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Rhipicephalus microplus poses a substantial threat to livestock health and agricultural economies worldwide. Its remarkable adaptability to diverse environments and hosts is a testament to its extensive genetic diversity. This review delves into the genetic diversity of R. microplus, employing three pivotal genetic markers: the cytochrome c oxidase I (COX1) gene, ribosomal genes, and microsatellites. The COX1 gene, a crucial tool for genetic characterization and phylogenetic clustering, provides insights into the adaptability of ticks. Ribosomal genes, such as internal transcribed spacer regions (ITS-1 and2) as well as 18S and 28S, are routinely utilized for species differentiation. However, their use is limited due to indels (insertions and deletions). Microsatellites and minisatellites, known for their high polymorphism, have been successfully employed to study populations and genetic diversity across various tick species. Despite their effectiveness, challenges such as null alleles and marker variations warrant careful consideration. Bm86, a well-studied vaccine candidate, exhibits substantial genetic diversity. This diversity directly influences vaccine efficacy, posing challenges for developing a universally effective Bm86-based vaccine. Moreover, the review emphasizes the prevalence of genes associated with synthetic pyrethroid resistance. Identifying single nucleotide polymorphisms in the acaricide-resistant genes of R. microplus has facilitated the development of molecular markers for detecting and monitoring resistance against synthetic pyrethroids. However, mutations in sodium channels, the target site for synthetic pyrethroid, correlate well with the resistance status of R. microplus, which is not the case with other acaricide target genes. This study underscores the importance of understanding genetic diversity in developing effective tick management strategies. The choice of genetic marker should be tailored based on the level of taxonomic resolution and the group of ticks under investigation. A holistic approach combining multiple markers and integrating additional molecular and morphological data may offer a more comprehensive understanding of tick diversity and relationships. This research has far-reaching implications in formulating breeding programs and the development of vaccine against ticks and tick-borne diseases (TTBDs) as well as strategies for the management of resistant ticks.

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Tick and tick-borne disease control have been a serious research focus for many decades. In a global climate of increasing acaricide resistance, host immunity against tick infestation has become a much-needed complementary strategy to common chemical control. From the earliest acquired resistance studies in small animal models to proof of concept in large production animals, it was the isolation, characterization, and final recombinant protein production of the midgut antigen Bm86 from the Australian cattle tick strain of Rhipicephalus (Boophilus) microplus (later reinstated as R. (B.) australis) that established tick subunit vaccines as a viable alternative in tick and tick-borne disease control. In the past 37 years, this antigen has spawned numerous tick subunit vaccines (either Bm86-based or novel), and though we are still describing its molecular structure and function, this antigen remains the gold standard for all tick vaccines. In this paper, advances in tick vaccine development over the past three decades are discussed alongside the development of biotechnology, where existing gaps and future directives in the field are highlighted.

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Control of complex parasites via vaccination remains challenging, with the current combination of vaccines and small drugs remaining the choice for an integrated control strategy. Studies conducted to date, are providing evidence that multicomponent vaccines will be needed for the development of protective vaccines against endo- and ectoparasites, though multicomponent vaccines require an in-depth understanding of parasite biology which remains insufficient for ticks. With the rapid development and spread of acaricide resistance in ticks, new targets for acaricide development also remains to be identified, along with novel targets that can be exploited for the design of lead compounds. In this study, we analysed the differential gene expression of Rhipicephalus microplus ticks that were fed on cattle vaccinated with a multi-component vaccine (Bm86 and 3 putative Bm86-binding proteins). The data was scrutinised for the identification of vaccine targets, small drug targets and novel pathways that can be evaluated in future studies. Limitations associated with targeting novel proteins for vaccine and/or drug design is also discussed and placed into the context of challenges arising when targeting large protein families and intracellular localised proteins. Lastly, this study provide insight into how Bm86-based vaccines may reduce successful uptake and digestion of the bloodmeal and overall tick fecundity.

