Immunoexpression of IL-33 in the different clinical aspects of canine atopic dermatitis.

Canine atopic dermatitis (CAD) is a chronic and inflammatory skin condition with a multifaceted origin, involving genetic factors, skin barrier abnormalities, immune responses, and hypersensitivity to various allergens. Interleukin 33 (IL-33), released by keratinocytes upon cellular injury, plays a crucial role in atopic dermatitis pathogenesis by inducing Th2 lymphocyte-mediated immune responses. This study aimed to evaluate IL-33 expression in dogs with atopic dermatitis and compare it to a control group. Forty-nine dogs were included, with 39 having atopic dermatitis, subdivided into groups based on clinical characteristics, and ten in the control group. Lesion and pruritus scores were assessed, and incisional biopsies were analyzed for dermatopathological characteristics. IL-33 expression was evaluated using immunohistochemistry, the analyses were blinded, based on the measurement of immunostaining areas using Image Pro-Plus software, version 4.5, relying on a semi-automatic color segmentation method, where the tissue immunostaining area for each biomarker was artificially delimited and quantified. Statistically significant differences in IL-33 immunostaining were found among groups (P=0.0005). Lichenified dogs (group 4) exhibited higher immunostaining compared to erythema (group 3) (P=0.0006), alesional pruritus (group 2) (P=0.0261), and the control group (group 1) (P=0.0079). IL-33 immunostaining increased with lesion progression, strongly correlating with lesion scores (P<0.0001), particularly in patients with chronic lesions characterized by erythema and lichenification. These findings suggest IL-33's significant role in canine atopic dermatitis pathogenesis and its association with lesion and inflammation scores during the chronic phase. This suggests potential therapeutic interventions targeting IL-33 or its receptors, though further studies are needed to explore these possibilities.

Effect of commercial tannins on parasitic infection and immunity of lambs naturally infected with Haemonchus contortus.

This study aimed to evaluate the effects of different doses of quebracho extract tannins (Schinopsis lorenzii) (EQ) included in the basal diet (DB) on the count of eggs per gram of feces (EPG), count of parasites in the abomasum, hematological, and immunological parameters in lambs naturally infected with Haemonchus contortus. Forty-eight lambs were evaluated for 42 days and divided into four groups (n = 12): CONT- lambs that received DB without EQ supplementation; G1%- DB with EQ at 1% of dry matter intake (DMI) level; G3%- DB with EQ at the 3% of DMI level; G6%- DB with EQ at the 6% DMI level. For EPG counts, feces were collected weekly. For counting abomasal parasites, at the end of the experiment (D42) half of the lambs in each group were slaughtered. For hematological and immunological analysis, venous blood samples were collected at D0, D21, and D42 to perform the erythrocyte count, mean cell volume (MCV), hematocrit, differential leukocyte count, total plasma protein dosage, and serum IgA class anti-H. contortus using an indirect enzyme immunoassay (ELISA). In the EPG count, there was no significant difference (p > 0.05) between the groups compared with the control. Analysis of the abomasal parasite count showed Haemonchus contortus in its entirety; however, there was no significant difference (p > 0.05) in the number of males, females, and total parasites. No differences (p > 0.05) were observed in erythrocyte count, MCV, hematocrit, and total plasma protein. The average total leukocyte, monocyte, eosinophil, and basophil counts showed no difference (p > 0.05). However, the averages of segmented neutrophils in the G1% and G3% groups presented a reduction in the percentage of cells (p < 0.05) between D21 and D42. Regarding the lymphocyte count, a difference was observed in the CONT and G3% groups at D21 and D42 (p < 0.05), when compared to D0. The analyses of the average absorbance of anti-Haemonchus contortus serum IgA showed no difference (p > 0.05), when the groups were analyzed over time or when compared between them. It can be concluded that the inclusion of different doses of tannins from EQ did not alter the parasitological, hematological, and immunological parameters of lambs infected with H. contortus.

Infestation of rabbits with just-molted adults of the cattle tick Rhipicephalus microplus: biological parameters and efficiency.

