ABSTRACT
The Anaplasma marginale is a bacterium that has obligate intraerythrocytic multiplication in cattle causing important economic loss. The A. marginale major surface protein 1 (MSP1) complex, heterodimer composed of MSP1a and MSP1b, has been identified as adhesins for bovine erythrocytes. The objectives of this study were to sequences the msp1beta gene and produce and characterize recombinant MSP1a and MSP1b from a Brazilian strain of A. marginale, PR1. The msp1alpha and msp1beta genes from the PR1 strain were cloned and expressed in E. coli BL21 Star using the vectors pET102 and pET101/D-TOPO. Antibodies were produced against the recombinant proteins and were shown to react with rMSP1a and rMSP1b demonstrating a molecular mass of 70kDa to 105kDa and 100kDa, respectively for these proteins. Bovine erythrocytes were agglutinated by BL21/rMSP1a and BL21/rMSP1b and, this agglutination was inhibited by the presence of the IgY anti-rMSP1a, confirming the adhesion function of these proteins. Additionally, using the IgY anti-rMSP1a and rMSP1b in a IFI, the presence of rMSP1a and rMSP1b was confirmed on the outer membrane of the recombinant E. coli BL21. Our results show that the msp1beta gene from the PR1 strain has both the conserved region and contain the defined polymorphism regions previously described for other strains of A. marginale. The results from this study confirm adhesive functions for rMSP1a and rMSP1b from PR1 strain in bovine erythrocytes invasion.
Subject(s)
Anaplasma marginale/genetics , Bacterial Outer Membrane Proteins/genetics , Cattle Diseases/microbiology , Amino Acid Sequence , Animals , Bacterial Adhesion , Bacterial Outer Membrane Proteins/biosynthesis , Base Sequence , Blotting, Western , Cattle , Cattle Diseases/blood , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Erythrocytes/microbiology , Escherichia coli/genetics , Escherichia coli/metabolism , Fluorescent Antibody Technique, Indirect/veterinary , Immunoglobulins/immunology , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction/veterinary , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Sequence AlignmentABSTRACT
This study evaluated sero-occurrence of toxoplasmosis in swine and ovine slaughtered in the municipality of Guarapuava in the state of Paraná. Blood samples from 117 swine and 157 ovine were collected. Indirect Immunofluorescence Assay (IFA) was used to detect IgG anti-Toxoplasma gondii antibodies. Serum samples were considered positive at dilutions >or=1:64. Out of 117 swine blood sera, 10 (8.54%) were positive, while 11 out of 157 ovine samples (7.0%) showed a reaction to T. gondii antibodies. Our data showed a decrease in the occurrence of toxoplasmosis in swine and ovine. This is due to improved technologies used to raise swine, and better management used to raise ovine as well as the age of the animals evaluated.
Subject(s)
Antibodies, Protozoan/blood , Sheep/blood , Swine/blood , Toxoplasma/immunology , Animals , BrazilABSTRACT
Anaplasmosis, caused by Anaplasma marginale, results in significant economic losses of cattle in tropical and subtropical regions worldwide. Six major surface proteins (MSPs) were well characterized and designated as MSP1, MSP2, MSP3, MSP4, and MSP5. The objective of this study was to evaluate the humoral immune response of BALB/c mice against the recombinant MSPs, incorporated into immunostimulating complex (ISCOM). The recombinant proteins purified by Ni-NTA columns were incorporated into ISCOM and ISCOMATRIX by the lipid film hydration method. BALB/c mice immunized with ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccines produced whole IgG, IgG1, and IgG2a, in contrast to the negative groups (PBS and ISCOMATRIX adjuvant). All groups that received antigen responded specifically against the rMSPs by Western blotting, showing the rMSP1a (60-105kDa), rMSP1b (100kDa), rMSP4 (47kDa), and rMSP5 (29kDa). Additional studies will have to be performed in cattle to evaluate the humoral and cellular mechanisms of this subunit vaccine and their possible use as protective vaccines against homologous and heterologous strains of A. marginale.
