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The Rhipicephalus microplus tick causes enormous economic losses in livestock farming around the world. Despite several promising studies carried out with plant extracts such as Achyrocline satureioides against this ectoparasite, a major obstacle is related to pharmaceutical presentation forms. There is no study showing xantan gum-based hydrogel and polycaprolactone nanoparticles containing A. satureioides extract against R. microplus larvae. The objective of this study was to incorporate A. satureioides extract to develop a nanoformulation (AScn) and a hydrogel (ASlh) and evaluate them against R. microplus larvae with the purpose of increasing the contact time of the extract with the larvae and improve the effectiveness. The ethanolic extracts were incorporated in polycaprolactone nanoparticles and characterized via analysis of the mean hydrodinamic diameter and polidispersity index. The xanthan gum-based hydrogel formulation was prepared with crude extract of A. satureioides 40 mg/mL, 0.25% xanthan gum, and 8% poloxamer, to determine the bioadhesiveness of the formulation in bovine leather and the flow rate of the formulation in the animal. The results in larvae demonstrated that when evaluated in the form of a hydrogel (ASlh), mortality was higher, with 91.48% mortality at a concentration of 20 mg/mL presenting itself as an interesting alternative for controlling this ectoparasite.

Nanoformulations containing zein nanoparticles (ZN) can promote the stability and protection of molecules with acaricidal activity. The present study sought to develop nanoformulations with ZN associated with cypermethrin (CYPE) + chlorpyrifos (CHLO) + a plant compound (citral, menthol or limonene), characterize them, and verify their efficacy against Rhipicephalus microplus ticks. Additionally, we aimed to assess its safety in nontarget nematodes found in soil at a site subjected to contamination by acaricides. The nanoformulations were characterized by dynamic light scattering and nanoparticle tracking analysis. Nanoformulations 1 (ZN+CYPE+CHLO+citral), 2 (ZN+CYPE+CHLO+menthol), and 3 (ZN+CYPE+CHLO+limonene) were measured for diameter, polydispersion, zeta potential, concentration, and encapsulation efficiency. Nanoformulations 1, 2, and 3 were evaluated in a range from 0.004 to 0.466 mg/mL on R. microplus larvae and caused mortality > 80% at concentrations above 0.029 mg/mL. The commercial acaricide Colosso® (CYPE 15 g + CHLO 25 g + citronellal 1 g) was evaluated also from 0.004 to 0.512 mg/mL and resulted in 71.9% larval mortality at 0.064 mg/mL. Formulations 1, 2, and 3 at 0.466 mg/mL showed acaricidal efficacy of 50.2%, 40.5%, and 60.1% on engorged females, respectively, while Colosso® at 0.512 mg/mL obtained only 39.4%. The nanoformulations exhibited long residual period of activity and lower toxicity to nontarget nematodes. ZN was able to protect the active compounds against degradation during the storage period. Thus, ZN can be an alternative for the development of new acaricidal formulations using lower concentrations of active compounds., Competing Interests: Declaration of Competing Interest The authors warrant that there are no any conflicts of interests among authors and between authors and other people, institutions or organizations., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nanocarriers of acaricidal compounds improve the bioavailability, absorption, and tissue distribution of active ingredients, releasing them in a slow, targeted way and protecting them against premature degradation. Thus, this study aimed to develop formulations from solid lipid nanoparticles (SLN), or nanostructured lipid carriers (NLC) associated with cypermethrin (cip) + chlorpyrifos (chlo) and vegetable compounds (citral, menthol, or limonene). Particles were then characterised, and their efficacy was verified on R. microplus in comparison to nanoformulations without the plant-based compounds. Six different formulations were developed and characterised by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Formulations 1 (SLN+cyp+chlo+citral), 2 (SLN+cyp+chlo+menthol), 3 (SLN+cyp+chlo+limonene), 4 (NLC+cyp+chlo+citral), 5 (NLC+cyp+chlo+menthol) and 6 (NLC+cyp+chlo+limonene) had mean diameters from 286 to 304 nm; polydispersion from 0.16 to 0.18; zeta potential from -15.8 to -20 mV, concentration from 3.37 ± 0.24 × 10 