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Augmentation of immunization of cattle Brucella abortus S19 or a B. abortus soluble protein extract (SPEBA) vaccine through administration of recombinant bovine IL 2 (rBoIL 2) was evaluated. Seventy-five heifers were divided among 6 groups that were treated with the following: Group 1, no treatment; Group 2, rBoIL 2 (1microg/kg) on day 0; Group 3, SPEBA (2 mg) on day 0 and week 9; Group 4, SPEBA + rBoIL 2 on day 0, SPEBA on week 9; Group 5, S19 (10(7) CFU) on day 0 and week 9; Group 6, S19 + rBoIL 2 on day 0, S19 only on week 9. Approximately, 6 months after vaccination, cattle were bred by natural service, and at mid-gestation pregnant cattle were challenged intraconjunctivally with 9.1 x 10(5) CFU of virulent B. abortus S2308. Pre- and post-challenge antibody responses were measured by an enzyme-linked immunosorbent assay, a particle concentration fluorescence assay, and the card test. Lymphoproliferation (LP) responses to gamma-irradiated B. abortus and SPEBA antigens were measured in peripheral blood mononuclear cells. After vaccination, antibody responses to B. abortus elevated rapidly in SPEBA- and S19-vaccinates with and without rBoIL 2, however, these responses were significantly (P < 0.05) higher in vaccinates which also received rBoIL 2. Antibody levels for all vaccinated groups had returned to those of negative control groups by the challenge date with the exception of the SPEBA/rBoIL 2 group. In general, LP responses were higher in vaccinated or rBoIL 2-treated cattle than for unvaccinated controls. Challenge of 48 pregnant heifers resulted in abortions in 4/9 of Group 1, 0/9 of Group 2, 4/8 of Group 3, 2/9 of Group 4, 1/7 of Group 5, and 0/6 of Group 6 cattle. Treatment with rBoIL 2 alone (Group 2) provided significant (P < 0.05) protection from infection, abortions and induction of sero-positive status compared to untreated (Group 1) cattle. Co-administration of rBoIL 2 with S19 resulted in significant (P < 0.05) augmentation in onset, duration and magnitude of LP responses to B. abortus antigens following challenge. Characterization of the cytokine response of bovine monocyte-derived macrophages by real-time polymerase chain reaction indicated that in vitro stimulation of these cells with rBoIL 2 resulted in a profound up-regulation of genes encoding tumor necrosis factor-alpha, IL 12p40, and interferon-gamma reflecting activation of the cells. Overall, rBoIL 2-treatment was associated with fewer infections, sero-conversions and a significant (P = 0.02) level of protection against abortion as compared to vaccination alone or no treatment.

The long-held paradigm of T lymphocyte-mediated activation of mononuclear phagocytes (Mø) as the major mechanism of protection against facultative intracellular pathogens such as Brucella has been modified to include killing of infected Mø by various subsets of T lymphocytes. Remnants of killed infected cells are phagocytosed by immunologically-activated Mø, which are much more efficient at killing such pathogens. Most of the detailed information regarding immunity in general and that of brucellosis specifically has been obtained using murine infection models rather than in cattle. However, there has been considerable definition of cellular phenotypes, cytokines and functional characteristics of T lymphocytes in cattle over the last decade. This was mainly due to development of monoclonal antibodies against cell surface markers and application of molecular cloning and polymerase chain reaction (PCR) for isolation, characterization and detection of genes encoding bovine cytokines. This review discusses cellular and molecular immunity in bovine brucellosis as pertains to T lymphocyte interactions with the Mø. Although current knowledge directly obtained from brucellosis immunity studies in the bovine host is limited and incomplete, the many parallels between the bovine and murine immune systems allow for some extrapolation in the description of bovine host defense mechanisms. Direct information from studies with immunized cattle supports the concepts of coordinate activation of uninfected Mø and killing of Brucella-infected Mø by antigen-specific T lymphocytes as major mechanisms of host defense in bovine brucellosis. There also appears to be a bias in the T lymphocyte compartment towards recognition of particular bacterial stress proteins following immunization with live Brucella vaccines., (Copyright 2002 Elsevier Science B.V.)

The adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA) alone or in combination with trehalose dimycolate (TDM) or muramyl dipeptide (MDP) on bovine humoral and cellular responses to a soluble protein extract of gamma irradiated Brucella abortus strain 19 (SPEBA) were investigated. Thirty-five beef steers were randomly allotted to nine groups. Three of these groups received SPEBA (2 mg protein per dose) subcutaneously in combination with adjuvants, one group received the reduced dose of B. abortus strain 19 (S19), and one group received SPEBA alone. Controls included groups receiving adjuvant preparations only or no vaccine. Immune responses to the various immunizations were assessed sequentially for 56 days using various in vitro and in vivo assays. The humoral response to B. abortus was measured using standard serologic tests, an enzyme-linked immunosorbent assay, and a quantitative fluorometric immunoassay. The cell-mediated immune (CMI) response was measured by antigen-specific lymphoproliferation (LP), interleukin 2 (IL 2) production, and soluble suppressor factor release. Skin testing at day 35 for delayed-type hypersensitivity (DTH) to SPEBA was also performed. Minimal humoral responses were induced with SPEBA alone. The highest and most sustained serum antibody responses to B. abortus antigens were elicited by the S19 vaccine. A combination of SPEBA with DDA + TDM induced higher antibody levels than SPEBA with DDA or SPEBA with DDA + MDP. Responses to DTH among the groups receiving SPEBA were most notable in the SPEBA with DDA + TDM groups. Increased IL 2 production was greatest in the S19 and SPEBA with DDA + TDM vaccinates. The results indicated that a combination of DDA + TDM best potentiated immune responses to a soluble B. abortus antigen preparation and may be useful as adjuvants for future vaccines.

Five different adjuvants were examined for potentiation of humoral and cell-mediated immune (CMI) responses in cattle to a Brucella abortus soluble antigen (BASA). Two separate experiments were performed involving a total of 64 steers, divided among six groups (Experiment 1) and 9 groups (Experiment 2). The adjuvants used were: muramyl dipeptide, Freund's incomplete adjuvant, dimethyl-dioctadecyl ammonium bromide (DDA), Bordetella pertussis and Propionibacterium acnes. In each experiment, three groups received BASA (2 mg protein) subcutaneously with adjuvant, one group received a reduced dose of B. abortus Strain 19 (S19), one group served as unvaccinated controls, and another group received BASA alone. Primary responses were studied following a single immunization in comparison to the single inoculation with S19. For each experiment serum antibody responses and CMI responses were sequentially determined over a period of 56 days. Antibody responses to B. abortus were measured using the brucellosis card, rivanol precipitation-plate agglutination, complement fixation, and fluorometric immunoassay tests, and as well as with an enzyme-linked immunosorbent assay. The CMI response was measured using antigen-specific lymphoproliferation (LP) and skin testing for delayed-type hypersensitivity (DTH) to BASA (Experiment 2). Specific aspects of induced CMI responses investigated were macrophage activation (IL-1 production), helper T cell activation (IL-2 production), and release of soluble suppressor factor(s). In general, mean antibody responses were significantly higher (P less than 0.05) in immunized steers than in control steers and those receiving BASA alone. The LP responses to heat-killed B. abortus were generally higher in immunized groups than in the controls. The LP and DTH responses were greatest in the groups receiving S19 and BASA + DDA. Increased induction of IL-1 was largest in the group receiving BASA + DDA whereas IL-2 release was greatest in S19 vaccinated steers. Suppressor T cell responses were most obvious in the groups receiving S19, BASA + B. pertussis, and P. acnes. These studies demonstrated that DDA potentiates CMI responses to a soluble B. abortus antigen and may be useful as an adjuvant for future vaccines, particularly subunit vaccines.