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Introduction: Hyalomma and Rhipicephalus ticks are important genera that can transmit diseases to both animals and humans, including Crimean-Congo hemorrhagic fever, tick-borne encephalitis, and several types of spotted fever. The accurate identification of tick species is essential for the effective control and prevention of tick-borne diseases. However, traditional identification methods based on morphology can be challenging and subjective, leading to errors. The development of DNA markers has provided more precise and efficient methods for tick species identification, but the currently available markers have limitations in their discriminatory power and sensitivity. To address this need for more sensitive and specific markers, this study aimed to identify two minimum sequence fragments required for tick Hyalomma and Rhipicephalus species identification using the Bm86 cDNA marker, which has previously been shown to be in perfect agreement with the current taxonomy of hard ticks based on its complete sequence. Methods: Based on our in silico determination that a minimum sequence of 398 bp for Rhipicephalus spp. (from 1487 to 1884) and 559 bp for Hyalomma species (from 539 to 1097) was necessary for species delineation, two distinct PCR assays were developed to apply these sequences in practice. Results and discussion: Discrimination between species within each genus was achieved through sequence homology and phylogenetic analysis following the sequencing of the two PCR products. Subsequently, their performance was evaluated by testing them on the field-collected ticks of the Hyalomma and Rhipicephalus genera obtained from various host animals in different geographic regions of Tunisia. The use of shorter partial sequences specific to the tick genera Rhipicephalus and Hyalomma, which target the tick's RNA banks, could represent a significant advance in the field of tick species identification, providing a sensitive and discriminatory tool for interspecific and intraspecific diversity analysis.

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Rhipicephalus microplus economically impacts cattle production in tropical and subtropical countries. Application of acaricides constitutes the major control method; however, inadequate use has increased resistant tick populations, resulting in environmental and cattle product contamination. Anti-tick vaccines based on the Bm86 antigen are an environmentally friendly, safe, and economically sustainable alternative for controlling R. microplus infestations. Nevertheless, variable efficacy has been experienced against different geographic tick strains. Herein, we evaluated the efficacy of a conserved polypeptide Bm86 derived from a Mexican R. microplus strain previously characterized. Twelve cows were assigned to three experimental groups and immunized with three doses of the polypeptide Bm86 (pBm86), adjuvant/saline alone, and Bm86 antigen (control +), respectively. Specific IgG antibody levels were measured by ELISA and confirmed by Western blot. In addition, the reproductive performance of naturally infested R. microplus was also determined. The more affected parameter was the adult female tick number, with a reduction of 44% by the pBm86 compared to the controls (p < 0.05), showing a vaccine efficacy of 58%. Anti-pBm86 IgG antibodies were immunogenic and capable of recognizing the native Bm86 protein in the eggs, larvae, and guts of R. microplus. The negative correlation between antibody levels and the reduction of naturally tick-infested cattle suggested that the effect of the polypeptide Bm86 was attributed to the antibody response in immunized cattle. In conclusion, the polypeptide Bm86 showed a specific immune response in cattle and conferred protection against R. microplus in a Mexican tropical region. These findings support further experiments with this antigen to demonstrate its effectiveness as a regional vaccine.

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The immunoprophylactic management of ticks is the most effective option to control tick infestations and counter spread the acaricide resistance problem worldwide. Several researchers reported an inconsistent efficacy of the single antigen-based immunization of hosts against different tick species. In the present study, to develop a multi-target immunization protocol, proteins from Rhipicephalus microplus BM86 and Hyalomma anatolicum subolesin (SUB) and tropomyosin (TPM) were targeted to evaluate the cross-protective potential. The sequence identities of the BM86, SUB, and TPM coding genes amongst Indian tick isolates of targeted species were 95.6-99.8%, 98.7-99.6%, and 98.9-99.9%, respectively, while at the predicted amino acid level, the identities were 93.2 to 99.5, 97.6 to 99.4, and 98.2 to 99.3%. The targeted genes were expressed in the eukaryotic expression system, pKLAC2-Kluyveromyces lactis, and 100 µg each of purified recombinant protein (Bm86-89 kDa, SUB-21 kDa, and TPM-36 kDa) mixed with adjuvant was injected individually through the intramuscular route at different sites of the body on days 0, 30, and 60 to immunize cross-bred cattle. Post-immunization, a statistically significant (p < 0.001) antibody response (IgG, IgG1, and IgG2) in comparison to the control, starting from 15 to 140 days, against each antigen was recorded. Following multi-antigen immunization, the animals were challenged twice with the larvae of R. microplus and H. anatolicum and theadults of H. anatolicum, and a significant vaccine efficacy of 87.2% and 86.2% against H. anatolicum larvae and adults, respectively, and 86.7% against R. microplus was obtained. The current study provides significant support to develop a multi-antigen vaccine against cattle tick species.

