Genetically modified live vaccine offers protective immunity against wild-type Anaplasma marginale tick-transmission challenge

Abstract

Anaplasma marginale is a tick-borne pathogen of cattle that causes bovine anaplasmosis in tropical and subtropical regions throughout the world. Killed vaccines derived from infected erythrocytes have been used for control of this disease with limited success. Recently, we described a targeted deletion mutation in the phage head-to-tail connector protein gene of A. marginale which caused bacterial attenuation in vivo and provided protection as a modified live vaccine (MLAV). Following intravenous injection of susceptible steers, the MLAV induced protective immunity against disease progression. In the current study, we demonstrated that the immunity resulting from MLAV in cattle prevents the disease progression resulting from virulent A. marginale intrastadial transmission from infected Dermacentor variabilis male ticks. The nonimmunized control steers receiving the infection from ticks developed fever, lethargy, and inappetence for several days post tick exposure with significant decreases in the packed cell volume and increases in bacteremia. In contrast, the MLAV immunized steers remained healthy after being challenged with infected ticks and this group of animals had a significant reduction in bacteremia as compared with the controls. This study demonstrated that the A. marginale MLAV provided protection against acute tick-transmitted anaplasmosis, in addition to protection documented in steers challenge-exposed with infected blood as reported previously.

Fig. 1.

A. marginale inclusions identified by TEM in ticks fed on steers. A) A. marginale organisms in a morula (phagosome) within the salivary gland region were observed in a tick recovered on day 4 following tick feeding on a steer. B) A large hemocyte containing an inclusion with A. marginale organisms was observed in an infected D. variabilis tick removed after day 5 post blood feeding. C). Higher magnification of the boxed section of the image B where discrete A. marginale organisms with defined inner and outer membranes are visible similar to the organisms in panel A.

Fig. 2.

TaqMan probe-based qPCR assay estimation of A. marginale numbers in ticks fed on steers. Genomic DNAs recovered from ticks fed from days 1 to 7 on unvaccinated and MLAV steers were assessed for the bacterial numbers by qPCR targeting to the A. marginale 16S rDNA gene. Total of 12 ticks per each time point were assessed. Differences noted for the bacterial numbers compared to day 1 to days 6 and 7 were identified with p values.

Fig. 3.

PCV changes over time following vaccination and tick transmission infection. A) Average PCV values for the vaccinated animals during vaccination phase and following tick transmission challenge were shown in black line. For unvaccinated animals, PCV were only measured following infection challenge and the data were presented in red line. (Horizontal dotted line at 24% represents the lowest standard cut-off value for PCV to represent the normal range). B) All PCV values for days 35 to 55 were plotted for vaccinated and unvaccinated animals following infection challenge to assess statistical significance using Student’s t-test.

Fig. 4.

Changes in the blood parameters in vaccinated and unvaccinated animals following tick infection. Differences in the counts of red blood cells (RBC) (A), reticulocytes (B), mean corpuscular volume (MCV) (C) and monocytes (D) over time post infection were plotted for vaccinated (black lines) and unvaccinated (red lines) steers. The panel to the right of each image reflect statical analysis data for the values over time from day 35 to the study end (data in red dots, infection controls; black dots, vaccinated animals). Significant differences noted in between the groups when assessed using the unpaired t-test were shown in the right panels of the Fig. To further document the significance, the two-way ANOVA with Tukey’s test was performed which yielded similar p values. The following of the values for when performing the two-way ANOVA. RBC, P < 0.0001; reticulocytes P = 0.0849; MCV, P = 0.0001; monocytes, P < 0.0001; and for PCV P < 0.0001.

Fig. 5.

Variations in the blood cells determined from blood smear analysis. Blood films for 8 time points during the bacteremic phage following tick transmission infection (from days 21 to 49) for all animals in vaccinated and unvaccinated groups were evaluated microscopically. Morphological changes were assessed for RBCs, neutrophils, and lymphocytes. In each slide, red blood cells were evaluated by viewing 10 oil immersion fields, whereas 10 lymphocytes and 20 neutrophils were assessed. Vaccinated animals had mostly normal morphology for all three cell types (example images in the upper panel) and notable abnormalities were observed in the infection controls (images in the lower panel). High numbers of A. marginale inclusions (thin arrowhead line), reticulocytosis and anisocytosis (thick arrowhead line) were apparent in unvaccinated infected animals (inset Table). High number of activated lymphocytes having expanded cytoplasm and binucleated cells were observed in unvaccinated animals (inset Table). Banded (immature) neutrophils were observed in high numbers in the infected steers not receiving the vaccine.

Fig. 6.

Systemic bacterial numbers assessed by real-time qPCR. The 16S rDNA TaqMan probe-based qPCR assays were performed using DNAs recovered from blood sampled over time for the unvaccinated (A) and vaccinated animals (B). C) The MLAV group steer blood samples were also assessed by a qPCR assay targeted to the mutant insertion-specific mCherry gene segment. D) Bacterial numbers for vaccinated (black) and unvaccinated (red) animals during the peak bacteremia days (days 28–50) were plotted to assess statistical significance by performing the Student’s t-test.

Fig. 7.

A. marginale-specific IgG response. A. marginale-specific IgG response assessed by ELISA. Plasma samples collected from different days post-vaccination and post tick transmission infection challenge were assessed in vaccinated (black) and unvaccinated (red) steers. A. marginale whole cell antigens recovered from ISE6 cell cultures were used to coat the ELISA plates. Average absorbance values of plasma collected from steers for both groups were plotted against plasma sampling days.