Fig 1.
Plasmid map of AM581-KO-tuf-mCherry-Gent construct.
Left and right homology arms for targeted allelic exchange are shown on the plasmid map. The segment containing the left and right homology arms separated by the E. chaffeensis tuf promoter segment to drive the expression of mCherry and gentamicin resistance cassette was amplified for use in allelic exchange homologous recombination.
Fig 2.
Targeted allelic exchange mutagenesis deleting A. marginale AM581 gene encoding phtcp.
A) A cartoon illustrating the genomic region selected for preparing the allelic exchange construct and construct design. Restriction enzyme sites used in defining the mutation development [EcoRV (E) and HindIII (H)] are presented with their genomic coordinates. The size of the inserted fragment (tuf-mCherry-Gent) was included. The PCR primer sused in the analysis were identified as numbers 1 to 4. B) The AM581 gene deletion mutant in ISE6 tick cell culture expressing mCherry. The mCherry expression was detected by confocal microscopy using 40x magnification lenses. (The length bar on the bottom left corner of the figure to define the size; 10 microns). C) PCR analysis to define the mutation. Three different PCRs were performed: in PCR I, primers targeting the genomic region upstream to the insertion and the mCherry coding sequence were used; PCR II primers targeted to the mCherry coding region and downstream to the inserted region; in PCRIII, primers were targeted to A. marginale sequence upstream and downstream to the inserted fragment. (L, 1 kb plus molecular weight DNA markers; W, wild-type A. marginale genomic DNA; M, phtcp mutant genomic DNA). D) Southern blot analysis of genomic DNAs (W, wild-type and M, mutant) digested with EcoRV (E) and HindIII (H). The DNA blot analysis was performed using an mCherry gene segment as the probe.
Table 1.
Clinical signs observed for all A. marginale vaccine study groups.
Fig 3.
PCV and RBC count assessed in all three groups of steers.
Average PCV and RBC values from animals sampled during the study period were presented for the non-vaccinated animals, WCAV and MLAV during vaccination phase and following infection challenge. Days of adjuvant injections (control group), vaccinations (WCAV and MLAV) and infection challenge day (all three groups) are identified in the figures. At significance level α = 0.05, One-way ANOVA with repeated measures showed significant differences for PCV (P = 0.0128*) and RBC (P = 0.0375*) levels for MLAV compared to infection controls or WCAV group animals for the days 60–70. However, no significant difference was observed for WCAV and infection control groups throughout the study period. Tukey’s multiple comparisons test also showed similar significant differences in PCV (P = 0.0056**) and RBC P = 0.0294*) for MLAV group animals compared to infection controls or WCAV animals between days 60 and 70. This method also did not yield significant difference between infection controls and WCAV animals.
Fig 4.
Thin blood smears assessed by light microscopy.
Blood smear images were presented from one animal each for day 42 post infection challenge from all three experimental group animals. The images were collected following viewing with a 40x magnification. A. marginale inclusions are shown by black arrows (observed only in infection controls and WCAV animals; panels A and B), but not in MLAV animals (C). The A. marginale inclusions (identified with arrowhead lines) and anisocytosis are evident in both non-vaccinated infection controls WCAV vaccinees, but not in MLAV vaccinees. The length bars on the bottom left corner of each panel represent 50 microns.
Fig 5.
Percent infected erythrocytes as measured by light microscopy.
Average percent infected erythrocytes following enumeration in 20 randomly chosen fields under 40x magnification and presented for all groups throughout the study period. Days of vaccinations and infection challenges were as in Fig 3. (significance for days 55–65) At significance level α = 0.05, One-way ANOVA with repeated measures showed that percent infected erythrocytes (bacteremia) were significantly different between the infection control, WCAV and MLAV groups (P = 0.0058**), for several days post-challenge. Additionally, Tukey’s multiple comparisons test showed significant differences between infection control and MLAV (P = 0.0334*) groups, as well as between WCAV and MLAV (P = 0.0117* groups. No significant difference between infection control and WCAV was observed.
Fig 6.
Bacterial numbers in blood assessed by real-time qPCR.
The 16S rDNA TaqMan probe-based qPCR assays were performed for samples collected from all three groups animals over the study period (A-C). The MLAV group animal samples were also assessed by mCherry qPCR assay (D). Significance for days 55–65; at significance level α = 0.05, one-way ANOVA with repeated measures showed that 16S rDNA copy numbers of wild-type A. marginale bacteria were significantly different between the infection control, WCAV and MLAV groups (P = 0.0375*), for several days post-challenge. Additionally, Tukey’s multiple comparisons test showed a significant difference between infection control and MLAV groups (P = 0.0294*). No significant difference was observed between infection control and WCAV, or WCAV and MLAV.
Fig 7.
The presence of wild-type and mutant A. marginale in ticks assessed following blood feeding on MLAV group animals and their subsequent molting.
Genomic DNAs from 20 ticks fed on each animal (10 males and 10 females) from steers 4506 and HH5 were tested for the presence of wild-type and phtcp mutant A. marginale by conventional PCR targeting to amplify the entire insertion-specific region; anticipated product size for wild-type and mutant are 2.4 kb and 3.4 kb, respectively. As a total of 60 randomly selected ticks (30 males and 30 females) fed on animal # DP324 were negative for both wild-type and mutant A. marginale by qPCR, 10 randomly selected ticks from this animal were also tested by conventional PCR. L, 1kb plus molecular weight markers; M, A. marginale mutant; W, Wild-type strain,—refers to no template containing negative control. The numbers with the letters M or F are to indicate the tick identification numbers and to indicate their sex; M, male and F, female.
Table 2.
TaqMan qPCR data (Ct values) for ticks fed on steers.
Fig 8.
Post infection challenge histopathology sections of bone marrow at 40x magnification.
From left to right panels: A) WCAV vaccinees (4491, 4502 and 4505); B) MLAV vaccinees (DP324, 4506 and HH5); C) Infection controls (4405, 4406 and HH6).
Fig 9.
A. marginale-specific IgG response in cattle.
Antigen-specific IgG antibodies were measured by ELISA in plasma samples collected from day zero (prior to infection) and multiple time points post vaccination and post challenge. Purified 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 within each group were plotted against plasma collection days. At, significance level α = 0.05, One-way ANOVA with repeated measures showed that IgG levels were significantly different between the infection control, WCAV and MLAV groups (P < 0.0001****), for several days post-challenge. Additionally, Tukey’s multiple comparisons test showed significant differences between infection control and WCAV groups (P = 0.0003***), infection control and MLAV groups (P = 0.0024**), and WCAV and MLAV groups (P = 0.0041**).
Table 3.
List of oligonucleotides used in this study.