American mitogenome reference for the tropical brown dog tick, Rhipicephalus linnaei (Audouin, 1826).

The brown dog tick, Rhipicephalus linnaei (Audouin, 1826), is distributed across the American continent and is formerly known as the "tropical lineage". It belongs to the Rhipicephalus sanguineus (Latreille, 1806) species complex, referred to as R. sanguineus (sensu lato). Mitochondrial genome sequences are frequently used for the identification and represent reference material for field studies. In the present study, the entire mitochondrial genomes of R. linnaei (∼15 kb) collected from dogs in Mexico were sequenced and compared with available mitogenomes of R. sanguineus (s.l.). The mitochondrial genome is ∼90% identical to the reference genome of R. sanguineus (sensu stricto, former "temperate lineage") and > 99% identical to R. linnaei mitogenome derived from the neotype. Two additional mitogenomes were obtained and described as R. linnaei and R. turanicus from dogs in Saudi Arabia. The present study delivers a molecular reference for R. linnaei from America and complements R. linnaei mitogenomes from Africa, Asia and Australia. We propose to consider the complete mitogenome, as the reference for American R. linnaei, even when partial mitochondrial cox1, 12S rRNA or 16S rRNA genes are characterised.

Determination of the discriminant doses to identify resistance to fipronil, flumethrin and coumaphos, in Rhipicephalus sanguineus s.l. (Acari: Ixodidae) from Mexico.

Rhipicephalus sanguineus s.l. is an intra- and peridomiciliary tick of medical and veterinary importance, whose control is carried out through the application of various ixodicides, to which it can develop resistance. The objective of this work was to determine the discriminant doses (d.d.) to identify resistance against three ixodicides - fipronil, flumethrin and coumaphos - in R. sanguineus s.l. larvae originating from Mexico. Engorged ticks were collected from naturally infested dogs residing in rural communities in five Mexican states; the dogs had no history of recent treatment with ixodicides. All ticks were identified as R. sanguineus s.l. They were incubated for 25 days and their progeny was used in the larval package test (LPT) to be exposed to d.d. as determined in Rhipicephalus microplus, and those that were susceptible were analyzed using the LPT with six concentrations, for fipronil from 0.1 to 0.003125%, for flumethrin from 0.02 to 0.000625% and for coumaphos from 0.4 to 0.0125%. Mortality was analyzed with Probit methodology, to calculate the 50 and 99% lethal concentrations (LC50 and LC99). The d.d. was determined as a consensus value by multiplying the LC99 value × 2. Nine double susceptible samples (fipronil + flumethrin) and one triple susceptible sample (fipronil + flumethrin + coumaphos) were found; the d.d. determined were fipronil 0.05%, flumethrin 0.01% and coumaphos 0.43%. These doses can be used to rapidly and inexpensively identify resistant populations in samples collected in the field.

Determination of a Discriminant Dose to Identify Resistance to Amitraz in Rhipicephalus sanguineus s.l. (Acari: Ixodidae) from Mexico.

The brown dog tick, Rhipicephalus sanguineus s.l., is considered the most widely distributed three-host tick in the world and has medical and veterinary importance; the control of infestation is carried out with acaricides, towards which it can develop resistance. This study aimed to determine the discriminant dose (d.d) of amitraz to identify resistance in R. sanguineus s.l. larvae natives from Mexico and to evaluate its application in field-collected ticks. Engorged ticks were collected from naturally infested dogs residing in rural communities and were incubated for 25 days, and their progeny was used in a larval immersion test (LIT) to be exposed to the d.d. determined in Rhipicephalus microplus, and those that were susceptible were analyzed using the LIT in six concentrations. Mortality was analyzed through probit methodology to calculate the lethal concentration (LC) 50 and 99. The d.d. was determined as a consensus value by multiplying the LC99 × 2, and then, we proceeded to evaluate it in in-field samples by using the LIT technique. The d.d. calculated was 4 ppm. The in-field evaluation found 64% of the resistant samples to amitraz with mortality percentages between 98.3% and 0.35%. This dose can be used to rapidly and inexpensively identify resistant populations in samples collected in the field.

Thymol and eugenol against Rhipicephalus sanguineus sensu lato engorged females: Biological, histopathological and bioinformatic analysis.