In this study, we report for the first time the successful infestation of rabbits with just-molted, unfed adults of Rhipicephalus microplus. Six New Zealand White rabbits were experimentally infested with 20 female and 20 male unfed adult ticks released into plastic chambers fixed on the shaved backs of each host. The attachment and feeding processes were successful. The biological characteristics of the ticks and the occurrence of adverse events in the tick-attachment area were studied. The average engorgement period was 10.7 days, and 33.3% of the engorged females completed the parasitic phase. The average weight of the recovered engorged females was 149.8 mg, with an average egg mass weight of 70.9 mg, a conversion efficiency index of 47.3%, and a hatching percentage of 88.31%. The adverse reactions found in the tick-attachment area were the usual inflammatory responses of the organism to infestation by these ectoparasites; however, it did not prevent the ticks from feeding and completing their life cycle. These data indicate that the infestation of rabbits with just-molted, unfed adult ticks could be a valuable, alternative animal model for rapid and economical evaluation of vaccine candidates and new molecules with acaricidal activity against Rhipicephalus microplus.

Functional and Mass Spectrometric Evaluation of an Anti-Tick Antigen Based on the P0 Peptide Conjugated to Bm86 Protein.

A synthetic 20 amino acid peptide of the ribosomal protein P0 from ticks, when conjugated to keyhole limpet hemocyanin from Megathura crenulata and used as an immunogen against Rhipicephalus microplus and Rhipicephalus sanguineus s.l. species, has shown efficacies of around 90%. There is also experimental evidence of a high efficacy of this conjugate against Amblyomma mixtum and Ixodes ricinus species, which suggest that this antigen could be a good broad-spectrum anti-tick vaccine candidate. In this study, the P0 peptide (pP0) was chemically conjugated to Bm86 as a carrier protein. SDS-PAGE analysis of this conjugate demonstrated that it is highly heterogeneous in size, carrying from 1 to 18 molecules of pP0 per molecule of Bm86. Forty-nine out of the 54 lysine residues and the N-terminal end of Bm86 were found partially linked to pP0 by using LC-MS/MS analysis and the combination of four different softwares. Several post-translational modifications of Bm86 protein were also identified by mass spectrometry. High immunogenicity and efficacy were achieved when dogs and cattle were vaccinated with the pP0-Bm86 conjugate and challenged with R. sanguineus s.l. and R. microplus, respectively. These results encourage the development of this antigen with promising possibilities as an anti-tick vaccine.

Comparative Proteomic Analysis of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) Tropical and Temperate Lineages: Uncovering Differences During Ehrlichia canis Infection.

The tick vector Rhipicephalus sanguineus is established as a complex of closely related species with high veterinary-medical significance, in which the presence of different genetic, morphological, and biological traits has resulted in the recognition of different lineages within taxa. One of the most striking differences in the "temperate" and "tropical" lineages of R. sanguineus (s.l.) is the vector competence to Ehrlichia canis, suggesting that these ticks tolerate and react differently to pathogen infection. The present study addresses the SG and MG proteome of the R. sanguineus tropical and temperate lineages and compares their proteomic profile during E. canis infection. Batches of nymphs from the two lineages were allowed to feed on naïve and experimentally E. canis infected dogs and after molting, adults were dissected, and salivary glands and midgut tissues separated. Samples were screened for the presence of E. canis before proteomic analyses. The representation of the proteins identified in infected and non-infected tissues of each lineage was compared and gene ontology used for protein classification. Results highlight important differences in those proteomic profiles that added to previous reported genetic, biological, behavioral, and morphological differences, strengthening the hypothesis of the existence of two different species. Comparing infected and non-infected tissues, the results show that, while in midgut tissues the response to E. canis infection is similar in the salivary glands, the two lineages show a different pattern of protein representation. Focusing on the proteins found only in the infected condition, the data suggests that the cement cone produced during tick feeding may be implicated in pathogen infection. This study adds useful information to the debate on the controversial R. sanguineus systematic status, to the discussion related with the different vectorial competence occurring between the two lineages and identifies potential targets for efficient tick and tick-borne disease control.

Cytotoxic Effects of Extract of Acmella oleracea in the Ovaries and Midgut of Rhipicephalus sanguineus Latreille, 1806 (Acari: Ixodidae) Female Ticks.

