Subject(s)
Anaplasmataceae Infections/veterinary , Dog Diseases/microbiology , Neorickettsia/isolation & purification , Anaplasmataceae Infections/microbiology , Anaplasmataceae Infections/pathology , Animals , Brazil , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Dog Diseases/pathology , Dogs , Histocytochemistry , Immunohistochemistry , Lymph Nodes/microbiology , Lymph Nodes/pathology , Male , Mesentery/microbiology , Mesentery/pathology , Microscopy , Neorickettsia/geneticsABSTRACT
Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paran , Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.
Subject(s)
Anaplasma marginale/genetics , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Bacterial Vaccines/genetics , Membrane Proteins/genetics , Anaplasma marginale/immunology , Anaplasma marginale/isolation & purification , Anaplasmosis/immunology , Anaplasmosis/prevention & control , Animals , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Bacterial Vaccines/immunology , Brazil , Cattle , Cattle Diseases/immunology , Cattle Diseases/prevention & control , Cloning, Molecular , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Escherichia coli/genetics , Gene Expression , Immunoblotting , Membrane Proteins/immunology , Polymerase Chain Reaction , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Analysis, DNAABSTRACT
Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paraná, Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO® vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinantclone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post- immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.
Subject(s)
Animals , Cattle , Rabbits , Anaplasma marginale/genetics , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Vaccines/genetics , Membrane Proteins/genetics , Membrane Proteins/immunology , Anaplasma marginale/immunology , Anaplasma marginale/isolation & purification , Anaplasmosis/immunology , Anaplasmosis/prevention & control , Antigens, Bacterial/immunology , Bacterial Vaccines/immunology , Brazil , Cattle Diseases/immunology , Cattle Diseases/prevention & control , Cloning, Molecular , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Escherichia coli/genetics , Gene Expression , Immunoblotting , Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Analysis, DNAABSTRACT
Antigenic characterization of A. marginale isolates has contributed to identifying the presence of common and restricts epitopes of major surface proteins (MSPs). The data may improve vaccine development to protect against A. marginale isolates from different regions. Brazilian A. marginale isolates were characterized antigenically by Western blot with monoclonal antibodies (MAbs) against MSPs and rabbit anti-MSP-4 from Florida strain. Six A. marginale isolates from MS, MG (AUFV1), SP, PR-L1, PR-HV, RS and Florida strain were tested with ANA22B1 to MSP-1a, AMR36A6 to MSP-1b, ANAF19E2 to MSP-2, AMG75C1 and AMG76B2 to MSP-3 and ANAF16C1 to MSP-5. ANA22B1 recognized MSP-1a epitope in all A. marginale isolates, and reacted with polypeptides of different size ranging 46-105kDa. MSP2 was not detected in MS and SP isolates by ANAF19E2, and only PR-L1 and MG (AUFV1) isolates reacted with MAbs which recognize MSP3 epitope. MSP4 and MSP5 were detected in all A. marginale isolates analyzed. The results revealed conservation of MSP-1a and MSP-5 epitopes among all Brazilian isolates, and showed antigenic variability to MSP-1b, MSP-2 and MSP-3 proteins, agreeing with recent data about the genetic diversity found in the polimorphic multigene family responsible for these proteins.
