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.

Action of permethrin on Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females: morpho-physiological evaluation of salivary glands.

Currently the most effective method of tick control is the use of acaricides, among which stands out permethrin (active ingredient of acaricide Advantage(®) Max3, Bayer), a neurotoxic pyrethroid. However, assessments of their effects on other tick systems such as glandular are still scarce. Thus, this study provides information, through histochemical techniques, about the toxic effect of this pyrethroid on the morphophysiology of salivary glands of semi-engorged Rhipicephalus sanguineus females exposed to different concentrations of permethrin (206, 1031, and 2062 ppm). The results showed that permethrin caused significant changes in the salivary gland metabolism accelerating the process of glandular degeneration, an event which would occur naturally and with great intensity only in the final engorgement stage. Furthermore, this study pointed out that permethrin reduces the salivary gland secretion ability through a drastic reduction of proteins, lipids, and polysaccharides in acinar cells. These changes impair the females to finalize the feeding process, what indirectly affects the reproductive process.

Degenerative process and cell death in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged female exposed to the acaricide permethrin.

Ticks are ectoparasites of great medical and veterinary importance around the world and synthetic chemicals such as permethrin have been used for their control. This study provides a cytochemistry analysis of both degenerative and cell death processes in salivary glands of the brown dog tick Rhipicephalus sanguineus semi-engorged females exposed to 206, 1,031, and 2,062 ppm of permethrin. The results presented herein demonstrate that permethrin is a potent chemical acaricide that would act on the glandular tissue's morphophysiology in this tick species by eliciting severe changes in the acinus shape, intense vacuolation of the acinar cells' cytoplasm, marked glandular tissue disorganization, culminating in an advanced degenerative stage with consequent formation of many apoptotic bodies (cell death). In addition, permethrin induced major changes in the acinar cells' nucleus, such as a change both in its shape and size, chromatin marginalization, nuclear fragmentation, and appearance of picnotic nuclei, especially when the highest concentrations of the product were used. Thus, permethrin induced early degeneration of this tissue characterized by significant changes in the structure of acinar cells and production of enzymes related to the cell death process, in addition to interfering directly in the genetic material of these cells.

Cytotoxic effects of permethrin in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females.

Because of the medical and veterinary importance of ticks and the wide use of synthetic chemical substances such as permethrin (active ingredient of Advantage® Max3 - Bayer)for their control, this study evaluated the effects of different concentrations (206, 1031 and 2062 ppm) of the acaricide on the salivary glands of Rhipicephalus sanguineus semi-engorged females. Results showed that permethrin is a potent substance that acts morpho-physiologically in the tick glandular tissue, causing changes in the acini shape intense vacuolation in acinar cells, and disruption of the tissue by cell death process, with subsequent formation of apoptotic bodies, especially at higher concentrations, thus precluding the accurate identification of different types of acini. Importantly, it is demonstrated that permethrin acts on salivary gland tissue, as well as affecting the nervous system, accelerating the process of glandular degeneration, and interfering with the engorgement process of female ticks, preventing them from completing the feeding process.