Brazil's battle against Rhipicephalus (Boophilus) microplus ticks: current strategies and future directions.

Ticks are parasitic arthropods that cause significant economic losses to livestock production worldwide. Although Rhipicephalus (Boophilus) microplus, the cattle tick, occurs throughout the Brazilian territory, there is no official program to control this tick, which is the vector of tick fever pathogens. We address the situation of R. (B.) microplus resistance to synthetic acaricides in Brazil, including cattle tick management; the status of tick resistance per Brazilian state; the history of resistance occurrence of different acaricides; multiple resistance occurrence; and the main strategies for integrated tick management. Tick control in Brazil is characterized by management errors. Local laboratories affiliated with federal and state research institutions and universities employ the Adult Immersion Test as a primary diagnostic method to assess acaricide resistance to topically applied drugs. Only three states (Acre, Amapá, and Amazonas) have no reports on resistant populations. Misinformation on tick control strategies, misuse of available products for tick control, no adoption of Integrated Parasite Management (IPM) practices, low technical support to producers, and the high-speed emergence of acaricide-resistant tick populations are the main problems. We also propose a list of needs and priorities for cattle tick control regarding communication, research, and policies.

Effects of Piper aduncum (Piperales: Piperaceae) Essential Oil and Its Main Component Dillapiole on Detoxifying Enzymes and Acetylcholinesterase Activity of Amblyomma sculptum (Acari: Ixodidae).

Amblyomma sculptum is a species of tick in the family Ixodidae, with equids and capybaras among its preferred hosts. In this study, the acaricidal activity of the essential oil (EO) from Piper aduncum and its main component, Dillapiole, were evaluated against larvae of A. sculptum to establish lethal concentration values and assess the effects of these compounds on tick enzymes. Dillapiole exhibited slightly greater activity (LC50 = 3.38 mg/mL; 95% CI = 3.24 to 3.54) than P. aduncum EO (LC50 = 3.49 mg/mL; 95% CI = 3.36 to 3.62) against ticks. The activities of α-esterase (α-EST), ß-esterase (ß-EST), and glutathione-S-transferase (GST) enzymes in A. sculptum larvae treated with Dillapiole showed a significant increase compared to the control at all concentrations (LC5, LC25, LC50 and LC75), similar results were obtained with P. aduncum EO, except for α-EST, which did not differ from the control at the highest concentration (LC75). The results of the acetylcholinesterase (AChE) activity show an increase in enzyme activity at the two lower concentrations (LC5 and LC25) and a reduction in activity at the two higher, lethal concentrations (LC50 and LC75) compared to the control. These results suggest potential mechanisms of action for these natural acaricides and can provide guidance for the future development of potential plant-derived formulations.

Enzootic stability of tick fever in Holstein calves grazing in a tropical region, subjected to strategic cattle tick control with fluralaner.

BACKGROUND: In 2022, fluralaner was launched on the market for use in the control of the cattle tick Rhipicephalus microplus after showing 100% efficacy in registration trials against the causative agents of cattle tick fever (TFAs). The aim of the present study was to determine whether a strategic control regimen against R. microplus using fluralaner (FLU) in Holstein calves grazing in a tropical region would alter the enzootic stability status of cattle tick fever, triggering outbreaks in these animals up to 22 months age. METHODS: In this study, a group of calves treated with FLU was compared with a control group treated with the regimen currently being used on the farm, which consisted of the fipronil + fluazuron formulation (FIFLUA). In the first experiment, the efficacy of the FIFLUA pour-on formulation was evaluated in a field study. In the second experiment, which lasted 550 days, two experimental groups (n = 30/group) of Holstein calves naturally infested with R. microplus were analyzed. Calves aged 4 to 10 months received either a specific treatment regimen with FLU (experimental group) or FIFLUA (control group). During this period, tick counts, animal weight measurement, feces collection (to determine eggs and oocysts per gram of feces), tick fever monitoring, blood smears (to ascertain enzootic stability of the herd), PCR testing for TFAs and serology (indirect enzyme-linked immunosorbent assay [iELISA]) were performed. All calves were evaluated for signs of tick fever between ages 11 and 22 months. RESULTS: FIFLUA showed an acaricidal efficacy of > 90% from post-treatment days 14 to 35. Regarding treatments against the TFAs, the average number of treatments was similar between groups, but animals treated with FLU had a smaller reduction in packed cell volume on some of the evaluation dates of the second and third treatment against TFAs. In calves aged 10 months in the FLU group, B. bovis was not detected by PCR (0/15 samples), 40% of the samples had antibody titers and 33% (10/30) of the samples had positive blood smears. Regarding B. bigemina, > 86% of the samples in both groups tested positive for B. bigemina DNA and antibodies; there was no difference in the antibody titers between the groups. There were no clinical cases of cattle tick fever in calves aged 11 to 22 months. CONCLUSIONS: In comparison with the control treatment, the strategic control regimen against R. microplus with FLU that was implemented in the present study did not negatively affect the enzootic stability status of A. marginale and B. bigemina in the herd up to 22 months of age. The enzootic stability status of B. bovis was not reached by either group. These results likely represent a characteristic of the local tick population, so further studies should be performed.

