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Background: Fluorescence expression systems adapted to the analysis of host-mycoplasma interactions were recently developed. The aim of this work was to apply them to study the colonisation and persistence capabilities of the bovine pathogens Mycoplasma mycoides subsp. mycoides (Mmm) and Mycoplasma bovis in bovine cells. Methods. Mini-transposons affording high-level expression of GFP2, mCherry, mKO2 and mNeonGreen were used to mark Mmm and M. bovis strains. The resulting fluorescent mutants were characterised by epifluorescence microscopy and fluorimetry and the sites of transposon insertion were identified by sequencing. Interactions of mNeonGreen mutants with bovine cells were analysed using flow cytometry and confocal microscopy. Results: The production, selection and characterisation of fluorescent clones were straightforward and compatible with the production of fluorescent mutant banks. M. bovis presented much higher adhesion, invasion and proliferation capacities than Mmm in culture with non-phagocytic cells and showed increased resistance to elimination by macrophages. Conclusion: The remarkable differences in the invasion and persistence capabilities of Mmm and M. bovis observed here are in agreement with the pathogenesis of the infections caused by these mycoplasmas. mNeonGreen fluorescent mutants have proven extremely useful for analysis of mycoplasma-host cell interactions. Furthermore, the fluorescence expression tools used in this study offer innumerable perspectives for the functional analysis of a wide range of mycoplasma species both in vitro and in vivo.

The tropical bont tick, Amblyomma variegatum, is a tick species of veterinary importance and is considered as one of major pest of ruminants in Africa and in the Caribbean. It causes direct skin lesions, transmits heartwater, and reactivates bovine dermatophilosis. Tick saliva is reported to affect overall host responses through immunomodulatory and anti-inflammatory molecules, among other bioactive molecules. The general objective of this study was to better understand the role of saliva in interaction between the Amblyomma tick and the host using cellular biology approaches and proteomics, and to discuss its impact on disease transmission and/or activation. We first focused on the immuno-modulating effects of semi-fed A. variegatum female saliva on bovine peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages in vitro. We analyzed its immuno-suppressive properties by measuring the effect of saliva on PBMC proliferation, and observed a significant decrease in ConA-stimulated PBMC lymphoproliferation. We then studied the effect of saliva on bovine macrophages using flow cytometry to analyze the expression of MHC-II and co-stimulation molecules (CD40, CD80, and CD86) and by measuring the production of nitric oxide (NO) and pro-or anti-inflammatory cytokines. We observed a significant decrease in the expression of MHC-II, CD40, and CD80 molecules, associated with decreased levels of IL-12-p40 and TNF-alpha and increased level of IL-10, which could explain the saliva-induced modulation of NO. To elucidate these immunomodulatory effects, crude saliva proteins were analyzed using proteomics with an Orbitrap Elite mass spectrometer. Among the 336 proteins identified in A. variegatum saliva, we evidenced bioactive molecules exhibiting anti-inflammatory, immuno-modulatory, and anti-oxidant properties (e.g., serpins, phospholipases A2, heme lipoprotein). We also characterized an intriguing ubiquitination complex that could be involved in saliva-induced immune modulation of the host. We propose a model for the interaction between A. variegatum saliva and host immune cells that could have an effect during tick feeding by favoring pathogen dissemination or activation by reducing the efficiency of host immune response to the corresponding tick-borne diseases.

Whole genome expression in diploid and doubled diploid citrus seedlings in intra and interspecific contexts.. International Citrus Congress.