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The presence in nature of species with genetic resistance to ticks, or with acquired resistance after repeated tick infestations, has encouraged the scientific community to consider vaccination as an alternative to the unsustainable chemical control of ticks. After numerous attempts to artificially immunize hosts with tick extracts, the purification and characterization of the Bm86 antigen by Willadsen et al. in 1989 constituted a revolutionary step forward in the development of vaccines against ticks. Previously, innovative studies that had used tick gut extracts for the immunization of cattle against Rhipicepahalus microplus (previously named Boophilus microplus) ticks, with amazingly successful results, demonstrated the feasibility of using antigens other than salivary-gland-derived molecules to induce a strong anti-tick immunity. However, the practical application of an anti-tick vaccine required the isolation, identification, and purification of the responsible antigen, which was finally defined as the Bm86 protein. More than thirty years later, the only commercially available anti-tick vaccines are still based on this antigen, and all our current knowledge about the field application of immunological control based on vaccination against ticks has been obtained through the use of these vaccines.

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The southern cattle fever tick (SCFT) Rhipicephalus (Boophilus) microplus, is considered the most important ectoparasite of livestock in the world because of high financial losses associated with direct feeding and transmission of the hemoparasites Babesia bovis, B. bigemina, and Anaplasma marginale. Unfortunately, SCFT in many parts of the world have evolved resistance to all market-available pesticides thus driving development of new control technologies. Vaccination against ticks using the tick gut protein Bm86 has been shown to be effective against acaricide-resistant ticks. This technique has been successfully implemented in Puerto Rico for the control of acaricide-resistant R. microplus on dairy and beef cattle. Observations from Puerto Rico indicate a potentially positive interaction between anti-tick vaccination when used in conjunction with systemic acaricide treatment. In this project, controlled animal studies were completed directly comparing efficacy of anti-tick vaccination with and without systemic acaricide. Results show that the Bm86 anti-tick vaccine in combination with the macrocyclic lactone, Moxidectin, expressed a synergistic interaction, providing greater and longer efficacy than either treatment alone.

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BACKGROUND: There have been ongoing efforts to identify anti-tick vaccine targets to protect cattle from infestation with cattle fever ticks Rhipicephalus (Boophilus) microplus. Two commercial vaccines based on the tick gut protein Bm86 have had variable effectiveness, which has led to poor acceptance, and numerous studies have attempted to identify vaccine antigens that will provide more consistently effective protection. Transcriptomic analysis of R. microplus led to identification of three aquaporin genes annotated to code for transmembrane proteins involved in the transport of water across cell membranes. Previous work showed that vaccination with full-length recombinant aquaporin 1 (RmAQP1) reduced tick burdens on cattle. Targeted silencing of aquaporin 2 (RmAQP2) expression suggested it might also be a good anti-tick vaccination target. METHODS: Three synthetic peptides from the predicted extracellular domains of RmAQP2 were used to vaccinate cattle. Peptides were conjugated to keyhole limpet hemocyanin (KLH) as an antigenic carrier molecule. We monitored the antibody response with ELISA and challenged vaccinated cattle with R. microplus larvae. RESULTS: There was a 25% reduction overall in the numbers of ticks feeding to repletion on the vaccinated cattle. Immune sera from vaccinated cattle recognized native tick proteins on a western blot and reacted to the three individual synthetic peptides in an ELISA. The vaccinated calf with the highest total IgG titer was not the most effective at controlling ticks; ratios of IgG isotypes 1 and 2 differed greatly among the three vaccinated cattle; the calf with the highest IgG1/IgG2 ratio had the fewest ticks. Ticks on vaccinated cattle had significantly greater replete weights compared to ticks on controls, mirroring results seen with RNA silencing of RmAQP2. However, protein data could not confirm that vaccination had any impact on the ability of the tick to concentrate its blood meal by removing water. CONCLUSIONS: A reduced number of ticks feed successfully on cattle vaccinated to produce antibodies against the extracellular domains of RmAQP2. However, our predicted mechanism, that antibody binding blocks the ability of RmAQP2 to move water out of the blood meal, could not be confirmed. Further study will be required to define the mechanism of action and to determine whether these vaccine targets will be useful components of an anti-tick vaccine cocktail.

