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Detection of antibodies to  Anaplasma  spp. using commercial competitive enzyme-linked immunosorbent assay (ccELISA) is based on the recombinant major surface protein 5 fused to maltose binding protein (MBP-MSP5) or glutathione S-transferase (GST-MSP5). To avoid false positive reactions due to the presence of antibodies against  E .  coli  MBP in cattle, previous sera absorption is required. This study evaluated the replacement of MBP-MSP5 or GST-MSP5 antigens by the truncate MSP5 (residues 28–210) of  A .  marginale  (tMSP5m),  A .  centrale  (tMSP5c) and fusion protein MSP5 (tMSP5cm), expressed without N-terminus transmembrane helix in the ccELISA test. Immunoreactivity was evaluated by western blot using monoclonal antibodies against the tMSP5 and by  in-house  cELISA (hcELISA) with purified tMSP5m, tMSP5c or tMSP5cm using sera from cattle infected with  A .  marginale  (n = 226) or vaccinated with  A .  centrale  (n = 173) and uninfected cattle (n = 216). Results of hcELISA were compared with those of ccELISA. Recombinant protein was expressed highly soluble (> 95%) in  E .  coli  without a molecular chaperone. Specificity of the hcELISA-tMSP5m, -MSP5c or -tMSP5cm was identical to (99.5%) and greater than that in ccELISA (96.3%). Sensitivity of hcELISA-tMSP5m and ccELISA was identical (95.5%), but lower than that of hcELISA-tMSP5cm (96.2%) and -tMSP5c (97.2%). The analysis of vaccinated cattle by hcELISA-tMSP5c showed sensitivity of 99.4%. In summary, the generation of fusion MSP5  A .  marginale - A .  centrale  protein without transmembrane helix was a very effective method to express the recombinant protein highly soluble in the bacterial cytoplasm and contributed to an increased test performance for detecting antibodies in cattle naturally infected with  A .  marginale  or vaccinated with  A .  centrale .

plos one

Development of a novel fusion protein with Anaplasma marginale and A. centrale MSP5 improved performance of Anaplasma antibody detection by cELISA in infected and vaccinated cattle