%) and Rickettsia parkeri (strain ApPR) in both A. parkeri (1/21; 5%) and haplotype Nazaré (42/77; 55%) ticks. In addition, we detected Rickettsia rhipicephali in 31 (40%) of the 77 haplotype Nazaré ticks. This is the first record of this rickettsial agent in a species of the genus Amblyomma. The pathogenic potential of this bacterium is undetermined, but the unprecedented association with Amblyomma ticks may represent a cause for concern for public and/or animal health.

Acaricidal activity of thymol against larvae of Rhipicephalus microplus (Acari: Ixodidae) under semi-natural conditions.

This is the first study to investigate the activity of thymol on Rhipicephalus microplus larvae under semi-natural conditions. For this purpose, tests were conducted in pots with Brachiaria decumbens seedlings containing cattle tick larvae. Thymol, diluted in ethanol 50° GL, was tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 mg/mL, along with the control group treated with the solvent alone. Each treatment was composed of five pots (1 pot = a repetition). The experiment was performed in three steps. On the first day, the larvae were applied at the base of the signalgrass. Twenty-four hours later, approximately 25 mL of the solution was applied with thymol on the top of the vegetation in each pot. The survival of the larvae was measured 24 h after application of the solutions. Each pot was analyzed individually, and the grass fillets contained larvae were cut with scissors, placed in Petri dishes, and taken to the laboratory to count the number of living larvae. At the highest concentrations (10, 15, and 20 mg /mL), the number of live larvae declined by more than 95 % in relation to the control group. The lethal concentration 50 % (LC50) and LC90 values were 3.45 and 9.25 mg/ml, respectively. The application of thymol in semi-natural conditions starting concentration of 10 mg/mL significantly reduced the number of living R. microplus larvae.

Entomopathogenic nematodes associated with essential oil of Lippia sidoides for control of Rhipicephalus microplus (Acari: Ixodidae).

In vitro effect of the association of the entomopathogenic nematodes Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 with the essential oil of Lippia sidoides on engorged females of Rhipicephalus microplus was assessed. Engorged females with homogeneous weights (p >0.05) were divided into six groups of ten ticks each (each female = an experimental unit). In the treated groups, the ticks were exposed to the oil (40 µl/ml=4%) and nematodes (300 entomopathogenic nematodes (EPNs/tick) separately and also to the nematodes together with the oil, while in the control group, the females were immersed in Tween 80 (3%). All the treatments caused a significant reduction in the egg mass weight (p <0.05). The hatching percentage of larvae was reduced (p <0.05) in the groups treated with H. bacteriophora HP88, H. indica LPP1, and H. bacteriophora HP88 associated with the oil, but in the group treated only with the oil, the hatching of percentage of larvae rate was similar to that of the control group (p >0.05). The percentage of control in the group exposed only to the oil was 50.7%, while in the other treated groups, the percentage of control was greater than 95 %, reaching 100 %in the group treated with H. indica LPP1 associated with the oil. The results of the L. sidoides essential oil in the concentrations tested in association with both H. bacteriophora HP88 and H. indica LPP1 indicate that these combinations can be used to control R. microplus.

Determination of the susceptibility of unengorged larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae) to different methods of dissolving thymol.

The present study evaluated efficacy of thymol against engorged females and unengorged larvae of Rhipicephalus microplus when diluted in 1 % dimethyl sulfoxide (DMSO) (formulation 1) and diluted in hydro-ethanol solutions of 50 % (formulation 2), 40 % (formulation 3), and 30 % (formulation 4). The immersion test was used on the engorged females for all dilution methods, while the larval packet method was applied to the larvae to test formulations 3 and 4 since the other two had already been tested before. Two control groups were established in both experiments; in one, ticks were treated with 1 % DMSO, and in the other, with 50 % ethanol, with ten repetitions for each treatment. The groups were maintained at 27±1 °C and RH>80 ± 10 %. For engorged females, the parameters weight before oviposition, egg mass weight, hatching percentage of larvae, and percentage of control were evaluated, while for larvae, percentage of mortality was evaluated. The egg mass weight of all treatments differed significantly (p<0.05) from the control groups; however, the reduction in egg mass weight was more accentuated (p<0.05) in the treatments with formulations 1, 3, and 4. The values of hatching percentage of the group treated with formulation 2 and control group were statistically similar (p>0.05). The group treated with formulation 2 attained the lowest percentage of control (66 %). In the other treatments, the control percentages were greater than 99 %. In larval test, the mortality was 100 % in all the treatments, and there was no mortality in the control groups.

Acaricidal activity of essential oil from Lippia sidoides on unengorged larvae and nymphs of Rhipicephalus sanguineus (Acari: Ixodidae) and Amblyomma cajennense (Acari: Ixodidae).

The aims of this work were to identify the compounds and to investigate the acaricidal activity of the essential oil of Lippia sidoides for unengorged larvae and nymphs of Rhipicephalus sanguineus and Amblyomma cajennense. The oil was analyzed by gas chromatography and gas chromatography/mass spectrometry. In total, 22 compounds comprising 98.5% of the total peak area were identified. The major constituent of the essential oil was thymol (69.9%). The acaricidal activity against larvae and nymphs was assessed using a modified larval packet test. In all experiments, oils were tested at concentrations of 2.35, 4.70, 9.40 14.10 and 18.80 mg/mL. The mortalities of larvae and nymphs of R. sanguineus were 20.6, 47.8, 73.6, 99.5 and 99.0% and 12.0, 50.0, 76.3, 96.0 and 96.1%, respectively. For larvae and nymphs of A. cajennense the rates of mortality were 41.9, 63.3, 77.8, 82.5 and 100.0% and 0.0, 32.8, 64.8, 71.1 and 94.0%, respectively. The LC 90 values of the L. sidoides oil were 11.56 and 12.97 mg/mL for larvae and nymphs of R. sanguineus and 15.70 and 18.52 mg/mL for larvae and nymphs of A. cajennense, respectively. The essential oil from L. sidoides has acaricidal activity on unengorged larvae and nymphs of R. sanguineus and A. cajennense.