The present study investigated the effects of different concentrations of Acmella oleracea extract on the germinative cells and digestive processes of semi-engorged Rhipicephalus sanguineus females. For this experiment, 150 ticks were divided into five groups (30 individuals each). The animals were immersed for 5 min in different concentrations of the extract, distilled water, or ethanol 50%/DMSO 1%, dried, and kept in biological oxygen demand incubator for 7 days. The alterations were associated with the size of germinative cells and yolk granules; presence, size, and location of vacuoles in the cytoplasm of germinative cells; nuclear modifications in the germinative cells; damages to the nucleus and cytoplasm of the midgut generative cells; size of digestive cells; number of captured blood elements; accumulated digestive residues and digestive vacuoles in the digestive cells cytoplasm; and the number and distribution of proteins and polysaccharides in all the cells of both organs. The concentrations used in this study prevented an efficient and complete blood digestion by the midgut epithelial cells of the treated animals, resulting in the absence of the necessary nutrients to maintain the physiological events in the ectoparasites. In advanced stages, This can lead the ectoparasite to death. The germinative cells were highly impaired and probably not able to advance developmental stages (I-V) or complete vitellogenesis to be released during ovulation, which would prevent the females from originating a new individual. Thus, it can be concluded that the effects of A. oleracea are similar to those caused by chemical products widely recognized as effective to control ticks.

Ticks as potential vectors of Mycobacterium leprae: Use of tick cell lines to culture the bacilli and generate transgenic strains.

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation.

Ticks as potential vectors of Mycobacterium leprae: use of tick cell lines to culture the bacilli and generate transgenic strains

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation.

Cellular response in the tick feeding site in crossbred cattle artificially infested by Rhipicephalus microplus.

Ticks and tick-borne diseases are of global importance and can cause serious economic losses to cattle-raising businesses. However, only few attempts have been made to describe the relationships between various cellular immune components and tick counts of, and therefore resistance to, Rhipicephalus microplus. The present study evaluated the late inflammatory cellular response and dermal dendritic cells at the attachment site in naturally presensitized bovines, artificially infested by R. microplus ticks. Twenty-two crossbred (Holstein × Gir) male experimental cattle were artificially infested with approximately 10,000 larvae of R. microplus. Parasitic infestation was determined by counting the engorged female ticks (4.5-8.0 mm in diameter) at the 21st day after artificial infestation. Biopsies (6 mm) were taken prior to the infestation and along with the tick count from the right pinna of each animal, and examined with histological techniques and immunohistochemistry using S100 protein. Inflammatory cell counts were conducted in the sections stained with the May-Grünwald Giemsa technique, and immunostained dermal dendritic cells were evaluated and classified in scores ranging from 0 to 4. An average of 39.2 ticks per animal was found. Eosinophils (47.8%) constituted the major portion of the cellular infiltrate, followed by mononuclear cells (28.3%), neutrophils (14.4%), and basophils (0% to approximately 2%). The dendritic cell count shows a considerable population in the dermis, with pre- and post-infestation mean scores of 1.54 and 1.89, respectively; these scores were not significantly different. Our results pointed out the importance of the cellular response in the cattle resistance to ticks.

Immunogenic potential of Rhipicephalus (Boophilus) microplus aquaporin 1 against Rhipicephalus sanguineus in domestic dogs / Potencial imunogênico da aquaporina 1 de Rhipicephalus (Boophilus) microplus contra o carrapato Rhipicephalus sanguineus em cães domésticos

Abstract This study evaluated a recombinant aquaporin 1 protein of Rhipicephalus (Boophilus) microplus (RmAQP1) as antigen in a vaccine against R. sanguineus. Five dogs were immunized with RmAQP1 (10 µg) + adjuvant (Montanide) (G1), and five were inoculated with adjuvant only (G2), three times. Twenty-one days after the last immunization, animals of both groups were challenged with R. sanguineus larvae, nymphs and adults, and their biotic potential was compared. Blood samples were collected before each immunization and every 28 days after the last immunization for 10 weeks. Serum antibody titers (IgG) were assessed by ELISA. We observed that: engorgement period of adult females from G1 was 12% shorter than G2; larvae from G1 had 8.7% longer engorgement period than G2 and weighed 7.2% less; nymphs from G1 had 4.5% shorter engorgement period than G2 and weighed 3.6% less; although the antibody titers increased following the second immunization, they rapidly decreased after the third immunization. Results indicated low immunoprotection of RmAQP1 against adult R. sanguineus ticks, and possible efficacy on larvae and nymphs fed on immunized dogs. Further studies should be performed for a full evaluation of the immunoprotection of RmAQP1 against R. sanguineus infestations in dogs.

