Michel Olivier

Mic1-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep

This study assessed the effectiveness of a mutant strain of Toxoplasma gondii (RH strain) lacking the mic1 and mic3 genes (Mic1-3KO) against Toxoplasma abortion in sheep. Ewes were inoculated subcutaneously with 105 Mic1-3KO tachyzoïtes in three independent experiments. Following vaccination, Mic1-3KO induced a mild febrile response and serum IgG antibodies, which persisted throughout the experiments. Tissue cysts formed in the sheep, but were not, under our experimental conditions, infectious when given orally. Ewes were mated two months after vaccination and were orally challenged with the PRU strain of T. gondii at mid-gestation (400 oocysts in Experiments 1 and 2; 100 oocysts in Experiment 3). Challenge of vaccinated pregnant ewes resulted in a slight febrile response, whereas unvaccinated ewes developed a more severe, characteristic febrile response of longer duration. After challenge, all unvaccinated ewes aborted whereas 62%, 91% and 64% (Experiments 1, 2 and 3 respectively) of the lambs from vaccinated ewes were viable, with no clinical signs of infection. Mic1-3KO was as effective as S48, the strain used as a live vaccine for sheep (Toxovax®). A dose of 105 Mic1-3KO tachyzoites was sufficient to induce protection (versus a dose of 2 × 106). Both subcutaneous and intraperitoneal injections were effective. Moreover, preliminary results showed the potential of Mic1-3KO to reduce the development of tissue cysts in lambs born to vaccinated ewes. This study demonstrates that Mic1-3KO is a potent vaccine candidate.

Nramp1 Is Not a Major Determinant in the Control of Brucella melitensis Infection in Mice

ABSTRACT Brucella, the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of the Nramp1 gene. The role of this gene in the control of Brucella infection was investigated. When BALB/c mice (Nramp1s) and C.CB congenic mice (Nramp1r) were infected with Brucella melitensis, the number of Brucella organisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). This Nramp1-linked susceptibility to Brucella was temporary, since similar numbers of Brucella were recovered from the two strains of mice 2 weeks p.i. The effect of Nramp1 expression occurred within splenocytes intracellularly infected by Brucella. However, there was no difference between in vitro replication rates of Brucella in macrophages isolated from the two strains of mice infected in vivo or in Nramp1 RAW264 transfectants. In mice, infection with Brucella induced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-α), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-γ), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-γ and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-α mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice to B. melitensis infection. In contrast to infections with Salmonella, Leishmania, and Mycobacterium, the expression of the Nramp1 gene appears to be of limited importance for the natural resistance of mice to Brucella.

Analysis of the immune response in sheep efferent lymph during Salmonella abortusovis infection

The efferent lymph duct of the ovine prescapular lymph node was cannulated, and Salmonella abortusovis (SAO), a specific pathogen for sheep inducing abortion and mortality of newborn lambs, was inoculated by the subcutaneous route in this lymph node drained area. While the prescapular lymph node draining the inoculation site represented an efficient barrier for the vaccinal SAO Rv6 strain spreading, SAO 15/5 virulent bacteria were steadily detected in efferent lymph of infected sheep. The inoculation of the virulent strain of SAO induced a greater increase of the cell output than did the attenuated vaccinal strain, but proportions of blast cells appearing in the efferent lymph were similar in both cases. Flow cytometry analysis showed that B and T cell outputs were both increased during SAO infections, but while T cell subset proportions slightly decreased, B cell percentages significantly rose, and, at the peak response, almost all of the lymphoblast cells were activated B cells. Typical antibody profiles characteristic of a primary immune response were observed, and antibody titres were greater in the efferent lymph of animals inoculated with the virulent strain of SAO. Many of the cytokine mRNAs we investigated were steadily detected by RT-PCR in efferent lymph cells of control sheep, but frequencies of detection of IL-2, IFN gamma, IL-1 beta and TNF alpha mRNAs were augmented in efferent lymph cells following inoculation of both SAO virulent or vaccinal strains. IL-10 and IL-8 mRNAs could only be detected after a SAO inoculation, while detection of IL-4 mRNAs was increased only in efferent lymph cells from SAO virulent strain-infected sheep. The efferent lymph cannulation technique thus appeared a very powerful way to study the in vivo development of the immune response to SAO, in its natural host, the sheep.

