Seasonal and circadian evaluation of Ageratum conyzoides essential oil and its nematicidal activity against Caenorhabditis elegans.

The aim of this study was to evaluate the circadian and seasonal variation of Ageratum conyzoides essential oil (EO) and its nematicidal effect on the free-living nematode Caenorhabditis elegans as a model for parasitic helminths. For the seasonal study, the plants were collected from January to December 2022, at 6 a.m., and to assess the circadian rhythm, the plants were collected in April (rainy season) and October (dry season), at 6, 9, 12 a.m. and 3 and 6 p.m. The fresh plants were then subjected to hydrodistillation, and their chemical composition was analyzed using gas chromatography coupled with mass spectrometry (GC-MS). The motility test with C. elegans was carried out. The primary constituent of the oils was precocene I (65.97 to 78.42 %, respectively), followed by E-caryophyllene (6.04 to 12.16 %), comprising an average of 79.87 % of the composition throughout the year. The average yields of EOs were slightly higher in the rainy season, at 0.68 %, compared to the dry season, at 0.62 %. High light hours in the rainy season (12 a.m., 0.96 %) and in the dry season (9 a.m., 0.88 %) seem to contribute to higher daily oil yields. It was observed that the variation between the main constituents of A. conyzoides occurs in inverse proportion when analyzing the main classes of compounds present in the oils: chromenes (CH) and sesquiterpene hydrocarbons (SH). And that the month of March had the highest content of E-caryophyllene (12.16 %) when compared to the other months of the year. On the other hand, January and December had the lowest levels of precocene I (65.97 and 66.85 %). The IC50 of the EO of A. conyzoides varied according to the month and time of collection. The EO obtained in January was the most effective against C. elegans, with an IC50 of 0.01 mg/mL. Thus, A. conyzoides EO could be an alternative for nematode control, exhibiting greater efficacy if extracted during specific seasonal periods.

Nemabiome metabarcoding shows a high prevalence of Haemonchus contortus and predominance of Camelostrongylus mentulatus in alpaca herds in the northern UK.

Gastrointestinal nematodes (GINs) are a common threat faced by pastoral livestock. Since their major introduction to the UK in the early 1990s, South American camelids have been cograzed with sheep, horses, and other livestock, allowing exposure to a range of GIN species. However, there have been no molecular-based studies to investigate the GIN populations present in these camelids. In the current study, we sampled nine alpaca herds from northern England and southern Scotland and used high-throughput metabarcoded sequencing to describe their GIN species composition. A total of 71 amplicon sequence variants (ASVs) were identified representing eight known GIN species. Haemonchus contortus was the most prevalent species found in almost all herds in significant proportions. The identification of H. contortus in other livestock species is unusual in the northern UK, implying that alpacas may be suitable hosts and potential reservoirs for infection in other hosts. In addition, the camelid-adapted GIN species Camelostrongylus mentulatus was identified predominantly in herds with higher faecal egg counts. These findings highlight the value of applying advanced molecular methods, such as nemabiome metabarcoding to describe the dynamics of gastrointestinal nematode infections in novel situations. The results provide a strong base for further studies involving cograzing animals to confirm the potential role of alpacas in transmitting GIN species between hosts.

Biology of the non-parasitic phase of the cattle tick Rhipicephalus (Boophilus) microplus in an area of Amazon influence.