Resumo Este estudo avaliou a proteína recombinante (aquaporina) do carrapato Rhipicephalus (Boophilus) microplus como antígeno em vacina contra Rhipicephalus sanguineus. Cinco cães foram imunizados com RmAQP1 (10 µg) + adjuvante (G1) e cinco foram inoculados apenas com adjuvante (G2), três vezes. 21 dias após a última imunização todos os animais foram desafiados com larvas, ninfas e adultos de R. sanguineus, e potencial biótico dos carrapatos foi comparado. Amostras de sangue foram coletadas antes de cada imunização e a cada 28 dias após a última imunização, durante 10 semanas. Títulos de anticorpos dos soros dos cães foram avaliados por ELISA. Resultados: o período de ingurgitamento das fêmeas do G1 foi 12% mais curto que o período de ingurgitamento de G2; o período de ingurgitamento das larvas do G1 8,7% foi mais longo e o peso 7,2% menor que no caso de G2; o período de ingurgitamento das ninfas do G1 4,5% foi mais curto e peso 3,6% menor que no caso do G2; aumento dos títulos de anticorpos do G1 após a segunda imunização e declínio após a terceira imunização. Os resultados indicaram baixo potencial de imunoproteção de RmAQP1 contra R. sanguineus adultos, e possível eficácia contra larvas e ninfas, na dose testada. Sugere-se desenvolver novos estudos para melhor avaliação da eficácia de RmAQP1 contra R. sanguineus em cães.

Immunogenic potential of Rhipicephalus (Boophilus) microplus aquaporin 1 against Rhipicephalus sanguineus in domestic dogs.

This study evaluated a recombinant aquaporin 1 protein of Rhipicephalus (Boophilus) microplus (RmAQP1) as antigen in a vaccine against R. sanguineus. Five dogs were immunized with RmAQP1 (10 µg) + adjuvant (Montanide) (G1), and five were inoculated with adjuvant only (G2), three times. Twenty-one days after the last immunization, animals of both groups were challenged with R. sanguineus larvae, nymphs and adults, and their biotic potential was compared. Blood samples were collected before each immunization and every 28 days after the last immunization for 10 weeks. Serum antibody titers (IgG) were assessed by ELISA. We observed that: engorgement period of adult females from G1 was 12% shorter than G2; larvae from G1 had 8.7% longer engorgement period than G2 and weighed 7.2% less; nymphs from G1 had 4.5% shorter engorgement period than G2 and weighed 3.6% less; although the antibody titers increased following the second immunization, they rapidly decreased after the third immunization. Results indicated low immunoprotection of RmAQP1 against adult R. sanguineus ticks, and possible efficacy on larvae and nymphs fed on immunized dogs. Further studies should be performed for a full evaluation of the immunoprotection of RmAQP1 against R. sanguineus infestations in dogs.

Dinotefuran-induced morphophysiological changes in the ovaries and midgut of semi-engorged females Rhipicephalus sanguineus Latreille, 1806 (Acari: Ixodidae) ticks.

The present study demonstrated the effects of dinotefuran (active compound of the Protetor Pet® acaricide) in germ cells and the digestive processes of semi-engorged females of R. sanguineus exposed to different concentrations of the chemical. For this purpose, 120 semi-engorged females were divided into four treatment groups with 30 individuals each: group I or control (distilled water), group II (5000 ppm), group III (6250 ppm), and group IV (8334 ppm of dinotefuran). All ticks were immersed in different concentrations of dinotefuran or in distilled water for 5 min and then were dried and stored in biological oxygen demand (BOD) incubator for 7 days. The results show the action of this compound, exhibiting morphohistologic and histochemical changes in the oocytes and the midgut cells of individuals of different groups, which were compared with those of group I (control). The alterations occurred mainly in relation to the size of the germ cells and yolk granules; presence, quantity, size, and location of vacuoles found in the cytoplasm of these germ cells; the damage occurred in the generative cells of the midgut; the size of the digestive cells; the quantity of blood elements captured, accumulated digestive wastes and digestive vacuoles found in the cytoplasm of the digestive cells of the midgut, as well as the amount and distribution of proteins, polysaccharides, lipids of all cells in both organs. So, it has demonstrated the effectiveness of dinotefuran in the reduction of fertility and digestive processes of semi-engorged females of R. sanguineus, data that points the possibility of employing this chemical to control these ectoparasites.

ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells.

In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides.

Lack of acquired resistance in dogs to successive infestations of Rhipicephalus sanguineus ticks from Brazil and Argentina.