The aim of this study was to evaluate the reproductive biology and histopathology of the ovaries of engorged females of Rhipicephalus sanguineus sensu lato exposed to combinations of thymol and eugenol, as well as to evaluate in silico the possible interactions of thymol and eugenol in tick cell membranes. To evaluate the tick reproductive biology, the adult immersion test (AIT) was performed, in which the engorged females were immersed in solutions of thymol and eugenol, combined or alone, at concentrations of 2.5 and 5.0 mg/mL. Two control groups (water and 3% DMSO) were also performed. The ticks were kept in a controlled chamber (B.O.D - 27 ± 1 °C and 80% ± 5% RH) to evaluate egg production and viability. To perform the ovaries histopathological evaluation, females were immersed in combination of thymol and eugenol (each at 2.5 and 5.0 mg/mL) and control (water and 3% DMSO) solutions. After immersion, the females were kept in B.O.D (27 ± 1 °C and RH of 80% ± 5%) for four days, they were dissected and the ovaries processed for histological analysis. In addition, an in silico analysis was performed using PASS online® software to predict probability activity (PA) of thymol and eugenol in cell membranes. The treatment with the combination of thymol and eugenol (each at 5.0 mg/mL) caused a reduction (p < 0.01) in oviposition, while the treatments with thymol (5.0 mg/mL) and combination of thymol and eugenol (5.0 mg/mL) reduced (p < 0.05) the egg viability. The treatment with combination of thymol and eugenol (5.0 mg/mL) resulted in a control percentage of 99.9%, while in the other treatments, control percentages below 56% were observed. Oocytes from the females exposed to the combinations of thymol and eugenol (each compound at 2.5 mg/mL) showed histopathological changes, except on oocyte V, while those treated with these compounds alone at 2.5 mg/mL, did not reveal any change. Changes in the shape of the oocyte, presence of vacuoles in the cytoplasm and germinal vesicle, reduction and fusion of yolk granules and rupture of some oocytes were observed. In silico analysis, showed that these compounds can act as membrane permeability inhibitors, membrane permeability agonists, membrane integrity antagonists and apoptosis agonists. We conclude that the combination of thymol and eugenol causes changes in the reproductive biology and morphophysiology of engorged females oocytes. The in silico analysis using thymol and eugenol revealed the possibility of disorganization in the cell membranes, a fact that may explain the histopathological alterations observed.

Liver and spleen of hosts of Rhipicephalus linnaei exposed to synthetic (afoxolaner) and natural acaricides (esters from castor oil). A comparative clinical-morphological study / Fígado e baço de hospedeiros de Rhipicephalus linnaei expostos aos acaricidas sintético (afoxolaner) e natural (ésteres do óleo de mamona). Estudo clínico-morfológico comparativo

In dogs, Rhipicephalus linnaei transmits pathogens such as Ehrlichia canis, Babesia vogeli, and Hepatozoon canis. The veterinary market has synthetic acaricides to ticks control. Esters derived from castor oil are efficient. However, there is little information about their effects on non-target organisms. This work consisted of a clinical (AST, ALT, and ALP) and histological and histochemical analysis (liver and spleen) of female rabbits exposed to these esters and afoxolaner. The rabbits were divided into three groups: control group (CG) received Bandeirante® rabbit feed; the afoxolaner treatment (TG1) received rabbit feed and two doses of afoxolaner; castor oil esters treatment (TG2) received rabbit feed enriched with esters (1.75 g esters/kg). No alterations were observed in the AST, ALT, and ALP enzymes in exposure to esters TG2. Rabbits from TG1 showed changes in AST. The liver of rabbits exposed to afoxolaner underwent histological and histochemical changes, such as steatosis and vacuolation, as well as poor protein labeling. Polysaccharides were intensely observed in the group exposed to esters. The spleen showed no changes in any of the exposure. Esters from castor oil caused fewer liver changes when incorporated into the feed and fed to rabbits than exposure to afoxolaner.(AU)

Carrapatos Rhipicephalus linnaei transmitem patógenos como Ehrlichia canis, Babesia vogeli e Hepatozoon canis, para os cães. O mercado veterinário dispõe de acaricidas sintéticos para o controle desses ectoparasitas. Ésteres derivados do óleo de mamona são eficientes, mas há pouca informação sobre seus efeitos em organismos não-alvos. Esse trabalho objetivou analisar clínica (AST, ALT e ALP), histológica e histoquímicamente (fígado e baço) coelhas expostas aos ésteres e ao afoxolaner, tendo sido as mesmas alocadas nos grupos: Controle (GC) que recebeu ração Bandeirante®; Tratamento 1 (TG1), além da ração foram expostas ao afoxolaner, Tratamento 2 (TG2) receberam ração enriquecida com os ésteres (1,75 g ésteres/kg). Não houve alterações nas enzimas AST, ALT e ALP na exposição aos ésteres (TG2). No TG1 houve alterações na AST. O fígado das coelhas do TG1 apresentou alterações histológicas e histoquímicas, tais como esteatose e vacuolização, bem como fraca marcação de proteínas. Polissacarídeos foi intensamente marcados no fígado do TG2. O baço não apresentou alterações em nenhum dos grupos. Os ésteres do óleo de mamona causaram menos alterações hepáticas nas coelhas do que quando elas foram expostas ao afoxolaner.(AU)