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Arthropod vectors account for a number of animal and human diseases, posing substantial threats to health and safety on a global scale. Ticks are considered as one of the most prominent vectors, as they can parasitize almost any vertebrate class and transmit a multitude of infectious diseases, particularly ones that affect humans and domestic animals. While various tick species elicit different tick-borne infections in specific geographic regions, single species can have widespread effects, such as blacklegged ticks, which are widely distributed across the eastern United States and can transmit a variety of infections, including Lyme borreliosis, anaplasmosis, relapsing fever disease, ehrlichiosis, babesiosis, and Powassan virus disease. Despite increasing awareness about ticks as serious disease vectors, effective vaccines against most tick-borne infections are not available. Previously, the successful development of an anti-tick vaccine for use in veterinary animals was based on an 86-kDa midgut antigen from Rhipicephalus (formerly Boophilus) microplus ticks. Herein we describe the fundamentals of vaccine development using protein antigens as model vaccinogen candidates, beginning with the cloning, expression, and purification of recombinant proteins, host immunization, and the assessment of protective efficacy in laboratory settings using a tick-borne murine model of Lyme borreliosis.

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Cattle tick (Rhipicephalus microplus) represents a severe problem causing substantial economic losses, estimated in billions of dollars annually. Currently, chemical acaricides represent the most widely used control method. However, several problems such as resistance have been described. Phage-based vaccines represent a fast and low-cost tool for antigen delivery. In this regard, the objective of the present work was to develop a candidate phage-based vaccine displaying a cattle tick antigen (Bm86-derived Sbm7462 antigen) on the surface of bacteriophage M13. Phage ELISA and dot blotting analysis confirmed the display of the antigen. Vaccine immunogenicity was evaluated using a bovine monocyte-derived dendritic cell-based ex vivo assay and a murine in vivo assay. The ex vivo model showed the maturation of dendritic cells after being pulsed with the phage-based vaccine. The humoral response was confirmed in the in vivo assay. These results demonstrated the capacity of the phage-based vaccine to induce both humoral and cellular immune-specific responses. Importantly, this is the first report describing a control method for cattle ticks using a candidate phage-based vaccine. Further studies to evaluate the immunogenicity in a bovine model are needed. The current approach represents a promising alternative to control cattle tick infestations.

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The control of cattle tick, Rhipicephalus microplus, is focused on repeated use of acaricides. However, due to growing acaricide resistance and residues problem, immunization of animals along with limited use of effective acaricides is considered a suitable option for the control of tick infestations. To date, more than fifty vaccine candidates have been identified and tested worldwide, but two vaccines were developed using the extensively studied candidate, Bm86. The main reason for limited vaccine commercialization in other countries is genetic diversity in the Bm86 gene leading to considerable variation in vaccine efficacy. India, with 193.46 million cattle population distributed in 28 states and 9 union territories, is suffering from multiple tick infestation dominated by R. microplus. As R. microplus has developed multi-acaricide resistance, an efficacious vaccine may provide a sustainable intervention for tick control. Preliminary experiments revealed that the presently available commercial vaccine based on the BM86 gene is not efficacious against Indian strain. In concert with the principle of reverse vaccinology, genetic polymorphism of the Bm86 gene within Indian isolates of R. microplus was studied. A 578 bp conserved nucleotide sequences of Bm86 from 65 R. microplus isolates collected from 9 Indian states was sequenced and revealed 95.6-99.8% and 93.2-99.5% identity in nucleotides and amino acids sequences, respectively. The identities of nucleotides and deduced amino acids were 94.7-99.8% and 91.8-99.5%, respectively, between full-length sequence (orf) of the Bm86 gene of IVRI-I strain and published sequences of vaccine strains. Six nucleotides deletion were observed in Indian Bm86 sequences. Four B-cell epitopes (D519-K554, H563-Q587, C598-T606, T609-K623), which are present in the conserved region of the IVRI-I Bm86 sequence, were selected. The results confirm that the use of available commercial Bm86 vaccines is not a suitable option against Indian isolates of R. microplus. A country-specific multi-epitope Bm86 vaccine consisting of four specific B-cell epitopes along with candidate molecules, subolesin and tropomyosin in chimeric/co-immunization format may provide a sustainable option for implementation in an integrated tick management system.

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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.

