Efficacy of essential oils of Egletes viscosa and Lippia schaueriana on the reproductive biology of Rhipicephalus sanguineus sensu lato engorged females.

The study analyzed the chemical composition and the acaricide effect of Egletes viscosa Less (macela-da-terra) and Lippia schaueriana Mart. (lipia-da-serra) essential oils (EOs) on Rhipicephalus sanguineus s. l. (Acari: Ixodidae) engorged females. The chemical analysis (GC-MS and GC-FID) identified 27 components in E. viscosa EO and 18 in L. schaueriana EO, which comprise more than 98% of its constituents. The effects of the oils on the reproductive biology of R. sanguineus ticks were assessed by adult immersion test. Both EOs significantly reduced (p < 0.05) the egg production index when the females were exposed to 25 and 50 mg/mL, also affecting the egg viability. During the laying process, the eggs produced by the females exposed to the EO showed several morphological alterations such as dehydrated, darkened, and disaggregated, and these alterations were more severe as the concentrations increased. The mortality percentages were 58.9%, 70.8% and 92.7% when the ticks were exposed to 12.5, 25 and 50 mg/mL of E. viscosa oil, respectively. In the same concentrations, the efficacy of L. schaueriana was 39.3%, 53.4%, and 84.6%. Therefore, it can be concluded that the essential oils of E. viscosa and L. schaueriana have acaricidal effect in females of R. sanguineus s.l ticks.

Establishment and multiapproach characterization of Amblyomma sculptum (Acari: Ixodidae) cell line (ASE-14) from Brazil.

The establishment and characterization of the ASE-14 cell line derived from embryos of Amblyomma sculptum is described here. Primary cultures were started, and after 60 days of culturing a confluent monolayer was formed and the first subculture was then carried out. After this, new subcultures were carried out every 4 weeks. Cryopreservation of cells was successful only after the 14th subculture. We compared the chromosomes of the ASE-14 cell line with those of parental ticks. Cytogenetic analysis revealed occurrences of variable and increased diploid numbers in the ASE-14 cell line in comparison with adult ticks, probably through polyploidization events, chromosome fusions and translocations, which allowed generation of cells with distinct diploid numbers. Confirmation of the origin of the A. sculptum cell line was obtained through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In addition, no DNA from Anaplasma marginale, Anaplasma spp., Babesia/Theileria spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Mycoplasma spp. or Rickettsia spp. was detected in the cells through PCR assays. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect polysaccharides and protein, respectively. In conclusion, a new cell line derived from embryos of A. sculptum was generated and characterized in this study. The ASE-14 cell line was deposited in the Tick Cell Biobank at the University of Liverpool, and in the Tick Cell Biobank South America Outpost at the Oswaldo Cruz Foundation (FIOCRUZ). The ASE-14 cell line is an important addition to the existing panel of tick cell lines and can be used as a tool for advancing research in various areas of the virology, bacteriology, biology and control of this tick.

Establishment and characterization of a cell line (RBME-6) of Rhipicephalus (Boophilus) microplus from Brazil.

Tick cell lines have already proved to be a useful tool for obtaining more information about possible vector species and the factors governing their ability to transmit a pathogen. Here, we established and characterized a cell line (RBME-6) derived from embryos of Rhipicephalus microplus from Brazil. Primary tick cell cultures were prepared in L-15B medium supplemented with 20% fetal bovine serum and 10% tryptose phosphate broth. The cell monolayers were subcultured when they reached a density of approximately 8 × 10 5 cells/mL (95% viability). Only after the sixth subculture were cells thawed from storage in liquid nitrogen successfully. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect total polysaccharides and total protein, respectively . No DNA from Anaplasma spp., Anaplasma marginale, Babesia spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Rickettsia spp. or Mycoplasma spp. was detected in the cells through PCR assays. In addition, we performed chromosomal characterization of the tick cell line and confirmed the R. microplus origin of the cell line through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In conclusion, we established and characterized a new cell line from a Brazilian population of R. microplus, which may form a useful tool for studying several aspects of ticks and tick-borne pathogens.

Rhipicephalus sanguineus salivary gland extract as a source of immunomodulatory molecules.