Acaricidal and repellent activity of the essential oils of Backhousia citriodora, Callistemon viminalis and Cinnamodendron dinisii against Rhipicephalus spp.

The ticks Rhipicephalus microplus and Rhipicephalus sanguineus sensu lato (s.l) are of great importance to agriculture, veterinary medicine and public health. Due to a number of problems related to the use of synthetic acaricides, natural products emerge as promising substances for alternative tick control. In the present study, essential oils of Backhousia citriodora, Callistemon viminalis and Cinnamodendron dinisii were extracted by hydrodistillation, characterized by GC-MS and GC-FID and biologically evaluated for acaricidal activity against R. microplus and repellent activity against R. sanguineus s.l. Inhibition of acetylcholinesterase (AChe) by the essential oils was also evaluated. The major constituent of B. citriodora is citral (98.9 %), whereas the essential oil from C. viminalis is rich in 1.8-cineole (78.1 %), α-pinene (12.5 %) and limonene (3.36 %), and that from C. dinisii contains α-pinene (30.8 %), ß-pinene (12.5 %) and sabinene (11.3 %) as the principal constituents. The median lethal concentrations (LC 50) estimated for the essential oils on engorged R. microplus females were 3.276 µL.mL-1 for B. citriodora, 8.195 µL.mL-1 for C. dinisii and 8.936 µL.mL-1 for C. viminalis. The essential oil of B. citriodora showed the best repellent effect against unfed R. sanguineus s.l. adults, demonstrating repellent action up to 3 h after application. The essential oil of C. viminalis was able to reduce AChe activity, with an average inhibitory concentration (IC 50) of 0.33 µg mL-1. Thus, these oils can be considered as sources of bioactive compounds for tick control.

Rhipicephalus sanguineus Complex in the Americas: Systematic, Genetic Diversity, and Geographic Insights.

The Rhipicephalus sanguineus group encompasses at least 12 validated species of Palearctic and Afrotropical hard ticks, which are relevant in veterinary medicine and public health. The taxonomy of R. sanguineus s.s., has been particularly intensely debated, due to its wide geographic distribution, morphological variants, parasite-host associations, and its capacity and vectorial competence for the transmission of several pathogens. By sequencing mitochondrial markers, it was possible to identify the existence of multiple lineages, among which the Tropical and the Temperate lineages stand out, particularly in America. However, the northern limit between these lineages is not clear due to the lack of extensive sampling across Mexico. For this reason, the aim of the present study was to determine the genetic diversity and structure of the R. sanguineus group in Mexico and to compare it with the populations reported in the Americas, in order to propose the northern limit of the R. sanguineus Tropical lineage and the potential regions of sympatry with R. sanguineus s.s. The findings of this study now confirm the presence of R. sanguineus s.s. in Mexico, showing a subtle genetic structure and high genetic diversity throughout its distribution in the Americas. In contrast, the Tropical lineage seems to be genetically less diverse in its overall distribution in the Americas. The genetic diversity of these two independent lineages could have important epidemiological implications in the transmission of tick pathogens.

Repellent activity of acetylcarvacrol and its effects on salivary gland morphology in unfed Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae).

Rhipicephalus sanguineus sensu lato (s.l.), commonly known as brown dog tick, is a widely distributed tick species that is substantially important for human and veterinary medicine. Therefore, it is the target of different control methods. Carvacrol and its semisynthetic derivative, acetylcarvacrol, are promising chemical compounds for alternative tick control. Thus, this study aimed to compare the repellent activities of carvacrol and acetylcarvacrol at different concentrations and drying times. Additionally, morphological alterations found in salivary glands were evaluated through histological techniques after exposure to acetylcarvacrol. The impact of the morphological changes on the development and survival of acini/cells in salivary glands was measured by a semiquantitative analysis. The repellent action of both compounds did not differ when evaluated at different concentrations, although acetylcarvacrol increased its effects as the concentration raised. Regarding the different drying times, acetylcarvacrol maintained its effects after 3 hours of exposure, while the efficacy of carvacrol decreased during this time period. Salivary glands of unfed R. sanguineus s.l. females showed dose-dependent alterations in the size and shape of acini as well as cytoplasmic vacuolization. Loss of the acinar cell limit, rupture of secretory granules and nuclear changes in the cells were also observed in the treated groups. Thus, our results demonstrated the potential of acetylcarvacrol to act as repellent against R. sanguineus s.l. Additionally, the morphological alterations found in salivary glands may interfere with the feeding process of ticks, which contributes to mitigate infestation by this species.