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Development of veterinary subunit vaccines comes with a spectrum of challenges, such as the choice of adjuvant, antigen delivery vehicle, and optimization of dosing strategy. Over the years, our laboratory has largely focused on investigating silica vesicles (SVs) for developing effective veterinary vaccines for multiple targets. Rhipicephalus microplus (cattle tick) are known to have a high impact on cattle health and the livestock industry in the tropical and subtropical regions. Development of vaccine using Bm86 antigen against R. microplus has emerged as an attractive alternative to control ticks. In this study, we have investigated the biodistribution of SV in a live animal model, as well as further explored the SV ability for vaccine development. Rhodamine-labeled SV-140-C18 (Rho-SV-140-C18) vesicles were used to adsorb the Cy5-labeled R. microplus Bm86 antigen (Cy5-Bm86) to enable detection and characterization of the biodistribution of SV as well as antigen in vivo in a small animal model for up to 28 days using optical fluorescence imaging. We tracked the in vivo biodistribution of SVs and Bm86 antigen at different timepoints (days 3, 8, 13, and 28) in BALB/c mice. The biodistribution analysis by live imaging as well as by measuring the fluorescent intensity of harvested organs over the duration of the experiment (28 days) showed greater accumulation of SVs at the site of injection. The Bm86 antigen biodistribution was traced in lymph nodes, kidney, and liver, contributing to our understanding how this delivery platform successfully elicits antibody responses in the groups administered antigen in combination with SV. Selected tissues (skin, lymph nodes, spleen, kidney, liver, and lungs) were examined for any cellular abnormalities by histological analysis. No adverse effect or any other abnormalities were observed in the tissues.

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BACKGROUND: Ticks are a problem for cattle production mainly in tropical and subtropical regions, because they generate great economic losses. Acaricides and vaccines have been used to try to keep tick populations under control. This has been proven difficult given the resistance to acaricides and vaccines observed in ticks. Resistance to protein rBm86-based vaccines has been associated with the genetic diversity of Bm86 among the ectoparasite's populations. So far, neither genetic diversity, nor spatial distribution of circulating Bm86 haplotypes, have been studied within the Mexican territory. Here, we explored the genetic diversity of 125 Bm86 cDNA gene sequences from R. microplus from 10 endemic areas of Mexico by analyzing haplotype distribution patterns to help in understanding the population genetic structure of Mexican ticks. RESULTS: Our results showed an average nucleotide identity among the Mexican isolates of 98.3%, ranging from 91.1 to 100%. Divergence between the Mexican and Yeerongpilly (the Bm86 reference vaccine antigen) sequences ranged from 3.1 to 7.4%. Based on the geographic distribution of Bm86 haplotypes in Mexico, our results suggest gene flow occurrence within different regions of the Mexican territory, and even the USA. CONCLUSIONS: The polymorphism of Bm86 found in the populations included in this study, could account for the poor efficacy of the current Bm86 antigen based commercial vaccine in many regions of Mexico. Our data may contribute towards designing new, highly-specific, Bm86 antigen vaccine candidates against R. microplus circulating in Mexico.

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BACKGROUND: Rhipicephalus microplus is a hard tick species that has a high impact on cattle health and production in tropical and subtropical regions. Recently, ribosomal DNA and morphological analysis resulted in the reinstatement of R. australis as a separate species from R. microplus. Both feed on cattle and can transmit bovine pathogens such as Anaplasma and Babesia species. The current treatment with acaricides is becoming increasingly less effective due to the emergence of resistant tick strains. A promising alternative can be found in the form of anti-tick vaccines. The available commercial vaccines can be used to control tick infestation, but the lack of a knockdown effect (> 90% reduction in tick numbers as seen with effective acaricides) hampers its widespread use, hence higher efficacious vaccines are needed. Instead of searching for new protective antigens, we investigated the efficacy of vaccines that contain more than one (partially) protective antigen. For screening vaccine formulations, a previously developed in vitro feeding assay was used in which R. australis larvae are fed sera that were raised against the candidate vaccine antigens. In the present study, the efficacy of the Bm86 midgut antigen and the cytosolic Subolesin (SUB) antigen were evaluated in vitro. RESULTS: Antiserum against recombinant Bm86 (rBm86) partially inhibited larval engorgement, whereas antiserum against recombinant SUB (rSUB) did not have any effect on feeding of larvae. Importantly, when larvae were fed a combination of antiserum against rBm86 and rSUB, a synergistic effect on significantly reducing larval infestations was found. Immunohistochemical analysis revealed that the rBm86 antiserum reacted with gut epithelium of R. australis larvae, whereas the antiserum against rSUB stained salivary glands and rectal sac epithelium. CONCLUSIONS: Combining anti-Bm86 and anti-subolesin antibodies synergistically reduced R. australis larval feeding in vitro. Rhipicephalus australis is a one host tick, meaning that the larvae develop to nymphs and subsequently adults on the same host. Hence, this protective effect could be even more pronounced when larvae are used for infestation of vaccinated cattle, as the antibodies could then affect all three developmental stages. This will be tested in future in vivo experiments.