BACKGROUND: Rhipicephalus (Boophilus) microplus is the most important tick species affecting cattle in the world. Under field conditions, the non-parasitic phase of R. (B.) microplus is unknown in the Amazon biome, including Brazil. The present study aimed to evaluate the non-parasitic phase of R. (B.) microplus in field (grass plots) and laboratory conditions. METHODS: The study was conducted from September 2020 to April 2022 in an Amazonian region (Maranhão State, Brazil). We evaluated the biological parameters of R. (B.) microplus under laboratory and field conditions. Engorged females were exposed to experimental conditions every 14 days, totaling 20 months of study. The following biological parameters were observed: pre-oviposition period, egg mass incubation period, and maximum larval survival period. RESULTS: Abiotic data (e.g., temperature and humidity) varied little throughout the year. Precipitation was the factor that varied the most throughout the year (dry ~ 30 mm3 and rain 400 mm3), and the parameters of pre-oviposition and pre-hatching are longer during the rainy season. A possible negative effect of the dry season on the percentage of hatched eggs was observed. Larval longevity in the plots of both control and free females was short (mean ~ 50-60 days), below that recorded for larvae under controlled conditions (mean ~ 95 days). CONCLUSIONS: Rhipicephalus (Boophilus) microplus was able to complete its non-parasitic phase by producing host-seeking larvae in the pasture during all months of the study. The results indicate that R. (B.) microplus can complete up to six generations per year in biome Amazon. To our knowledge, this is the highest number of annual generations for R. (B.) microplus in Latin America.

Polymeric films of corn starch enhance the lethal effects of thymol and carvacrol terpenes upon Rhipicephalus microplus ticks.

The tick Rhipicephalus microplus is a parasite of great importance in cattle breeding. It is responsible for huge economic losses. The application of synthetic acaricides is used as a form of control. However, resistant strains have been selected over the years, making it necessary to search for new alternative formulations. The present study aimed to formulate biodegradable films impregnated with the terpenes carvacrol and thymol and evaluate their efficacy on larvae and adults of R. microplus through in vitro tests. The following formulations were prepared: Film 1 (starch based); Film 2 (based on starch and glycerol); Film 1 + Carvarcol or Thymol; Film 2 + Carvarcol or Thymol. Terpenes had a final concentration of 5.0 mg/mL. To evaluate the formulations on larvae, the immersion test was performed by dividing into six groups according to the concentration of terpenes: 5.0, 2.5, 1.25, 0.625, 0.313, 0.156 mg/mL and the control groups: 1% ethanol solution; 10% ethanol solution; Film 1; and Film 2. For the evaluations on adult ticks, ten experimental groups (n = 10) were used: 1) Carvacrol; 2) Film 1 + Carvacrol; 3) Film 2 + Carvacrol; 4) Thymol; 5) Film 1 + Thymol; 6) Film 2 + Thymol; 7) Distilled water; 8) 10% ethanol solution; 9) Film 1; and 10) Film 2. In experimental groups 1-6, carvacrol and thymol (free or incorporated in two different biodegradable film formulations) were evaluated at the same concentration (5.0 mg/mL). Each group of ticks was immersed in their respective solutions for five minutes. The results of the tests on larvae showed that the Film 1 + thymol and Film 2 + carvacrol formulations had the lowest lethal concentrations (0.076 and 0.255 mg/mL, respectively), values up to 9.0-fold lower than the monoterpenes tested outside the formulation. Carvacrol and thymol at the concentrations tested were effective in controlling engorged females with a percentage of 32.2% and 63.8%, respectively. When incorporated into biodegradable film formulations, these monoterpenes showed much greater efficacy. Film 1 + carvacrol and Film 2 + carvacrol with control percentages of 71.6% and 97.2%, respectively, while the formulations Film 1 + thymol and Film 2 + thymol showed values of 96.9% and 100.0%. The tick control activity of the biopolymer formulations with thymol and carvacrol was demonstrated through the high mortality rates of larvae and engorged females of the tick R. microplus. Therefore, the results obtained indicate that these formulations have great potential for tick control mainly because of the percentage of control up to 100% in engorged females in in vitro tests.

Does parity order or cow antibody level influence the humoral immunity of calves for cattle anaplasmosis?

