Flora Satiko Kano

Antigenic characterization of Anaplasma marginale isolates from different regions of Brazil.

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.

Neorickettsia helminthoeca in Brazilian dogs: a cytopathological, histopathological and immunohistochemical study

Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD,USAINTRODUCTIONNeorickettsia helminthoeca is an intracellular bacte-rium of reticuloendothelial cells of dogs thatcauses salmon poisoning disease (SPD), whichwas considered endemic only to specific geo-2graphical locations of North America [1–3].However, 20 mature dogs demonstrated lesionsconsistent with this infection in Southern Braziland intracytoplasmic and intralesional neorickett-sial bodies were identified within reticuloendo-thelial cells by Giemsa stain [4]. Additionally, bymolecular biology methods, it was shown that themesenteric lymph node of one dog containedDNA that was similar to N. helminthoeca as knownin the USA [5].This article describes the cytological, histopath-ological and immunohistochemical findings asso-ciated with N. helminthoeca in additional casesfrom Brazil.MATERIALS AND METHODS

Induced immune response of DNA vaccine encoding an association MSP1a, MSP1b, and MSP5 antigens of Anaplasma marginale

The outer membrane proteins of Anaplasma marginale have been the focus of research to obtain an improved vaccine against bovine anaplasmosis. We evaluated the capacity of the recombinant plasmids pcDNA-msp1alpha, pcDNA-msp1beta, and pcDNA-mp5 to express MSP1a, MSP1b, and MSP5 proteins, and to determine the immunogenicity of BALB/c mice immunized with these plasmids individually or in association. Expression of proteins was confirmed in Vero cells by IFA. The combination of recombinant plasmids showed high antibodies response, produced better induction of Th1 response than individual plasmids, and induced significant proliferation of splenocytes. The mice sera immunized with A. marginale showed seroconversion and reacted with all native MSPs, but demonstrated predominance of the humoral IgG1 isotype and did not induce significant proliferation of splenocytes. The use of association of recombinant plasmid can be an effective strategy for the immunoprophylaxis of anaplasmosis.

Cloning, expression, and characterization of the MSP1a and MSP1b recombinant proteins from PR1 Anaplasma marginale strain, Brazil

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.