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.

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.

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.

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.

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.

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.

Assessment of lipid profile in fat body and eggs of Rhipicephalus microplus engorged females exposed to (E)-cinnamaldehyde and α-bisabolol, potential acaricide compounds.

In the present study, the lipid profile from the fat body and eggs of Rhipicephalus microplus was evaluated after exposure of engorged females to (E)-cinnamaldehyde and α-bisabolol, substances which have acaricide potential according to the literature. Engorged females collected from artificially infested cattle were immersed in a concentration of 10.0 mg/mL of each substance. Dissection of the female fat bodies was performed at different times (72 h and 120 h), for subsequent lipid extraction. In addition, on the fifth day of oviposition, were collected 50.0 ml50.0 mL aliquots of the egg mass of each treatment to perform the same lipid extraction procedure. To assess the lipid profiles, the samples were submitted to the thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GCMS) analysis. Furthermore, an in silico analysis was performed using PASS online® software to predict the possible molecular targets of (E)-cinnamaldehyde and α-bisabolol. As result, the main lipids identified from the fat body were triacylglycerides, fatty acids, and cholesterol, whereas, triacylglycerides (TAG), fatty acids (FA), and cholesterol (CHO) and cholesterol esters (CHOE), were identified in the eggs. The results also showed a significant increase (p < 0.05) of CHO in the fat body in the group exposed to (E)-cinnamaldehyde at 72 h (0.12 µg/fat body) and 120 h (0.46 µg/fat body), in the eggs from females treated with this same substance, there was a significant reduction (p < 0.05) in the amount of CHO (0.21 µg), compared to the water control group (0.45 µg). In the GCMS technique, 5 chemical classes were found, and variations were observed between these substances, mainly hydrocarbons and steroids, in the different groups, and (E)-cinnamaldehyde promoted the greatest changes. From the predictions of the in silico study, 38 and 20 targets were selected, respectively, which are mainly related to alterations in lipid metabolism, immune system and nervous system. This study provides the first report of changes in lipid metabolism of R. microplus exposed to (E)-cinnamaldehyde and α-bisabolol, as well as presenting possible activity on the molecular targets of these substances, expanding knowledge for the potential use of these compounds in the development of botanical acaricides.

Acaricide activity of extract and an isolated compound of Lithraea brasiliensis on Rhipicephalus microplus and selectivity actions against a non-target organism.

Rhipicephalus microplus, known as the cattle tick, is a cause of great economic losses for dairy cattle farming because of its high frequency of occurrence and the difficulty in controlling it. This research characterized the chemical profile and evaluated the in vitro toxicity of crude Lithraea brasiliensis extract and its isolated compound against acaricide-resistant and acaricide-susceptible R. microplus strains. Acaricidal activity was evaluated using a larval immersion test and the selectivity against non-target organisms was assessed on Artemia salina assay. The chemical investigation by high-performance liquid chromatography coupled with mass spectrometry (i.e., HPLC-MS) analysis showed the presence of hydrolysable tannins as well as urushiol derivatives. Column chromatography (CC) was carried out on the extract to obtain fractions and an isolated compound. The extract exhibited significant activity against acaricide-resistant (LC50 0.64 mg/mL) and acaricide-susceptible (LC50 0.76 mg/mL) strains of R. microplus larvae. The isolated compound from the extract (urushiol II), exhibited LC50 of 1.11 mg/mL for acaricide-resistant larvae. For acute toxicity in A. salina, the extract showed LC50>100 µg/mL. Thus, our findings represent the first effort to demonstrate the potential of L. brasiliensis extract and urushiol II as potential natural acaricides to replace or to be integrated into the conventional control of R. microplus larvae.

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 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.

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.

Exposure of Rhipicephalus sanguineus sensu lato Latreille, 1806 (Acari: Ixodidae) to hexane extract of Acmella oleracea (Jambu): semi-engorged and engorged ticks.

