RESUMO

Tick saliva has a pivotal function in parasitism. It has pharmacological and immunomodulatory properties, with several proteins reported in its composition. Thyroglobulin type-1 domain protease inhibitor (thyropin)-like proteins are found in tick saliva, but their function, properties and structures are poorly characterized. It has been reported that thyropins are capable of inhibiting cysteine peptidases present in antigen-presenting cells. To elucidate the role of thyropin-like proteins in ticks, we conducted in silico analysis and cloned an open reading frame from a thyropin-like protein found in Rhipicephalus microplus. The recombinant protein was successfully expressed, followed by immunological characterization and a vaccine trial against Rhipicephalus sanguineus in rabbits. Several differences are observed between thyropin-like proteins from hard and soft ticks, especially the number of thyroglobulin domains and predicted glycosylation pattern. Thyropin-like proteins also differ between postriata and metastriata ticks, the latter having a coil-domain at the C-terminal region and high number of predicted glycosylation sites. Overall, the data suggested divergence in thyropin-like proteins functions among ticks. The recombinant thyropin-like protein is immunogenic and the antibodies against it are able to recognize the native protein in tick saliva and tissues. While the recombinant protein does not elicit a protective response against R. sanguineus infestation, its characterization paves the way for further investigations aimed at determining the precise function of this protein in tick physiology.

Assuntos

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BACKGROUND: When feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding. METHODS: Ticks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species. RESULTS: Changes in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as "sialome switching." This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva. CONCLUSIONS: Overall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods.

Assuntos

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Studies evaluating candidate tick-derived proteins as anti-tick vaccines in natural hosts have been limited due to high costs. To overcome this problem, animal models are used in immunization tests. The aim of this article was to review the use of rabbits as an experimental model for the evaluation of tick-derived proteins as vaccines. A total of 57 tick proteins were tested for their immunogenic potential using rabbits as models for vaccination. The most commonly used rabbit breeds were New Zealand (73.8%), Japanese white (19%), Californians (4.8%) and Flemish lop-eared (2.4%) rabbits. Anti-tick vaccines efficacy resulted in up to 99.9%. Haemaphysalis longicornis (17.9%) and Ornithodoros moubata (12.8%) were the most common tick models in vaccination trials. Experiments with rabbits have revealed that some proteins (CoAQP, OeAQP, OeAQP1, Bm86, GST-Hl, 64TRP, serpins and voraxin) can induce immune responses against various tick species. In addition, in some cases it was possible to determine that the vaccine efficacy in rabbits was similar to that of experiments performed on natural hosts (e.g., Bm86, IrFER2, RmFER2, serpins and serine protease inhibitor). In conclusion, results showed that prior to performing anti-tick vaccination trials using natural hosts, rabbits can be used as suitable experimental models for these studies.

RESUMO

Rhipicephalus (Boophilus) microplus, the Cattle Fever Tick, causes significant economic losses in livestock in tropical and subtropical regions of the world. As the usual control strategy based on chemical acaricides presents different drawbacks, alternative control strategies have been considered for tick control. In recent decades, several tick proteins have been evaluated as targets for the development of anti-tick vaccines. Thus, in the present work, coding sequences from three different proteins present in tick saliva were employed together to construct a recombinant chimeric protein that was evaluated as an antigen in rabbit immunization. Then, the elicited antibodies were tested in a tick artificial feeding experiment to verify the protective effect against the parasites. In addition to Rhipicephalus microplus subtilisin inhibitor 7 (RmSI-7), a serine protease inhibitor member of the TIL (Trypsin Inhibitory Like) family, an interdomain region from the Kunitz inhibitor BmTI-A, and a new cysteine-rich AMP-like microplusin, called RmSEI (previously identified as an elastase inhibitor), were selected to compose the chimeric protein. Anti-chimeric IgG antibodies were able to affect R. microplus female egg production after artificial feeding. Moreover, antibodies elicited in infested tick-resistant and tick-susceptible cattle recognized the recombinant chimera. Additionally, the functional characterization of recombinant RmSEI was performed and revealed antimicrobial activity against gram-positive bacteria. Moreover, the antimicrobial protein was also recognized by antibodies elicited in sera from cattle previously exposed to R. microplus bites. Together, these data suggest that the chimeric protein composed of three salivary antigens is suitable for anti-tick vaccine development.