Identification of a non-host semiochemical from miniature pinscher, Canis lupus familiaris, that repels Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

It is already known that the beagle breed of domestic dogs produces semiochemicals capable of repelling the brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.). With a view to discovering new non-host semiochemicals as tick repellents, we compared the semiochemicals produced by a putative tick-resistant breed of dog, miniature pinscher, with known tick-resistant (beagle) and tick-susceptible (English cocker spaniel) breeds. Two non-host compounds produced by beagles, i.e. 2-hexanone and benzaldehyde, were shown to be present in samples collected from all three breeds. Furthermore, two compounds, 6-methyl-5-hepten-2-one and 1,2,4-trimethylbenzene, were found in higher amounts in samples collected from miniature pinscher dogs. The mean amounts of benzaldehyde, 2-hexanone and 1,2,4-trimethylbenzene were similar for beagles and miniature pinschers (P > 0.05) and higher than the means observed for cocker spaniels (P < 0.05), whereas the mean amount of 6-methyl-5-hepten-2-one produced by miniature pinschers was significantly higher (P < 0.05) than for the other breeds of dogs. In Petri-dish assays with adult R. sanguineus s.l., 6-methyl-5-hepten-2-one was repellent for all observation periods evaluated for the two highest concentrations (0.100 and 0.200 mg.cm-2, P < 0.01). The obtained results support our hypothesis that miniature pinschers are a tick-resistant dog breed and agree with previous observations of miniature pinschers being the breed least parasitized by ticks. Furthermore, the non-host semiochemical 6-methyl-5-hepten-2-one has potential to be developed for use as a repellent for the protection of susceptible dogs from R. sanguineus s.l. ticks.

Analysis on the prokaryotic microbiome in females and embryonic cell cultures of Rhipicephalus sanguineus tropical and temperate lineages from two specific localities in Brazil / Análise do microbioma procarionte de fêmeas e cultura celular embrionária de Rhipicephalus sanguineus linhagens tropical e temperada de duas localidades no Brasil

Two lineages of Rhipicephalus sanguineus are known in Brazil: the temperate or southern and the tropical or northern populations. The distribution patterns of both lineages of R. sanguineus have epidemiological implications that can affect vectorial competence concerning Ehrlichia canis, the agent of canine monocytic ehrlichiosis. Intending to identify the microbiomes of both lineages and compare microorganisms in R. sanguineus, we used the 16S rRNA (V4-V5 region) gene-based metataxonomic approach, through NGS sequencing on the MiSeq Illumina platform. We selected specimens of females from the environment and samples of primary embryonic cell cultures, from both lineages, and this was the first study to investigate the prokaryotic microbiome in tick cell cultures. The results showed that many bacterial taxa detected in the samples were typical members of the host environment. A significant diversity of microorganisms in R. sanguineus females and in embryonic cell cultures from both lineages was found, with emphasis on the presence of Coxiella in all samples, albeit in different proportions. The Coxiella species present in the two lineages of ticks may be different and may have co-evolved with them, thus driving different patterns of interactions between ticks and the pathogens that they can harbor or transmit to vertebrate hosts.(AU)