Cellular composition of Corynebacterium pseudotuberculosis pyogranulomas in sheep

The cellular composition of 46 pyogranulomas experimentally induced with Corynebacterium pseudotuberculosis in sheep was determined by immunohistochemistry. Lesions localized in inoculation sites or draining lymph nodes consisted of macrophage and lymphocyte layers distributed around a necrotic center and surrounded by a fibrous capsule. In immature lesions, T cells of the CD4+ subset predominated, whereas in mature lesions proportions of CD8+ T lymphocytes and cells expressing the γ/δ chains for the T cell receptor increased. Numerous pyogranuloma cells expressed the interleukin‐2 receptor. In addition to these general characteristics, a large individual variability in the proportions of macrophage and T cell subsets was observed for lesions of the same age, in particular for epithelioid macrophages. This heterogeneity suggests a different cellular pattern in relation to the persistence or the elimination of bacteria by the host. J. Leukoc. Biol. 56: 666–670; 1994.

Establishment and characterisation of ovine blood monocyte-derived cell lines

Studies of the important functions in host defense assured by macrophages, both as functional elements and as potential targets for intracellular pathogens, are often inhibited by the lack of a source of large numbers of uniform, well-characterised cells. To address this lack for ovine studies, we have established cell lines from spontaneously-proliferating adherent mononuclear cells from sheep blood. Eight such lines which have been continuously cultured for over 400 passages have phagocytic activities and cytochemical characteristics indicating that they retain the nature of mononuclear phagocytes. They display typical functional membrane proteins such as CD14, Fc receptors and MHC class II. Such cells can facilitate in vitro studies of pathogen-monocyte interactions and can furnish copious amounts of cells for transfer experiments.

Natural killer (NK) activity and interferon (IFN) production by a fraction of spleen and blood lymphocytes in swine

Abstract After carbonyl iron treatment and gradient isolation, spleen and blood pig lymphocytes exhibited NK activity and produced IFN after viral induction. Removal of plastic-adherent cells, including the majority of B cells, did not change these activities. The plastic-non-adherent cells were further separated into two subsets of roughly similar size by panning using a monoclonal, anti-T, and anti-null cell antibodies (81+cells). NK activity and IFN production were found in the 81 − cell fraction. A significantly higher proportion of null lymphocytes from blood and of splenic Fc-gamma receptor-bearing lymphocytes was also found among the 81 − cell fraction as compared to the 81 + fraction, without any change among other subsets. Similar proportions of helper (PT4+), cytotoxic (PT8+) and total T cells (MSA4+) were found among lymphocytes bound to target K562 cells and among the whole lymphocyte population. In contrast, lymphocytes that bound K562 cells demonstrated a striking increase in the proportion of Fc-gamma receptor-positive cells of high affinity. These results show that NK cells and IFN-producing cells are mainly included in the same blood and spleen fraction, and suggest that among 81 − cells only those expressing an Fc-gamma receptor of high affinity are active.

Humoral immune response to Salmonella abortusovis in sheep: in vitro induction of an antibody synthesis from either sensitized or unprimed lymph node cells

Abstract In vitro culture conditions were determined to induce an anti-Salmonella abortusovis antibody synthesis from lymph node leucocytes of three immunized sheep and two unprimed lambs maintained in culture in the presence of heat-inactivated bacteria for 2 weeks. Humoral immune responses were assessed by enumerating specific antibody-secreting cells using ELISASPOT and by titrating immunoglobulins secreted into culture supernatants using ELISA techniques. Optimal secondary antibody response was observed from cultures performed with fetal calf serum (compared with horse serum) and with an antigen concentration of one to ten bacteria per cell. This kind of antigenic stimulation allowed induction of numerous antibody-secreting cells without adsorption of the secreted antibodies. Maximal numbers of antibody-secreting cells could reach a rate of 1% of the sheep leucocytes initially put into culture. Kinetic profiles of antibody production from boosted lymph node cells were characterized by an ascending phase from the sixth to the twelfth day of culture and then showed a plateau phase until Day 14. Most of the responses were composed of IgM and IgG1 antibodies, traces of IgG2 being detected at the end of experiments. From the twelfth day of antigenic stimulation, the IgM isotype was preferentially expressed with high antigen concentration (100 bacteria per cell), whereas the highest amounts of IgG1 were detected at lower concentration (one to ten bacteria per cell). While anti-Salmonella IgM appeared to be mainly specific for the lipopolysaccharide (LPS) cell wall fraction, some IgG1 recognized other bacterial antigens. Kinetic profiles and magnitudes of primary antibody responses induced in vitro from lamb lymph node cells did not differ from those defined in cultures of sheep boosted leucocytes. But these immune reactions were mainly made up of anti-LPS IgM. Few anti-Salmonella IgG1 were detected from the tenth day of culture. So these in vitro assays allowed induction of an antibody synthesis from either in vivo sensitized or unprimed sheep lymph node leucocytes. This methodology would permit achievement of more detailed studies on interactions between Salmonella and lymph node leucocytes, leading to a better understanding of the mechanisms controlling bacterial dissemination through the lymphoid tissue.

Capacities of Migrating CD1b+ Lymph Dendritic Cells to Present Salmonella Antigens to Naive T Cells

Dendritic cells (DCs) are well known as professional antigen-presenting cells (APC) able to initiate specific T-cell responses to pathogens in lymph nodes (LN) draining the site of infection. However, the respective contribution of migratory and LN-resident DCs in this process remains unclear. As DC subsets represent important targets for vaccination strategies, more precise knowledge of DC subsets able to present vaccine antigens to T cells efficiently is required. To investigate the capacities of DCs migrating in the lymph (L-DCs) to initiate a specific T-cell response, we used physiologically generated DCs collected from a pseudoafferent lymphatic cannulation model in sheep. The CD1b+ L-DCs were assessed for presenting antigens from the vaccine attenuated strain of Salmonella enterica serovar Abortusovis. CD1b+ L-DCs were able to phagocytose, process and to present efficiently Salmonella antigens to effector/memory T cells in vitro. They were shown to be efficient APC for the priming of allogeneic naive T cells associated with inducing both IFN-γ and IL-4 responses. They were also efficient in presenting Salmonella antigens to autologous naive T cells associated with inducing both IFN-γ and IL-10 responses. The capacities of L-DCs to process and present Salmonella antigens to T cells were investigated in vivo after conjunctival inoculation of Salmonella. The CD1b+ L-DCs collected after inoculation were able to induce the proliferative response of CD4+ T cells suggesting the in vivo capture of Salmonella antigens by the CD1b+ L-DCs, and their potential to present them directly to CD4+ T cells. In this study, CD1b+ L-DCs present potential characteristics of APC to initiate by themselves T cell priming in the LN. They could be used as target cells for driving immune activation in vaccinal strategies.

6.24 Reactivity of workshop monoclonal antibodies with normal and pathological ovine lymph nodes

The monoclonal antibodies (82 mAbs) included in the Second Workshop that belonged to agreed named bovine CD and workshop clusters were tested for their reactivity and tissue distribution on (1) normal sheep lymph nodes and (2) pathological sheep lymph nodes. Pathological lymph nodes were induced following experimental infection with Corynebacterium pseudotuberculosis and were characterized by the presence of typical pyogranulomas. Amongst the 82 mAbs tested, 38 reacted with sheep lymph nodes. The results of these tests are presented and discussed.

IMMUNOHISTOCHEMICAL STUDY ON THE REACTIVITY OF WORKSHOP MONOCLONAL ANTIBODIES WITH SHEEP LYMPH NODES

Of the 302 monoclonal antibodies submitted to the Third Workshop on Ruminant Leukocyte Antigens, 167 have been tested for their reactivity on sheep lymph node sections, according to the APAAP immunohistochemical technique. Of the 57 monoclonal antibodies able to react on ovine tissue sections, only 37 induced a clear and well-contrasted staining. The more striking results are presented in this paper.