The tropical bont tick, Amblyomma variegatum, is a major pest of ruminants, causing direct skin lesions, vectoring the obligate intracellular ricketsiale Ehrlichia ruminantium, the causative agent of heartwater, and being able to reactivate dermatophilosis. In general, tick salivary proteins are the result of host blood feeding adaptation and are known to contain inhibitors of blood clotting, platelet aggregation and angiogenesis, as well as vasodilators and immunomodulators. The general objective of this study was to better understand the role of the saliva in the tick interaction with the host but also in pathogen transmission or diseases activation. We therefore first analysed the immunomodulating properties of semi-fed A. variegatum female saliva on bovine peripheral blood mononuclear cells (PBMCs) in vitro. Flow cytometry and cytokine ELISAs have been used to evaluate the saliva impact on PBMCs. We focused on immunosuppressive properties by analysing both lymphocytes proliferation and monocyte-derived macrophages phenotype characterised by inhibition or induction of stimulatory and co-stimulatory molecules such as MHC-II, CD40, CD80 or CD86, and production of pro- or anti-inflammatory cytokines such as interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p40, and tumor necrosis factor (TNF)-α. Moreover, a proteomics exhaustive molecular characterisation of A. variegatum saliva was performed to cluster data set according to the evidenced immunomodulatory properties, and allowed refined characterisation of Amblyomma sialome. Bioinformatics functional analysis of tick saliva highlighted molecular determinants that could, at least in part, explain the biological effects observed on bovine PBMCs. Our results bring new insights for a better understanding of tick-ruminant interactions, and open new perspectives to develop integrative strategies to interfere with the infectious pathoimmunological process of such tick-borne infections.

African swine fever (ASF) is a lethal haemorrhagic swine disease with disastrous consequences for pig production. Ornithodoros ticks such as O. erraticus in Spain and Portugal, and O. moubata sensu lato in eastern and southern Africa are able to maintain and transmit the virus, and are competent vectors and reservoirs for ASFV (1). These ticks can maintain ASFV for years and transmit the virus through different routes such as transovarial and/or sexual transmission from tick to tick, as well as horizontal transmission to suids via contaminated saliva or coxal fluid. Vector competence in virus transmission can be related to extrinsic or intrinsic factors. Here we present the intrinsic effect of O. porcinus tick salivary gland extract on the African swine fever virus infection in domestic pig. During the early stage of pig infection with ASFV, mononuclear phagocytic cells are the main targets for viral replication (2). Tick saliva has been shown to modulate the host physiological and immunological responses during feeding on skin, thus affecting viral infection. To better understand the interaction between soft tick, ASFV and pig at the bite location and the possible influence of tick saliva on pig infection by ASFV, salivary gland extract (SGE) of Ornithodoros porcinus, co-inoculated or not with ASFV, was used for intradermal auricular inoculation. Our observations focused both on the pig systemic immune response and on pig skin inflammation and cellular modulation (especially LCs and macrophages) at the tick bite location. Unlike previous studies, the assessment of such immune modulations was conducted on the natural host, domestic pigs, and a highly adapted tick-virus association with O. porcinus ticks collected from Madagascar and a Madagascan ASF virus strain. Our results showed that, after the virus triggered the disease, pigs inoculated with virus and SGE presented greater hyperthermia than pigs inoculated with virus alone. The density of Langerhans cells was modulated at the tick bite or inoculation site, either through recruitment by ASFV or inhibition by SGE. Additionally, SGE and virus induced macrophage recruitment each. This effect was enhanced when they were co-inoculated. Finally, the co-inoculation of SGE and virus delayed the early local spread of virus to the first lymph node after the inoculation site. This study has shown that the effect of SGE was powerful enough to be quantified in pig, both on the systemic and local immune response (3). We believe this model should be developed with infected tick and could improve knowledge of both tick vector competence and tick saliva immunomodulation.

Amblyomma variegatum, the tropical bont tick, is a species of veterinary importance being considered as one of major pest of ruminants in Africa and in the Caribbean. It is associated with severe dermatophilosis and transmits the rickettsiale Ehrlichia ruminantium, the agent of heartwater, to ruminants. Tick's saliva contains a complex mixture of bioactive molecules including proteins that modulate host responses to ensure successful blood feeding. The limited amount of saliva that can be obtained from ticks has hampered characterisation of salivary proteins using traditional protein chemistry, but recent advances in proteomics (especially mass spectrometry and bioinformatics) provide new tools to finely investigate tick sialomes. The general objective of this study was to better understand the role of A. variegatum saliva in tick/host/pathogen interactions. Salivation of semi-fed females was induced after detachment by injection of pilocarpin. Crude saliva proteins were pre-fractioned by SDS-PAGE and generated peptides were analysed online by nano-flow Ultimate 3000 HPLC–nanoelectrospray ionization using an Orbitrap Elite mass spectrometer. Raw data analysis was performed using the MaxQuant software and proteins identified according to UniProt Knowledgebase. Among the 336 proteins identified in A. variegatum saliva, a bioinformatics functional analysis of molecular determinants highlighted immunomodulators (e.g. heme lipoprotein, serpins, phospholipase A2 or ubiquitin) that could, at least in part, explain the biological effects observed on bovine immune cells in vitro. Our results bring new insights for a better understanding of tick-ruminant interactions at the molecular level, and pave the way towards integrative strategies to interfere with both the immunomosuppressive and infectious processes in corresponding tick-borne diseases.