The present study analyzed the efficacy of hexane extract of Acmella oleracea against Rhipicephalus sanguineus sensu lato. After exposure of R. sanguineus s. l. female ticks to 14 different concentrations of the hexane extract of A. oleracea, the LC50 was established as 18.135 mg/mL (limits: 16.251-22.675). The effects of the extract were more significant in the ovary of the semi-engorged females, that presented damages in most oocytes. Since such changes would not be repaired, the oocytes could not advance to further stages of development (I-V), thereby causing the inhibition of ovary development, interruption of vitellogenesis, oocyte death and, consequently, infertility. The semi-engorged females also presented extensive damages in the midgut cells, which would prevent (totally or partially) these cells from functioning properly (blood intake and release of nutrients), impairing the ectoparasite nutrition and the viability of the individual. Thus, the hexane extract of A. oleracea can be considered as a promising alternative to chemical control of pests of medical and veterinary importance, due to its efficient acaricidal activity and lesser environmental impact, when used against female ticks in the middle feeding stage.

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus.

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC50 varying from 0.61 to 0.26 mg P mL-1. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.

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.

Effects of essential oils on native and recombinant acetylcholinesterases of Rhipicephalus microplus / Efeito de óleos essenciais sobre acetilcolinesterases nativa e recombinante de Rhipicephalus microplus

Abstract 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.

Resumo Este estudo relata a ação de óleos essenciais de cinco plantas na atividade de acetilcolinesterases (AChE) nativas e recombinantes de Rhipicephalus microplus. A atividade enzimática do extrato de acetilcolinesterase nativa suscetível (S.AChE) e resistente (R.AChE) e da enzima recombinante (rBmAChE1) foi determinada. Um teste de inibição da AChE foi utilizado, para verificar o efeito dos óleos essenciais sobre a atividade enzimática. Óleos essenciais de Eucalyptus globulus, Citrus aurantifolia, Citrus aurantium var. dulcis inibiram a atividade de S.AChE e R.AChE. Os óleos das duas espécies de Citrus inibiram S.AChE e R.AChE de maneira semelhante, mas mostraram maior inibição sobre R.AChE. O óleo de E. globulus inibiu a AChE nativa, mas sem diferença entre a S.AChE e a R.AChE; no entanto, 71% de inibição para rBmAChE1 foi observada. O óleo de Mentha piperita também inibiu S.AChE e R.AChE, mas houve inibição significativa apenas nas concentrações mais altas testadas. O óleo de Cymbopogon winterianus não inibiu a AChE. Estudos adicionais são necessários com os óleos das duas espécies de Citrus que inibiram a R.AchE, devido ao problema de R. microplus resistente aos organofosforados ter como alvo AChE. O óleo de C. winterianus pode ser usado contra populações de R. microplus, que são resistentes a organofosforados, porque suas propriedades acaricidas agem por mecanismos diferentes.

Practices employed by veterinary practitioners for controlling canine gastrointestinal helminths and ectoparasites / Práticas realizadas por veterinários para controle de helmintos gastrintestinais e ectoparasitos de cães

Abstract 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.

Resumo O presente estudo avaliou os métodos de controle empregados por médicos veterinários clínicos para o controle de parasitos de cães no Brasil. Vinte e uma perguntas foram feitas a 403 veterinários de diferentes regiões do país. O uso de associações de compostos ativos em intervalos de três meses foi o mais recomendado para profilaxia de helmintos gastrointestinais, repetido após 15 dias. A profilaxia de rotina contra ectoparasitos foi recomendada por 82,4% dos veterinários, e 46,6% mudam rotineiramente o composto indicado. Tratamentos profiláticos mensais para ectoparasitos, com produtos sistêmicos, tópicos e / ou impregnados com colar, foram recomendados por 21,5% dos veterinários. Os efeitos colaterais das coleiras impregnadas com ectoparasiticidas foram relatados por 58% dos médicos veterinários. As isoxazolinas foram o grupo químico mais utilizado para tratar ectoparasitos em cães. A baixa eficácia do fipronil no controle de carrapatos foi suspeitada por 79,5% dos médicos veterinários. As isoxazolinas e a associação de compostos anti-helmínticos são os medicamentos mais comuns para prevenir ou tratar ectoparasitos e nematoides gastrointestinais, respectivamente. A suspeita da ineficácia dos antiparasitários é compartilhada entre os médicos veterinários de algumas regiões do Brasil. Orientações são necessárias, especificamente para o controle de helmintos e ectoparasitos gastrointestinais em cães no Brasil.