What is the optimal timing to initiate strategic control of Rhipicephalus microplus in taurine cattle in a tropical region?

Cattle tick control poses a significant challenge for livestock in tropical and subtropical regions. The objective of this study was to determine the most suitable timing to initiate a strategic tick control program and to identify the ideal number of acaricide treatments for adult taurine cattle (Bos taurus taurus) in a tropical region throughout the year. Three groups with 10 bovines each were performed: T01 (strategic treatment in late autumn/winter/late spring, every 28 days), T02 (strategic treatment to act in the "first tick generation" - early spring/summer/early autumn, every 28 days) and T03 (control). Tick counts (females 4-8 mm) were conducted every 14 days. If the tick burden in any group reached 30 or more during these counts, we applied an additional treatment. Over the course of a year, T02 required significantly fewer (p < 0.05) acaricide treatments than T01, with nine treatments for T02 and eleven for T01. Furthermore, during the tick counts, animals in T02 showed a lower tick burden compared to those in T01. Initiating the strategic tick control program in early spring, corresponding to the first tick generation, proved more effective than starting in autumn. This approach not only required fewer acaricide treatments but also resulted in a reduced tick burden. These benefits are particularly valuable in terms of animal welfare and managing acaricide resistance issues.

Daylight photodynamic inactivation of cattle tick Rhipicephalus microplus by porphyrins: An alternative for the ectoparasite control.

The bovine tick Rhipicephalus microplus, a primary ectoparasite of veterinary concern, contributes significantly to disease transmission and reduced cattle productivity, resulting in substantial economic losses. The overuse of chemical acaricides has led to the emergence of resistant strains, posing a considerable challenge to veterinary medicine. Consequently, the development of alternative parasite control methods is essential to ensure livestock quality and enhance food safety worldwide. Our study introduces an innovative approach to photodynamic inactivation (PDI) of the bovine tick, harnessing natural daylight for a potential field application. Reproductive parameters (female and egg mass, egg production index, and larval hatch) were evaluated in engorged female ticks under photodynamic action using the hematoporphyrin (HP) and tetra-cationic porphyrins free-base meso-tetra-ruthenated (4-pyridyl) (RuTPyP) and its zinc(II) complex (ZnRuTPyP) as photosensitizers (PS). The results showed that there was no significant difference between the groups treated with tetra­ruthenium porphyrins and the control group. However, HP exhibits a control percentage of 97.9% at a concentration of 2.5 µmol.L-1, aligning with the expected control rates achieved by conventional chemical acaricides. Photophysical and physicochemical parameters such as the number of singlet oxygen produced and lipophilicity were discussed for each PS and related to tick control percentages. Furthermore, the interaction between HP and chitin, an important macromolecule presents in the tick's cuticle, considered as the primary target tick structure during PDI was observed by the absorption and fluorescence emission spectroscopic techniques. Therefore, the results presented here extend the potential for controlling R. microplus through photodynamic inactivation while utilizing sunlight as a source of natural irradiation.

Two protocols using fluralaner for Rhipicephalus microplus strategic control on taurine cattle in a tropical region.

