Abstract
Anaplasma marginale is the most prevalent tick-borne livestock pathogen with worldwide distribution. Bovine anaplasmosis is a significant threat to cattle industry. Anaplasmosis outbreaks in endemic areas are prevented via vaccination with live A. centrale produced in splenectomized calves. Since A. centrale live vaccine can carry other pathogens and cause disease in adult cattle, research efforts are directed to develop safe recombinant subunit vaccines. Previous work found that the subdominant proteins of A. marginale type IV secretion system (T4SS) and the subdominant elongation factor-Tu (Ef-Tu) were involved in the protective immunity against the experimental challenge in cattle immunized with the A. marginale outer membrane (OM). This study evaluated the immunogenicity and protection conferred by recombinant VirB9.1, VirB9.2, VirB10, VirB11, and Ef-Tu proteins cloned and expressed in E. coli. Twenty steers were randomly clustered into four groups (G) of five animals each. Cattle from G1 and G2 were immunized with a mixture of 50 μg of each recombinant protein with Quil A® or Montanide™ adjuvants, respectively. Cattle from G3 and G4 (controls) were immunized with Quil A and Montanide adjuvants, respectively. Cattle received four immunizations at three-week intervals and were challenged with 107 A. marginale-parasitized erythrocytes 42 days after the fourth immunization. After challenge, all cattle showed clinical signs, with a significant drop of packed cell volume and a significant increase of parasitized erythrocytes (p<0.05), requiring treatment with oxytetracycline to prevent death. The levels of IgG2 induced in the immunized groups did not correlate with the observed lack of protection. Additional strategies are required to evaluate the role of these proteins and their potential utility in the development of effective vaccines.
Highlights
The VirB9.1 and VirB9.2 proteins were expressed without their signal peptide, whereas VirB10, VirB11, and elongation factor-Tu (Ef-Tu) proteins were expressed with their full-length sequence
The recombinant proteins tVirB9.1 and Ef-Tu were expressed in soluble form in the cytoplasm of E. coli with a yield of 20 and 15 mg per liter of culture after their purification, respectively
TVirB9.1, tVirB9.2, VirB10, VirB11, and Ef-Tu but not major surface protein 5 (MSP5) were recognized by antibodies present in serum samples of all G1 and G2 cattle obtained 7 days after fourth immunization, when they were evaluated by Western blot (WB) (Fig 3)
Summary
Immunization of cattle with the native purified outer membrane (OM) of A. marginale has induced complete protection against infection and clinical disease [4,9,10]. Complete genome sequencing and proteomic studies of A. marginale allowed the identification of subdominant proteins, which are present in low abundance on the OM [13] These proteins remain invariant during infection and are highly conserved among different strains, making them attractive potential candidates for vaccines [12,18]. The immune protection against A. marginale induced by a vaccine based on the recombinant proteins VirB9.1, VirB9.2, VirB10, VirB9.1, and Ef-Tu was evaluated in cattle
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