Encapsulation of carvacrol and thymol with yeast cell wall and its repellent activity against Amblyomma sculptum and Rhipicephalus sanguineus (Sensu Lato).

The main way to avoid contact with ticks and consequently tick-borne disease is the use of synthetic repellents. The search of new repellent compounds to increase the possibilities of use in strategies controls are necessary. The present study evaluated the repellent activity of two natural terpenes carvacrol and thymol in each one two different formulation (encapsulated and nonencapsulated with yeast cell wall) against the ticks Amblyomma sculptum and Rhipicephalus sanguineus sensu lato nymphs. Nymphs of A. sculptum and R. sanguineus s.l. of a single generation were used. The vertical filter paper repellency assay were performed with different concentration of both terpenes encapsulated and nonencapsulated in yeast cell wall. The repellent concentration 50% (RC50) were calculated to each compound formulation. Both carvacrol and thymol (encapsulated and nonencapsulated), had a repellent activity against A. sculptum and R. sanguineus s.l nymphs. Amblyomma sculptum was more sensitive to nonencapsulated carvacrol (RC50 values: 0.0032 to 0.0082 mg/cm2 after 1 and 15 min) (P < 0.05), while R. sanguineus s.l. was more sensitive to encapsulated carvacrol (RC50 values: 0.00008 to 0.0035 mg/cm2 after 1 and 15 min) (P < 0.05). Among tick species, R. sanguineus s.l. was more sensitive for most compounds than A. sculptum (P < 0.05). Although with distinct repellent activities, carvacrol and thymol encapsulated can be a promising alternative to synthetic repellents against A. sculptum and R. sanguineus s.l.

Anonaine from Annona crassiflora inhibits glutathione S-transferase and improves cypermethrin activity on Rhipicephalus (Boophilus) microplus (Canestrini, 1887).

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) is one of the most important ectoparasites of cattle, causing severe economic losses in tropical and subtropical regions of the world. The selection of resistance to the most commonly used commercial acaricides has stimulated the search for new products for tick control. The identification and development of drugs that inhibit key tick enzymes, such as glutathione S-transferase (GST), is a rational approach that has already been applied to other parasites than ticks. In this context, alkaloids such as anonaine display several biological activities, including an acaricidal effect. This study aimed to assess the specific inhibition of the R. microplus GST by anonaine, and analyze the effect on ticks when anonaine is combined with cypermethrin. For this purpose, a molecular docking analysis was performed using an R. microplus GST three-dimensional structure model with anonaine and compared with a human GST-anonaine complex. The absorption, distribution, metabolism, excretion, and toxicity properties of anonaine were also predicted. Then, for in vitro analyses, anonaine was isolated from Annona crassiflora (Martius, 1841) leaves. The inhibition of purified recombinant R. microplus GST (rRmGST) by anonaine and the effect of this alkaloid on cypermethrin efficacy towards R. microplus were assessed. Anonaine has a higher affinity to the tick enzyme than to the human enzyme in silico and has moderate toxicity, being able to inhibit, in vitro, rRmGST up to 37.5% in a dose-dependent manner. Although anonaine alone has no activity against R. microplus, it increased the cypermethrin effect on larvae, reducing the LC50 from 44 to 22 µg/mL. In conclusion, anonaine is a natural compound that can increase the effect of cypermethrin against R. microplus.

Effects of Acacia mearnsii supplementation on nutrition, parasitological, blood parameters and methane emissions in Santa Inês sheep infected with Trichostrongylus colubriformis and Haemonchus contortus.