Macrophages are major cells of the innate immunity. Macrophages derived from monocyte precursors undergo specific differentiation depending on the local tissue environment. Similar to the T helper type 1 and T helper type 2 polarization, two distinct states of polarized activation for macrophages have been defined in mouse and humans: the classically activated (M1) macrophage and the alternatively activated (M2) macrophage phenotypes. On the other hand, these different patterns of macrophage differentiation drive adaptive responses during the stages of infection, hence restraining inflammation and favoring tissue repair. In vitro generation and characterization of these subpopulations are essential to perform relevant studies understanding the host-pathogen interactions. Currently, several in vitro differentiation and polarization protocols are used to induce M1 or M2 mouse and human macrophages but none have been developed for the bovine species. We developed a method for in vitro differentiation and polarization of bovine macrophages using GM-CSF and IFNg to induce M1, and IL-4 to induce M2 phenotype. We characterized M1/M2 macrophages by specific morphology, production of cytokines (IL-10, IL-12, TNF-a), NO production, and phenotypic markers such as CD206 and TLR2.

Effect of increasing saliva concentrations on total cell viability in the reverse transmigration transwell protocol. The viability of SIRP1-α+ monocyte-derived cells collected from monocyte/BAEC cultures grown on collagen-coated transwells according to the reverse-transmigration protocol was assessed following 48 h of incubation with zymosan and saliva at amounts varying from 0 to 55 μg/mL. (A) Relative number of dead SIRP1-α+ cells in the bottom of the transwell. (B) Relative number of dead SIRP1-α+ cells in the top of the transwell.

Morphology of infiltrated leukocytes next to the suture position within skin biopsies. Representative hematoxylin and eosin staining (H&E) (10X & 40X) of histological sections of skin biopsies close to the localization of the surgical monofilament coinjected with control solution (t = 24 h). Some infiltrated cells displayed a typical morphology with large round mononuclear cells characteristic of monocytes or macrophages and some display a multilobed nuclei characteristic of neutrophils (Zoom 40x, white arrow).

How tick bite prevent dendritic cells from triggering an appropriate immune response? Development of an in vitro model for the study of dendritic cell immunosuppression. 9. Colloque du réseau français d’Immunologie des Animaux Domestiques (IAD)

A better understanding of the basis of protective immunity and immunopathologic reactions associated with contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides small colony (MmmSC), is needed to develop better vaccines. Here we report on the characterization of T-cell memory responses associated with protective immunity in cattle and their use thereof to identify immuno-active proteins of MmmSC. Both effector memory (em) and central memory (cm)-like MmmSC specific CD4+ T cells were present ex vivo and capable of proliferation and IFN-[gamma] production upon re-stimulation in vitro with inactivated MmmSC. It has been shown in mice and humans that priming of T(cm) is required for long-lived protective immunity, which is also an essential requirement of any improved vaccine against CBPP. Pre-sensitized T-cells were used to screen recombinant proteins of MmmSC produced as his-tagged proteins_and purified by chromatography. Three of them were found to trigger the proliferation of both CD4+ T(em) and T(cm) and induce the production of IFN-[gamma]: lipoprotein A (LppA), a glucose-specific component of the PTS system (ptsG) and to a lesser extent a substrate-binding component of the ABC transporter (ABC). In contrast, although 1ppQ had no effect on its own, it strongly inhibited the proliferation of CD4 in response to MmmSC by a mechanism apparently independent of direct cytotoxicity. In conclusion, the T-cell reagents and methodology described in this study will help identify immuno-protective antigens of MmmSC that have potential for the development of a subunit vaccine against CBPP. In addition, these cellular tools can also be used to screen for immuno-inhibitory proteins whose corresponding genes could be targets for deletion/inactivation in the aim of developing improved attenuated vaccines. (Texte intégral)