BACKGROUND: The present study aimed to evaluate the effects of different treatment strategies using a new commercial formulation containing pour-on fluralaner on Rhipicephalus microplus infestation in cattle and in pastures in a tropical climate region where up to five generations of this tick species can occur per year. METHODS: Forty-five naturally infested cattle were divided into three experimental groups: T01, treated with fluralaner (2.5 mg/kg) pour-on every 42 days; T02, the cattle received the first treatment with fluralaner on Day 0 but the next treatment involved a weekly visual evaluation; T03, control, received palliative treatment with a spray formulation when the group mean was ≥ 30 ticks. Counts of female R. microplus were performed weekly until day 343, and larval counts on pasture were performed on Days 0, 30, and 60 and every 30 days until Day 330. RESULTS: Using fluralaner, six applications were performed in Group T01, and four were performed in Group T02. In the control group (T03), it was necessary to perform eight palliative acaricide treatments with the spray formulation. The animals in T01 and T02 showed lower mean tick counts (p ≤ 0.05) than the control group (T03) on 28 and 27 of the 49 evaluated dates, respectively. In the paddock where the animals were kept as controls, the R. microplus larvae counts increased to 1458. In the paddocks where the animals were treated with fluralaner, the number was ≤ 19 per paddock during the study. CONCLUSIONS: The different strategic treatment protocols performed with pour-on fluralaner (2.5 mg/kg) over a year in taurine cattle in a tropical region with a history of up to five annual generations of cattle ticks were effective, maintaining levels of R. microplus infestations in animals and in pastures close to zero in most of the study. Depending on the retreatment criterion adopted, the number of applications per year may be lower, resulting in a reduction in the mean cost of acaricide treatment per year and lower exposure of R. microplus populations to the active ingredient, resulting in lower resistance and selection pressure.

Essential oils and isolated compounds for tick control: advances beyond the laboratory.

BACKGROUND: Tick control is a worldwide challenge due to its resistance to acaricides. Essential oils (EOs) and isolated compounds (EOCs) are potential alternatives for tick control technologies. METHODS: A review with EOs and EOCs, under field and semi-field conditions, was performed based on Scopus, Web of Science and PubMed databases. Thirty-one studies published between 1991 and 2022 were selected. The search was performed using the following keywords: "essential oil" combined with "tick," "Ixodes," "Argas," "Rhipicephalus," "Amblyomma," "Hyalomma," "Dermacentor," "Haemaphysalis" and "Ornithodoros." The words "essential oil" and "tick" were searched in the singular and plural. RESULTS: The number of studies increased over the years. Brazil stands out with the largest number (51.6%) of publications. The most studied tick species were Rhipicephalus microplus (48.4%), Ixodes scapularis (19.4%), Amblyomma americanum and R. sanguineus sensu lato (9.7% each). Cattle (70%) and dogs (13%) were the main target animal species. Regarding the application of EOs/EOCs formulations, 74% of the studies were conducted with topical application (spray, pour-on, foam, drop) and 26% with environmental treatment (spray). Efficacy results are difficult to evaluate because of the lack of information on the methodology and standardization. The nanotechnology and combination with synthetic acaricides were reported as an alternative to enhance the efficacy of EOs/EOCs. No adverse reactions were observed in 86.6% of the studies evaluating EOs/EOCs clinical safety. Studies regarding toxicity in non-target species and residues are scarce. CONCLUSIONS: This article provides a comprehensive review on the use of EOs and EOCs to reduce tick infestations, in both the hosts and the environment. As future directions, we recommend the chemical characterization of EOs, methodology standardization, combination of EOs/EOCs with potential synergists, nanotechnology for new formulations and safety studies for target and non-target organisms, also considering the environmental friendliness.

Alterations in the metabolism of Pseudosuccinea columella (Mollusca: Gastropoda) caused by Heterorhabditis bacteriophora HP88 (Rhabditida: Heterorhabditidae).

The gastropod Pseudosuccinea columella participates in the dissemination of Fasciola hepatica in the environment, acting as the main intermediate host of this parasite in Brazil. The present study sought to elucidate the possible pathogenic effects of the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora on P. columella, by evaluating the influence of infection on alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as the concentrations of total protein, uric acid, and urea in the snail's hemolymph. For this, the snails were exposed to EPNs for 24 and 48 h, and for each exposure time, 20 snails were dissected after 7, 14 and 21 days for hemolymph collection. The primary findings suggest a significant proteolysis alongside elevated levels of uric acid and urea in P. columella infected individuals. These findings reveal that H. bacteriophora HP88 infection induced serious changes in the snail's metabolism, triggering important deleterious effects.