Gastrointestinal nematodes are a main problem for ruminant production, reducing animal performance and increasing environmental impact per unit of animal product generated. Tannin supplementation may lead to positive results regarding aspects such as parasitic infections and methane (CH4) emissions. Therefore, the objective of this experiment was to evaluate the effects of the condensed tannins (CT) extract made of powdered Acacia mearnsii bark (PAB) on nutrition, parasitic status and CH4 emissions in sheep artificially infected with Trichostrongylus colubriformis and Haemonchus contortus. Twenty 10-month old Santa Inês lambs (24.7 ± 3.14 kg of initial body weight) were used in a 50-day trial. Animals were divided in four treatment groups according to parasitic infection and PAB supplementation: two control groups without infections, one without PAB (C-) (n = 4) and one with PAB (C+) (n = 4); two infected groups, one without PAB (I-) (n = 6) and another receiving PAB (I+) (n = 6). Initially, animals were kept in individual pens where they were fed ad libitum chopped tifton 85 hay (Cynodon spp.) and 210 g/animal/day of concentrate. On the first day of experiment, animals of I- and I+ groups were artificially infected with infective larvae (L3) of T. colubriformis and H. contortus. Lambs were weighed fortnightly to calculate average daily body weight gain (ADG). Blood and faeces samples were also collected in the same moment of weighing for the evaluation of blood parameters and faecal egg count (FEC) respectively. After 40 days of experiment, measurements of CH4 emissions in small chamber system started and following that, apparent total tract digestibility (ATTD) assay was carried out in metabolic cages. In the end of experimental period (50 days), lambs were slaughtered and samples of abomasum and small intestine content were collected for worm count, identification, and eggs/female count. No significant (p > 0.05) treatment effects were verified for ADG, ATTD and worm count. Blood parameters were affected in both infected groups (p < 0.05) from the 28th experimental day onwards, when these animals started to show reduced red blood cells, haemoglobin and packed cell volume when compared to C- and C+. Decreased FEC was verified in I+ when compared to I- and also, H. contortus eggs/female worm for I+ was lower than for I- (p < 0.05). Both infected groups showed higher CH4 emissions than the control groups (p < 0.05). Results highlighted the anthelmintic potential of PAB and indicated methanogenic effect of parasitic nematode infections.

Seasonal analysis and acaricidal activity of the thymol-type essential oil of Ocimum gratissimum and its major constituents against Rhipicephalus microplus (Acari: Ixodidae).

The tick Rhipicephalus microplus affects cattle health, with production loss in tropical and subtropical regions. Moreover, the use of commercial acaricides has been reduced due to the resistance of this parasite. Although alternatives such as plant bioactive molecules have been sought, essential oils present variations in their chemical constituents due to environmental factors, which can interfere with their acaricidal activity. The objective of the present study was to evaluate the seasonal influence of the essential oil of Ocimum gratissimum and its major constituents on acaricidal activity against R. microplus larvae. A high-yield essential oil of O. gratissimum and its major constituents were used, and a plant with a thymol-type oil was selected for seasonal analysis and acaricidal activity against R. microplus. Gas chromatography (GC) and GC-mass spectrometry (MS) were employed to identify 31 oil constituents (average yield of 6.26%). The main compounds were found to be thymol (33.4 to 47.9%), γ-terpinene (26.2 to 36.8%), and p-cymene (4.3 to 17.0%). Concerning acaricidal activity, the December (LC50 0.84 mg/mL) and September (LC50 1.58 mg/mL) oils obtained in the dry season were the most active, and assays performed with commercial standards revealed LC50 values of p-cymene, thymol, and γ-terpinene of 1.41, 1.81, and 3.08 mg/mL, respectively. Overall, lower acaricidal activities were found for oils produced from plants harvested in the rainy season. The results showed that seasonal variation in the chemical composition of the O. gratissimum essential oil influences its acaricidal activity. The seasonal variations in the thymol-type essential oil of O. gratissimum can represent an important strategy for the control of R. microplus.

Acaricide activity of different extracts from Piper tuberculatum fruits against Rhipicephalus microplus.

Rhipicephalus microplus is the main tick that affects cattle. Plant bioactive molecules can be used to control this ectoparasite. The aim of this study was to evaluate the in vitro efficacy of Piper tuberculatum fruit extracts obtained with different solvents on R. microplus larvae and engorged females. Hexane, ethyl ether, ethanolic, and methanolic extracts of P. tuberculatum fruits were evaluated. After extraction, all of the extracts were dried. Adult immersion tests and larval packet tests were performed with five different concentrations of each of the extracts. The hexane extracts of P. tuberculatum showed the highest larvicidal activity against R. microplus (lethal concentration (LC50 = 0.04 mg/mL), followed by the ethyl ether (LC50 = 0.08 mg/mL), ethanolic (LC50 = 2.73 mg/mL), and methanolic (LC50 = 4.49 mg/mL) extracts. The P. tuberculatum fruit extracts were also effective against R. microplus-engorged females. Ethyl acetate extracts showed the highest efficiency (LC50 = 18.4 mg/mL), followed by the methanolic (LC50 = 105.6 mg/mL), ethanolic (LC50 = 140.0 mg/mL), and hexane (LC50 = 297.4 mg/mL) extracts. All of the extracts showed similar chromatographic profiles containing 24% piperine. The P. tuberculatum fruit extracts contain bioactive compounds with great potential to improve the standard formulations of acaricides for the control of R. microplus.

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.

