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United States Department of Agriculture

Agricultural Research Service

Research Project: IMMUNOLOGIC AND PHARMACOLOGICAL INTERVENTIONS OF VECTOR-BORNE BABESIOSIS Title: The novel protein BboRhop68 is expressed by intraerytrhocytic stages of Babesia bovis

Authors
item Baravalle, M -
item Thompson, C -
item Torioni DE Echaide, S -
item Palacios, C -
item Valentini, B -
item Suarez, Carlos
item Florin Christensen, M -
item Echaide, I -

Submitted to: Parasitology International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 26, 2010
Publication Date: N/A

Interpretive Summary: This manuscript describes the identification of a Babesia bovis gene that is homologous to the P. falciparum rhoptry protein PfRhop148 gene. The intron-less 1830 bp novel gene, predictive of a 68 kDa protein, is highly conserved among different B. bovis isolates, and it was denominated bborhop. The deducted protein from the B. bovis T2Bo strain, named BboRhop-68, showed two putative transmembrane domains, at least seven B-cell epitopes, and a well conserved DUF501 super family domain. The BboRhop-68gene was amplified, analyzed and compared among different B. bovis strains and isolates showing overall high sequence conservation among strains. A fragment of BboRhop68 was expressed as a recombinant fusion protein (rBboRhop68) and inoculated in mice to generate specific antibodies. The anti-rBboRhop68 mice serum identified the novel protein in intraerythrocytic trophozoites and merozoites by WB and ELISA, but not in free merozoites. Sera from naturally and experimentally infected bovines also recognized BboRhop68, suggesting that it is expressed and immunogenic during B. bovis infection of cattle. In addition, fluorescence microscopy analysis using anti-rBboRhop68 antibodies showed a rod shape strong reactivity associated to trophozoites and merozoites-infected erythrocytes, but this pattern of reactivity was not observed in free merozoites. Additionally, in contrast with B. bovis infected erythrocytes, expression of BboRhop68 was not detected in free merozoites using ELISA. Thus, at least three independent lines of evidence support differential expression of BboRhop68 in intraerythrocytic stages of B. bovis and its possible functional role immediately after B. bovis erythrocyte invasion. Taken together, the data suggest that BboRhop68 could be considered as a novel additional target for developing improved methods to control bovine babesiosis.

Technical Abstract: The apical complex of intracellular hemoparasites contains organelles like micronemes and rhoptries, specialized structures required for adherence and invasion of host cells. Several molecules discharged from rhoptries have been identified from Plasmodium spp., but only a single rhoptry associated protein-1 (RAP-1) has been characterized from Babesia bovis. In silico search of the B. bovis genome allowed to identifying a sequence homologous to the gene that encodes a P. falciparum rhoptry protein PfRhop148. The intron-less 1830 bp novel gene, predictive of a 68 kDa protein, is highly conserved among different B. bovis isolates, and it was denominated bborhop. The deducted protein from the B. bovis T2Bo strain, named BboRhop-68, showed two putative transmembrane domains, at least seven B-cell epitopes, and a well conserved DUF501 super family domain. The BboRhop-68gene was amplified, analyzed and compared among different B. bovis strains and isolates showing overall high sequence conservation among strains. A fragment of BboRhop68 was expressed as a recombinant fusion protein (rBboRhop68) and inoculated in mice to generate specific antibodies. The anti-rBboRhop68 mice serum identified the novel protein in intraerythrocytic trophozoites and merozoites by WB and ELISA, but not in free merozoites. Sera from naturally and experimentally infected bovines also recognized BboRhop68, suggesting that it is expressed and immunogenic during B. bovis infection of cattle. In addition, fluorescence microscopy analysis using anti-rBboRhop68 antibodies showed a rod shape strong reactivity associated to trophozoites and merozoites-infected erythrocytes, but this pattern of reactivity was not observed in free merozoites. Additionally, in contrast with B. bovis infected erythrocytes, expression of BboRhop68 was not detected in free merozoites using ELISA. Thus, at least three independent lines of evidence support differential expression of BboRhop68 in intraerythrocytic stages of B. bovis and its possible functional role immediately after B. bovis erythrocyte invasion. Taken together, the data suggest that BboRhop68 could be considered as a novel additional target for developing improved methods to control bovine babesiosis.

Last Modified: 11/22/2014
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