Assuntos

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The cattle tick Rhipicephalus microplus is a significant problem for livestock, causing losses of billions of dollars per year. This work aimed to determine the chemical composition of essential oils obtained from Laurus nobilis and Copaifera officinalis and evaluate activity against engorged R. microplus females. Chemical composition analyzed by GC-MS revealed the presence of 39 components accounting for 95.38% of the oil in L. nobilis, the most abundant being 1,8-cineol (25.7%), trans-sabinene-hydrate (20.8%), and α-terpinil acetate (15.0%). Chemical analysis of C. officinalis oil identified 25 components corresponding to 80.5% of the total constituents, where the major compounds were ß-caryophyllene (21.1%), caryophyllene oxide (10.7%), and α-trans-bergamotene (9.3%). Adult immersion test (AIT) showed that L. nobilis essential oil at 5% or 10% caused 80.5% mortality of engorged females after 24 h and reached 96.9% and 100% mortality on the third day after treatment, respectively. While the essential oil from C. officinalis caused 84.7% mortality after six days at 10% and at 5%, achieved approximately 100% mortality rate at the end of the experiment (day 15). Both essential oils and the combination significantly inhibited egg-laying; however, the combination treatment showed higher effectiveness than the isolated oils at 2.5%. A possible synergic action of L. nobilis and C. officinalis against the cattle tick R. microplus is therefore suggested. The present work introduces a potential alternative for the development of a formulation environment-friendly (green pesticide) used to control cattle tick infestations.

Assuntos

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Rhipicephalus microplus is a cattle ectoparasite found in tropical and subtropical regions around the world with great impact on livestock production. R. microplus can also harbor pathogens, such as Babesia sp. and Anaplasma sp. which further compromise cattle production. Blood meal acquisition and digestion are key steps for tick development. In ticks, digestion takes place inside midgut cells and is mediated by aspartic and cysteine peptidases and, therefore, regulated by their inhibitors. Cystatins are a family of cysteine peptidases inhibitors found in several organisms and have been associated in ticks with blood acquisition, blood digestion, modulation of host immune response and tick immunity. In this work, we characterized a novel R. microplus type 1 cystatin, named Rmcystatin-1b. The inhibitor transcripts were found to be highly expressed in the midgut of partially and fully engorged females and they appear to be modulated at different days post-detachment. Purified recombinant Rmcystatin-1b displayed inhibitory activity towards typical cysteine peptidases with high affinity. Moreover, rRmcystatin-1b was able to inhibit native R. microplus cysteine peptidases and RNAi-mediated knockdown of the cystatin transcripts resulted in increased proteolytic activity. Moreover, rRmcystatin-1b was able to interfere with B. bovis growth in vitro. Taken together our data strongly suggest that Rmcystatin-1b is a regulator of blood digestion in R. microplus midgut.

Assuntos

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The cattle tick Rhipicephalus microplus is one of the most important ectoparasites causing significant economic losses for the cattle industry. The major tool of control is reducing the number of ticks, applying acaricides in cattle. However, overuse has led to selection of resistant populations of R. microplus to most of these products, some even to more than one active principle. Thus, exploration for new molecules with acaricidal activity in R. microplus has become necessary. Triosephosphate isomerase (TIM) is an essential enzyme in R. microplus metabolism and could be an interesting target for the development of new methods for tick control. In this work, we screened 227 compounds, from our in-house chemo-library, against TIM from R. microplus. Four compounds (50, 98, 14, and 161) selectively inhibited this enzyme with IC50 values between 25 and 50 µM. They were also able to diminish cellular viability of BME26 embryonic cells by more than 50% at 50 µM. A molecular docking study showed that the compounds bind in different regions of the protein; compound 14 interacts with the dimer interface. Furthermore, compound 14 affected the survival of partially engorged females, fed artificially, using the capillary technique. This molecule is simple, easy to produce, and important biological data-including toxicological information-are available for it. Our results imply a promising role for compound 14 as a prototype for development of a new acaricidal involving selective TIM inhibition.