An alkaloidal fraction of Annona tomentosa possesses anthelmintic activity and induces surface modifications in Haemonchus contortus.

The nematode Haemonchus contortus is, as a parasite, responsible for most mortality of small ruminants, causing significant economic losses. Numerous plant-derived compounds have exhibited promising anthelmintic activities against this nematode. Notably, the Annona genus stands out for demonstrated anthelmintic effects by extracts from several of its species against different nematodes. This study aimed to assess the effect of an Annona tomentosa fraction, rich in alkaloids, on H. contortus. This fraction, named Alk.F, is derived from the methanolic extract of the plant's stem bark. Chemical characterization of Alk.F was performed by liquid chromatography coupled with mass spectrometry. Among the nine predominant peaks obtained, seven alkaloids were identified: reticuline, reticuline N-oxide, reticuline N-oxide isomer, cyclanoline, asimilobine, tetrahydropalmatine and anonaine. Alk.F inhibited the larval development of H. contortus with an IC50 of 0.026 mg/mL, inhibited larval exsheathment with an IC50 of 0.38 mg/mL, and displayed low hemolytic activity towards sheep erythrocytes. Furthermore, atomic force microscopy revealed that Alk.F altered adhesive forces and the height profile on the surface of H. contortus larvae. In conclusion, A. tomentosa alkaloids alter the cuticle structure of H. contortus, inhibiting larval development and exsheathment, thus offering possibilities for contributing to the development of new anthelmintic drugs.

In silico and In vitro Assessment of Dimeric Flavonoids (Brachydins) on Rhipicephalus microplus Glutathione S-transferase.

INTRODUCTION: Rhipicephalus microplus, an important cattle ectoparasite, is responsible for a substantial negative impact on the economy due to productivity loss. The emergence of resistance to widely used commercial acaricides has sparked efforts to explore alternative products for tick control. METHOD: To address this challenge, innovative solutions targeting essential tick enzymes, like glutathione S-transferase (GST), have gained attention. Dimeric flavonoids, particularly brachydins (BRAs), have demonstrated various biological activities, including antiparasitic effects. The objectives of this study were to isolate four dimeric flavonoids from Fridericia platyphylla roots and to evaluate their potential as inhibitors of R. microplus GST. RESULTS: In vitro assays confirmed the inhibition of R. microplus GST by BRA-G, BRA-I, BRA-J, and BRA-K with IC50 values of 0.075, 0.079, 0.075, and 0.058 mg/mL, respectively, with minimal hemolytic effects. Molecular docking of BRA-G, BRA-I, BRA-J, and BRA-K in a threedimensional model of R. microplus GST revealed predicted interactions with MolDock Scores of - 142.537, -126.831, -108.571, and -123.041, respectively. Both in silico and in vitro analyses show that brachydins are potential inhibitors of R. microplus GST. CONCLUSION: The findings of this study deepen our understanding of GST inhibition in ticks, affirming its viability as a drug target. This knowledge contributes to the advancement of treatment modalities and strategies for improved tick control.

Phenotypic and genotypic characterization of acaricide resistance in Rhipicephalus microplus field isolates from South Africa and Brazil.

Rhipicephalus (Boophilus) microplus is one of the most successful ticks infesting cattle around the world. This highly-invasive species transmits cattle parasites that cause cattle fever leading to a high socio-economic burden. Tick eradication programs have often failed, due to the development of acaricide resistance. Here we characterize acaricide resistance in a large number of tick isolates from regions in South Africa (KwaZulu Natal, Mpumalanga, Western & Eastern Cape provinces) and two Brazilian regions. By means of Larval Packet Tests (LPT's) acaricide resistance was evaluated against five commonly used acaricides (chlorfenvinphos, fipronil, deltamethrin, amitraz, and ivermectin). Furthermore, the coding region containing the knock down resistance (kdr) mutation, known to result in pyrethroid resistance, was sequenced. Resistance to at least one acaricide class was reported in each of the five regions, and a high proportion of tick isolates exhibited multi-resistance to at least two acaricide classes (range: 22.2-80.0%). Furthermore, resistance ratios (RR) showed high spatial variation (intercontinental, as well as regional) but low regional spatial autocorrelation. Previous and current acaricide use correlated with current RR, and several combinations of acaricide RR were positively correlated. Moreover, fipronil resistance tended to be higher in farms with more intense acaricide use. The kdr-mutations provided the ticks a fitness advantage under the selection pressure of synthetic pyrethroids based on population (kdr-allele frequency) and individual level data (genotypes). The data show the threat of acaricide (multi-)resistance is high in Brazil and South Africa, but acaricide specific levels need to be assessed locally. For this purpose, gathering complementary molecular information on mutations that underlie resistance can reduce costs and expedite necessary actions. In an era of human-caused habitat alterations, implementing molecular data-driven programs becomes essential in overcoming tick-induced socio-economic losses.