Rhipicephalus sanguineus sensu lato (s.l.), popularly known as 'brown dog tick', is the primary vector of pathogens affecting dogs worldwide. To enter the host's organism, these pathogens utilise the anticoagulant, antiplatelet, anti-inflammatory and immunomodulatory actions of compounds present in the tick's saliva; such compounds are released by the ectoparasite in order to attach and feed on dogs. Nitric oxide (NO) is one of the regulatory factors in inflammation, apoptosis and immunomodulation. Here, we evaluated the in vitro activity of salivary gland extract of female dog ticks on the macrophage-derived J774 cell line, with and without lipopolysaccharide (LPS) stimulation. Cultures were evaluated for possible morphological alterations caused by exposure to the extract. There was no apparent in vitro cytotoxicity of the extract. Also, the NO secretory response in the non-LPS-stimulated cells was not inhibited. On the other hand, the extract presented modulatory action in the cultures of LPS-stimulated cells at a concentration of 0.1 µg/mL, possibly through macrophage activation, and induced a significant decrease in NO secretion. These results confirm the modulatory potential of bioactive molecules in the salivary glands of R. sanguineus ticks.

Immunomodulatory and morphophysiological effects of Rhipicephalus sanguineus s. l. (Acari: Ixodidae) salivary gland extracts.

Rhipicephalus sanguineus s. l. is popularly known as the "brown dog tick" since dogs are its preferential hosts, but the species has been reported to parasitize other mammals, including humans, with significant medical-veterinary importance since it transmits several important pathogenic agents during the feeding period. The tick saliva is a complex mixture that has several functions, including the capability to modulate the hemostatic, inflammatory and immunologic systems of the host, allowing pathogens to settle. Despite knowledge about the immunosuppressive action of tick saliva, little is known about the mechanisms involved in this process and the morphophysiological effects caused by exposure to the salivary gland extract, taking into consideration the different periods of the glandular cycle. Thus, the objective of this study was to analyze the in vitro effects of salivary gland extracts obtained from R. sanguineus s. l. females fed on host rabbits for two (SGE2 - Salivary Gland Extracts of 2 days) and four days (SGE4 - Salivary Gland Extracts of 4 days) on J774 cells (monocyte macrophage cell line) and verify the occurrence of morphological and immunomodulatory alterations in these cells when exposed to different concentrations of these extracts. The results showed that: (i) SGE2 and SGE4 at the concentration of 4 µg/mL presented cytotoxicity to the J774 cells exposed for 24 and 48 hours; (ii) SGE2 at the concentrations of 2 µg/mL(48-hour exposure) and 1 µg/mL (24-hour exposure) and SGE4 at the concentrations of 2 and 1 µg/mL (48-hour exposure) showed proinflammatory activity, confirmed by the increased secretion of NO and proinflammatory cytokine (IL-2), and the presence of morphological characteristics detected by microscopy; and (iii) SGE2 and SGE4 at the concentrations of 0.5 and 0.1 µg/mL had immunomodulatory activity, demonstrated by decreases in the secretion of NO and proinflammatory cytokines (IL2, IL-6 and TNF-α) and increase in the synthesis of IL-10, confirmed by the morphophysiological analysis. These unprecedented data are extremely relevant for future research to identify the processes involved in the ectoparasite-host relationship, as well to develop more efficient tick control strategies.

Detrimental effect of deltamethrin on the central nervous system (synganglion) of Rhipicephalus sanguineus ticks.

Ticks are ectoparasites of medical and veterinary importance, which transmit many infectious agents, causing significant damage to the hosts. The "dog tick" Rhipicephalus sanguineus is responsible for transmitting several pathogens to dogs, motivating researchers to investigate efficient and sustainable control methods. Currently, chemical acaricides currently in use target the central nervous system (synganglion), which is responsible for controlling all the systemic functions of the ticks. Here, the neurotoxic potential of deltamethrin on the synganglion of unfed R. sanguineus female ticks was investigated. The results showed that the synganglion of the females belonging to the control group presented intact morphological characteristics; however, the ones from the treatment group (exposed to 1.5, 3.12 and 6.25 ppm of deltamethrin) displayed alterations, which were increasingly intense as the concentration increased. Observed alterations were mainly in the cortex region and in the neuropile, indicating that the deltamethrin is neurotoxic.