13 to 5.44 ± 0.18 × 10 13 particles/mL and encapsulation efficiency (EE) > 98.01 % for all active ingredients. All formulations were evaluated for their acaricidal potential by the larval packet test (LPT) and compared with nanoformulations without the plant-based compounds. Formulations were also compared with positive (Colosso® at 512 µg/mL) and negative controls (distilled water and nanoparticles without active ingredients). The SLN (1, 2 and 3) and NLC (4, 5 and 6) formulations, at 7 µg/mL, resulted in 90.4 % , 75.9 % , 93.8 % , 100 % , 95.1 % and 72.7 % mortality. The data demonstrated that the addition of citral, menthol or limonene in the formulations improved their acaricide action against tick larvae. Except for formulation 4, for which it was not possible to determine lethal concentrations (LC). Formulations, 1, 2, 3, 5 and 6 reached LC 50 and LC 90 values of 3.3 and 7.2, 5.4 and 9.2, 4.0 and 8.1, 2.3 and 5.4 as well as 5.5 and 9.4 µg/mL, respectively. It was possible to encapsulate the active ingredients and characterise the lipid carrier systems. SLN and NLC protected the active ingredients against degradation in solution and increased the overall stability. A stabile solution is necessary for synthesizing commercial acaricidal products. It is hoped that these findings may contribute to new studies focused on the use of nanocarriers in tick formulations. By reducing the amount or concentration of active ingredients within commercial products, the risk of residues presents in food of animal origin or remaining in the environment is reduced. Nanocarriers help prevent these challenges, while still maintaining effective parasitic control. Utilizing a combination of natural and synthetic products can be part of integrated management solutions and can help overcome widespread acaricide resistance in populations of cattle ticks., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The tick Rhipicephalus microplus (Canestrini) is a major economic hurdle to the global livestock industry. The incorrect and indiscriminate control carried out by synthetic chemical compounds has contributed to the emergence of parasite resistance present today to many products available in the veterinary market. Effective, economically viable and potentially safe alternatives need to be investigated, including herbal medicine and biological control by entomopathogenic fungi. In this study, extracts from Achyrocline satureioides (Lam.) DC. obtained by extraction with solvents of different polarities associated and not associated with the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin were evaluated on different stages of the life cycle of R. microplus. The activity of plant extracts, fungus and their associations in ticks was analyzed in vitro through the adult immersion test, larval packet test and larval immersion test. The highest efficacy on engorged females (43.3%) was reached by the hexanic partition (ASh) at 5 mg/mL + fungus, reducing larval hatchability. ASh associated with B. bassiana also caused high mortality of larvae, reaching LC 50 of 6.23 and 2.65 mg/mL in the packet and immersion tests, respectively. The results obtained showed that extracts, when used in combination with the fungus, have their action enhanced, reaching 100% mortality of larvae. Among the evaluated methodologies, it was observed that the larval immersion test allows better assessment, due to the time of contact with the larvae. The results obtained with A. satureioides associated with the fungus are considered promising and open new perspectives for future product development., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Although Brazilian livestock is considered one of the most productive in the world, the losses caused by parasites still greatly limit these activities and its incorrect control through traditional synthetic chemicals has caused problems such as parasitic resistance. The present study evaluated the anthelmintic and acaricidal action of ethanolic extract (EE) and essential oil (OE) obtained from Achyrocline satureioides inflorescences against an endo and an ectoparasite of great importance in sheep and cattle. The action against Haemonchus contortus was evaluated by the Larval Development Test (LDT) and the Egg Hatching Test (EHT), while the acaricidal potential against Rhipicephalus (Boophilus) microplus was evaluated by the Adult Immersion Test (AIT) and