RESUMO

Metalloproteases (MPs) have been considered essential for blood feeding and other physiological functions in several hematophagous animals, including ticks. We report the characterization of MP sequences of three important ticks from Asia, Africa and America: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) and R. microplus (BrRm-MPs). Amino acid sequence identity between R. microplus and R. sanguineus MPs ranged from 76 to 100 %, and identities among I. persulcatus, I. ricinus and I. scapularis MP sequences ranged from 88 to 97 %. This high sequence identity and typical functional motifs show that all sequences are MPs. The presence of a zinc binding site, a Met-turn and cysteine rich domain at the C-terminal region indicates that these proteins belong to the reproplysin family of MPs. Differences in amino acid sequences of BrRm-MP1, BrRm-MP2, BrRm-MP4 and BrRm-MP5 (from Porto Alegre strain ticks) were 6, 2, 7 and 5 %, respectively, when compared with sequences deposited in GenBank for the same genes from other R. microplus isolates. Analyses of MPs predicted that they have various highly antigenic regions. Semi-quantitative RT-PCR analysis revealed the presence of transcripts in salivary glands of partially and fully fed female ticks. None of these transcripts were observed in males (except BrRm-MP4) and eggs. These enzymes may be functional components required during tick feeding to manipulate host defenses and support tick hematophagy.

Assuntos

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Background: The hard tick Rhipicephalus (Boophilus) microplus is an hematophagous ectoparasite that causes important economic losses in the cattle raising. The control of the parasite is usually accomplished through the use of acaricides. Even though these chemicals substances present effectiveness, their continuous use can lead to the selection of resistant parasites. When not used properly acaricides can cause environmental damages in addition to risks for animal and human health. Because of these drawbacks there are efforts to the development of alternatives control methods. The immunological control is a promissing method due to target specifi city, which increases the environmental and animal safety. The development of such approach relies on the discovery and characterization of molecules involved in the metabolism of the parasite different life stages. The embryogenesis is the stage where the metabolites of maternal origin are mobilized through catabolic and anabolic pathways necessary for the development of the embryo. The study of the substrates and enzymes involved in metabolic pathways of the embryogenesis allows the directed search to potential antigens to the development of an anti-R. microplus vaccine.Review: The embryo, during its development, passes through the stages of a sincicial blastoderm, celular blastoderm and segmentation, and both morfological changes a

Background: The hard tick Rhipicephalus (Boophilus) microplus is an hematophagous ectoparasite that causes important economic losses in the cattle raising. The control of the parasite is usually accomplished through the use of acaricides. Even though these chemicals substances present effectiveness, their continuous use can lead to the selection of resistant parasites. When not used properly acaricides can cause environmental damages in addition to risks for animal and human health. Because of these drawbacks there are efforts to the development of alternatives control methods. The immunological control is a promissing method due to target specifi city, which increases the environmental and animal safety. The development of such approach relies on the discovery and characterization of molecules involved in the metabolism of the parasite different life stages. The embryogenesis is the stage where the metabolites of maternal origin are mobilized through catabolic and anabolic pathways necessary for the development of the embryo. The study of the substrates and enzymes involved in metabolic pathways of the embryogenesis allows the directed search to potential antigens to the development of an anti-R. microplus vaccine.Review: The embryo, during its development, passes through the stages of a sincicial blastoderm, celular blastoderm and segmentation, and both morfological changes a

RESUMO

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by fl ies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale.Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the fi rst line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire o

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by fl ies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale.Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the fi rst line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire o