Maarten Sileghem

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. The production of haemopoietic chimaeras from trypanotolerant and susceptible twin calves and comparison of their responses after infection with singleton calves, allowed an assessment of the role of the haemopoietic system in trypanotolerance. An in vivo depletion of CD4 cells in the two breeds allowed an appraisal of the role of T and B lymphocytes in trypanotolerance. The results of the two experiments suggest that natural resistance comprises at least two mechanisms, an innate mechanism that controls parasite growth, and another, involving the haemopoietic system, that is able to limit anaemia. This supports the hypothesis that innate mechanisms in trypanotolerant cattle are more efficient in controlling disease, making them less reliant on antibody responses.

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

Abstract Monoclonal antibodies and IgG purified from rabbit polyclonal antiserum, raised against recombinant bovine tumor necrosis factor-alpha (TNF-α), have been employed in ELISA procedures to quantitate bovine TNF-α. These antibodies were potent in neutralizing the biological activity of recombinant as well as natural bovine TNF-α. The monoclonal antibodies were used as capture antibodies and were either passively adsorbed or covalently linked to ELISA plates. Polyclonal rabbit anti-TNF IgG was used as the detecting antibody in combination with a biotinylated anti-rabbit serum and a streptavidin-horseradish peroxidase conjugate. The detection limit for recombinant TNF-α medium was 10 pg ml−1 and in bovine or ovine serum was 35 pg ml−1. A good correlation was found between the ELISA and the WEHI-164 Clone 13 biologic assay when TNF-α was measured in medium containing serum or in serum. This capture ELISA was also capable of detecting ovine, but not porcine. TNF in supernatants from cultures of lipopolysaccharide-stimulated pulmonary alveolar macrophages.

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 &bgr;‐galactosidase, ovalbumin and ferritin. The parasite non‐specific IgM antibodies appear around the same time as the parasite‐specific antibodies, but their origin and function are not clear. We tested the hypothesis that CD5+ B cells (or B‐1 cells), which increase during trypanosome infections in cattle, are responsible for production of antibodies to non‐trypanosome antigens. Splenic CD5+ and CD5− B cells from infected cattle were sorted and tested in a single cell blot assay. The numbers of immunoglobulin‐secreting cells were similar in both B‐cell populations. However, antibodies with reactivity for non‐trypanosome antigens were significantly more prevalent in the CD5+ B‐cell fraction and were exclusively IgM. The preference for production of these antibodies by CD5+ B cells and the expansion of this subpopulation during infections in cattle, strongly suggest that CD5+ B cells are the main source of trypanosome non‐specific antibodies. We propose that these antibodies are natural, polyreactive antibodies that are predominantly secreted by CD5+ B cells. Since B‐1 cells are up‐regulated in many states of immune insufficiency, the immunosuppression associated with trypanosome infections may be responsible for the increase of this subset and the concomitant increase in trypanosome non‐specific antibodies.

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. Rapid depletion of T cells was associated with complement-binding antibody isotypes (IgG2a, IgM), suggesting a complement-mediated mechanism. Clearance of T cells after several days was observed with a non complement-binding isotype (IgG1), suggesting phagocytosis or induction of apoptosis as possible mechanisms. Clearance of the cells in peripheral blood and spleen was obtained with 10-20 mg of anti-CD4 or anti-CD8, but almost ten times as much was needed to obtain depletion of the cells in lymph nodes and Peyers patches. Depletion lasted for 12 days for CD4 T cells and 3 weeks for CD8 T cells. Successful and lasting depletion (at least 2 weeks) was also obtained with other T cell reagents, such as anti-CD2 and anti-WC1 (gamma/delta T cells). Although B lymphocytes could be removed by a complement-binding antibody, complete depletion of these cells only lasted for a few hours, probably because B cells regenerate faster than T cells. T cell function was severely inhibited when CD4+ T cells were depleted. Stimulation of T cells with foot and mouth disease viral antigen (FMDV) in vaccinated calves was non-existent after depletion. Even 2 months after restoration of normal CD4 T cell levels in blood, activity to FMDV was low. This suggested that the depleted T cells were replaced by naive cells.

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. The dimyristylglycerol moiety of the anchor was released by phospholipase C cleavage. Integrity of the anchor and efficiency of cleavage was verified by autoradiography and methanol:hexane extraction. For analysis of biological function, bovine monocytes were used which had been incubated with bovine interferon gamma (primed) or with culture medium (unprimed). The VSG purified in its membrane-associated form was found to stimulate both primed and unprimed cells to secrete TNFalpha. The same preparation from which the dimyristylglycerol moiety had been cleaved was no longer able to stimulate unprimed cells but could still stimulate primed cells. Our data indicate that the presence of the dimyristylglycerol is not an absolute requirement for induction of TNFalpha production but can substitute for the interferon gamma priming. Therefore, we favor the hypothesis that stimulation of macrophages to secrete TNFalpha by the mfVSG is mediated by an as yet unknown trigger moiety and is facilitated by the dimyristylglycerol anchor.

1 – African Trypanosomiasis

Publisher Summary This chapter describes African trypanosomes as protozoan parasites that cause disease in humans and livestock. Both Trypanosoma brucei rhodesiense and T. brucei gambiense cause sleeping sickness in humans. Tsetse flies only occur in Africa and Saudi Arabia. However, some trypanosome species can be transmitted in the absence of the tsetse flies and can be found far outside the African tsetse belt. Trypanosoma evansi only exists as bloodstream forms and is transmitted through mechanical transfer by biting flies. Most trypanosomes do not manifest a strict host tropism and can infect a variety of livestock species. They also infect wild animals, which form a reservoir from which the tsetse flies continuously reinfect livestock. The infected animals develop fever, lose weight, and progressively become weak and unproductive. Both Trypanosoma brucei gambiense and T. b. rhodesiense cause sleeping sickness in humans whereas T. b. brucei is unable to infect humans and is lysed by human serum in vitro.

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. The number of B cells increased between 2 and 6 days post-infection, and thereafter returned to pre-infection values. Little change was observed within the afferent lymph veiled cell population. Examination of activation markers on the lymphocyte fraction of afferent lymph revealed a decrease in the number of cells expressing the Interleukin-2 receptor alpha-chain from Day 5 post-infection. At this time the expression of ACT 1, another early activation marker, was seen to increase. Afferent lymph cells collected pre-infection and on the first 4 days post-infection proliferated in response to stimulation with Concanavalin A in vitro. This response to mitogenic stimulation was completely abrogated from day five post-infection. However, these cells were not capable of suppressing the capacity of normal peripheral blood mononuclear cells to respond to mitogenic stimulus in co-culture assays. These studies suggest that although a degree of lymphocyte activation occurs in the afferent lymph following tsetse-transmitted infection with T. congolense, this may be sub-optimal owing to the immunosuppression which appears to operate at the level of the skin and the lymph nodes.

Identifying the Mechanisms of Trypanotolerance in Cattle

Some West African cattle breeds such as N’Dama have survived in tsetse-infected areas for thousands of years and are productive under trypanosome challenge, a trait known as trypanotolerance. Elucidation of the mechanisms of trypanotolerance could lead to new options for disease control. This review describes responses in trypanotolerant and trypanosusceptible breeds of cattle, and compares them with responses in mouse models. It focuses on the roles of haemopoietic tissue, T lymphocytes and antibodies in resistance to trpanosomiasis.