Duas linhagens de Rhipicephalus sanguineus são conhecidas no Brasil: populações da linhagem temperada ou do sul, e tropical ou do norte. Os padrões de distribuição de ambas as linhagens de R. sanguineus têm implicações epidemiológicas, podendo afetar a competência vetorial de Ehrlichia canis, o agente etiológico da erliquiose monocítica canina. Com a intenção de identificar os microbiomas de ambas as linhagens e comparar microrganismos de R. sanguineus, foi utilizada a metataxonomia, baseada no gene 16S rRNA (região V4-V5), por meio do sequenciamento de nova geração na plataforma MiSeq Illumina. Foram selecionadas amostras de fêmeas do ambiente e cultivo primário de células embrionárias, considerando-se as duas linhagens conhecidas do Brasil. Este é o primeiro estudo que investiga o microbioma procariótico de células de cultura de carrapato. Os resultados mostram que muitos grupos de bactérias detectadas nas amostras são membros típicos do ambiente do hospedeiro. Uma diversidade significativa de microrganismos em fêmeas e cultura de células embrionárias nas duas linhagens de R. sanguineus foi encontrada, com ênfase na presença de Coxiella em todas as amostras, ainda que em diferentes proporções. Possivelmente, as espécies de Coxiella presentes nas duas linhagens de carrapatos são diferentes e co-evoluíram com essas linhagens, conduzindo a diferentes padrões de interação entre carrapatos e patógenos que podem abrigar ou transmitir aos hospedeiros vertebrados.(AU)

Analysis on the prokaryotic microbiome in females and embryonic cell cultures of Rhipicephalus sanguineus tropical and temperate lineages from two specific localities in Brazil / Análise do microbioma procarionte de fêmeas e cultura celular embrionária de Rhipicephalus sanguineus linhagens tropical e temperada de duas localidades no Brasil

Abstract Two lineages of Rhipicephalus sanguineus are known in Brazil: the temperate or southern and the tropical or northern populations. The distribution patterns of both lineages of R. sanguineus have epidemiological implications that can affect vectorial competence concerning Ehrlichia canis, the agent of canine monocytic ehrlichiosis. Intending to identify the microbiomes of both lineages and compare microorganisms in R. sanguineus, we used the 16S rRNA (V4-V5 region) gene-based metataxonomic approach, through NGS sequencing on the MiSeq Illumina platform. We selected specimens of females from the environment and samples of primary embryonic cell cultures, from both lineages, and this was the first study to investigate the prokaryotic microbiome in tick cell cultures. The results showed that many bacterial taxa detected in the samples were typical members of the host environment. A significant diversity of microorganisms in R. sanguineus females and in embryonic cell cultures from both lineages was found, with emphasis on the presence of Coxiella in all samples, albeit in different proportions. The Coxiella species present in the two lineages of ticks may be different and may have co-evolved with them, thus driving different patterns of interactions between ticks and the pathogens that they can harbor or transmit to vertebrate hosts.

Resumo Duas linhagens de Rhipicephalus sanguineus são conhecidas no Brasil: populações da linhagem temperada ou do sul, e tropical ou do norte. Os padrões de distribuição de ambas as linhagens de R. sanguineus têm implicações epidemiológicas, podendo afetar a competência vetorial de Ehrlichia canis, o agente etiológico da erliquiose monocítica canina. Com a intenção de identificar os microbiomas de ambas as linhagens e comparar microrganismos de R. sanguineus, foi utilizada a metataxonomia, baseada no gene 16S rRNA (região V4-V5), por meio do sequenciamento de nova geração na plataforma MiSeq Illumina. Foram selecionadas amostras de fêmeas do ambiente e cultivo primário de células embrionárias, considerando-se as duas linhagens conhecidas do Brasil. Este é o primeiro estudo que investiga o microbioma procariótico de células de cultura de carrapato. Os resultados mostram que muitos grupos de bactérias detectadas nas amostras são membros típicos do ambiente do hospedeiro. Uma diversidade significativa de microrganismos em fêmeas e cultura de células embrionárias nas duas linhagens de R. sanguineus foi encontrada, com ênfase na presença de Coxiella em todas as amostras, ainda que em diferentes proporções. Possivelmente, as espécies de Coxiella presentes nas duas linhagens de carrapatos são diferentes e co-evoluíram com essas linhagens, conduzindo a diferentes padrões de interação entre carrapatos e patógenos que podem abrigar ou transmitir aos hospedeiros vertebrados.

A case of gynandromorphism in Rhipicephalus sanguineus s.l. from Mexico.

We report the presence of a brown dog tick (Rhipicephalus sanguineus s.l.) gynandromorph collected inside a house in the state of Guanajuato, Mexico. This work provides the first report of gynandromorphism in a tick from Mexico, and represents the third report of this condition in R. sanguineus s.l. in the world.

Combination of thymol and eugenol for the control of Rhipicephalus sanguineus sensu lato: Evaluation of synergism on immature stages and formulation development.