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.

Influence of exposure Heterorhabditis bacteriophora HP88, (Rhabditida: Heterorhabditidae) on biological and physiological parameters of Pseudosuccinea columella (Basommatophora: Lymnaeidae).

Many studies about fasciolosis control have been carried out, whether acting on the adult parasite or in Pseudosuccinea columella, compromising the development of the larval stages. The present study aimed to evaluate, under laboratory conditions, the susceptibility of P. columella to Heterorhabditis bacteriophora HP88, during for 24 and 48 hours of exposure. The snails were evaluated for 21 days for accumulated mortality; number of eggs laid; hatchability rate; biochemical changes; and histopathological analysis. We found that exposure induced a reduction in glucose and glycogen levels, characterizing a negative energy balance, due to the depletion of energy reserves as a result of the direct competition established by the nematode/endosymbiont bacteria complex in such substrates. A mortality rate of 48.25% and 65.52% was observed in the group exposed for 24 h and 48 h, respectively, along with significant impairment of reproductive biology in both exposed groups in relation to the respective controls. The results presented here show that P. columella is susceptible to the nematode H. bacteriophora, with the potential to be used as an alternative bioagent in the control of this mollusk, especially in areas considered endemic for fascioliasis, in line with the position expressed by the World Health Organization Health.

Effect of a preventive strategic control program, with imidocarb dipropionate, against tick fever agents in dairy calves.

Chemoprophylaxis with dipropionate imidocarb (IMD) is a method adopted to prevent cattle tick fever (TF). Sixty weaned dairy heifers (±60 days old), without previous exposure to Rhipicephalus microplus ticks, were housed in Tifton paddocks and were subsequently exposed to R. microplus ticks and monitored up to 315 days old. Thirty animals were kept as controls (T01) and 30 received five preventive strategic treatments with IMD at 21-day intervals (T02). The heifers were monitored weekly by means of packed cell volume (PCV) and blood smears to evaluate the presence of TF agents. Salvage treatments (ST) with diminazene and enrofloxacin were administered when animals showed PCV ≤ 24%. The A. marginale prevalence was 39.3% and 37.7%, B. bovis 6.0%, and 7.3%, and B. bigemina 16.3% and 13.7% for T01 and T02, respectively. Regarding PCV values, group T01 showed lower PCV than group T02, between 119 and 161 days of life, but when animals were 196, 210, 217, and between 252 to 301 days old, an inversion occurred. The IMD treatment protocol was effective in group T02 from day 91 to 175 while treatment was being administered, but from day 182 to 315 after the IMD treatment protocol was completed, the number of salvage treatments against TF agents performed in T02 group increased significantly. The sequential application of IMD treatments with intervals less than 21 days is not recommended.

Efficiency of Experimental Formulation Containing Duddingtonia flagrans and Pochonia chlamydosporia against Moniezia expansa Eggs.

This study aimed to evaluate the effectiveness of the experimental formulation containing chlamydospores of Duddingtonia flagrans and Pochonia chlamydosporia fungi, against Moniezia expansa. Two experiments were carried out. The first experiment evaluated the in vitro efficacy using 1 g of the experimental formulation (V1) added to 100 M. expansa eggs and the control (V2) (without the fungal formulation). Intact eggs or eggs with alterations were counted in order to evaluate their effectiveness. The second experiment evaluated the action of the fungal formulation on M. expansa eggs after passing through the gastrointestinal tract of goats. Three groups were identified as B1, B2, and B3, which received 1.0, 1.5 g of experimental fungal formulation, and placebo, respectively. In experiment 1, all the eggs in V1 were subjected to the predatory action of fungi, while in V2, the eggs remained intact. In experiment 2, the reduction of eggs in groups B1 and B2 were 49% and 57% 24 h after ingestion, 60% and 63% 48 h after, and 48% and 58% 72 h after. The predatory capacity against M. expansa eggs shown in the tests demonstrated that experimental fungal formulation has the potential to be used on integrated helminth control programs.

Design and Synthesis of New Anthranyl Phenylhydrazides: Antileishmanial Activity and Structure-Activity Relationship.

Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. A centenary approach to antimonial-based drugs was first initiated with the synthesis of urea stibamine by Upendranath Brahmachari in 1922. The need for new drug development led to resistance toward antimoniates. New drug development to treat leishmaniasis is urgently needed. In this way, searching for new substances with antileishmanial activity, we synthesized ten anthranyl phenylhydrazide and three quinazolinone derivatives and evaluated them against promastigotes and the intracellular amastigotes of Leishmania amazonensis. Three compounds showed good activity against promastigotes 1b, 1d, and 1g, with IC50 between 1 and 5 µM. These new phenylhydrazides were tested against Leishmania arginase, but they all failed to inhibit this parasite enzyme, as we have shown in a previous study. To explain the possible mechanism of action, we proposed the enzyme PTR1 as a new target for these compounds based on in silico analysis. In conclusion, the new anthranyl hydrazide derivatives can be a promising scaffold for developing new substances against the protozoa parasite.

Selective versus strategic control against Rhipicephalus microplus in cattle: A comparative analysis of efficacy, animal health, productivity, cost, and resistance management.

This study compared selective control versus strategic control against Rhipicephalus (Boophilus) microplus, under conditions of a real dairy farm. Strategic control requires that all animals be treated with acaricide at regular pre-determined intervals. Selective control only requires treatment of infested animals and only when they are at or above a pre-determined threshold. Tick counts on animals and in pasture were performed and the susceptibility of tick populations to the different treatment methods was evaluated at the beginning and end of the study using the Larval Packet Test, Larval Immersion Test, and Adult Immersion Test. Over the four years of the study strategic control was more advantageous than the selective control as the group experienced lower tick burden on animals and in pasture, absence of skin lesions and myiases, lower operating costs, treatments concentrated in fewer months of the year and lower resistance pressure.

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.

Acaricidal activity of a 2-methoxy-clovan-9-ol rich fraction from Eugenia copacabanensis (Myrtaceae) extract on Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus (Ixodidae, canestrini, 1888) is an invasive ectoparasite of cattle which causes high economic losses in emerging countries such as Brazil. Phytochemical compounds have been tested as an alternative to synthetic acaricides due to potentially lower mammalian toxicity. This study evaluated the acaricidal activity against R. microplus of the 2-methoxy-clovan-9-ol rich fraction obtained from Eugenia copacabanensis Kiaersk.leaves, a little known Myrtaceae species from the Brazilian Atlantic Forest. This fraction was obtained by maceration with methanol, partition with n-hexane and purification by normal-phase column chromatography. GC-MS, 1H and 13C NMR and IR analysis contributed to the identification of a major compound as the sesquiterpene 2-methoxy-clovan-9-ol, reported for the first time for the Myrtaceae family. The fraction was tested against R. microplus unfed larvae and engorged females and a 93% larval mortality was observed at the concentration of 50 mg mL-1. Lower concentrations of the solution tested demonstrated a significant difference in egg mass weight, hatching and control percentage. Experiments with 50.0 mg mL-1 showed significative results, with lower concentration and maximum efficacy for both assays. The IC50 values for unfed larvae and engorged females were 21.76 and 11.13 mg mL-1, respectively. These results were similar to those obtained in other studies with isolated botanical compounds and essential oils. The lower IC50 for engorged females than for unfed R. microplus larvae had also been described for other plant materials, including plants from the same Myrtaceae family. The present result suggested different mechanisms of action of the compound on the reproductive biology of engorged females, improving its effect against egg viability. These results are important for tick control, suggesting that 2-methoxy-clovan-9-ol could be a potential natural acaricidal product against both R. microplus unfed larvae and engorged females.

Influence of the acaricide emulsion pH on the effectiveness of spray products to control the cattle tick: laboratory and field investigations.

The current work evaluated the efficacy of 10 commercial acaricides in different pHs (4.5, 5.5, and 6.5) in laboratory (adult immersion tests (AIT), pH evaluation over time) and field assays (tick counts and efficacy). In the AIT (n=70), higher efficacies were obtained when the acaricide emulsion had a more acidic pH (4.5), mainly for two combinations of pyrethroids + organophosphate (acaricide 3 and acaricide 9). For amidine, a higher pH (6.5) showed a higher efficacy. Over time, there was a trend in the pH of these emulsions increasing. When the efficacy of chlorpyrifos + cypermethrin + piperonyl butoxide (acaricide 3) at different pHs was evaluated over time (0, 6, 12, and 24h) by AIT, the less acidic pH (6.5) showed a strongly variation in the acaricide efficacy range. The mean pH of the water samples from different regions of Brazil was 6.5. In the field, the association of pyrethroid + organophosphates (acaricide 9) with pH of 4.5 and 5.5 were more effective in tick control than the emulsion prepared with this same spray formulation at pH 6.5. The pH of the acaricide emulsions is an important point of attention and is recommended that the veterinary industry start to develop/share information regarding how the pH can affect the acaricide efficacy.