Anaplasmosis is a widely distributed vector-borne disease of cattle caused by the bacteria Anaplasma marginale, which may lead to severe losses in beef and dairy cattle production. Anecdotal information among farmers suggested that some cows may deliver calves more resilient to anaplasmosis. To investigate this, we tested two hypotheses: (i) whether the parity order or (ii) cow antibody levels may influence the humoral immunity of pre-weaning calves against cattle anaplasmosis. For this study, we sampled 170 cattle (Bos taurus taurus, Angus breed) on a farm in Southern Brazil, comprising 85 cows (50 multiparous and 35 primiparous) and their 85 calves (50 days old). Antibodies against A. marginale were investigated using iELISA. Eighty percent of the animals were considered seropositive (100% of the cows and 60% of the calves). There was no significant difference in seroprevalence between calves from primiparous and multiparous cows. However, calves from multiparous cows exhibited higher antibody levels. To address the second question, we classified cows based on their antibody levels to A. marginale (high or low). Calves from cows with high antibody levels also showed elevated antibody levels against A. marginale. Furthermore, calves from cows with high antibody levels had approximately four times greater odds of being seropositive for A. marginale at 50 days old than those born to mothers with low antibody levels. In conclusion, the calf's antibody level against A. marginale appears to be related to the cow's parity order and the mother's antibody level.

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

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

Essential oil Ageratum conyzoides chemotypes and anti-tick activities.

Rhipicephalus (Boophilus) microplus represents a significant obstacle to animal productivity in tropical and subtropical areas, leading to considerable economic losses for the dairy and meat production industries. Essential oils (EO) extracted from Ageratum conyzoides are known to cause death and induce morphogenetic abnormalities in several insect species. This plant, however, presents morphological flower variations, which range from white to purple, associated to different chemotypes. In this context, this study aimed to conduct a novel assessment on the effects of EO extracted from two A. conyzoides chemotypes in the control of the bovine tick R. microplus. The primary constituents of the oil obtained from white flower samples (WFs) were precocene I (80.4 %) and (E)-caryophyllene (14.8 %), while purple flower oil samples (PFs) contained predominantly ß-acoradiene (12.9 %), γ-amorphene (12.3 %), α-pinene (9.9 %), bicyclogermacrene (8.9 %), α-santalene (8.7 %), and andro encecalinol (5.6 %). Interestingly, only the EO chemotype from A. conyzoides PFs displayed acaricidal activity towards R. microplus larvae, with an LC50 of 1.49 mg/mL.

Evaluation of a combination of Citrus aurantium var. Dulcis essential oil and albendazole for the treatment of sheep gastrointestinal nematodes.

Citrus fruits are consumed all over the world and their by-products are used for animal feed and essential oils production. This study aimed to evaluate the in vitro and in vivo activity of Citrus aurantium var. Dulcis essential oil (CaEO) combined with ABZ against benzimidazole resistant Haemonchus contortus. In vitro egg hatching assays (EHA) were performed using CaEO and ABZ to estimate the effective concentration to achieve 50% egg death (EC50) values and calculate the test essential oil and drug combinations using a simplex-centroid mixture design. These concentrations were used for a second round of EHAs. Sixteen sheep were randomly allocated into two groups and treated with ABZ and the combination of CaEO and ABZ, and faecal egg count reduction tests were performed. In the first round of EHA, CaEO and ABZ showed EC50 values of 0.57 and 0.0048 mg mL-1, respectively. The H. contortus strain used in the study was shown to be highly benzimidazole resistant, with only 1.5% of parasites having susceptible ß-tubulin SNP genotypes. The ABZ reduced the shedding of nematode eggs by 78%, however, its combination with CaEO reduced faecal egg counts by only 9%. The present study is important to highlight the interferences of natural products in anthelmintic metabolism and consequently in drug efficacy.

In vitro assessment of the acaricidal activity of a carvacrol shampoo on tick larvae.

Ticks are a widely distributed arthropod of veterinary importance. Resistance of ticks to synthetic acaricides has become widespread, warranting the development of new drugs for tick management. Carvacrol is a volatile monoterpene, with promising results against various species of ticks; however, to be used for therapeutic purposes, carvacrol must be included in a formulation that makes its application feasible. This study aims to develop a formulation of a carvacrol-containing shampoo that is effective against two species of ticks: Rhipicephalus sanguineus and R. microplus. Shampoo sensory characteristics and pH were evaluated at 37, 25 and 5 °C, for a maximum of 15 days. The shampoo remained stable at 25 and 5 °C. The efficacy of the carvacrol-containing formulation against two species of ticks was assessed by the larval immersion test. Mortality of both tick species was significantly higher for the carvacrol shampoo than for a carvacrol hydroalcoholic solution. In conclusion, the carvacrol-containing shampoo showed larvicidal efficacy on ticks.