Larval Package Test (LPT). Chemical analyzes were performed by liquid chromatography coupled to mass of spectrometry (LC-MS) and gas chromatography coupled to mass spectrometry (GC-MS) in order to evaluate the chemical composition of EE and EO, respectively. Luteolin (m/z 285), quercetin (m/z 301) and methyl quercetin (m/z 315) were the major compounds detected in the A. satureioides EE, while 41 compounds were identified in EO. Among them, the major compounds were caryophyllene oxide (15.4%), limonene (10.9%), trans-calamenene + δ-cadinene (8.0%), (E)-caryophyllene (5.5%), spathulenol (5.1%) and α-pinene (4.5%). Against H. contortus, by EHT, the EE and EO showed LC 50 of 83.14 mg mL -1 and 10.42 mg mL -1 , respectively. In LDT, LC 50 were 0.16 mg mL -1 and 0.42 mg mL -1 , respectively by EE and EO. In the AIT assay for R. (B.) microplus tick LC 50 obtained for EE and EO were 76.43 mg mL -1 and 114.35 mg mL -1 , respectively. EE at 100 mg mL -1 completely inhibited egg laying, while the EO at 50 mg mL -1 completely inhibited larval hatching. No significant larvicidal effect was observed in LPT for both extracts. The EE and EO presented a high efficacy at low concentrations in the inhibition of egg hatching and larval development of H. contortus. Although both extracts did not show consistent larvicidal action, the results were significant in inhibiting the oviposition of R. (B.) microplus engorged females., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The synthesis of thiophenic compounds, previously identified in Tagetes patula, revealed that 4-(5'-(hydroxymethyl)-[2,2'-bithiophene]-5-yl)but-3-yn-1-ol), or simply Thio1, has a pronounced in vitro anthelmintic effect against Haemonchus contortus, showing 100% efficacy in the egg hatch and larval development tests presenting EC 50  = 0.1731 mg.mL -1 and EC 50  = 0.3243 mg.mL -1 , respectively. So, this compound was selected to preparation of a nanostructured formulation to be orally administered to Santa Inês sheep. In general, from the fecal egg count reduction test (FECRT), it was observed that the product kept the parasitic load in the digestive tract of the hosts stable, with eggs per gram of faeces (EPG) counts having a mean value < 3,000 (EPG mean  = 2167.1, efficacy = 36,45%), thus protecting the animals from health risks caused by a massive nematode infestation. To better understand the mode of action of this thiophene derivative, in silico molecular modeling studies were carried out with the glutamate-activated chloride channel (GluCl), a well-known molecular target of anthelmintic compounds. Based on the affinity score (GlideScore = -5.7 kcal.mol -1 ) and the proposed binding mode, Thio1 could be classified as a potential GluCl ligand, justifying the promising results observed in the anthelmintic assays., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The cattle tick Rhipicephalus microplus is a great sanitary problem and causes huge losses to livestock, being a vector of important diseases. The aim of this work was to verify the action of plant-derived material obtained from Tagetes patula on the life cycle of R. microplus, as well as to analyze the action of these extracts on ovary cells of engorged females. In the Adult Immersion Test, the crude ethanolic extract (Tp EtOH ) showed an efficacy of 99.2% (LC 50  = 18.60 mg mL -1 ). From the repellency test, it was found that both the Tp EtOH and essential oil were 100% efficient on the larval stage. In addition, in the Larval Package Test, a mortality of 98.37% was obtained with Tp EtOH (LC 50  = 3.798 mg mL -1 ). From the microscopic analysis of ovary cells, morphological changes in the chorion and cytoplasm of oocytes were observed, with vacuolization around the germinal vesicle and disorganization of the pedicular cells, suggesting an interference in the normal embryogenic formation of the larvae. These results demonstrate that T. patula extracts interfere at all stages of development of the ixodid, from eggs to adults forms, and have a pronounced repellent effect. In addition, the results of the cytotoxicity assays performed on keratinocytes, as well as previous information on oral and dermal acute toxicity (LD 50  > 4000 mg kg -1 ), attest that T. patula can be a safe alternative for ectoparasitic control, representing an alternative for development of a formulation used to help control populations of ticks in the field.

