Changes in saliva protein profile throughout Rhipicephalus microplus blood feeding.

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.

Rhipicephalus microplus thyropin-like protein: Structural and immunologic analyzes.

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.

Rabbits as Animal Models for Anti-Tick Vaccine Development: A Global Scenario.

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.

Stable internal reference genes for quantitative RT-PCR analyses in Rhipicephalus microplus during embryogenesis.

Studies on the transcriptional control of gene expression are crucial to understand changes in organism's physiological or cellular conditions. To obtain reliable data on mRNA amounts and the estimation of gene expression levels, it is crucial to normalize the target gene with one or more internal reference gene(s). However, the use of constitutive genes as reference genes is controversial, as their expression patterns are sometimes more complex than previously thought. In various arthropod vectors, including ticks, several constitutive genes have been identified by studying gene expression in different tissues and life stages. The cattle tick Rhipicephalus microplus is a major vector for several pathogens and is widely distributed in tropical and subtropical regions globally. Tick developmental physiology is an essential aspect of research, particularly embryogenesis, where many important developmental events occur, thus the identification of stable reference genes is essential for the interpretation of reliable gene expression data. This study aimed to identify and select R. microplus housekeeping genes and evaluate their stability during embryogenesis. Reference genes used as internal control in molecular assays were selected based on previous studies. These genes were screened by quantitative PCR (qPCR) and tested for gene expression stability during embryogenesis. Results demonstrated that the relative stability of reference genes varied at different time points during the embryogenesis. The GeNorm tool showed that elongation factor 1α (Elf1a) and ribosomal protein L4 (Rpl4) were the most stable genes, while H3 histone family 3A (Hist3A) and ribosomal protein S18 (RpS18) were the least stable. The NormFinder tool showed that Rpl4 was the most stable gene, while the ranking of Elf1a was intermediate in all tested conditions. The BestKeeper tool showed that Rpl4 and cyclophilin A (CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDH are suitable internal controls for normalizing qPCR during R. microplus embryogenesis. These genes were consistently identified as the most stable in various analysis methods employed in this study. Thus, findings presented in this study offer valuable information for the study of gene expression during embryogenesis in R. microplus.

A multiepitope chimeric antigen from Rhipicephalus microplus-secreted salivary proteins elicits anti-tick protective antibodies in rabbit.

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.

Evaluation of essential oils as an ecological alternative in the search for control Rhipicephalus microplus (Acari: Ixodidae).

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.

A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases.

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.

Novel and Selective Rhipicephalus microplus Triosephosphate Isomerase Inhibitors with Acaricidal Activity.

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.

Reprolysin metalloproteases from Ixodes persulcatus, Rhipicephalus sanguineus and Rhipicephalus microplus ticks.

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.

[In vitro analysis of amitraz efficacy against Rhipicephalus (Boophilus) microplus (Canestrini, 1887) populations of southern region of Rio Grande do Sul state]. / Análise in vitro da eficácia do amitraz sobre populações de Rhipicephalus (Boophilus) microplus (Canestrini, 1887) da região sul do Rio Grande do Sul.

Commercial formulation of the amitraz is the most used acaricide in the southern region of Rio Grande do Sul State, however scientific data about the efficacy of these products in this region are sparse. The aim of the present study was to evaluate the frequency of R. (B.) microplus populations resistant to amitraz acaricides in the Southern region of Rio Grande do Sul State. So, the efficacies of five commercial products containing amitraz were compared. Results showed that the efficacy of amitraz acaricides was lower than 69% in 23% of studied populations. Nearly 29% of the tick samples showed an acaricide efficacy equal to zero, suggesting the presence of a high level of resistance to amitraz. Moreover, the most used commercial product in the region showed the lowest efficacy index. These results alert about the possible spreading of resistant on tick populations and justify research directed toward the search for alternative products to control this parasite.

Análise in vitro da eficácia do amitraz sobre populações de Rhipicephalus (Boophilus) microplus (Canestrini, 1887) da região sul do Rio Grande do Sul

Commercial formulation of the amitraz is the most used acaricide in the southern region of Rio Grande do Sul State, however scientific data about the efficacy of these products in this region are sparse. The aim of the present study was to evaluate the frequency of R. (B.) microplus populations resistant to amitraz acaricides in the Southern region of Rio Grande do Sul State. So, the efficacies of five commercial products containing amitraz were compared. Results showed that the efficacy of amitraz acaricides was lower than 69% in 23% of studied populations. Nearly 29% of the tick samples showed an acaricide efficacy equal to zero, suggesting the presence of a high level of resistance to amitraz. Moreover, the most used commercial product in the region showed the lowest efficacy index. These results alert about the possible spreading of resistant on tick populations and justify research directed toward the search for alternative products to control this parasite.

Formulações comerciais à base de amitraz são muito utilizadas como acaricidas na Região Sul do Rio Grande do Sul, contudo sem maiores estudos da sua eficácia nessa região. O presente estudo objetivou estimar a frequência de populações de R. (B.) microplus resistente ao amitraz na Região Sul do Rio Grande do Sul, e comparar a eficácia de cinco produtos comerciais à base desse princípio ativo. Os resultados demonstraram que, em 23% das populações estudadas, o amitraz teve uma eficácia inferior a 69%. Em 29% dessas amostras de carrapatos, o grau de eficácia foi igual a zero, sugerindo a presença de altos níveis de resistência ao amitraz. Além disso, o produto comercial mais utilizado na região demonstra o menor índice de eficácia. Esses resultados alertam sobre a possibilidade da dispersão de populações de carrapatos resistentes, o que justifica a necessidade de direcionar pesquisas visando a novas alternativas e produtos para o controle desse parasito.

A serine protease inhibitor (serpin) from Haemaphysalis longicornis as an anti-tick vaccine.

The application of anti-tick vaccine has been shown to be the most promising alternative tick control strategy compared to the current use of acaricides that suffer from a number of limitations. The success of this strategy is dependent on the cloning, and characterization of tick molecules involved in the mediation of tick central physiological roles. Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a cDNA with high similarity in the reactive center loop (RCL) to representative serpin, heparin cofactor II. We have named this novel gene as Haemaphysalis longicornis serpin-2 (HLS2). RT-PCR analysis showed that HLS2 mRNA transcripts are not expressed in salivary glands but in hemolymph by feeding ticks. HLS2 was not introduced into the bite site as measured by Western blot analysis. The activated partial thromboplastin time (APTT) and the thrombin inhibitory assay using recombinant HLS2 (rHLS2) demonstrated prolonged coagulation time and inhibition of thrombin activity. These results suggested that HLS2 is present only in hemolymph of the feeding ticks and the function of HLS2 is homeostasis in tick physiological compartment. Vaccination of rabbits with rHLS2 conferred protective immunity against ticks, resulting in 44.6 and 43.0% mortality in nymphal and adult ticks, respectively. These results show that rHLS2 could be an important candidate as a component of a cocktail anti-tick vaccine.