Comparative studies between brown dog tick Rhipicephalus sanguineus populations from Brazil (Jaboticabal, São Paulo) and Argentina (Rafaela, Santa Fé) showed significant biological, morphological and genetic differences between them. This work aimed to study, in a comparative way, the acquisition of resistance in domestic dogs to R. sanguineus from Jaboticabal and Rafaela, after successive and controlled infestations. Ticks were kept in a BOD incubator under controlled conditions (27 °C, 80 % relative humidity, 12-h photoperiod). Ten dogs, Dachshund breed, males and females, 6 months old, short- or long-haired, without prior contact with ticks, were used as hosts. They were distributed into two experimental groups composed of five animals each: G1 infested with ten adult couples of R. sanguineus (Jaboticabal) per animal, and G2 infested with ten adult couples of R. sanguineus (Rafaela) per animal. Ticks' biological parameters and titration of antibodies from the dogs' sera by ELISA test were used for comparison between the strains. Results of the biological parameters showed that the dogs did not acquire immunity to either of the R. sanguineus strains after repeated infestations. The ELISA test showed low antibody titers in sera of dogs from G2, in successive infestations, and higher antibody responses post second and third infestations in G1. It also demonstrated cross-reactivity between sera of dogs infested with R. sanguineus (Jaboticabal) and antigens from R. sanguineus (Rafaela) and vice versa. We conclude that Dachshund dogs did not develop resistance against neither Jaboticabal nor Rafaela strains of R. sanguineus.

Potential of the chemical dinotefuran in the control of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged female ticks.

Ticks are vectors of several pathogens to vertebrates, including the human being. They produce lesions on the hosts during the blood feeding and great economic losses. Several chemical acaricides have been used in an attempt to control tick infestations; however these substances are harmful to both the human being and non-target organisms, and to the environment. Therefore, there is a need to fight these ectoparasites through less harmful methods, less aggressive to the environment, non-target organisms and to the human health. The present study examined the efficacy of dinotefuran on the susceptibility of Rhipicephalus sanguineus semi-engorged females exposed to different concentrations of the product. Its lethal concentration of 50% (LC50) at 95% confidence interval was determined. The ticks were immersed in Petri dishes containing different concentrations of dinotefuran or distilled water for 5 minutes and then dried and maintained in an incubator for 7 days. The results showed the daily number of dead R. sanguineus semi-engorged females after being treated with different concentrations of dinotefuran. The mortality data in bioassay 2 were subjected to Probit analysis, where a LC50 of 10,182.253 ppm (8725.987-13,440.084) and 95% confidence interval were estimated. The susceptibility of R. sanguineus semi-engorged females to dinotefuran in higher concentrations of the acaricide was demonstrated, indicating that its effect is probably dose-dependent. In addition, the action of dinotefuran was slow and gradual, interfering in the development and growth of the individuals throughout the observation period (7 days).

Morpho-histochemical characterization of the salivary glands of semi-engorged Amblyomma triste (Koch, 1844) (Acari: Ixodidae) female ticks.

This study presents the morphological and physiological characterization of the salivary glands of semi-engorged Amblyomma triste females. Unfed individuals were placed on New Zealand White rabbits for feeding and the females, after 4 days, were collected, dissected and the salivary glands were submitted to the application of histological (hematoxylin-eosin technique) and histochemical tests for the detection of protein (bromophenol blue technique, polysaccharides (periodic acid-Schiff technique), lipid (Nile blue technique) and calcium (von Kossa technique). The histological results show that the glandular tissue is composed by a system of ducts and three types of acini (I, II and III). The acini I are formed by a large central cell surrounded by several smaller agranular peripheral cells. Acini II are formed by cells a, c1, c2, c3 and c5, which are full of secretion granules. Acini III are constituted by cells d, e and f; the former two contain secretion granules, the latter is agranular. The glandular histochemical composition was also verified. Data obtained here will certainly help in the understanding of the cellular morphology and of the general physiology of these organs in this specie, providing important information for the creation of scientific bases which will contribute for the development of more specific and efficient methods of control.

Effects of andiroba (Carapa guianensis) oil in ticks: Ultrastructural analysis of the synganglion of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae).

The present study performed an analysis of the ultrastructural changes induced by andiroba seed oil in the synganglion of Rhipicephalus sanguineus female ticks, aiming to provide scientific grounds to help in the creation of more specific and efficient methods of control. The synganglion consists of a mass of fused nerves externally covered by the neural lamella, a uniform and acellular layer. Just below, the perineurium is found, formed by glial cells. Internally, the synganglion is subdivided into an outer cortical region (cortex), which contains the cellular bodies of the neural cells and an inner region, the neuropile, formed by a set of nerve fibers (extensions of the neural cells). The results showed that the synganglion of females exposed to andiroba oil showed structural changes, such as: irregular and apparently thinner neural lamella, perineurium glial cells presenting large cytoplasmic vacuoles, decrease in the extensions of glial cells, separation of cortex cells, which were formerly attached through their membranes, neural cells presenting irregular plasma membranes and cytoplasm with autophagic vacuoles and mitochondria with disorganized cristae and in process of degeneration. This study confirmed the neurotoxic action of the andiroba oil, which would probably be able to impair the neural functions. Thus, it is suggested that this product has the potential to be used as an alternative method to control ticks.

Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) nymphs: an ultrastructural study of the integument and midgut.

This is an ultrastructural study of two important organs of ticks: the integument, covering the body and involved in the molting process, and the midgut, the first organ to have contact with the ingested host blood. The integument of Rhipicephalus sanguineus sensu lato freshly engorged nymphs consists of a layer of epidermal cells rich in organelles and a cuticle, subdivided in epicuticle and procuticle. The latter presents two distinct regions: the exocuticle (with several pore canals) and the endocuticle (secreted in highly organized overlaid lamellae). The midgut of R. sanguineus sensu lato freshly engorged nymphs consists of an epithelial wall formed by two types of cells: the generative cells, with few organelles; and the digestive cells, with several endosomes, digestive vacuoles, hematin residual bodies, and small drops of lipid.

Changes in the synganglion of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) female ticks exposed to permethrin: an ultrastructural overview.

This study performed the ultra-structural analysis of the changes caused by permethrin in the synganglion of semi-engorged Rhipicephalus sanguineus females, aiming to understand the toxic action of this substance at cellular level. The results showed that the neural lamella had its structure changed, allowing the influx of the toxic agent into the nervous tissue. The glial cells of the perineurium, as well as the neural cells of the cortex showed great changes, such as: irregular nuclei with chromatin margination, cytoplasmic vacuolation and degenerating mitochondria. These changes showed that the permethrin would be able to induce the degeneration of the synganglion through an atypical death process, involving apoptosis and autophagy. In addition, a dilated rough endoplasmic reticulum was observed in the neural cells, suggesting an intense synthesis of the hydrolytic enzymes that would be used in the processes of degradation of the damaged cellular structures (formation of lysosomes). The subperineurium and the neuropile also showed changes in their structures. Thus, it is suggested that permethrin is a dose-dependent compound able to impair the metabolism of the organism as a whole, once all the other organs and body structures are directly dependent of the neural control. The information obtained in this study can be used in the improvement of the control methods which use permethrin as active substance, helping in the creation of a really efficient acaricide product to fight this important urban plague.

Fluazuron-induced morphological changes in Rhipicephalus sanguineus Latreille, 1806 (Acari: Ixodidae) nymphs: An ultra-structural evaluation of the cuticle formation and digestive processes.

Rhipicephalus sanguineus is a species of tick which is widely distributed in America, Africa and Australia and is probably the most prevalent among all the other ixodid tick species. The present study demonstrated the effects of the arthropod growth regulator fluazuron (Acatak(®)), in the formation of the integument and the digestive processes of R. sanguineus nymphs fed on rabbits treated with different doses of this chemical acaricide. For this, three different doses of fluazuron (20mg/kg, 40mg/kg and 80mg/kg) were applied "pour on" to the hosts divided into three different treated-groups (II, III, IV) of three animals each. A fourth group (I) of rabbits (n=3) was given distilled water as control. On the first day after treatment (24h), the hosts were artificially infested with R. sanguineus nymphs. After full engorgement (7 days), the nymphs were removed and placed on labeled Petri dishes and kept in BOD incubator for 7 days. The engorged nymphs were then taken to ultra-structural analysis. Results revealed the following main ultra-structural changes in the nymphs integument and midgut of the different treated groups (II, III, IV): cuticle disorganization and the absence of subdivisions, damages in the integument epithelial cells, size of digestive cells, amount of endosomes, autophagic and digestive vacuoles, accumulated digestive residues, lipid droplets and organelles found in the digestive cells' cytoplasm, as well as the presence of microvilli in their plasma membranes. It is concluded that fluazuron may act on the integument and midgut cells of R. sanguineus engorged nymphs by impairing the synthesis of the new cuticle and the digestive processes (absorption of the blood ingested from the host, digestion - hemolysis, formation of digestive residues and release of nutrients to be converted into lipid, as well as for the synthesis of structural protein), which interfere in the development of nymphs, being able to prevent the emergence of adults after periodical ecdysis. These data indicate the possibility to use this arthropod growth regulator (AGR) in the control of R. sanguineus, at least in the nymphal stage of its biological cycle.