Phytochemicals in Gastrointestinal Nematode Control: Pharmacokinetic-Pharmacodynamic Evaluation of the Ivermectin plus Carvone Combination.

A wide variety of plant-derived phytochemicals with anthelmintic effects have been described. Most of them have shown activity against parasites in vitro but have not been extensively explored in vivo. The aim of the current work was to study the pharmacokinetic-pharmacodynamic relationship of the combined administration of carvone (R-CNE) and ivermectin (IVM) to lambs. Three trials were conducted to evaluate the pharmacological interaction between R-CNE and IVM in lambs infected with resistant nematodes. Drug concentrations were measured in plasma, target tissues, and H. contortus by HPLC with fluorescent (IVM) and ultraviolet (R-CNE) detection. The effect of both compounds on parasites was estimated by the fecal egg count reduction. Coadministration with R-CNE significantly increased the plasma bioavailability of IVM. R-CNE showed a moderate anthelmintic effect, which was greater on the susceptible isolate of H. contortus. After the combination of R-CNE and IVM as an oral emulsion, both compounds were quantified in H. contortus recovered from infected lambs. However, R-CNE concentrations were much lower than those reported to achieve anthelmintic effects in the in vitro assays. Optimization of the pharmaceutical formulation, dose rate, and administration schedule is needed to take advantage of the intrinsic anthelmintic activity of phytochemicals.

Efficacy of lotilaner against myiasis caused by Cochliomyia hominivorax (Diptera: Calliphoridae) in naturally infested dogs.

BACKGROUND: The New World screwworm fly, Cochliomyia hominivorax, is widely distributed across South America. This parasitic insect is a significant cause of primary myiasis in animals, including dogs. There is an urgent need for a rapid and efficient treatment to improve the recovery of affected animals. In the present study we evaluated the potential of lotilaner for the treatment of myiasis caused by C. hominivorax larvae in naturally infested dogs. Lotilaner belongs to the isoxazoline class of chemical compounds and is marketed as Credelio™ for use against ticks and fleas in dogs and cats. METHODS: Eleven dogs with naturally acquired myiasis were enrolled in this study based on the severity of lesions and the number of identified larvae. All animals received a single oral administration of lotilaner at a minimum dose of 20.5 mg/kg body weight. After treatment, the number of expelled larvae, live or dead, was determined at 2, 6 and 24 h, and the larval expulsion rate, larvicidal effect and overall efficacy were calculated. After 24 h, the remaining larvae were removed, counted and identified. The lesions were cleaned, and palliative treatment was administered when necessary, according to the animal's health status. RESULTS: All larvae were identified as C. hominivorax. The larval expulsion rate was 80.5% and 93.0% at 2 and 6 h post-treatment, respectively. Lotilaner showed an overall efficacy of 100% at 24 h post-treatment. CONCLUSIONS: Lotilaner demonstrated a rapid onset of action and a high efficacy against C. hominivorax. We therefore recommend lotilaner for the effective treatment of myiasis in dogs.

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.

Seasonal dynamics of Amblyomma cajennense (Fabricius, 1787) sensu stricto in a degraded area of the Amazon biome, with notes on Rickettsia amblyommatis infection.