The aim of this study was to investigate whether the combination of thymol with eugenol has a synergistic effect on the immature life stages of Rhipicephalus sanguineus sensu lato (s.l.), to evaluate the cost-benefit ratio of using these compounds in combination, and to develop a formulation combining thymol with eugenol with activity on immature stages of R. sanguineus s.l. To evaluate synergism, thymol and eugenol, combined (ratio 1:1) or not, were tested at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL on unfed larvae and nymphs using a larval packet test, and 0.625, 1.25, 2.5, 5.0 and 10 mg/mL on engorged larvae and nymphs using an immersion test. A cost estimate was calculated to produce 1 L of a solution containing a concentration of thymol and eugenol, combined or not, that could cause a tick mortality rate greater than 95 %. Finally, a formulation was developed, consisting of a micellar dispersion containing polymers (MDP), with thymol + eugenol (1:1), at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL, and the activity was evaluated on unfed and engorged larvae and nymphs. For unfed larvae and nymphs, concentrations of 2.5 and 5.0 mg/mL and 2.5, 5.0 and 10.0 mg/mL, respectively, presented synergistic effects. In tests with engorged larvae and nymphs, respective concentrations of 0.625, 1.25 and 2.5 mg/mL and 2.5 and 5.0 mg/mL had synergistic effects. The estimated costs for producing a solution of 1 L with efficacy greater than 95 % was $5.97 using only thymol (15 mg/mL), $ 5.93 using only eugenol (15 mg/mL), and $ 3.97 using thymol + eugenol (1:1 - 5,0 mg/mL). In tests with MDP, the combination of thymol + eugenol resulted in a mortality rate higher than 95 % at concentration of 10 mg/mL for unfed and engorged larvae and nymphs. Thus, the combination of thymol + eugenol, depending on the concentration, has synergistic effects and this combination lowers the cost for the active ingredients thymol and eugenol. The combination of thymol + eugenol in MDP had acaricidal activity against immature life stages of R. sanguineus s.l.

Low concentrations of acetylcarvacrol induce drastic morphological damages in ovaries of surviving Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae).

Rhipicephalus sanguineus sensu lato (s. l.) ticks are targets of acaricidal treatments due to their great veterinary and medical importance. Acetylcarvacrol stands out as a promising acaricidal substance for its increased biocidal activity and stability. Additionally, its toxicity to mammals is reduced when compared to the parent molecule, carvacrol. The present study aimed to evaluate the effects of acetylcarvacrol on the morphology of ovaries of engorged R. sanguineus s. l. ticks. The animals were subjected to the Adult Immersion Test to calculate the lethal concentrations (LC50 and LC90) of acetylcarvacrol. Subsequently, the surviving ticks were dissected for collection of the ovaries. The samples were processed through routine histological techniques. The histological sections were stained with hematoxylin and eosin, bromophenol blue (protein detection) and PAS (polysaccharide detection). The alterations found in the ovaries were evaluated by morphometric and semiquantitative analysis. The LC50 and LC90 were 17.805 and 26.164 µL/mL, respectively. The most severe morphological alterations were disappearance of the female germ cells (oocytes), nucleolus vacuolization, thicker and irregular chorion, and decrease in size of the oocytes. Also, the content of proteins and carbohydrates in the oocytes were heavily affected by the chemical, as evidenced by a non-homogeneous staining pattern. The group exposed to the highest concentration of acetylcarvacrol (20 µL/mL) exhibited a statistically greater score in the semiquantitative analysis when compared to the other groups. The morphological changes in the ovaries may reduce the ticks' offspring production or generate descendants that will struggle to carry out essential biochemical processes during their lives. Thus, acetylcarvacrol may be a promising alternative to control tick infestation by impairing the reproduction of this parasite.

Successful Infection of Tick Cell Cultures of Rhipicephalus sanguineus (Tropical Lineage) with Ehrlichia canis.

