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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Faecal egg counts and nemabiome metabarcoding highlight the genomic complexity of equine cyathostomin communities and provide insight into their dynamics in a Scottish native pony herd.

Understanding the composition of gastrointestinal nematode communities may help to mitigate or exploit parasite adaptations within their host. We have used nemabiome deep amplicon sequencing of internal transcribed spacer-2 (ITS-2) ribosomal DNA to describe the temporal and host species composition of gastrointestinal nematode communities following sampling of six Scottish ponies across 57 months. In the absence of parasite control, each horse showed seasonal trends of increases and decreases in faecal egg counts, consistent with the epidemiology of equine strongylid parasites, however, the composition of parasites within individuals changed over time. Sixteen presumptive strongylid species were identified in each of the horses, 13 of which were distributed in a complex clade together with small numbers of amplicon sequences which could not be classified beyond the Cyathostominae subfamily level. Egg shedding of seven trichostrongylid species, which had previously been identified in co-grazed Soay sheep, was identified during the early spring. Faecal egg counts and the percentage of amplicon sequences assigned to each gastrointestinal nematode species were combined to describe their relative abundance across both host and time. Significant differences in species diversity between horses and between months were observed, being greatest from March to May and least from October to December. The magnitude of the individual horse effect varied between months and, conversely, the magnitude of the seasonal effect varied between individual horses. The most abundant gastrointestinal nematode in each of the horses was Cylicostephanus longibursatus (46.6% overall), while the abundance of the other strongylid species varied between horses and relative to each other. Patent C. longibursatus infections over the winter months might represent a genetic adaptation towards longer adult worm survival, or a lower rate of developmental arrest in the autumn. This study provides insight into highly complex phylogenetic relationships between closely related cyathostomin species; and describes the dynamics of egg shedding and pasture contamination of co-infecting equine gastrointestinal nematode communities. The results could be applied to determine how climatic and management factors affect the equilibrium between hosts and their parasites, and to inform the development of sustainable gastrointestinal nematode control strategies for different host species.

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.