Abstract
Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequence SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences' variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized: one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system.
Highlights
The tick-borne intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) is the causal agent of anaplasmosis, a hemoparasitic disease of cattle
Epitope mapping of major surface protein 1a (MSP1a) by Phage Display Thirty-nine randomly selected MSP1a mimotopes were obtained after three rounds of biopanning using a phage displayed 12-mer random peptide library against the anti-MSP1a monoclonal antibody 15D2 (Figure 1A)
This sequence corresponds to tandem repeats located in the amino-terminal region of MSP1a, and may be considered part of the antigenic determinant region
Summary
The tick-borne intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) is the causal agent of anaplasmosis, a hemoparasitic disease of cattle. A. marginale is distributed worldwide in tropical and subtropical regions of the world [1], resulting in considerable economic loss to both dairy and beef industries. A. marginale is transmitted horizontally by ixodid ticks, while mechanical transmission occurs when infected blood is transferred to susceptible cattle by fly bites or blood-contaminated fomites [3]. Six major surface proteins (MSPs) have been characterized on the erythrocytic stage of A. marginale. The major surface protein 1 (MSP1) has been extensively studied [4]. The MSP1a has been shown that is involved in the adhesion, infection and tick transmission of A. marginale, as well as to contribute to protective immunity in cattle [5,6]. MSP1a contains a variable number of tandemly repeated peptides in the amino-terminal region, which are exposed extracellularly for interaction with host cell receptors [7,8]
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