La péripneumonie contagieuse bovine est une maladie respiratoire due à Mycoplasma mycoides subsp. mycoides SC (MmmSC) et caractérisée par une forte réaction inflammatoire. La vaccination actuelle, basée sur l'utilisation d'une souche atténuée (T1/44), présente une efficacité modérée et une virulence résiduelle. L'amélioration des vaccins serait facilitée par une meilleure connaissance des mécanismes immunologiques développés lors de la maladie ou de la vaccination. Les cellules présentatrices d'antigènes (CPA), cellules dendritiques et macrophages, sont les premières cellules à entrer en action pour la mise en place de la réponse immunitaire. Cette étude porte donc sur l'analyse des effets de différentes souches de MmmSC sur les CPA. Nous avons mesuré l'induction des molécules de maturation ou d'activation des CPA ainsi que la production de cytokines inflammatoires lors d'un contact in vitro avec des souches de MmmSC atténuées ou virulentes. Les résultats montrent une corrélation entre la virulence connue de la souche et la réponse de ces CPA, suggérant des répercussions importantes sur la réponse immunitaire qui sera ensuite mise en place par l'animal. (Texte intégral)

The pivotal role of central memory (cm) CD4 in long term protective immunity has recently been confirmed in a murine model of leishmaniasis (Zaph et al, Nature Medicine 2004). Upon secondary infection, T(cm) cells proliferate in draining lymph nodes before differentiating into tissue-homing effector T cells and mediating protection. Because of their capacity to persist after antigen clearance, T (cm) cells are privileged targets for vaccine development. Here we report on the characterization of CD4+ T(cm) elicited in cattle during contagious bovine pleuropneumonia (CBPP). CFSE and flow cytometric analysis of antigen recall responses in draining lymph nodes of cattle developing chronic CBPP indicated that both CD62L-CD45RO+CD4+ and CD62L+CD45RO+CD4+ T cells proliferate to a similar extent during the first 5 days of stimulation in vitro. However, after 7 days, CD62L+ cells that have proliferated (CFSEdim) represent up to 80% of total CD4 which coincides with a substantial increase in absolute numbers of CD4. IFN-[gamma] secretion (ELISA) was only marginally affected by the enrichment of cultures for CD62L+ CD4. Finally, pathogen-induced IFN-[gamma] synthesis was detected primarily in CD62L-CD4+ T cells as early as day 2 by direct intracellular staining. High CD62L expression and proliferation but low cytokine production in response to Ag are characteristics of T(cm) cells in mice. In summary, the approach described here allows direct characterization of pathogen-derived bovine CD4+T(cm) in uncloned populations. This has direct applications for the development of vaccines inducing long-term protection against CBPP and will also provide better insight into the regulation of CD4+ T(cm) during infection in cattle. (Texte intégral)

La compréhension des mécanismes immunitaires impliqués dans la résistance et dans la pathogénie de la PPCB, maladie bactérienne classée dans la liste A de l'OIE, est nécessaire au développement de vaccins recombinants. Chez les bovins contrôlant la maladie (forme chronique de la PPCB) une forte prolifération des lymphocytes provenant des ganglions pulmonaires médiastinaux est observée in vitro en réponse aux mycoplasmes inactivés. Cette réponse est due principalement aux CD4 avec un rôle négligeable pour les CD8 et les lymphocytes [gamma][delta]TCR+. La population de CD4 répondeurs est composée de 2 sous-populations: CD25+CD45RO+62L- et CD25+CD45RO+62L+ mais plus de 80% des CD4 qui prolifèrent sont CD62L+. Par contre, chez les animaux qui meurent, il n'y a pas de prolifération des CD4 mais une activation (blastogenèse et expression du rIL-2) de lymphocytes majoritairement CD62L-. De même, en présence de mycoplasmes vivants, les CD4 d'animaux contrôlant la maladie s'activent mais ne prolifèrent pas. Enfin, une réponse IFN-[gamma] est détectée chez tous les animaux quel que soit l'issue de la maladie. Ces résultats suggèrent que les CD4 de la mémoire centrale pourraient être impliqués dans une réponse anamnestique protectrice contre la PPCB. De plus, l'activation des CD4 en l'absence de prolifération chez les animaux qui meurent de PPCB ainsi qu'en présence de MmmSC vivants in vitro suggère l'existence d'un mécanisme immuno-inhibiteur développé par ce pathogène. (Texte intégral)

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