Records and altitudinal assessment of Amblyomma aureolatum and Amblyomma ovale (Acari: Ixodidae) in the State of Rio de Janeiro, southeast Brazil.

Information on the altitudinal distribution of the hard ticks Amblyomma aureolatum and Amblyomma ovale in Brazil is scarce and mainly limited to occasional records. In this study we report our evaluation of records on the altitudinal distribution of A. aureolatum and A. ovale collected from dogs and humans and directly from the environment (host-questing ticks), based on active or passive procedures. The collections were conducted in rural areas of municipalities in the state of Rio de Janeiro, Brazil between 2013 and 2017. Active procedures consisted of dragging or flagging, visual examination of vegetation and removal of ticks present on the authors' clothing or on infested dogs. Overall, 222 ticks were collected. The altitudes at the collection sites ranged from 98 to 1220 m a.s.l. We noted a significant difference in the altitudinal distribution of A. aureolatum and A. ovale (Mann-Whitney U-test, U = 518.5, P < 0.001). The overlap of these two species occurred at altitudes of between 650 and 900 m a.s.l. The results indicated that the higher the altitude, the greater the probability for the occurrence of A. aureolatum and, conversely, the lower the likelihood for the occurrence of A. ovale. The findings of this study improve currrent knowledge on the bioecology of these tick species and have implications for studies on the epidemiology of spotted fever in Brazil.

A loop-mediated isothermal amplification (LAMP) assay to identify isotype 1 ß-tubulin locus SNPs in synthetic double-stranded Haemonchus contortus DNA.

Development of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 ß-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 ß-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 ß-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 ß-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-021-01414-w.

Effects of carvacrol and thymol on the antioxidant and detoxifying enzymes of Rhipicephalus microplus (Acari: Ixodidae).

The present study was conducted to evaluate the effects of carvacrol and thymol on the antioxidant and detoxifying enzymes of larvae from two populations of R. microplus: Jaguar (tick population resistant to six classes of acaricides) and Porto Alegre (susceptible tick population). Carvacrol and thymol were tested at concentrations ranging from 0.14 to 5.0 mg mL-1 in both populations to determine the LC50. In addition, the LC1, LC25, and LC75 were estimated using the LC50 and HillSlope of each compound. Larvae of both populations of R. microplus were then treated with the LC1, LC25, LC50, and LC75 of each monoterpene, and those that survived were processed to evaluate the effects of the compounds on the antioxidant and detoxifying systems of larvae; these effects were assessed by determining the activity of the enzymes, glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX). Larvae from the Jaguar population treated with different lethal concentrations of carvacrol and thymol displayed a dose-dependent increase in CAT, GPX, SOD, and GST after treatment with LC25. Further, larvae treated with the LC75 had the highest levels of enzyme activity for carvacrol (1.76 mg mL-1) and thymol (1.32 mg mL-1). CAT, GPX, SOD, and GST activity in Porto Alegre population larvae treated with carvacrol and thymol also increased significantly up to the LC50 of each monoterpene. However, at the LC75 of carvacrol and thymol, a decrease in the activity of all enzymes was observed for this tick population. These findings indicate that carvacrol and thymol induced increased activity of all evaluated enzymes at different lethal concentrations in R. microplus larvae from two populations. Such findings unveil the possible mechanisms of action of these natural acaricides.

Development and validation of software that quantifies the larval mortality of Rhipicephalus (Boophilus) microplus cattle tick.