Sheep breeding has suffered economic losses due to parasitism by gastrointestinal nematodes, particularly Haemonchus contortus. The use of natural products, specifically Tagetes patula, has been suggested as an alternative method of combatting this issue. Chemical analyses of the extracts of this species described in the literature report the presence of important classes of secondary metabolites such as thiophenes, flavonoids, alkaloids, and benzofurans, some of which were identified and isolated in this study. The aim of this work was to test the effect of the essential oil (EO) and the ethanolic extract of the aerial parts (Tp Et OH ) of T. patula on eggs and larvae of H. contortus, through an egg hatch test (EHT) and a larval development test (LDT). In the EHT, the EO showed 100% inhibition at 0.75 mg mL -1 (LC 50 = 0.0780 mg mL -1 ), and the Tp Et OH showed 100% inhibition at 100 mg mL -1 (LC 50 = 12.8 mg mL -1 ). In the LDT, the EO showed 100% inhibition at 0.375 mg mL -1 (LC 50 = 0.0400 mg mL -1 ), and the Tp Et OH showed 100% inhibition at 1.56 mg mL -1 (LC 50 = 0.340 mg mL -1 ). Compared to available literature data, the results presented here suggest that the crude extracts of T. patula have substantial potential for controlling this nematode by interrupting its life cycle and/or preventing it from reaching the infective stage., (© 2018 Wiley-VHCA AG, Zurich, Switzerland.)

The anthelminthic activity of the essential oil (EO) of Piper aduncum L. was tested in vitro on eggs and larvae of resistant (Embrapa2010) and susceptible (McMaster) isolates of Haemonchus contortus. The EO was obtained by steam distillation and its components identified by chromatography. EO concentrations of 12.5 to 0.02 mg/mL were used in the egg hatch test (EHT) and concentrations of 3.12 to 0.01 mg/mL in the larval development test (LDT). Inhibition concentrations (IC) were determined by the SAS Probit procedure, and significant differences assessed by ANOVA followed by Tukey's test. In the EHT, the IC50 for the susceptible isolate was 5.72 mg/mL. In the LDT, the IC50 and IC90 were, respectively, 0.10 mg/mL and 0.34 mg/mL for the susceptible isolate, and 0.22 mg/mL and 0.51 mg/mL for the resistant isolate. The EO (dillapiole 76.2%) was highly efficacious on phase L1. Due to the higher ICs obtained for the resistant isolate, it was raised the hypothesis that dillapiole may have a mechanism of action that resembles those of other anthelmintic compounds. We further review and discuss studies, especially those conducted in Brazil, that quantified the major constituents of P. aduncum-derived EO.

The present study aimed to evaluate the acaricidal action of the chemically modified essential oil of Cymbopogon spp. and Corymbia citriodora on Rhipicephalus (Boophilus) microplus. Citronellal was converted into N-butylcitronellylamine and in N-prop-2-inylcitronellylamine, analogs of juvenoids, by reductive amination using butylamine (N1 to N3) and propargylamine (N4 to N7). In vitro assays included the adult immersion, and larval packet tests. Engorged females were weighed in groups of 10 and tested in three replicates for six concentrations. They were immersed in the modified oils or control solution and incubated. In the larval packet test, the same substances and concentrations were evaluated in three replicates. In the in vivo test, six pastured heifers naturally infested with R. (B.) microplus were used per treatment: negative control, positive control (amitraz, Triatox(®)), original oil of C. citriodora at 1.5%, and modified oil containing 0.9% N-prop-2-inylcitronellylamine (N7). Ticks were counted in the right side of the body in 24 animals from day D-3 to D21. LC50 and LC90 were obtained by Probit analysis, while the in vivo results were log transformed and compared using the Tukey test. Among the nitrocellylamines tested in vitro, N6 was most effective on the engorged females (100% efficacy at 50mg/mL) and N7 on the larvae (100% efficacy at 6.25mg/mL). In the test with larvae, the original oil of C. citriodora was less effective than the counterpart modified oil (N7), proving that the chemical modification optimized its effect. In the in vivo test, no significant difference was observed between N7 and the negative control. The average numbers of ticks on the animals' right side were 32.8, 8.1, 37.9 and 35.4 for the negative control, positive control, original oil and N7, respectively. The chemical modification improved the efficacy in vitro, but it was not observed in vivo, perhaps due to the low stability of the amines under field conditions. The evaluation carried out here has not been previously studied, so this concept expands the horizon for research into chemically modified substances for parasite control