Investigation of activity of monoterpenes and phenylpropanoids against immature stages of Amblyomma cajennense and Rhipicephalus sanguineus (Acari: Ixodidae).

The objective of this study was to assess the acaricidal activity of carvacrol, thymol, eugenol, and (E)-cinnamaldehyde on unengorged larvae and nymphs of Amblyomma cajennense and Rhipicephalus sanguineus, using the modified larval packet test. Carvacrol, eugenol, and (E)-cinnamaldehyde were tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 µl/ml, while thymol was tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 mg/ml, in all cases with 10 repetitions per treatment. For the A. cajennense larvae, mortality rates caused by carvacrol, thymol, eugenol, and (E)-cinnamaldehyde at the lowest concentration were 45.0, 62.7, 10.2, and 81.6%, respectively, reached 100% at the concentration of 5.0 µl/ml for carvacrol and (E)-cinnamaldehyde and 5.0 mg/ml for thymol, while this mortality was observed at 15.0 µl/ml for eugenol. For the nymphs of this species, carvacrol and thymol caused 100% mortality starting at a concentration of 5.0 µl/ml and 10.0 mg/ml, respectively, while eugenol caused 100% mortality at 20.0 µl/ml and the mortality caused by (E)-cinnamaldehyde did not exceed 64%. In the tests with R. sanguineus larvae, the lowest concentration of carvacrol and (E)-cinnamaldehyde resulted in 100% mortality, while this percentage was observed starting at 10.0 µl/ml for eugenol. For nymphs, carvacrol and thymol at the smallest concentration caused 100% lethality, unlike the results for eugenol and (E)-cinnamaldehyde, where 100% mortality was only observed starting at the concentration of 10.0 µl/ml. The results obtained indicate that the tested substances have acaricidal activity on unengorged larvae and nymphs of A. cajennense and R. sanguineus.

Repellent activity of eugenol on larvae of Rhipicephalus microplus and Dermacentor nitens (Acari: Ixodidae).

The repellent activity of eugenol was evaluated on Rhipicephalus microplus and Dermacentor nitens larvae in a bioassay based on the vertical migration of these tick larvae during the host-seek phase. Solutions were used at concentrations of 10, 20, 30, 40, and 50 µl/ml and a control only with the solvent ethanol 50 GL. The larvae's reaction of climbing the rods of filter paper was observed each hour for 12 h and once more at 24 h. The repellence was calculated as a percentage through visual estimation, from the tendency of the larvae to climb the rods when the top part was impregnated with the eugenol solutions. For R. microplus larvae, the four highest concentrations produced repellence levels were greater than 80 % for up to 9 h. The repellence rates after 10 h for the concentration of 10 µl/ml was lower than 2 % (p > 0.05); and after 24 h, the values observed for the concentrations of 40 and 50 µl/ml were 77 and 80 % (p < 0.05). For the D. nitens larvae, the repellence was greater than 80 % for up to 5 h at the concentrations of 40 and 50 µl/ml; and after 9 h, the repellence fell for all treated groups were significantly different than the control were found only at the three highest concentrations. Therefore, eugenol has repellent activity on the larval stage of these two tick species, with the R. microplus larvae being more sensitive.

Spectroscopic evaluation of thymol dissolved by different methods and influence on acaricidal activity against larvae of Rhipicephalus microplus (Acari: Ixodidae).

The acaricidal activity of three thymol formulations was investigated at five concentrations (1.25, 2.5, 5.0, 7.5, and 10.0 mg/ml) on Rhipicephalus microplus larvae, and the behavior of its solubility in these formulations was analyzed. The thymol was dissolved in distilled water plus 1 % dimethylsulfoxide as adjuvant under two heating regimes (water bath in formulation 1 and hot plate in formulation 2) as well as without heating in 50 % ethanol and 50 % water (v/v). The acaricidal activity was assessed by the modified larval packet test, and the solubilization behavior was investigated by ultraviolet-visible spectroscopy, based on the Beer-Lambert law. With formulations 1 and 2, the mortality was greater than 95 % starting at the thymol concentrations of 5.0 and 7.5 mg/ml, respectively, while with formulation 3, this mortality level was reached starting at a concentration of 2.5 mg/ml, showing that the addition of ethanol in the solution enhanced the acaricidal action of thymol. This result was supported by the LC 90 values, which were 3.3, 2.4, and 1.6 mg/ml of thymol for formulations 1, 2, and 3, respectively. This result is related to the better solubility of thymol in the hydroethanolic formulation, since the spectroscopic analysis revealed that the thymol dissolved more completely in this formulation. This fact was evident once the R (2) obtained from the linear regression analysis of the relation absorbance × concentration of the formulations 1, 2, and 3 approached the optimal value (R (2) = 1) in the following sequence: 1, 2, and 3 (0.717, 0.901, and 0.968, respectively).