Evaluation of Type 1 Immune Response in Naïve and Vaccinated Animals following Challenge with Leptospira borgpetersenii Serovar Hardjo: Involvement of WC1+ γδ and CD4 T Cells

The spirochete bacterium Leptospira spp. serovar Hardjo is a pathogen that causes disease in cattle and humans throughout the world. Infected cattle are the maintenance host for Leptospira borgpetersenii serovar Hardjo (subtype hardjobovis) (20) and Leptospira interrogans serovar Hardjo (subtype hardjo prajitno) and have a variety of clinical illnesses including abortion, infertility, and mastitis, while their calves may be stillborn, weak, or clinically normal but infected (see references 14-16, 21, 25, and 26). Infection is commonly transmitted by contact of urine or reproductive fluids from infected animals with the mucosal membranes of uninfected humans or animals, either directly or through fomites. Zoonotic infection of humans with leptospires including those of the serovar Hardjo group (1) poses a significant public health problem of increasing concern since leptospirosis in humans may be fatal due to involvement of multiple organs including liver, lungs, kidney, and brain (see reference 23). It was previously thought that protective immunity against leptospirosis was sufficiently provided by antibodies (20), since anti-leptospiral lipopolysaccharide (LPS) antibodies have been shown elsewhere to provide passive immunity in some animal models, protecting against a number of strains and species of Leptospira (30, 36). However, Bolin et al. (4, 6) showed that high titers of anti-LPS antibody induced by conventional leptospiral vaccines may not be protective against L. borgpetersenii serovar Hardjo. Moreover, recently developed vaccines that protect against L. borgpetersenii serovar Hardjo including renal colonization and urinary shedding (7, 41) and protect against transplacental infection of the fetus (D. Alt, R. Hornsby, and C. A. Bolin, submitted for publication) induce a type 1, or cell-mediated, immune response (18, 41). Cell-mediated or type 1 immunity is generally regarded as including production of gamma interferon (IFN-γ) and generation of cytotoxic CD8 T cells. While cytotoxic CD8 T cells and IFN-γ are both particularly important in control or clearance of infections with viruses and intracellular bacteria and protozoa, IFN-γ may also have a role in protection against extracellular microbes through its ability to activate macrophages and promote production of immunoglobulin G2 (IgG2) classes of antibodies, as has been suggested previously for immunity to extracellular stages of the protozoan parasite Babesia bigemina (9). While bovine IgG2 and IgG1 are both able to fix complement, which may be an important effector mechanism for control of leptospires in its own right, bovine IgG2 antibodies also act as opsonins (37), thereby potentially increasing the number of leptospires phagocytosed. Moreover, although leptospires are not considered to be intracellular pathogens that survive phagocytosis, IFN-γ activation of macrophages may increase the efficiency of killing. For example, Candida albicans, largely an extracellular organism that also targets the kidney, suppresses macrophage nitric oxide production through a soluble factor (10), and thus, IFN-γ is required for optimal production of nitric oxide by macrophages in vivo during a Candida infection (31). Because of the increasing incidence of infection in cattle and the zoonotic nature of human infections, it was of interest to examine the immune response of naive cattle following challenge with L. borgpetersenii serovar Hardjo during the periparturient period. This experimental design was chosen because of the effects that L. borgpetersenii serovar Hardjo infections have on events associated with pregnancy and transmission of the infection to the calves. Since natural infection is chronic in cattle, it was of interest to determine if it established itself due to the absence of a type 1 immune response or an insufficient one. While many bacterial infections induce type 1 immune responses, some are associated with induction of a type 2 response, such as that which occurs in patients with lepromatous leprosy (48), while other infections such as those with Brucella abortus may initially induce a type 1 response but after the first few weeks of infection show a hiatus of IFN-γ production (40). Here the cellular immune response of nonvaccinated cattle following challenge was compared with that of cattle that had been immunized prior to challenge with a vaccine protective against L. borgpetersenii serovar Hardjo infections (7) and which induces a type 1 cell-mediated immune response (41). Production of IFN-γ by peripheral blood mononuclear cells (PBMC) in response to antigen was measured by enzyme-linked immunosorbent assay (ELISA), and since any of the major T-cell populations including αβ CD4, αβ CD8, or γδ T cells may produce IFN-γ, two-color flow cytometry was used to determine the contribution by individual T-cell subpopulations. Generation of antigen-specific IgG1 and IgG2 antibodies in sera was also evaluated since the lack of IgG2 is correlated with a type 2 immune response.