Rhipicephalus (Boophilus) microplus is controlled almost exclusively using synthetic acaricides, and reports of resistant populations have been described worldwide. Several time-consuming and laborious toxicological in vitro tests have been used to diagnose acaricidal resistance, especially those that require differential counting of live and dead larvae. Larval mortality is currently done manually and subjectively, which can limit the performance of a large number of tests and comparing results between different laboratories. The present study aimed to develop and validate a new automatic counting method to evaluate tick larval mortality. A software for differentiation of live and dead larvae was developed using different steps: obtaining videos; image segmentation using the firefly algorithm; detection of larvae with the fast radial symmetry transform technique (FRST); and tracking of the larvae at a given time. Larval immersion tests with ivermectin, cypermethrin, and fipronil were performed to validate the developed software. The larval mortality evaluation was performed by (1) recording for 60 s for each package and (2) manual counts of the same sample using three different analysts, each responsible for counting one replicate of each test. All videos obtained were copied and cut at 60, 40, and 20 s for later analysis in the counting software. The median lethal doses (LD50) of the different compounds in each test were calculated for each method (automatic and manual) for different video times. There was no statistical difference in LD50 between manual and automatic count techniques for ivermectin and fipronil. The LD50 of cypermethrin calculated with manual evaluation was up to 2.2 times lower than that of automatic evaluation. The acquisition time of the videos was 2.9-4.4 times faster than the manual evaluation. The average processing time for each video was 5.73 min, regardless of their duration. Thus, the method developed for automatic counting of tick larvae was validated, and although it still has points to be optimized, it can be considered a viable alternative for determining the percentage of tick larvae mortality and could be applied to toxicological in vitro tests with acaricides, assisting in the diagnosis of resistant tick populations and studies of novel acaricide development.

Use of agro-industrial by-products containing tannins for the integrated control of gastrointestinal nematodes in ruminants.

Previous studies have illustrated that different bioactive legume fodders containing condensed tannins might represent one of the options for integrated sustainable control of gastrointestinal nematodes (GIN) in ruminants, which may help address the worldwide development of resistance to synthetic anthelmintics. More recently, impetus has been given to assess the potential antiparasitic activity of less conventional resources, represented by different agro-industrial by-products (AIBPs). This review presents in vitro and in vivo results obtained with a range of tannin-containing AIBPs of various geographical and botanical origins, namely AIBP of nuts, temperate and tropical barks, carob, coffee and cocoa. They tend to confirm the "proof of concept" for their antiparasitic effects and also for other aspects of ruminant production in an agro-ecological context. Socio-economic aspects of the exploitation of such non-conventional resources are also discussed as potential models of the circular economy, by using waste. The different modes of use of these resources are presented in this review, as well as strengths, weaknesses, opportunities, and threats (SWOT) analyses to illustrate the advantages and limitations of on-farm use.

TITLE: Utilisation de sous-produits agro-industriels contenant des tanins pour le contrôle intégré des nématodes gastro-intestinaux chez les ruminants. ABSTRACT: Plusieurs études antérieures ont illustré le fait que des légumineuses bioactives contenant des tannins condensés peuvent représenter une des alternatives à intégrer avec d'autres options pour une maitrise durable des nématodes gastro-intestinaux en réponse au développement constant et à l'expansion continue à l'échelle mondiale des résistances aux anthelminthiques de synthèse. Des recherches plus récentes se sont intéressées au potentiel d'application de ressources moins conventionnelles que représentent des coproduits agroindustriels (CPAI). Cette revue vise à présenter des résultats in vitro et in vivo obtenus avec une gamme de CPAI d'origines géographiques et botaniques diversifiées (coproduits de l'industrie des noix, du bois (en régions tempérées et tropicales), du caroubier, du café et du cacao). Ces résultats ont confirmé la preuve de concept pour les effets antiparasitaires, et aussi pour d'autres volets de la production des ruminants dans un contexte agro écologique de l'élevage. Par ailleurs, les aspects socio-économiques d'exploitation de ces ressources, considérées jusqu'à présent comme des déchets, dans un modèle de circuits courts sont aussi évoqués. Les avantages et inconvénients des différentes modalités d'exploitation des CPAI sont aussi discutés dans le cadre d'une analyse SWOT.

Anthelmintic evaluation and essential oils composition of Hyptis dilatata Benth. and Mesosphaerum suaveolens Kuntze from the Brazilian Amazon.

