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

Abstract

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.

Development and evaluation of a double-antigen sandwich ELISA to identify Anaplasma marginale–infected and A. centrale–vaccinated cattle

“…Soluble truncated MSP5 protein, without N-terminus transmembrane helix (tMSP5, residues 28-210) and without molecular chaperones, improved the specificity of the cELISA. 20 The fusion protein of MSP5 of A. centrale and A. marginale (MSP5c-MSP5m) increased the sensitivity of the cELISA when evaluated in a cattle population from endemic areas in which the live vaccine is used regularly. 20 In recent years, the double-antigen sandwich ELISA (das-ELISA) has been used successfully to detect low levels of antibodies to several pathogens.…”

“…20 The fusion protein of MSP5 of A. centrale and A. marginale (MSP5c-MSP5m) increased the sensitivity of the cELISA when evaluated in a cattle population from endemic areas in which the live vaccine is used regularly. 20 In recent years, the double-antigen sandwich ELISA (das-ELISA) has been used successfully to detect low levels of antibodies to several pathogens. 8,12,17,21,24,25,27 In this test, specific antibodies present in serum samples bind to the immobilized antigen on the solid phase with one antigenbinding site.…”

“…Complementary DNA encoding residues 28-210 with a 6-histidine tag at the C-terminus of the A. marginale MSP5 (tMSP5m) or A. centrale MSP5 (tMSP5c) were amplified by PCR and cloned in a pET9b vector (Novagen, Madison, WI) to build ptMSP5m and ptMSP5c vectors. 20 tMSP5m and tMSP5c were expressed in E. coli BL21 RIL (DE3)pLysS cells (Novagen) and purified by pseudo-affinity with Ni 2+ -NTA agarose (Qiagen, Hilden, Germany). Briefly, E. coli BL21 RIL (DE3)pLysS competent cells transformed with ptMSP5m or ptMSP5c were cultured to an optical density at 600 nm (OD 600nm ) = 1 at 37°C in 500 mL of Luria-Bertani medium supplemented with 50 µg/mL of kanamycin and 34 µg/mL of chloramphenicol.…”

A vaccine using Anaplasma marginale subdominant type IV secretion system recombinant proteins was not protective against a virulent challenge

“…All the animals were confirmed to be free of Anaplasma spp. infection by cELISA and nested PCR (nPCR) before the start of the experiment [23,24]. All procedures were approved by the Animal Care Committee of the Faculty of Veterinary Sciences, National University of Litoral (Protocol number 243/15).…”

“…The clinical parameters of each group were expressed as the mean of the maximum percent (%) drop of PCV, mean of the maximum PPE and mean of the cumulative T above 39.5˚C. Cattle were bled weekly during five weeks after challenge; the whole blood was analyzed by nPCR and the serum samples were tested by cELISA [23,24].…”