Identification of mechanisms of natural resistance to African trypanosomiasis in cattle.

Natural resistance to African trypanosomiasis in certain Bos taurus cattle in West Africa, called trypanotolerance, may hold solutions for control of this economically crippling disease. Comparison of immune responses between trypanotolerant and trypanosusceptible cattle have shown some differences in antibody response, complement level and cytokine expression, but it is not known whether these differences are the cause of resistance. Two experiments were carried out to assess the contribution of the immune and haemopoietic systems to trypanotolerance.

An accessory role for the diacylglycerol moiety of variable surface glycoprotein of African trypanosomes in the stimulation of bovine monocytes.

The membrane-associated form of the variable surface glycoprotein (mfVSG) from African trypanosomes is a potent macrophage activator capable of inducing production of tumor necrosis factor alpha (TNFalpha) in both bovine and murine models. Using a bovine model, we have re-investigated the hypothesis that the diacylglycerol moiety of the glycosylphosphatodylinositol (GPI) anchor is involved in macrophage activation and might be the actual parasite toxin. The anchor of the variable surface glycoprotein (VSG) was labeled with (3)H-myristic acid and VSG purified in its membrane-associated form.

Effective in vivo depletion of T cell subpopulations and loss of memory cells in cattle using mouse monoclonal antibodies.

Conditions were established to obtain depletion of T lymphocyte subsets in lymphoid tissues of calves by injection of mouse monoclonal antibodies to T cell antigens. Adverse reactions were avoided by injecting small quantities of antibody, until target cells had disappeared from blood. Two different mechanisms appeared to be responsible for elimination of the target cells.

CD5+ B lymphocytes are the main source of antibodies reactive with non-parasite antigens in Trypanosoma congolense-infected cattle.

Mice infected with African trypanosomes produce exceptionally large amounts of serum IgM, a major part of which binds to non-trypanosome antigens such as trinitrophenol and single-strand DNA. In this paper, we describe that in cattle infected with Trypanosoma congolense and T. vivax, similar antibodies are found, although they bind mainly to protein antigens, such as beta-galactosidase, ovalbumin and ferritin.

Differential and strain-specific triggering of bovine alveolar macrophage effector functions by mycoplasmas.

Mycoplasma strains being considered as pathogenic or non-pathogenic for cattle were tested on their capacity to activate bovine alveolar macrophages in vitro. Of particular interest was the behaviour of Mycoplasma mycoides ssp. mycoides small colony type (M.

Neutralization of bovine concanavalin-A T cell supernatant-mediated anti-Cowdria ruminantium activity with antibodies specific to interferon gamma but not to tumor necrosis factor.

In an earlier study we demonstrated that Concanavalin-A stimulated bovine T cell supernatants inhibited the growth of Cowdria ruminantium in bovine endothelial cells in vitro. An investigation was conducted to identify the cytokines which were responsible for this growth inhibition. Addition of antiserum against bovine interferon gamma (IFN gamma) reproducibly neutralized the inhibitory effect of the T cell supernatants, whereas addition of antisera against bovine tumor necrosis factor alpha (TNF alpha) had no effect.

In vitro simulation of immunosuppression caused by Trypanosoma brucei: active involvement of gamma interferon and tumor necrosis factor in the pathway of suppression.

Experimental infections of mice with the African trypanosome Trypanosoma brucei lead to a profound state of T-cell unresponsiveness in the lymph node cell (LNC) compartment. This suppression is mediated by macrophage-like cells which inhibit interleukin 2 (IL-2) secretion and down-regulate IL-2 receptor expression (M. Sileghem, A.

Cytokine regulation by virus infection: bovine viral diarrhea virus, a flavivirus, downregulates production of tumor necrosis factor alpha in macrophages in vitro.

Bovine bone marrow-derived macrophages were infected in vitro with noncytopathic or cytopathic strains of bovine viral diarrhea virus. Infection with both biotypes resulted in a decreased production of tumor necrosis factor alpha upon stimulation with heat-inactivated Salmonella dublin or lipopolysaccharide. Other macrophage functions were not downregulated, indicating that the observed effect was not due to a loss in macrophage viability.

Effect of bovine leukemia virus infection on bovine peripheral blood monocyte responsiveness to lipopolysaccharide stimulation in vitro.

