Susceptibility of Amblyomma sculptum, Vector of Rickettsia rickettsii, Ticks from a National Park and an Experimental Farm to Different Synthetic Acaricides.

Amblyomma sculptum is a relevant tick species from a One Health perspective, playing an important role as a vector of Rickettsia rickettsii, the main agent of spotted fever rickettsiosis in Brazil. In this study, we evaluated the susceptibility of two A. sculptum populations from Goiás state (midwestern Brazil) to different acaricides. The first tick population (GYN strain) originated from an experimental farm, where the ticks are annually exposed to acaricides. The second (PNE strain) was collected in a national park (Emas National Park), where the ticks had not been exposed to acaricides. Immersion tests were conducted with 21-day-old laboratory-reared larvae and nymphs originating from adult ticks collected in the areas mentioned above. The chosen acaricides were two synthetic pyrethroids (cypermethrin and deltamethrin), one organophosphate (chlorfenvinphos), one formamidine (amitraz), and two combinations of pyrethroids and organophosphates (cypermethrin, chlorpyrifos and citronellal; cypermethrin, fenthion and chlorpyrifos). Mortality data were used to determine the lethal concentration (LC) values at which 50%, 90%, and 99% of the ticks died (LC50, LC90, and LC99, respectively), and resistance ratios (RR) were calculated based on the LC values. The RR revealed differences between the acaricide-exposed (GYN) and unexposed (PNE) tick strains. The PNE strain larvae and nymphs were susceptible to all the tested acaricides. The GYN strain larvae were tolerant to cypermethrin, whereas the nymphs were tolerant to deltamethrin, chlorfenvinphos, and the combination of cypermethrin, chlorpyrifos, and citronellal (2 < RR ≤ 10). The GYN strain nymphs were resistant to amitraz (RR > 10). This is the first report of A. sculptum nymphs with resistance to amitraz and tolerance to deltamethrin, chlorfenvinphos, and the combination of cypermethrin, chlorpyrifos, and citronellal.

Combinations of amitraz with essential oils from Lippia sidoides and Thymus vulgaris, thymol and thymol acetate for Rhipicephalus microplus control: studies under laboratory and field conditions.

This study aimed to assess the effect of combining amitraz with essential oils (EOs) from Thymus vulgaris and Lippia sidoides, as well as the monoterpenes thymol and thymol acetate, on Rhipicephalus microplus in laboratory conditions, and to select the most effective combination for testing in field conditions. The chemical analysis showed that EOs were mainly composed of monoterpenes, with thymol and p-cymene as the major compounds. In larval (LIT) and adult (AIT) immersion tests using different concentrations of the oils and terpenes mixed with amitraz, the results showed that both EOs and thymol improved the efficacy of amitraz against larvae and engorged females of R. microplus, whereas thymol acetate only enhanced activity against larvae. The most favorable outcome was obtained with the EO of L. sidoides combined with amitraz, resulting in 99 % and 100 % efficacy against larvae and engorged females, respectively. Furthermore, the combination of amitraz with thymol showed presented an efficacy of 94 % and 91 % against larvae and engorged females, respectively. Thus, for the other tests, the combination of thymol + amitraz was chosen due to the ease of working with pure thymol in bioassays, and easier standardization. The immersion test (thymol + amitraz) with semi-engorged females showed 100 % efficacy for the combination of thymol + amitraz, while in tests with different solvents (thymol + amitraz), ethanol being the most effective solvent among those tested (ethanol, Triton, and Tween), resulting in 95 % efficacy on engorged females. In the field test, in treatments with amitraz and thymol + amitraz, efficacy of 54 % and 74 % was observed on day + 3 and 33 % and 43 % on day + 7, respectively. Assessing the reproductive biology of females recovered from animals treated with amitraz or amitraz + thymol, in day + 7, efficacies of 33 % and 52 %, respectively, were observed. EOs from T. vulgaris and L. sidoides and thymol improved the acaricidal activity of amitraz on larvae and engorged females of R. microplus under laboratory conditions, while thymol acetate only enhanced activity against larvae. Thymol increased the efficacy of amitraz under field conditions, however for the development of a commercially available acaricide to R. microplus control, additional studies are needed to increase the efficacy. Further research is needed (by changing concentrations, adding other compounds and/or developing formulations) to increase acaricidal efficacy and develop new effective products to combat R. microplus infestations in cattle.

Thymol and eugenol against Rhipicephalus sanguineus sensu lato engorged females: Biological, histopathological and bioinformatic analysis.