BACKGROUND: The tick Amblyomma cajennense sensu stricto (A. cajennense s.s.) frequently parasitizes animals and humans in the Amazon biome, in addition to being a vector of Rickettsia amblyommatis. In the present study, we evaluated both the population dynamics of A. cajennense s.s. in a degraded area of the Amazon biome and the presence of rickettsial organisms in this tick population. METHODS: The study was carried out in a rural area of the Santa Inês municipality (altitude: 24 m a.s.l.), Maranhão state, Brazil. Ticks were collected from the environment for 24 consecutive months, from June 2021 to May 2023. The region is characterized by two warm seasons: a rainy season (November-May) and a dry season (June-October). We characterized the temporal activity of A. cajennense s.s. on the vegetation by examining questing activity for each life stage (larvae, nymphs, adults [males and females]) in relation to the dry and rainy season. Ticks collected in this study were randomly selected and individually tested by a TaqMan real-time PCR assay that targeted a 147-bp fragment of the rickettsial gltA gene. RESULTS: Overall, 1843 (62.4%) adults (52.6% females, 47.4% males), 1110 (37.6%) nymphs and 398 larval clusters were collected. All adult females and nymphs were morphologically identified as A. cajennense s.s. Larval activity was observed from April to December, with a peak from June to September (dry season); nymph abundance peaked from September to November (transition period between dry and rainy seasons); and adult ticks were abundant from October to May (spring/summer/early autumn). The infection rate by R. amblyommatis in A. cajennense s.s. ticks was at least 7% (7/99). CONCLUSION: Our data suggest a 1-year generation pattern for A. cajennense s.s., with a well-defined seasonality of larvae, nymphs and adults in the Amazon biome. Larvae predominate during the dry season, nymphs are most abundant in the dry-rainy season transition and adults are most abundant in the rainy season. The presence of R. amblyommatis in adult ticks suggests that animals and humans in the study region are at risk of infection by this species belonging to the spotted fever group of Rickettsia.

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.

Pharmacological characterization of geraniol in sheep and its potential use in the control of gastrointestinal nematodes.

Geraniol (GNL) was effective against gastrointestinal nematodes in vitro; nevertheless, the anthelmintic effect of phytochemicals combined with synthetic drugs has been little explored in vivo. This article characterized in vitro / in vivo the pharmacological features of GNL in sheep as well as its pharmacokinetic interaction with albendazole (ABZ). Additionally, the in vivo efficacy of GNL against Haemonchus contortus was evaluated in lambs. Liver microsomes from lambs were incubated in the absence or presence of GNL to analyze CYP1A1, CYP1A2 and FMO metabolic pathways. The effect of GNL on the hepatic sulfoxidation and sulfonation of ABZ and the ruminal sulforeduction of albendazole sulfoxide (ABZSO) was assessed. The in vivo pharmacokinetic interaction of ABZ and GNL was evaluated in lambs. The effect of GNL on the fecal egg count was evaluated in lambs infected with a resistant isolate of H. contortus. In sheep liver microsomes, the presence of 2 mM GNL reduced the CYP1A1, CYP1A2 and FMO pathways by 77.9, 90.8 and 84.5%, respectively, with respect to control (P < 0.05). In the presence of 2 mM GNL, the ABZ sulfoxidation decreased from 114.4 ± 8.49 (control) to 50.24 ± 11.1 nmol/min.mg, and ABZSO2 production decrease from 0.52 ± 0.14 to 0.09 ± 0.03 nmol/h.mg. No changes in the pharmacokinetic behavior of ABZ were observed in the presence of GNL. The in vivo efficacy of four doses of GNL was 40.5%. These findings highlight the importance of integrated in vitro / in vivo pharmaco-parasitological studies to develop new pharmacological tools for controlling gastrointestinal parasites.

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.

Inhibition of Protease and Egg Hatching of Haemonchus contortus by Soybean Seed Exudates.

Gastrointestinal nematode infection of small ruminants causes losses in livestock production. Plant compounds show promises as alternatives to commercial anthelmintics that have been exerting selective pressures that lead to the development of drug-resistant parasites. Soybean (Glycine max) is an economical value crop, with a higher protein content compared to other legumes. The objective of this study was to evaluate whether the protease inhibitors exuded from the G. max mature seeds have anthelmintic activity against Haemonchus contortus. To obtain the soybean exudates (SEX), mature seeds were immersed in 100 mM sodium acetate buffer, pH 5.0, at 10 C, for 24 hr. Then the naturally released substances present in SEX were collected and exhaustively dialyzed (cutoff 12 kDa) against distilled water. The dialyzed seed exudates (SEXD) were heated at 100 C for 10 min and centrifuged (12,000 g, at 4 C for 15 min). The supernatant obtained was recovered and designated as the heat-treated exudate fraction (SEXDH). The protein content, protease inhibitor activity, and the effect of each fraction on H. contortus egg hatch rate were evaluated. The inhibition extent of SEX, SEXD, and SEXDH on H. contortus egg proteases was 31.1, 42.9, and 63.8%, respectively. Moreover, SEX, SEXD, and SEXDH inhibited the egg hatching with EC50 of 0.175, 0.175, and 0.241 mg ml-1, respectively. Among the commercial protease inhibitors tested, only EDTA and E-64 inhibited the H. contortus hatch rate (79.0 and 28.9%, respectively). We present evidence demonstrating that soybean exudate proteins can effectively inhibit H. contortus egg hatching. This bioactivity is displayed by thermostable proteins and provides evidence that protease inhibitors are a potential candidate for anthelmintic use.