The effects of bovine leukemia virus (BLV) infection on cytokine activity of bovine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined. Compared to supernatants of LPS-stimulated monocytes from BLV-negative cows, supernatants from BLV-positive cows contained about four times more interleukin-1 beta (IL-1 beta) (as measured by an enzyme-linked immunosorbent assay (ELISA) for bovine IL-1 beta). Despite their higher IL-1 beta concentration, supernatants from BLV-positive cows stimulated proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-biphenyl tetrazolium bromide) assay similar to supernatants from BLV-negative cows, but showed about 30% less IL-1 activity than supernatants from BLV-negative cows on the IL-1-dependent cell line LBRM-33 1 A-5, and about five times more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929.

Are CD8 T cells involved in control of African trypanosomiasis in a natural host environment?

Murine models have suggested that CD8 T cells might play a major parasite-promoting role in African trypanosomiasis. To assess the role of these cells in a natural host environment, we have depleted CD8 cells from Boran cattle in vivo and subsequently infected these animals with Trypanosoma congolense by tsetse fly challenge. Following administration of a mouse monoclonal anti-bovine CD8 antibody, we have been able to achieve a depletion of more than 99.

Involvement of gamma delta T cells in immunity to trypanosomiasis.

In this study the involvement of peripheral gamma delta T cells, prepared by flow cytometry, in the immune response of cattle to primary infection with Trypanosoma congolense was assessed. Negligible in vitro proliferative responses were observed in gamma delta T cells isolated from trypanosusceptible Boran (Bos indicus) cattle at all stages examined post-infection when stimulated in vitro with parasite antigens. In contrast, both CD8+ T cells and gamma delta T cells from trypanotolerant N'Dama (Bos taurus) cattle proliferated markedly when stimulated in vitro with a complex of invariant trypanosome antigens with MW between 100,000 and 140,000 (100,000 MW complex).

Modulation of the phenotype and function of bovine afferent lymph cells during infection with Trypanosoma congolense.

Alterations in the phenotype and function of cells isolated from bovine afferent lymph were studied following tsetse-transmitted Trypanosoma congolense infection. Little alteration was observed in the output of the CD2+ T cells in the lymph, and within this population the CD4:CD8 ratio remained relatively constant. By contrast, a marked decrease was observed in the output of gamma delta T cells over the first 7 days following infection.

Tumour necrosis factor production by monocytes from cattle infected with Trypanosoma (Duttonella) vivax and Trypanosoma (Nannomonas) congolense: possible association with severity of anaemia associated with the disease.

Plasma of cattle infected with Trypanosoma vivax IL2337 was analysed for the presence of bovine tumour necrosis factor (TNF) by EIA in which TNF was captured by a monoclonal antibody (MoAb BC9) and detected by a rabbit polyclonal antiserum. At week 2-3 post infection (p.i.

A colorimetric assay for trypanosome viability and metabolic function.

We have adapted a tetrazolium salt (MTT) colorimetric cytotoxicity assay to the assessment of viability and metabolic function in cultured African trypanosomes. Trypomastigotes of Trypanosoma congolense and T. brucei rhodesianse were harvested from the blood of parasitemic rats and cultured under axenic conditions that support trypanosome viability and growth.

Immunosuppression in trypanotolerant N'Dama cattle following Trypanosoma congolense infection.

Tsetse-transmitted Trypanosoma congolense infection causes an impairment of in vitro T cell proliferative responses in Boran (Bos indicus) cattle. To assess the importance of this phenomenon as it may relate to the ability of trypanotolerant cattle to control infection with trypanosomes, T cell proliferative responses to mitogenic stimulus with Concanavalin A were measured in N'Dama (Bos taurus) cattle throughout infection. The responses of peripheral blood mononuclear cells from Boran and N'Dama cattle were similar.

Inhibition of T-cell responsiveness during experimental infections with Trypanosoma brucei: active involvement of endogenous gamma interferon.

Lymph node cells (LNC) from mice infected with Trypanosoma brucei contain macrophage-like cells that inhibit interleukin-2 receptor (IL-2R) expression (M. Sileghem, A. Darji, R.

Secretion of co-stimulatory cytokines by monocytes and macrophages during infection with Trypanosoma (Nannomonas) congolense in susceptible and tolerant cattle.