Hyptis dilatata and Mesosphaerum suaveolens belong to Lamiaceae, are aromatic and medicinal subshrubs with antiparasitic potential and occurrence in the Amazon Region. The essential oils of both species were extracted, analyzed, and identified by GC and GC-MS and then evaluated their anthelmintic activities against the nematode Caenorhabditis elegans. Primary constituents of the samples of H. dilatata oils were limonene (72.6%), myrcene (11.5%), and p-cymene (10.3%) to PAMA19 sample, and camphor (25.5%), α-pinene (25.4%), 1,8-cineole (18.8%), ß-pinene (12.0%), and limonene (5.9%) to PAMA108 sample, while in the oil of M. suaveolens, PAMA131 sample, predominated bicyclogermacrene (23.5%, 1,8-cineole (23.0%), germacrene D (17.2%), and (E)-caryophyllene (10.4%). The sample oil of H. dilatata (PAMA108) exhibited the lower anthelmintic inhibitory concentration, with an IC50 value of 2.09 mg/mL for C. elegans Bristol N2 strain, while the oils of H. dilatata (PAMA19) and M. suaveolens (PAMA131) showed an IC50 up of 4 mg/mL for C. elegans IVR15 and Bristol N2 strains. These results suggest that the H. dilatata and M. suaveolens oils constituents' combination can be helpful as a nematicidal agent due to their synergistic action.

Combination of cypermethrin and thymol for control of Rhipicephalus microplus: Efficacy evaluation and description of an action mechanism.

The cattle tick Rhipicephalus microplus is one of the most important ectoparasites in the tropical and subtropical regions of the world. Synthetic pyrethroids are widely used to control this tick, and the selection of resistant populations is a huge problem worldwide. The activity of thymol, a natural monoterpene, free or in combination with other compounds, has been demonstrated against different species of ticks. However, the mode of action is not fully understood. The present study aimed to evaluate the efficacy and the potential mode of action of the combination of cypermethrin and thymol on ticks from two populations with different levels of susceptibility to cypermethrin (low and high susceptibility). The isolated acaricidal activity of cypermethrin and thymol on larvae was carried out in different concentrations. The combination with different concentrations of cypermethrin and fixed concentrations of thymol (1300 µg/mL for the low susceptibility population; 690 µg/mL for the high susceptibility population) were performed. Adult engorged females were divided into five experimental groups (n = 20): 1) Control group untreated; 2) Control group: 2.0% (v/v) DMSO; 3) Thymol group: 1300 µg/mL thymol; 4) Cypermethrin group: 3700 µg/mL cypermethrin; 5) Association of cypermethrin (3700 µg/mL) + thymol (1300 µg/mL). A subgroup was used to study the efficacy of the reproductive parameters and another subgroup, with ten adults from each treatment, was used to quantify thymol and cypermethrin by HPLC chromatographic analysis. All compounds tested were effective on larvae from both populations, and the combination with thymol decreased the LC50 of cypermethrin (232.4 to 52.7 µg/mL) on the low-susceptibility population. The combination of thymol and cypermethrin was effective in both populations of R. microplus (reproductive performance of engorged females) when compared to the untreated control group, even with higher percent control values (pop. 1: 93.5 ± 5.6% and pop. 2: 92.7 ± 1.1%) than the group treated only with cypermethrin (pop. 1: 87.3 ± 7.3% and pop. 2: 83.5 ± 1.2%). From the HPLC analyzes, a higher concentration of cypermethrin (pop. 1: 30.3 ± 6.9 and pop. 2: 45.4 ± 17.7 ng/mg) was detected in the tissues of engorged females treated with the combination compared to analyte concentrations in groups treated with cypermethrin only (pop. 1: 12.4 ± 4.4 pop. 2: 25.5 ± 9.4 ng/mg). This was the first study to investigate the acaricidal efficacy of the combination of thymol + cypermethrin on R. microplus and demonstrate that the presence of thymol increases the concentration of cypermethrin in the internal tissues of engorged females through a possible mechanism for increasing the penetration of cypermethrin at the cuticular level.