There are two distinct lineages of ticks, Rhipicephalus sanguineus, in South America: tropical and temperate lineages. Only the tropical lineage is recognized as competent vector for Ehrlichia canis. The epidemiological data of canine monocytic ehrlichiosis is congruent with the distribution of the two lineages of R. sanguineus. Herein, we report the infection of R. sanguineus (tropical lineage) cell cultures with E. canis, after cryopreservation. R. sanguineus (tropical lineage) cell identity was confirmed by sequencing using a 16S rDNA gene fragment. Tick cell cultures were prepared in L-15B medium supplemented with 10%, 15%, and 20% Fetal Bovine Serum (FBS), and 10% of Tryptose Phosphate Broth (TPB). Cell cultures developed better at the concentration of 20% of FBS. Cultures in the fifth harvest (approximately 7 months later) were selected for the first infections. Optimal R. sanguineus cell growth and adhesion was observed (5.0 × 106 cells/mL, and the population doubling time every 57 h). Once infected with E. canis, the cultures were maintained in L-15B medium supplemented with 2% and 5% of FBS fortified with iron and 10% TPB. Infected cells were also cryopreserved. DNA was extracted from infected and noninfected cells and analyzed using quantitative real-time PCR targeting the E. canis-dsb gene. Primary culture of the fifth passage was infected by E. canis and it maintained the pathogen for at least 40 days before partial cell destruction. Subcultures of infected cells (fresh and cryopreserved cultures) onto new tick cell cultures were successful. The E. canis infection was confirmed by real-time PCR and light and transmission electron microscopy. The R. sanguineus (tropical lineage) cells infected with E. canis successfully infected new tick cell cultures, showing that these cells could be an alternative substrate for maintenance of this pathogen.

Successful infection of tick cell cultures of Rhipicephalus sanguineus (Tropical Lineage) with Ehrlichia canis

There are two distinct lineages of ticks, Rhipicephalus sanguineus, in South America: tropical and temperate lineages. Only the tropical lineage is recognized as competent vector for Ehrlichia canis. The epidemiological data of canine monocytic ehrlichiosis is congruent with the distribution of the two lineages of R. sanguineus. Herein, we report the infection of R. sanguineus (tropical lineage) cell cultures with E. canis, after cryopreservation. R. sanguineus (tropical lineage) cell identity was confirmed by sequencing using a 16S rDNA gene fragment. Tick cell cultures were prepared in L-15B medium supplemented with 10%, 15%, and 20% Fetal Bovine Serum (FBS), and 10% of Tryptose Phosphate Broth (TPB). Cell cultures developed better at the concentration of 20% of FBS. Cultures in the fifth harvest (approximately 7 months later) were selected for the first infections. Optimal R. sanguineus cell growth and adhesion was observed (5.0?×?106 cells/mL, and the population doubling time every 57?h). Once infected with E. canis, the cultures were maintained in L-15B medium supplemented with 2% and 5% of FBS fortified with iron and 10% TPB. Infected cells were also cryopreserved. DNA was extracted from infected and noninfected cells and analyzed using quantitative real-time PCR targeting the E. canis-dsb gene. Primary culture of the fifth passage was infected by E. canis and it maintained the pathogen for at least 40 days before partial cell destruction. Subcultures of infected cells (fresh and cryopreserved cultures) onto new tick cell cultures were successful. The E. canis infection was confirmed by real-time PCR and light and transmission electron microscopy. The R. sanguineus (tropical lineage) cells infected with E. canis successfully infected new tick cell cultures, showing that these cells could be an alternative substrate for maintenance of this pathogen.

Evaluation of the acaricidal activity of thymol incorporated in two formulations for topical use against immature stages of Rhipicephalus sanguineus sensu lato (Latreille, 1806) (Acari: Ixodidae).

The objective of this study was to assess, for the first time, the in vitro acaricidal activity of two topical formulations containing thymol, on immature stages of Rhipicephalus sanguineus sensu lato. For this purpose, two base formulations were prepared: an oil-in-water (O/W) emulsion and a hydroalcoholic solution, containing different thymol concentrations (0.5 to 20 mg/mL). We used the larval packet test for non-engorged larvae and nymphs, and the immersion test for engorged larvae and nymphs. For emulsion, a mortality rate of 94.2% was achieved at 0.75 mg/mL in non-engorged larvae. For engorged larvae, there was 95.0% mortality at 5.0 mg/mL. Non-engorged nymphs showed 83.3% mortality at 2.5 mg/mL, and for engorged nymphs, 86.0% mortality was verified at 5.0 mg/mL. For the hydroalcoholic solution, the mortality found for non-engorged larvae was 88.1% at 2.5 mg/mL. For engorged larvae, the highest mortality was 25.0% at 20 mg/mL; non-engorged nymphs had 91.0% mortality at 1.0 mg/mL and for engorged nymphs; the maximum value verified was 18.3% mortality at 20 mg/mL. Preliminary stability tests were carried, and the hydroalcoholic solution remained stable under all the conditions analyzed. The O/W emulsion showed signs of early instability at the concentration of 5.0 mg/mL. The results obtained indicate that the acaricidal activity of thymol, when included in the proposed formulations, was enhanced against non-engorged larvae with topical treatment in comparison with data in the literature. Although there were variations in toxicity between the different stages, these formulations are promising for future therapeutic use.