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In this work, we developed a general perturbation theory and machine learning method for data mining of proteomes to discover new B-cell epitopes useful for vaccine design. The method predicts the epitope activity εq(cqj) of one query peptide (q-peptide) under a set of experimental query conditions (cqj). The method uses as input the sequence of the q-peptide. The method also uses as input information about the sequence and epitope activity εr(crj) of a peptide of reference (r-peptide) assayed under similar experimental conditions (crj). The model proposed here is able to classify 1 048 190 pairs of query and reference peptide sequences from the proteome of many organisms reported on IEDB database. These pairs have variations (perturbations) under sequence or assay conditions. The model has accuracy, sensitivity, and specificity between 71 and 80% for training and external validation series. The retrieved information contains structural changes in 83 683 peptides sequences (Seq) determined in experimental assays with boundary conditions involving 1448 epitope organisms (Org), 323 host organisms (Host), 15 types of in vivo process (Proc), 28 experimental techniques (Tech), and 505 adjuvant additives (Adj). Afterward, we reported the experimental sampling, isolation, and sequencing of 15 complete sequences of Bm86 gene from state of Colima, Mexico. Last, we used the model to predict the epitope immunogenic scores under different experimental conditions for the 26 112 peptides obtained from these sequences. The model may become a useful tool for epitope selection toward vaccine design. The theoretical-experimental results on Bm86 protein may help the future design of a new vaccine based on this protein.

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Tick gut glycoprotein, designated as Bm86, found on the luminal surface of the plasma membrane of gut epithelial cells of Boophilus microplus, which is a concealed antigen, has been used as vaccine candidate molecule for immunization against ticks. To better understand the molecular diversity of Bm86 gene in ticks, a portion of the cDNA was sequenced from an Indian isolate of B. microplus. Comparison of nucleotide sequence revealed that Indian isolate had 97 % homology (18 polymorphisms) with that of the Australian isolate and 96 % homology (20 polymorphisms) with that of the Cuban vaccine strain. Further, the Indian isolate differed from the Cuban vaccine isolate at 7 amino acid loci, including 5 substitutions (at residues 88, 94, 175, 176 and 177) and 2 deletions (at 183 and 184). However, protein prediction studies did not show any difference in the putative antigenic epitopes of the protein expressed.

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Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5-6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30mg semi-engorged ticks fed more reliably, ticks as small as 15mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

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The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B.) microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B.) microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG) strain of R. (B.) microplus was produced using the Pichiapastoris expression system to test its ability to immunoprotect cattle against tick infestation. Secretion of rBm86-CG by P. pastoris through the bioprocess reported here simplified purification of the antigen. A specific humoral immune response was detected by ELISA in vaccinated cattle. Immunoblot results revealed that polyclonal antibodies from vaccinated cattle recognized a protein in larval extracts with a molecular weight corresponding to Bm86. The rBm86-CG antigen showed 31% efficacy against the Campo Grande strain of R. (B.) microplus infesting vaccinated cattle. The rBm86-CG is an antigen that could be used in a polyvalent vaccine as part of an integrated program for the control of R. (B.) microplus in the region that includes Mato Grosso do Sul.

O carrapato Rhipicephalus (Boophilus) microplus é, sem dúvidas, o ectoparasito economicamente mais importante para o gado a nível mundial. A utilização inadequada de acaricidas tem impulsionado a evolução da resistência em populações de R. (B.) microplus. Vacinas contra o carrapato representam uma tecnologia que pode ser combinada com acaricidas em programas de controle integrado para diminuir o impacto de R. (B.) microplus. A forma recombinante da Bm86 da cepa Campo Grande (rBm86-CG) de R. (B.) microplus foi produzido utilizando o sistema de expressão em Pichia pastoris para testar sua capacidade de imunoproteção ao gado contra a infestação de carrapatos. A secreção de rBm86-CG em P. pastoris pelo bioprocesso, simplificou a purificação do antígeno. A resposta imune humoral específica foi detectada por ELISA em soros de bovinos vacinados. Resultados de "imunoblot" revelaram que anticorpos policlonais de bovinos vacinados reconheceram uma proteína em extratos de larvas com um peso molecular correspondente à Bm86. O antígeno rBm86-CG mostrou eficácia de 31% contra a amostra CG de R. (B.) microplus utilizada para infestar os bovinos vacinados. Pelos resultados obtidos, concluímos que a rBm86-CG é um antígeno que pode ser usado em uma vacina polivalente, como parte de um programa integrado para o controle de R. (B.) microplus no estado do Mato Grosso do Sul, Brasil.

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