Fig 1.
Body temperatures, clinical signs, nasal shedding and viremia after experimental infection of weanlings with the EHV-1 strain NY03.
The groups (n = 5 per group) were established based on IgE-bio and/or EHV-1 gC antigen treatments of the foals at birth: group 1 received IgE-bio and Sav-gC/IL-4 antigen; group 2 received Sav-gC/IL-4 antigen; control group 3 no treatment at birth. EHV-1 challenge infection occurred at seven months of age (day 0; arrow) and was performed under identical conditions for all three groups. On day 0, measurements and samples were taken before infection. (A) Body temperatures; the dotted horizontal line shows the cut-off value for fever (101.5°F); (B) clinical scores; (C) nasal shedding expressed as viral copy numbers of the EHV-1 gene gB per ml nasal secretion sample and (D) viremia (gB Ct-value) per 5 x 106 PBMC. Nasal shedding and viremia were evaluated by real-time PCR. The dotted horizontal line shows the positive PCR Ct-value cut-off value. All negative PCR values were set to this value. All graphs show means and standard errors per group. Significant differences between groups: a = groups 1 and 3; c = groups 1 and 2.
Fig 2.
Antibodies to gC and gD in serum of weanlings after experimental EHV-1 infection (day 0; arrow).
The weanling groups correspond to IgE-bio and/or EHV-1 gC antigen treatments during the neonatal period. At weanling age all three groups of weanlings were infected with the same dose of EHV-1 strain NY03. Antibodies in serum were determined by a EHV-1 Multiplex assay. The graphs show: (A) total serum anti-EHV-1 gC antibodies and (B) total serum anti-EHV-1 gD antibodies. The graphs represent means and standard errors by group. Significant differences between groups: a = groups 1 and 3; b = groups 2 and 3.
Fig 3.
Anti-gC isotypes in the serum of weanlings after experimental EHV-1 infection (arrow).
Weanlings in group 1 received IgE and EHV-1 gC antigen at birth. Weanlings in group 2 received EHV-1 gC antigen at birth. Weanlings in the control group 3 did not receive any treatment at birth. Antibody isotypes to gC were measured in a EHV-1 Multiplex assay. (A) anti-IgM; (B) anti-IgG6; (C) anti-IgG1; (D) anti-IgG1/3; (E) anti-IgG4/7; (F) anti-gC IgG3/5. The graphs show means and standard errors by group. Significant differences between groups: a = groups 1 and 3; b = groups 2 and 3.
Fig 4.
Cytokine secretion from PBMC of weanlings after experimental EHV-1 infection in vivo (arrow).
Weanlings in group 1 received IgE and EHV-1 gC antigen as neonates and weanlings in group 2 received EHV-1 gC antigen. Weanlings in the control group 3 did not receive any neonatal. PBMC were isolated up to 5 months post EHV-1 infection. Cells were re-stimulated ex vivo with EHV-1 for 48 hour to provoke cytokine production. PBMC in medium served as non-stimulated control. Cytokines in cell culture supernatants were determined by cytokine multiplex analysis. The cytokine secretion values in graphs A-D were corrected by the non-stimulated control values of each weanling and day. The graphs show means and standard errors by group. Significant differences between groups: a = groups 1 and 3; b = groups 2 and 3; c = groups 1 and 2.
Fig 5.
EHV-1-specific IFN-g producing T-cells in weanlings (n = 15) after EHV-1 infection (arrow).
PBMC were isolated at different times after infection and were re-stimulated for 48hours with EHV-1 (MOI = 1). Cells were fixed, stained for intracellular IFN-g and cell surface CD4 and CD8 expression, and were analyzed by flow cytometry. A) Representative images of IFN-g producing CD8+ T-cells after EHV-1 stimulation. The images from left to right show the cell morphology and the lymphocyte gate; weanling PBMC on day 61 pi cultured in medium; PBMC from the same weanling re-stimulated with EHV-1; and EHV-1 re-stimulated cells from an adult horse EHV vaccinated horse. B) Total EHV-1-specic IFN-g producing T-cells and CD4+ or CD8+ IFN-g producing T-cells in the peripheral blood of the 15 weanlings until day 220 pi. Significant increases compared to the pre-infection control: d = total IFN-g T-cells, e = CD8+/IFN-g+, f = CD4+/IFN-g+.