In vitro repellency of DEET and ß-citronellol against the ticks Rhipicephalus sanguineus sensu lato and Amblyomma sculptum.

Rhipicephalus sanguineus sensu lato and Amblyomma sculptum can parasite humans and domestic animals and are vectors of pathogens, including zoonoses. Repellents are an important tool of tick control. This study aimed to compare the efficacy of N,N-diethyl- 3-methylbenzamide (DEET), a standard repellent, versus ß-citronellol in a Petri dish bioassay. A semicircle of filter paper (31.8cm2) was treated with 87µl of one of four concentrations (0.200, 0.100, 0.050 and 0.025mg/cm2) of ß-citronellol, DEET or solvent (ethanol). A head-to-head test was developed treating one side with increasing concentrations of ß-citronellol as above mentioned, against the highest concentration of DEET. Besides that a blank assay was performed. Three males and three females were placed in the middle of the plate and their location was evaluated 5, 10 and 30min after the test was initiated. As a result, the time had no significant effect on repellency response of the ticks exposed to both compounds and their concentrations. The repellency response raised according with the increase of concentration. Additionally, our findings indicate that the tick A. sculptum was more sensitive to the compounds tested and ß-citronellol showed a higher efficacy than DEET. In addition, ß-citronellol could be formulated to protect humans and other animals from R. sanguineus s. l. and A. sculptum infestation, as well as the diseases transmitted by these species.

First documentation of ivermectin resistance in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille, 1806), is an ectoparasite and disease vector of significant veterinary and public health importance that is distributed widely around the world. The intensive use of synthetic acaricides for tick control exerts a strong selective pressure for brown dog ticks to become resistant to them. Here, we investigated claims from the field regarding treatment failure associated with the use of veterinary products containing ivermectin (IVM) to control brown dog ticks infesting dogs in Yucatan state, Mexico. Dogs in six state municipalities were inspected to sample 15 R. sanguineus s.l. POPULATIONS: Interviews were conducted with dog owners to gather information on the history of dog treatment with conventional acaricides and IVM. The larval immersion test was used on the progeny of adult female ticks infesting dogs to test for susceptibility to IVM. Dose-mortality regressions, lethal concentrations (LC), their confidence intervals, and slope were estimated by probit analysis. Ten tick populations (66.7%) were classified as resistant compared with the most susceptible population. A high inter-population variation in the phenotypic level of IVM resistance was evident (resistance ratio at LC50% and LC99% ranged from 1.0 to 30.5, and from 1.0 to 458.8, respectively). Tick populations classified as resistant were collected from dogs known to have been treated with IVM. To our knowledge, this is the first report of IVM resistance in R. sanguineus s.l. worldwide. Veterinary and pet owner education on integrated tick management practices is required to avoid widespread resistance and increased treatment failure with products containing IVM and other macrocyclic lactones used to control endo- and ectoparasites affecting dogs. Integrated tick management will also help mitigate the burden of brown dog tick-borne diseases on human and animal populations.

Morphological alterations in salivary glands of Rhipicephalus sanguineus ticks (Acari: Ixodidae) exposed to neem seed oil with known azadirachtin concentration.

Neem (Azadirachta indica) has attracted the attention of researchers worldwide due to its repellent properties and recognized effects on the morphology and physiology of arthropods, including ticks. Therefore, this study aimed to demonstrate the effects of neem seed oil enriched with azadirachtin on salivary glands of Rhipicephalus sanguineus ticks, targets of great veterinary interest because of their ability to transmit pathogens to dogs. For this, R. sanguineus semi-engorged females were subjected to treatment with neem seed oil, with known azadirachtin concentrations (200, 400 and 600ppm). After dissection, salivary glands were collected and evaluated through morphological techniques in light microscopy, confocal scanning laser microscopy and transmission electron microscopy, so that the possible relation between neem action and further impairment in these ectoparasites feed performance could be established. Neem oil demonstrated a clear dose-dependent effect in the analyzed samples. The agranular (type I) and granular acini (types II and III) showed, particularly in individuals treated with the highest concentrations of the product, cells with irregular shape, intense cytoplasmic disorganization and vacuolation, dilation of rough endoplasmic reticulum lumen, besides alterations in mitochondrial intermembrane space. These morphological damages may indicate modifications in salivary glands physiology, demonstrating the harmful effects of compounds present in neem oil on ticks. These results reinforce the potential of neem as an alternative method for controlling R. sanguineus ticks, instead of synthetic acaricides.