Anthelmintic effect of essential rhizome oil from Hedychium coronarium Koenig (Zingiberaceae) introduced in Northeastern Brazil.

Hedychium coronarium is native to Tropical Asia and has been introduced into several Brazilian biomes. Significant biological properties described for the essential oil (EO) from this species' rhizomes include antimicrobial, larvicidal, anti-inflammatory, antioxidant, phytotoxic, and anthelmintic activities. The primary constituents identified in this study by GC-MS in the EO were monoterpenes 1,8-cineole (33.5%), ß-pinene (17.0%), α-terpineol (7.7%), α-pinene (7.3%), limonene (5.2%), and p-cymene (4.9%), comprising 75.6% of total oil compounds. The main monoterpenes' EO and standards were tested against N2 (susceptible) and UVR15 (resistant) adult nematode Caenorhabditis elegans strains, with varying dead rates in motility tests.. Nematocidal activity was not attributed to 1,8-cineole and ß-pinene, the main H. coronarium rhizome oil components, as both exhibited an inhibitory concentration (IC50) ≥ 5 mg/mL. On the other hand, the α-pinene (IC50, 1.69 mg/mL) and (S)-(-)-limonene (IC50, 1.66 mg/mL) standards demonstrated more efficient action than rhizome oil in motility tests, with significant adult C. elegans nematode mortality rates. These results support the hypothesis that the combination of H. coronarium EO constituents can be helpful as a nematicidal product, due to their synergistic action.

Combination of synthetic anthelmintics and monoterpenes: Assessment of efficacy, and ultrastructural and biophysical properties of Haemonchus contortus using atomic force microscopy.

The resistance of Haemonchus contortus to synthetic anthelmintics is of increasing concern; and different strategies are being evaluated to improve parasite control. The present study investigated the in vitro effects of combinations of synthetic compounds and monoterpenes. Additionally, the chemical association of the best combinations and their impact on the ultrastructural and biophysical properties of H. contortus eggs was evaluated. We assessed the efficacy of the monoterpenes, carvacrol, thymol, r-carvone, s-carvone, citral, and p-cymene and the anthelmintics, albendazole and levamisole using the egg hatch test (EHT) and the larval migration inhibition test (LMIT), respectively. The minimum effective concentrations of the monoterpenes, according to the EHT (efficacy ranging from 4.4%-11.8%) and LMIT (efficacy ranging from 5.6%-7.4%), were used in combination with different concentrations of synthetic compounds, and the IC50 and synergism rate (SR) were calculated. Fourier-transform infrared spectroscopy (FTIR) was used to analyze the chemical association between the best combinations as revealed by the in vitro tests (albendazole and levamisole with r-carvone or s-carvone). Atomic force microscopy (AFM) was used to assess the ultrastructural and biophysical properties of H. contortus eggs treated with the albendazole and r-carvone combination. Among the monoterpenes, the highest efficacies were exhibited by carvacrol (IC50 = 185.9 µg/mL) and thymol (IC50 = 187.0 µg/mL), according to the EHT, and s-carvone and carvacrol (IC50 = 1526.0 and 1785.3 µg/mL, respectively), according to the LMIT. According to the EHT, albendazole showed a slight statistically significant synergism in combination with r-carvone (SR = 3.8) and s-carvone (SR = 3.0). According to the LMIT, among the monoterpenes, r-carvone (SR = 1.7) and s-carvone (SR = 1.7) showed an increase in efficacy with levamisole; however, this was not statistically significant. The FTIR spectra of albendazole and levamisole, in association with r-carvone and s-carvone, indicated the presence of chemical interactions between the synthetic and natural molecules, contributing to the possible synergistic effects of these associations. Eggs treated with albendazole and r-carvone showed an increase in roughness and a decrease in height, suggesting that the treatment induced damage to the egg surface and an overflow of its internal contents. Overall, the combination of albendazole with r-carvone and s-carvone was efficacious against H. contortus, demonstrating a chemical association between the compounds; the significant changes in the egg ultrastructure justify this efficacy.

