A vaccine using Anaplasma marginale subdominant type IV secretion system recombinant proteins was not protective against a virulent challenge

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 10 7 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.

Sensitivity of Anaplasma marginale genotypes to oxytetracycline assessed by analyzing the msp1α gene in experimentally infected cattle

“…The two multidrug resistance efflux pumps identified in the genome of A. marginale could explain the different susceptibility of the isolate to the antibiotic (Brayton et al, 2005). In those cattle whose infection was chemosterilized, it still was frequently to detect specific DNA after the disappearance of antibodies (Coetzee et al, 2006a;Goff et al, 1990;Sarli et al, 2020). Therefore, the genotypes identified in S3…”

“…Blood samples stored at -20°C, previously obtained from steers born and raised in an anaplasmosis-free Holstein dairy herd located in a tick-free area, involved in a chemosterilization protocol using OTC, were selected (Sarli et al, 2020;Sarli et al, 2021). Briefly, the experiment consisted of i) multiplication of the A. marginale S1P in a splenectomized calf (donor); ii) inoculation of 10 normal steers (2-year-old) with 2-mL of the donor blood contained 10 7 parasitized erythrocytes by subcutaneous injection (day 0); iii) treatment of steers with acute anaplasmosis using OTC, between 21 and 30 days post-infection (dpi); iv) A. marginale OTC sterilizing treatment at 78, 85, and 92 dpi, and v) subinoculation of 50 ml of blood from 2 steers refractory to A. marginale sterilization procedure into 2 splenectomized calves (receptors) to confirm the presence of the genotypes in steers at 182 dpi (Fig.…”

“…infection by cELISA and nested PCR (nPCR). Steers were maintained in different isolation pens and sprayed weekly with flumethrin (Bayticol1 Pour-On, Bayer) to protect them from biting flies and rule out the possibility of A. marginale cross-infection during the experiment (Sarli et al, 2020). All procedures were approved by the Animal Care Committee of the Faculty of Veterinary Sciences, National University of Litoral (Protocol number 243/15).…”

Immune Response to Tick-Borne Hemoparasites: Host Adaptive Immune Response Mechanisms as Potential Targets for Therapies and Vaccines

“…Alternative subunit vaccines for A. marginale would include the use of subdominant proteins present in the outer membrane, showing the vantage of not changing during the infection and being highly conserved among different strains. They include the proteins of A. marginale type IV secretion system, a 1.05-MDa complex that spans the outer and inner bacterial membranes, whose role is mainly the host cell adhesion/invasion, and the elongation factor-Tu (Ef-Tu), a membrane-associated protein belonging to the family of hydrolases involved in protein synthesis [ 26 ]. Two other proteins of the A. marginale type IV secretion system, VirB9.1 and VirB9.2 induced a humoral and cellular immunity, stimulating CD4 + T-lymphocyte proliferation, IFN-γ secretion and IgG2 production in outer membrane-immunized cattle [ 27 ].…”

“…However, the vaccine based on the recombinant proteins VirB9.1, VirB9.2, VirB10, VirB11 and Ef-Tu was able to elicit high IgG2 titers in the immunized calves but failed to protect against A. marginale challenge. All the animals showed clinical symptoms and required treatments, suggesting that further studies are required to identify and express, in the native form, the main epitopes of these antigens [ 26 ].…”