Bovine macrophages and monocytes were cultured in vitro and analyzed for their capacity to secrete co-stimulatory cytokines. To this end, the culture medium was titrated on suboptimally stimulated murine thymocytes. A low residual release by normal monocytes was noted which usually remained below the detection limit of the assay.

Capture immunoassay for ruminant tumor necrosis factor-alpha: comparison with bioassay.

Monoclonal antibodies and IgG purified from rabbit polyclonal antiserum, raised against recombinant bovine tumor necrosis factor-alpha (TNF-alpha), have been employed in ELISA procedures to quantitate bovine TNF-alpha. These antibodies were potent in neutralizing the biological activity of recombinant as well as natural bovine TNF-alpha. The monoclonal antibodies were used as capture antibodies and were either passively adsorbed or covalently linked to ELISA plates.

Detection and neutralization of bovine tumor necrosis factor by a monoclonal antibody.

Monoclonal antibodies (MAbs) have been produced which are specific for bovine tumor necrosis factor (TNF). MAb BC9 detects bovine TNF in a radioimmunoassay with a detection limit of 24 pg/ml. BC9 also neutralizes the in vitro biological function of bovine recombinant TNF.

Selection of BoCD25 monoclonal antibodies by screening mouse L cells transfected with the bovine p55-interleukin-2 (IL-2) receptor gene.

The bovine interleukin-2 receptor-alpha (IL-2R alpha) gene has been isolated and a rabbit antiserum against a fusion protein of the gene has been produced. However, the antiserum does not inhibit IL-2-dependent proliferation. Since a panel of monoclonal antibodies (mAb) to bovine activation antigens was available, we transfected the gene into mouse L fibroblasts, selected stable transfectants with the rabbit antiserum, and screened for antibodies that bound the transfected cells but not the untransfected cells.

Parasite-specific T-cell responses of trypanotolerant and trypanosusceptible cattle during infection with Trypanosoma congolense.

During primary tsetse-transmitted challenge of Boran (Bos indicus) cattle with Trypanosoma congolense ILNat 3.1, a transient parasite antigen-specific T-cell proliferative response was observed in peripheral blood mononuclear cells and splenic mononuclear cells stimulated in vitro. A response was also observed with cells of N'Dama (Bos taurus) cattle, but in this case higher stimulation indices were observed and the response was maintained until the termination of the experiment at 40 days post-infection (p.

Suppression of interleukin 2 secretion and interleukin 2 receptor expression during tsetse-transmitted trypanosomiasis in cattle.

Infection with Trypanosoma congolense in cattle was found to be associated with a profound suppression of the host's immune system. Lymph node cells from infected cattle were unable to secrete interleukin 2 (IL 2) in vitro following mitogenic stimulation and the exogenous supply of IL 2 did not restore T cell proliferative responses. This was associated with an impaired expression of the alpha chain of the IL 2 receptor (IL 2R alpha).

Mechanisms underlying trypanosome-elicited immunosuppression.

T-cell proliferative responses of lymph node cells are profoundly suppressed during experimental infections of mice with Trypanosoma brucei. The active suppression of lymph node T-cell proliferative responses is attributed to the coexistence of at least two unlinked suppressive mechanisms that block different T-cell regulatory steps and operate through different effector mechanisms. The generation of prostaglandin-producing macrophages is entirely responsible for the suppression of IL-2 production whereas the induction of a prostaglandin-independent suppressive mechanism accounts for the suppression of the expression of IL-2 receptors (IL-2R).

Suppression of T-cell responsiveness during tsetse-transmitted trypanosomiasis in cattle.

In the present study, we demonstrate that lymph node cells from cattle infected with T. congolense through tsetse fly challenge were unable to proliferate in vitro following activation with the T-cell mitogen Concanavalin A. This was associated with a simultaneous suppression of interleukin 2 (IL-2) production and interleukin 2 receptor (IL-2R) expression.

The role of the macrophage in induction of immunosuppression in Trypanosoma congolense-infected cattle.

Impairment of T-cell function in Boran (Bos indicus) cattle during primary infection with Trypanosoma congolense ILNat 3.1 was found to occur in peripheral blood, spleen and, in particular, the lymph nodes. Lymph node cells from infected cattle failed to proliferate in response to mitogenic stimulus and suppressed proliferation of both normal peripheral blood mononuclear cells and lymph node cells in co-culture assays.