Local and systemic humoral immune responses to Histophilus somni recombinant antigens administered intranasally and subcutaneously to dairy calves

Abstract

Bovine respiratory disease (BRD) causes significant economic losses in dairy calves. Induction of an early immune response via parenteral vaccination is complicated by the interference of colostral immunity. In this study, we investigated early immunization against selected conserved bacterial antigens. Calves were vaccinated twice intranasally and then subcutaneously with Histophilus somni recombinant proteins (rOMP40 or rHsp60) mixed with one of two adjuvants: CpG ODN2007 or MPLA. The control group (Con) was treated with PBS. The first immunization was done between 24 and 48 h of life and then twice in two weeks intervals. Blood, nasal, and saliva secretion samples were collected directly before vaccination (S1-S3) and then on 42-44 (S4) and 59-61 (S5) day of life. Antibodies (IgG₁/IgG₂/IgM/IgA in serum; IgG₁/IgA in secretions) against both vaccine antigens were quantified in all samples. Intranasal and subcutaneous vaccinations using the described formulas did not increase antibody reactivity against the tested proteins. The reactivity of serum IgG₁, IgM, and IgA anti-rOMP40 antibodies was significantly higher in S1 in all groups than that in the other samplings (p˂0.01). Significant differences in the reactivity of serum anti-rOMP40 antibodies between groups were identified in S1 (IgA reactivity was higher in the CpG vs. MPLA group; p < 0.05), S4 (IgM reactivity was higher in Con vs. CpG group; p < 0.05), and S5 (IgG1 reactivity was higher in MPLA vs. Con group; p < 0.05). The lack of consistent changes in antibodies after immunization (S4 and S5) hinders the drawing of conclusions regarding the effect of immunization on antibody reactivity. In the future, establishing a proper immunization window and adjuvants for nasal vaccines against bacterial pathogens causing BRD in calves remains to be determined.

Keywords: CpG ODN2007; MPLA; rHsp60 H. somni; rOMP40 H. somni.

Fig. 1

Determination of nasal secretion IgA and IgG₁ antibody reactivity against H. somni rOMP40 and rHsp60. Box-plot graphs of IgA (a,c) and IgG₁ (b,d) antibodies reactivity against H.somni rHsp60 (a,b), and H.somni rOMP40 (c,d) antigens in ELISA. Nasal secretions were obtained as follows: S1: before the first intranasal immunization (24–48 h of life); S2: two weeks after the first immunization (14–16 days of age); S3: two weeks after the second intranasal immunization (28–30 days of age); S4: two weeks after the third subcutaneous immunization (42–44 days of age); S5: two weeks after S4 (59–61 days of age). Individual study groups were labelled as CpG (vaccine formulation rHsp60 + rOMP40 + CpG), Con (control group), and MPLA (vaccine formulation rHsp60 + rOMP40 + MPLA). Significant differences within individual study groups between consecutive samplings (e.g., CpG S1-S2, S2-S3, S3-S4, and S4-S5) are labelled with the same uppercase letters (A) for p ≤ 0.01 or small letters (a, b) for p ≤ 0.05. Significant differences within individual study groups between S1 and other samplings (e.g., CpG S1-S2, S1-S3, S1-S4, and S1-S5) are labelled with two asterisks (**) for p ≤ 0.01, or an asterisk (*) for p ≤ 0.05. The median line across the box, lower, and upper boxes indicate the 25th percentile to the 75th percentile, and whisker non-outlier range.

Fig. 2

Determination of serum IgG₁, IgG₂, IgM, and IgA antibody reactivity against H. somni rHsp60. Box-plot graphs of serum IgG₁ (a), IgG₂ (b), IgM (c) and IgA (d) antibodies reactivity against H. somni rHsp60 antigen in ELISA. Serum samples were obtained as follows: S1: before the first intranasal immunization (24–48 h of life); S2: two weeks after the first immunization (14–16 days of age); S3: two weeks after the second intranasal immunization (28–30 days of age); S4: two weeks after the third subcutaneous immunization (42–44 days of age); S5: two weeks after S4 (59–61 days of age). Individual study groups were labelled as CpG (vaccine formulation rHsp60 + rOMP40 + CpG), Con (control group), and MPLA (vaccine formulation rHsp60 + rOMP40 + MPLA). Significant differences within individual study groups between consecutive samplings (e.g., CpG S1-S2, S2-S3, S3-S4, S4-S5, etc.) are labelled with the same lowercase letters (a) for p ≤ 0.05. Significant differences within individual study groups between S1 and other samplings (e.g., CpG S1-S2, S1-S3, S1-S4, and S1-S5) are labelled with two asterisks (**) for p ≤ 0.01, or an asterisk (*) for p ≤ 0.05. The serum dilution was 1:50 for IgA, 1:100 for IgG₂ and IgM; 1:1000 for IgG₁. The median line across the box, lower, and upper boxes indicate the 25th percentile to the 75th percentile, and whisker non-outlier range.

Fig. 3

Determination of serum IgG₁, IgG₂, IgM and IgA antibody reactivity against H. somni rOMP40 antigen. Box-plot graphs of serum IgG₁ (a), IgG₂ (b), IgM (c) and IgA (d) antibodies reactivity against H. somni rOMP40 antigen in ELISA. Serum samples were obtained as follows: S1: before the first intranasal immunization (24–48 h of life); S2: two weeks after the first immunization (14–16 days of age); S3: two weeks after the second intranasal immunization (28–30 days of age); S4: two weeks after the third subcutaneous immunization (42–44 days of age); S5: two weeks after S4 (59–61 days of age). Individual study groups were labelled as CpG (vaccine formulation rHsp60 + rOMP40 + CpG), Con (control group), and MPLA (vaccine formulation rHsp60 + rOMP40 + MPLA). Significant differences within individual study groups between consecutive samplings (e.g., CpG S1-S2, S2-S3, S3-S4, and S4-S5) are labelled with the same uppercase letters (A-C) for p ≤ 0.01, or small letters (a-c) for p ≤ 0.05. Significant differences within individual study groups between S1 and other samplings (e.g., CpG S1-S2, S1-S3, S1-S4, and S1-S5) are labelled with two asterisks (**) for p ≤ 0.01, or an asterisk (*) for p ≤ 0.05. Significant differences between the study groups in the respective samples are labelled with brackets. Serum dilutions were 1:50 for IgA and 1:100 for IgG₁, IgG₂ and IgM. The median line across the box, lower, and upper boxes indicate the 25th percentile to the 75th percentile, and whisker non-outlier range.