New insights from molecular characterization of the tick Rhipicephalus (Boophilus) microplus in Brazil

Abstract The Rhipicephalus (Boophilus) microplus complex currently consists of five taxa, namely R. australis, R. annulatus, R. (B.) microplus clade A sensu, R. microplus clade B sensu, and R. (B.) microplus clade C sensu. Mitochondrial DNA-based methods help taxonomists when they are facing the morpho-taxonomic problem of distinguishing members of the R. (B.) microplus complex. The purpose of this study was to perform molecular characterization of ticks in all five regions of Brazil and infer their phylogenetic relationships. Molecular analysis characterized 10 haplotypes of the COX-1 gene. Molecular network analysis revealed that haplotype H-2 was the most dispersed of the studied populations (n = 11). Haplotype H-3 (n = 2) had the greatest genetic differentiation when compared to other Brazilian populations. A Bayesian phylogenetic tree of the COX-1 gene obtained strong support. In addition, it was observed that the population of R. (B.) microplus haplotype H-3 exhibited diverging branches among the other Brazilian populations in the study. The study concludes that the different regions of Brazil have R. (B.) microplus tick populations with distinct haplotypes

Analysis of Bm86 conserved epitopes: is a global vaccine against Cattle Tick Rhipicephalus microplus possible?

Abstract The cattle tick Rhipicephalus microplus causes significant economic losses in agribusiness. Control of this tick is achieved mainly through the application of chemical acaricides, often resulting in contamination of animal food products and of the environment. Another major concern associated with acaricide use is the increasing reports of resistance of this tick vector against the active ingredients of many commercial products. An alternative control method is vaccination. However, the commercially available vaccine based on a protein homologous to Bm86 exhibits variations in efficacy relative to the different geographical locations. This study aimed to identify antigenic determinants of the sequences of proteins homologous to Bm86. Phylogenetic analyses were performed to determine the extent of divergence between different populations of R. microplus to identify the sequence that could be used as a universal vaccine against the multiple geographically distinct populations of R. microplus and related tick species. Considering the extensive sequence and functional polymorphism observed among strains of R. microplus from different geographical regions, we can conclude that it may be possible to achieve effective vaccination against these cattle ticks using a single universal Bm86-based antigen

Prediction of Conserved Peptides of Paracoccidioides for Interferon-γ Release Assay: The First Step in the Development of a Lab-Based Approach for Immunological Assessment during Antifungal Therapy

Impaired antigen-specific cell-mediated immunity (CMI) is a primary immunological disturbance observed in individuals that develop paracoccidioidomycosis (PCM) after exposure to Paracoccidioides spp. Restoration of Paracoccidioides-specific CMI is crucial to stop the antifungal treatment and avoid relapses. A convenient and specific laboratory tool to assess antigen specific CMI is required for the appropriate clinical treatment of fungal infections, in order to decrease the time of antifungal therapy. We used an interferon-γ release assay strategy, used in the diagnosis of latent tuberculosis infection, to address our aims in this study. Information on proteins secreted by two well-studied representative strains—Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb-01)—were explored using PubMed or MEDLINE. From 26 publications, 252 proteins were identified, of which 203 were similar according to the Basic Local Alignment Search Tool. This enabled a selection of conserved peptides using the MEGA software. The SignalP-5.0, TMHMM, IEDB, NetMHC II, and IFNepitope algorithms were used to identify appropriate epitopes. In our study, we predicted antigenic epitopes of Paracoccidioides that could bind to MHC class II and induce IFN-γ secretion. These T cell epitopes can be used in the development of a laboratory tool to monitor the CMI of patients with PCM

Amino acid sequences used to construct the recombinant protein.

<p>Amino acid sequences used to construct the recombinant protein.</p

Schematic representation of the 3,877-bp plasmid encoding the chimeric protein.

<p><b>A.</b> Expression cassette representation of pAE/RmTI-CG-LTB construction. The chimeric protein was fused at the 6xHis-tag of the pAE vector at the N-terminal region. Each chimeric subunit protein is connected by a 2x SerGly flexible linker, which is presented in gray. <b>B.</b> <i>In silico</i> prediction of the chimeric protein conformational structure.</p

Effects on females and their progeny, and efficacy of vaccination with rRmTI-CG-LTB antigen against <i>R</i>. (<i>B</i>.) <i>microplus</i> that infest cattle.

<p>Effects on females and their progeny, and efficacy of vaccination with rRmTI-CG-LTB antigen against <i>R</i>. (<i>B</i>.) <i>microplus</i> that infest cattle.</p

Bioreactor standardization and RmLTI-BmCG-LTB chimera yield.

<p><b>A.</b> pH variation of the cultures in the TB and 2x (YT) culture media. <b>B.</b> Absorbance (A600 nm) variation of the TB and 2x (YT) culture media. <b>C.</b> TB culture medium in the fermenter (blue circle), TB and shaker (black square), 2x (YT) culture medium in the fermenter (green triangle) and shaker (inverted gray triangle). *Growth of the pre-inoculum in the fermenter. §Induction (1 mM IPTG).</p

Purification of the recombinant protein using the ÄKTA purifier system (GE Healthcare Life Sciences).

<p><b>A.</b> Purification of the protein expressed in the shaker. In the 10% polyacrylamide gel, wells 1 to 7 represent the imidazole-eluted fractions of the same recombinant protein. Well 8–0.5 μg of BSA. Well 9–1 μg of BSA. Well 10–2 μg of BSA. <b>B.</b> Purification of the protein expressed in the fermenter. In the 10% polyacrylamide gel, wells 1 to 8 represent the imidazole-eluted fractions of the recombinant protein (well 9 contains 1 μg of BSA, and well 10 contains 2 μg of BSA).</p

Antigenic analysis of the chimeric protein.

<p><b>A.</b> Characterization by western blotting. The chimeric protein was specifically recognized by the anti-6xHis mAb (diluted 1:5000), the LTB mAb (anti-CT) (diluted 1:5000), and anti-RmLTI-BmCG-LTB bovine sera (diluted 1:50). <b>B.</b> Characterization by ELISA. The chimeric protein was tested against anti-RmLTI-BmCG-LTB bovine sera (diluted 1:800).</p