Practices employed by veterinary practitioners for controlling canine gastrointestinal helminths and ectoparasites.

The present study attempted to evaluate the practical experience and methods employed by Brazilian veterinary practitioners for control of parasites. Twenty-one questions were asked of 403 veterinary practitioners based in different climatic zones with reference to parasite epidemiology from the country. Administration of a combination of drugs at three-month intervals was the most common regime recommended for prophylaxis against gastrointestinal helminths, with a single treatment repeated after 15 days. Routine prophylaxis against dog ectoparasites was recommended by 82.4% veterinary practitioners, and 46.6% changed the drug compound used. Monthly prophylaxic treatments for ectoparasites, using systemic, topical and/or collar-impregnated drugs, was recommended by 21.5% veterinary practitioners. Side-effects of ectoparasiticide-impregnated collars were suspected by 58% of the veterinary practitioners. Isoxazolines were the most frequently used chemical group to treat ectoparasites in dogs. Poor efficacy of fipronil in controlling ticks was suspected by 79.5% of the veterinary practitioners. The isoxazolines and combination of anthelmintic compounds are the most common drugs to prevent or treat ectoparasites and gastrointestinal nematodes, respectively. The suspect of the inefficacy of antiparasitic drugs is shared among the veterinary practitioners from part of Brazil. Guidelines are needed, specifically for the control of gastrointestinal helminths and ectoparasites in Brazilian dogs.

Effects of essential oils on native and recombinant acetylcholinesterases of Rhipicephalus microplus.

This study reports the action of essential oils (EO) from five plants on the activity of native and recombinant acetylcholinesterases (AChE) from Rhipicephalus microplus. Enzyme activity of native susceptible AChE extract (S.AChE), native resistant AChE extract (R.AChE), and recombinant enzyme (rBmAChE1) was determined. An acetylcholinesterase inhibition test was used to verify the effect of the EO on enzyme activity. EO from Eucalyptus globulus, Citrus aurantifolia, Citrus aurantium var.dulcis inhibited the activity of S.AChE and R.AChE. Oils from the two Citrus species inhibited S.AChE and R.AChE in a similar way while showing greater inhibition on R.AChE. The oil from E. globulus inhibited native AChE, but no difference was observed between the S.AChE and R.AChE; however, 71% inhibition for the rBmAChE1 was recorded. Mentha piperita oil also inhibited S.AChE and R.AChE, but there was significant inhibition at the highest concentration tested. Cymbopogon winterianus oil did not inhibit AChE. Further studies are warranted with the oils from the two Citrus species that inhibited R.AChE because of the problem with R. microplus resistant to organophosphates, which target AChE. C. winterianus oil can be used against R. microplus populations that are resistant to organophosphates because its acaricidal properties act by mechanism(s) other than AChE inhibition.

Effects of acaricidal essential oils from Lippia sidoides and Lippia gracilis and their main components on vitellogenesis in Rhipicephalus microplus (Canestrini, 1888) (Acari: Ixodidae).

Rhipicephalus microplus is an important cattle tick, and resistant strains to synthetic compounds have been widespread. The combined effects of different essential oil compounds enhance biological activity and reduce selection for the development of target organism resistance. Essential oils of two different genotypes of each of Lippia sidoides and Lippia gracilis and their main components, the isomers thymol and carvacrol, have acted as acaricides against R. microplus. Little is known about the effects of the essential oils of L. sidoides and L. gracilis and thymol and carvacrol on the morphophysiology of R. microplus ovaries. This study aimed to identify the morphological changes in the ovaries of R. microplus females treated with essential oils from two different genotypes of each of L. sidoides (102 and 103) and L. gracilis (106 and 201) and the terpenes thymol and carvacrol through histological techniques. The LC50 and LC75 of essential oils and thymol and carvacrol were used for Adult Immersion Test (AIT) with groups of five fully engorged females of R. microplus. A negative control (DMSO 3% solution) was performed. Seven days after the AIT, the ticks were dissected to collect ovaries and their histologic analysis. Only the group treated with the essential oil of L. gracilis genotype 106 at the LC50 had no change compared with the control. The other groups showed the following changes in oocytes I to V: vacuolation, chorion deformation, disorganization of yolk granules, and irregularities at the cell periphery, causing incomplete process of vitellogenesis. Thus, the essential oils tested in this study may be potent products for the control of cattle ticks and thereby preventing further life cycles.