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.

Comparison of the efficacy of different methods to apply acaricides for control of Rhipicephalus (Boophilus) microplus.

The present study compared the efficacy of different methods to apply an acaricide formulation to control Rhipicephalus (Boophilus) microplus. To compare the methods, an acaricide blend containing three active ingredients (a pyrethroid and two organophosphates) was used. In experiment 1 (farm 1: Goiânia, GO, Brazil), three methods were tested: a backpack sprayer (BS), power sprayer (PS) and spray race (SR). In experiment 2 (farm 2: São José do Rio Pardo, SP, Brazil), two methods were tested: BS and PS. In both experiments, 10 cattle with similar tick burdens were used. On day 0 in both experiments, the animals were treated with the acaricide. On day +1 (only in experiment 1), +3, +7, +14, +21, +28 and +35 (only in experiment 2), tick counts were performed to determine the control efficacy. The time application, pressure (KPa), volume applied (L) and ergonomic aspects of each spraying system were also evaluated. The adult immersion test (AIT) using three different acaricide blends (combinations of pyrethroid + organophosphate) was performed to compare the susceptibility of strains of each farm. In experiment 1, all treatments significantly reduced (p < 0.05) the number of ticks on the animals, and PS resulted in the greatest acaricide efficacy since day +1. In experiment 2, both treatments (PS and BS) reduced (p < 0.05) the tick burden, and as observed in experiment 1, PS resulted in the best reduction. The application times were 4.5, 150 and 330 s, while pressures were 306.8, 4,826.3 and 220.6 KPa for SR, PS and BS, respectively. In the AIT, the efficacy values were between 99.8 and 100% for the tick strain form farm 1 (Goiânia), while for tick strain from farm 2 (São José do Rio Pardo), the efficacy was between 67.2 and 80.9%. We conclude that the sprayer methods chosen influences the efficacy of the acaricide. All sprayer methods were efficient for acaricide application; the best efficacy was obtained using the PS, while the SR resulted in good efficacy and lower application time. The strain from farm 2 was less susceptible to all acaricides tested.

Automatic method based on deep learning to identify and account Rhipicephalus microplus larval hatching.

Reports of Rhipicephalus microplus resistant populations worldwide have increased extensively, making it difficult to control this ectoparasite. The adult immersion test, commonly used to screen for acaricide resistance, produces the results only after 40 days of the tick collection because it needs the eggs to be laid and larvae to hatch. The present study aims to develop an automatic method, based on deep learning, to predict the hatching of R. microplus larva based on egg morphology. Initially, the time course of embryonic development of tick eggs was performed to discriminate between viable and non-viable eggs. Secondly, using artificial intelligence deep learning techniques, a method was developed to classify and count the eggs. The larval hatching rate of three populations of R. microplus was evaluated for the software validation process. Groups of three and six images of eggs with 12 days of embryonic development were submitted to the software to predict the larval hatching percent automatically. The results obtained by the software were compared with the prediction results of the hatching percentage performed manually by the specialist and with the results of the hatching percentage of larvae obtained in the biological assay. The group with three images of each population submitted to the software for automatic prediction of the larval hatching percent presented mean values of 96.35% ± 3.33 (Piracanjuba population), 95.98% ± 3.5 (Desterro population) and 0.0% ± 0.0 (Barbalha population). For groups with six images, the values were 94.41% ± 3.84 (Piracanjuba population), 95.93% ± 2.36 (Desterro population) and 0.0% ± 0.0 (Barbalha population). Biological assays showed the following hatching percentage values: 98% ± 1.73 (Piracanjuba population); 96% ± 2.1 (Desterro population); and 0.14% ± 0.25 (Barbalha population). There was no statistical difference between the evaluated methods. The automatic method for predicting the hatching percentage of R. microplus larvae was validated and proved to be effective, with considerable reduction in time to obtain results.