Nemabiome metabarcoding reveals differences between gastrointestinal nematode species infecting co-grazed sheep and goats.

Our current understanding of differences in the epidemiology of gastrointestinal nematode (GIN) species in co-grazed sheep and goats is inadequate with reference to the development of sustainable control strategies. The next-generation metabarcoding sequencing method referred to as the 'nemabiome' allows some of these differences to be explored to describe the intensity of co-infecting GIN species. We applied this platform to study sheep and goats that were co-grazed on Guinea grass pasture in northeastern Brazil. Co-grazed goats and sheep were treated with a monepantel anthelmintic, then exposed to the same gastrointestinal nematode species. Overall, there were differences in the prevalence of GIN species identified in the sheep and goats; Trichostrongylus colubriformis and Teladorsagia circumcincta predominated in goat kids, while Haemonchus contortus predominated in adult does, ewes and lambs once burdens became re-established after anthelmintic treatment. Description of the pattern of re-infection following anthelmintic treatment was prevented by the unpredicted poor efficacy of 2.5 mg/kg and 5 mg/kg, respectively, of monepantel against O. columbianum and T. circumcincta in lambs, and T. circumcincta adult does. Differences in drug efficacy between host age and species groups may be important when considering sustainable GIN control strategies for co-grazed animals. The aggregated FECs of the adult does and goat kids representing re-established GIN burdens, were higher than those of the co-grazed adult ewes and lambs. This implies that there are inherent differences in GIN species adaptation to the two naïve small ruminant host species, and shows the need for better understanding of the factors giving rise to this situation associated with exposure to infective larvae and host responses. At the start of the study, the adult does were co-infected with several GIN species, with the highest intensity of T. circumcincta, contrasting with the situation in the adult ewes, in which H. contortus predominated. However, once burdens became re-established after treatment, H. contortus predominated in both adult does and ewes. This demonstrates the potential for host burdens of H. contortus to establish and predominate after anthelmintic treatment when burdens of co-infecting GIN species are low.

Acaricidal activity of (E)-cinnamaldehyde and α-bisabolol on populations of Rhipicephalus microplus (Acari: Ixodidae) with different resistance profiles.

This study aimed to investigate the acaricidal activity of (E)-cinnamaldehyde and α-bisabolol on populations of Rhipicephalus microplus with different resistance profiles. The adult immersion test (AIT) was used to characterize the susceptibility of tick populations (50 field populations) to synthetic acaricides: deltamethrin, amitraz, and chlorfenvinphos. The larval packet test (LPT) was used to determine the LC50 values for (E)-cinnamaldehyde (populations 1-25) and α-bisabolol (populations 26-50) at the concentrations of 0.31, 0.62, 1.25, 2.0, 2.5, 5.0 and 10.0 mg/mL. The susceptible strain Porto Alegre (POA) was used as a reference for calculating the resistance ratio (RR). In the AIT, deltamethrin did not show efficacy >95 % for any of the populations, whereas amitraz and chlorfenvinphos have presented efficacy >95 % for three (6 %) and 15 (30 %) populations, respectively. In the LPT, the LC50 values of (E)-cinnamaldehyde and α-bisabolol varied from 0.23 to 2.36 mg/mL and 1.57-3.01 mg/mL, respectively. The RR50 for (E)-cinnamaldehyde showed 20 (80 %) populations with values <1.0 and no population with values>1.5. As for α-bisabolol, only two (8%) populations have presented RR50 <1.0, whereas three (12 %) populations showed incipient resistance to this sesquiterpene (RR50 between 1.5 and 2.0). The results indicate that all studied tick populations showed low susceptibility to at least one of the commercial acaricides tested. In addition, comparison between the LC50 values of (E)-cinnamaldehyde and α-bisabolol for the field populations and the susceptible strain POA suggests that there is no cross-resistance of (E)-cinnamaldehyde and α-bisabolol for the tick populations evaluated, and that the differences in the LC50 values are due to population variations.