The aim of this study was to evaluate the reproductive biology and histopathology of the ovaries of engorged females of Rhipicephalus sanguineus sensu lato exposed to combinations of thymol and eugenol, as well as to evaluate in silico the possible interactions of thymol and eugenol in tick cell membranes. To evaluate the tick reproductive biology, the adult immersion test (AIT) was performed, in which the engorged females were immersed in solutions of thymol and eugenol, combined or alone, at concentrations of 2.5 and 5.0 mg/mL. Two control groups (water and 3% DMSO) were also performed. The ticks were kept in a controlled chamber (B.O.D - 27 ± 1 °C and 80% ± 5% RH) to evaluate egg production and viability. To perform the ovaries histopathological evaluation, females were immersed in combination of thymol and eugenol (each at 2.5 and 5.0 mg/mL) and control (water and 3% DMSO) solutions. After immersion, the females were kept in B.O.D (27 ± 1 °C and RH of 80% ± 5%) for four days, they were dissected and the ovaries processed for histological analysis. In addition, an in silico analysis was performed using PASS online® software to predict probability activity (PA) of thymol and eugenol in cell membranes. The treatment with the combination of thymol and eugenol (each at 5.0 mg/mL) caused a reduction (p < 0.01) in oviposition, while the treatments with thymol (5.0 mg/mL) and combination of thymol and eugenol (5.0 mg/mL) reduced (p < 0.05) the egg viability. The treatment with combination of thymol and eugenol (5.0 mg/mL) resulted in a control percentage of 99.9%, while in the other treatments, control percentages below 56% were observed. Oocytes from the females exposed to the combinations of thymol and eugenol (each compound at 2.5 mg/mL) showed histopathological changes, except on oocyte V, while those treated with these compounds alone at 2.5 mg/mL, did not reveal any change. Changes in the shape of the oocyte, presence of vacuoles in the cytoplasm and germinal vesicle, reduction and fusion of yolk granules and rupture of some oocytes were observed. In silico analysis, showed that these compounds can act as membrane permeability inhibitors, membrane permeability agonists, membrane integrity antagonists and apoptosis agonists. We conclude that the combination of thymol and eugenol causes changes in the reproductive biology and morphophysiology of engorged females oocytes. The in silico analysis using thymol and eugenol revealed the possibility of disorganization in the cell membranes, a fact that may explain the histopathological alterations observed.

Acaricidal activity of carvacrol and thymol on acaricide-resistant Rhipicephalus microplus (Acari: Ixodidae) populations and combination with cypermethrin: Is there cross-resistance and synergism?

This study evaluated the acaricidal activity of thymol and carvacrol on Rhipicephalus microplus populations with different resistance profiles and investigated the synergistic effect of combinations of these monoterpenes with cypermethrin. The adult immersion test (AIT) was used to characterize the susceptibility of tick populations (45 field populations) to synthetic acaricides: deltamethrin, amitraz and chlorfenvinphos. The larval packet test (LPT) was used to determine the LC50 values for thymol (25 tick populations) and carvacrol (20 tick populations). The susceptible strain Porto Alegre (POA) was used as a reference for calculating the resistance ratio (RR). Subsequently, larval immersion tests (LIT) were performed with combinations of cypermethrin with thymol or carvacrol to assess a synergistic effect. In the AIT, deltamethrin showed efficacy > 90% in one (2.2%) population tested (mean: 12.1 and 11.1 for populations 1-25 and 26-40, respectively), whereas amitraz and chlorfenvinphos showed efficacy > 90% for two (4.4%) populations (mean: 61.3 and 47.3 for populations 1-25 and 26-40, respectively) and eight (17.7%) populations (mean: 69.7 and 59.7 for populations 1-25 and 26-40, respectively). In the LPT, the LC50 values for thymol and carvacrol varied from 0.67 to 2.12 mg/mL and 0.55-3.21 mg/mL, with an average LC50 for populations of 1.49 and 1.75 mg/mL, respectively. For thymol, no resistance was observed in any of the populations, values of RR50 > 1.5. There was no correlation between the LC50 values for thymol and the efficacy of the chemical acaricides tested. Regarding carvacrol, for only one tick population had the value of RR50 > 1.5, indicating an incipient resistance. No correlation was observed between the LC50 values for carvacrol and the efficacy of tested acaricides. The combination of thymol and carvacrol with cypermethrin showed a synergistic effect in the resistant population (Jaguar - thymol 4.19 and carvacrol 3.67), and no synergistic interaction were showed in the susceptible population. Answering the questions we conclude that: 1 - The comparison between the LC50 values for thymol and carvacrol in field populations and the susceptible strain POA suggests the absence of cross-resistance (ticks and terpenes), and the differences between the LC50 values for thymol and carvacrol in the different R. microplus populations are inherent to the characteristics of each population tested; 2 - the combination of thymol or carvacrol with cypermethrin showed a synergistic effect with different activity according to the population of ticks.

High Occurrence of Chelonid Alphaherpesvirus 5 (ChHV5) in Green Sea Turtles Chelonia mydas with and without Fibropapillomatosis in Feeding Areas of the São Paulo Coast, Brazil.

Chelonid alphaherpesvirus 5 (ChHV5) has been consistently associated with fibropapillomatosis (FP), a neoplastic disease that affects sea turtles globally. The DNA of ChHV5 has been detected in cutaneous and noncutaneous tissues (e.g., lung) of green sea turtles Chelonia mydas with (FP+) and without (FP-) clinical signs of FP, indicating a persistent ChHV5 infection. Previously published and custom primer pairs were used to amplify the fragments of ChHV5 unique long (UL) partial genes (UL30 and UL18) through end-point PCR from cutaneous tumors (n = 31), nontumored skin (n = 49), and lungs (n = 26) from FP+ (n = 31) and FP- (n = 18) green sea turtles. The DNA of ChHV5 was detected in cutaneous tumors (80.6%, 25/31), nontumored skin (74.2%, 23/31 FP+; 27.8%, 5/18 FP-), and lung samples (91.7%, 11/12 FP+; 100%, 14/14 FP-). The high occurrence of ChHV5 observed in lung samples from FP- individuals was unexpected (14/14), providing the first evidence of ChHV5 DNA presence in lungs of individuals without FP. Our results also revealed high ChHV5 occurrence among the tested cohort (93.9%, 46/49) and suggested that a large proportion (83.4%, 15/18) of FP- green sea turtles had subclinical ChHV5 infections. Hence, our findings support the hypothesis that ChHV5 requires one or more possibly environmental or immune-related co-factors to induce FP.