Bovine Immune Response to Vaccination and Infection with Leptospira borgpetersenii Serovar Hardjo

Abstract

This study examined the humoral and cellular response of cattle vaccinated with two commercial leptospiral vaccines, Leptavoid and Spirovac, and a novel bacterin vaccine using Seppic Montanide oil emulsion adjuvant. Vaccination was followed by experimental challenge. All vaccinated cattle were protected from colonization of the kidney and shedding of Leptospira in urine, as detected by culture and immunofluorescence assay. Agglutinating antibody titers were detected in vaccinated cattle at 4 weeks following vaccination, with small anamnestic response detected following experimental challenge. Only animals vaccinated with the oil emulsion-adjuvanted bacterin produced significant IgG2 titers following vaccination, and nonvaccinated animals produced serum IgA titers after experimental challenge. CD4⁺ and γδ T cells from vaccinated cattle proliferated when cultured with antigen ex vivo Cellular responses included a marked proliferation of γδ T cells immediately following experimental challenge in vaccinated cattle and release of gamma interferon (IFN-γ), interleukin 17a (IL-17a), and IL-12p40 from stimulated cells. Proliferative and cytokine responses were found not just in peripheral mononuclear cells but also in lymphocytes isolated from renal lymph nodes at 10 weeks following experimental challenge. Overall, effects of leptospirosis vaccination and infection were subtle, resulting in only modest activation of CD4⁺ and γδ T cells. The use of Seppic Montanide oil emulsion adjuvants may shorten the initiation of response to vaccination, which could be useful during outbreaks or in areas where leptospirosis is endemic.IMPORTANCE Leptospirosis is an underdiagnosed, underreported zoonotic disease of which domestic livestock can be carriers. As a reservoir host for Leptospira borgpetersenii serovar Hardjo, cattle may present with reproductive issues, including abortion, birth of weak or infected calves, or failure to breed. Despite years of study and the availability of commercial vaccines, detailed analysis of the bovine immune response to vaccination and Leptospira challenge is lacking. This study evaluated immunologic responses to two efficacious commercial vaccines and a novel bacterin vaccine using an adjuvant chosen for enhanced cellular immune responses. Antigen-specific responsive CD4 and γδ T cells were detected following vaccination and were associated with release of inflammatory cytokines IFN-γ and IL-17a after stimulation. CD4 and γδ cells increased in the first week after infection and, combined with serum antibody, may play a role in clearance of bacteria from the blood and resident tissues. Additionally, these antigen-reactive T cells were found in the regional lymph nodes following infection, indicating that memory responses may not be circulating but are still present in regional lymph nodes. The information gained in this study expands knowledge of bovine immune response to leptospirosis vaccines and infection. The use of oil emulsion adjuvants may enhance early immune responses to leptospiral bacterins, which could be useful in outbreaks or situations where leptospirosis is endemic.

Keywords: Leptospira; adjuvant; adjuvants; cattle; immune response; vaccine; vaccines; veterinary vaccine development.

FIG 1

Depiction of experimental timeline with vaccination, challenge (Δ), and blood collection for immune response evaluation (↑, black arrows) marked at approximate weeks after vaccination or experimental/infectious challenge (postinfection [PI]). A total of 24 crossbred heifers were assigned to 4 groups, vaccine or nonvaccinated control group, with 6 cattle per group. Due to space constraints in the agricultural biosafety level 2 (Ag-BSL-2) containment facility, half of the cattle were challenged in replicate 1 and half of the cattle were challenged in replicate 2.

FIG 2

Microscopic agglutinating test. Serum antibody titer to strain 203 was measured at the given time points postvaccination and postinfectious challenge (PI, shaded bars). Group means (n = 6) + standard error of the mean (SEM). An asterisk (*) indicates statistical difference from week 0 within vaccine group and statistical difference between vaccine groups and control group within a time point (week); P ≤ 0.05.

FIG 3

Serum antibody enzyme-linked immunosorbent assay (ELISA) for IgM, IgA, IgG1, and IgG2 isotypes binding to plate-bound serovar Hardjo 203 whole-cell sonicate antigen at weeks 0, 4, 13, and 33 postvaccination and postinfection (PI) weeks 4 and 10 (shaded bars). Mean (n = 6) + SEM. An asterisk (*) indicates statistical difference from week 0 within vaccine group; a indicates statistical difference between control and vaccine groups within time point (week); P ≤ 0.05. (A, B, C, and D) y axis scales are not all equivalent.

FIG 4

Proliferation of in vitro-stimulated peripheral blood mononuclear cells (PBMCs). Percentage of cellular phenotype proliferating in response to 203 antigen for (A) CD4-expressing population, (B) CD8-expressing population, (C) γδ T-cell receptor (γδ TCR)-expressing population, and (D) B cell- or CD21-expressing population. Mean (n = 6) + SEM; an asterisk (*) indicates a significant difference (P < 0.05) between control and vaccine groups within a time point (week).

FIG 5

Cytokine release by antigen-stimulated PBMCs. Supernatants from PBMCs stimulated with 203 antigen were collected 48 h after stimulation and assayed for cytokines. Mean + SEM depicted. An asterisk (*) indicates a significant difference between control and vaccine groups within a time point (week) (P < 0.05). # indicates a significant difference between time points within treatment group (P < 0.05).

FIG 6

Cross-reactive response to other Leptospira strains. [³H]-Thymidine incorporation measurement of proliferation when stimulated with different Leptospira strains, serovars, and species. PBMCs collected from vaccinated and control cattle 32 weeks postvaccination. Stimulation index (SI) of pokeweed mitogen (PWM; positive-control mitogen) values between 50 and 300 (not shown). Mean (n = 6) + SEM; an asterisk (*) indicates that a group is significantly different (P < 0.05) from other groups.

FIG 7

(A) Proliferation of lymphocytes from renal lymph node. Percentage of proliferating lymphocytes, by phenotype, isolated from renal lymph node of strain 203-infected cattle, 10 weeks postinfection, stimulated ex vivo with 203 antigens. (B) Cytokine release from cultured renal lymphocytes collected for analysis 48 h poststimulation. Mean ± SEM. #, statistical difference (P < 0.05) between vaccine group and nonvaccinated control group; *, statistical difference from nonstimulated well within group (nonstimulated not shown).