and shed light on the challenges to find effective formulations and application methods., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The development of resistance to anthelmintics has prompted research into alternative methods of controlling intestinal nematodes in ruminants. This study aimed to assess the activity of Ananas comosus on Haemonchus contortus in Santa Inês sheep. The aqueous extract of pineapple skin (AEPS), bromelain from pineapple stems (B4882) and residue from pineapple processing was evaluated in in vitro and in vivo tests. The enzymatic activity of substances was analyzed by the azocasein method. The egg hatch test (EHT) and larval development test (LDT) were performed using the Embrapa2010 isolate of H. contortus. In the in vivo test, 36 sheep artificially infected with H. contortus were divided into six groups: G1: 2 g/kg BW of the aqueous extract administered for three days; G2: 2 g/kg BW of the industrial pineapple residue for 60 days; G3: 180 mg/animal of bromelain in a single dose; G4: negative control I; G5: positive control (levamisole phosphate); and G6: negative control II. The eggs per gram (EPG) in the feces were counted till 28 days after treatment. LC₅₀ and LC₉₀ were obtained by the probit procedure, while the in vivo test results were analyzed by GLM. The aqueous extract in the in vitro and in vivo test, the bromelain and industrial residue presented 0.102, 0.157, 1.864 and 0.048 enzyme units/mL, respectively. In the egg hatch test, the LC₅₀ and LC₉₀ were respectively 31 and 81 mg/mL for the aqueous extract and 0.50 and 2 mg/mL for bromelain. In the larval development test, the LC₅₀ and LC₉₀ were respectively 1.7 and 7.3 mg/mL for the aqueous extract and 0.019 and 0.086 mg/mL for bromelain. In the in vivo test, the general efficacies of the treatments in relation to the negative control were 22.6%, 42.2%, 3.65% and 89% for the aqueous extract, industrial pineapple residue, bromelain and positive control respectively. The transformed EPG values were 3.19 ± 0.59, 3.32 ± 0.25, 2.85 ± 0.66, 3.44 ± 0.50, 2.28 ± 0.93 and 2.75 ± 0.94 for the aqueous extract, industrial residue, bromelain, negative control I, positive control and negative control II respectively. The results for all the treated groups differed significantly (p<0.05) from the positive control, and although the residue presented efficacy of 42.2%, there was no statistical difference (p>0.05) in relation to the negative control. Therefore, both the aqueous extract and bromelain were effective in vitro, but showed reduced anthelmintic efficacy in vivo. For the pineapple residue, the 42.2% in vivo efficacy in reducing the EPG and the possibility of reducing environmental contamination through reuse of industrial residue indicate it can also be useful for control of this parasite., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Measures to control the cattle tick, Rhipicephalus (Boophilus) microplus, based only on chemical products are becoming unsustainable, mainly because of the development of resistance. The objective of this study was to test the effect of the aqueous extract of pineapple skin (AEPS) and bromelain extracted from the stem (Sigma-Aldrich®, B4882) on engorged females and larvae of R. (B.) microplus in vitro. These substances were diluted in water and evaluated at eight concentrations. Engorged females were collected and distributed in groups of 10, with three repetitions for each treatment. After immersion in the solutions, the females were placed in an incubator for observation of survival, oviposition and larval hatching. The larval packet method was used, also with three repetitions with about 100 larvae each. The packets were incubated and the readings were performed after 24 h. The estimated reproduction and efficacy of the solutions were calculated. The LC(50) and LC(90) were estimated using the Probit procedure of the SAS program. The eight concentrations were compared within each treatment by the Tukey test. For the experiment with engorged females, the most effective concentrations were 125, 250 and 500 mg/mL: 33%, 48% and 59% for the AEPS and 27%, 51% and 55% for the bromelain. The LC(50) and LC(90) values were, respectively, 276 and 8691 mg/mL for AEPS and 373 and 5172 mg/mL for bromelain. None of the dilutions tested was effective against the larvae of R. (B.) microplus. This is the first report of the action of pineapple extracts or their constituents on cattle ticks. The results demonstrate that further studies regarding composition of tick cuticle, with evaluation of other solvents and formulations, should be conducted seeking to enhance the effect of pineapple extracts and compounds against this ectoparasite., (Copyright © 2013 Elsevier Inc. All rights reserved.)