Research Article: 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

Date Published: January 23, 2019

Publisher: Public Library of Science

Author(s): María E. Primo, Carolina S. Thompson, Beatriz S. Valentini, Macarena Sarli, María B. Novoa, Atilio J. Mangold, Susana T. de Echaide, Ulrike Gertrud Munderloh.


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.

Partial Text

Bovine anaplasmosis is an infectious disease caused by the obligate intraerythrocytic bacterium Anaplasma marginale [1] and is transmitted biologically to susceptible cattle by ticks or mechanically by biting flies and fomites [2–4]. The disease is endemic in mainly tropical and subtropical areas of the world. In Argentina, A. marginale is prevalent north of 33°S; nevertheless, anaplasmosis outbreaks have been detected to the south of the endemic zone due to the movement of carrier cattle to non-endemic areas [5,6]. Acute anaplasmosis affects mostly adult bovines and is characterized by severe anemia with destruction of erythrocytes, abortion, weight loss, reduced milk production and death. Cattle that recover from acute disease remain carriers and serve as reservoirs for transmission to other animals [7]. Immunization with live A. centrale, a species closely related to A. marginale, is used to prevent acute anaplasmosis in several countries worldwide, including Argentina. The immunogen causes only mild clinical signs and does not prevent infection with A. marginale, but reduces disease severity and prevents death [8]. The absence of anaplasmosis outbreaks in endemic areas is achieved after a high proportion of calves become naturally infected with A. marginale or by inoculation of young cattle with the A. centrale live vaccine, along with avoidance of the entry of A. marginale-infected cattle to anaplasmosis-free zones of Argentina. Implementation of appropriate control measures requires highly sensitive and specific serological tests that could provide mainly information on: i) precise identification of carriers, ii) epidemiological status of anaplasmosis in calves from enzootic regions, and iii) the immune response to A. centrale vaccine.

In this work, truncated MSP5 from A. marginale and A. centrale was expressed without a chaperone protein, with high yield, solubility and purity, without losing the immunoreactivity. The replacement of MBP-MSP5 of A. marginale in the ccELISA with either of the three variants of the truncated MSP5 of A. marginale (tMSP5m) or A. centrale (tMSP5c) or both together as fusion protein (tMSP5cm) improved the performance of hcELISA, particularly by increasing the specificity, which was achieved by eliminating cross-reactions against MBP.




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