A Trifecta of New Insights into Ovine Footrot for Infection Drivers, Immune Response and Host Pathogen Interactions.

Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industry’s biggest welfare problems. The complex aetiology of footrot makes in-situ or in-vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved, how they may interact with the host and ultimately providing a way to identify targets for future hypotheses driven investigative work. Here we present the first combined global analysis of the bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intra tissue and surface bacterial populations and the most abundant bacterial transcriptome were analysed, demonstrating footrot affected skin has a reduced diversity and increased abundances of, not only the causative bacteria Dichelobacter nodosus, but other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica. Host transcriptomics reveals a suppression of biological processes relating to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot affected interdigital skin comparted to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome which could lead to the identification of future therapeutic targets

Phenotypic and functional analysis of monocyte populations in cattle peripheral blood identifies a subset with high endocytic and allogeneic T-cell stimulatory capacity

International audienceAbstractCirculating monocytes in several mammalian species can be subdivided into functionally distinct subpopulations based on differential expression of surface molecules. We confirm that bovine monocytes express CD172a and MHC class II with two distinct populations of CD14+CD16low/-CD163+ and CD14−CD16++CD163low- cells, and a more diffuse population of CD14+CD16+CD163+ cells. In contrast, ovine monocytes consisted of only a major CD14+CD16+ subset and a very low percentage of CD14−CD16++cells. The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115. However, further mRNA analyses revealed that the CD14−CD16++ monocytes were CX3CR1highCCR2low whereas the major CD14+ subset was CX3CR1lowCCR2high. The former were positive for CD1b and had lower levels of CD11b and CD86 than the CD14+ monocytes. The more diffuse CD14+CD16+ population generally expressed intermediate levels of these molecules. All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1β, with the CD16++ subset expressing higher levels of IL-12 and lower levels of IL-10. The CD14−CD16++ cells were more endocytic and induced greater allogeneic T cell responses compared to the other monocyte populations. Taken together the data show both similarities and differences between the classical, intermediate and non-classical definitions of monocytes as described for other mammalian species, with additional potential subpopulations. Further functional analyses of these monocyte populations may help explain inter-animal and inter-species variations to infection, inflammation and vaccination in ruminant livestock

New challenges for vaccination to prevent chlamydial abortion in sheep

Ovine enzootic abortion (OEA) is caused by the obligate intracellular Gram-negative bacterium Chlamydia abortus. OEA remains a common cause of infectious abortion in many sheep-rearing countries despite the existence of commercially available vaccines that protect against the disease. There are a number of confounding factors that influence the uptake and use of these vaccines, which includes an inability to discriminate between infected and vaccinated animals (DIVA) using conventional serological diagnostic techniques. This suggests that the immunity elicited by current vaccines is similar to that observed in convalescent, immune sheep that have experienced OEA. The existence of these vaccines provides an opportunity to understand how protection against OEA is elicited and also to understand why vaccines can occasionally appear to fail, as has been reported recently for OEA. Interferon-gamma (IFN-gamma), the cytokine that classically defines Th1-type adaptive immunity, is a strong correlate of protection against OEA in sheep and has been shown to inhibit the growth of C. abortus in vitro. Humoral immunity to C. abortus is observed in both vaccinated and naturally infected sheep, but antibody responses tend to be used more as diagnostic markers than targets for strategic vaccine design. A future successful DIVA vaccine against OEA should aim to elicit the immunological correlate of protection (IFN-gamma) concomitantly with an antibody profile that is distinct from that of the natural infection. Such an approach requires careful selection of protective components of C. abortus combined with an effective delivery system that elicits IFN-gamma-producing CD4+ve memory T cells

Hemagglutinin protein of Peste des Petits Ruminants virus (PPRV) activates the innate immune response via Toll-like receptor 2 signaling

Centro de Investigación en Sanidad Animal (CISA)The toll-like receptor (TLR) family comprises both cell-surface and intracellular receptors that recognize different types of pathogen-associated molecular patterns (PAMPs) leading to the production of pro-inflammatory cytokines and subsequent development of adaptive immunity. TLR2 is a cell-surface receptor initially thought to act as a bacterial sentinel but also shown to recognize a number of viral glycoproteins. In this study, we sought to characterize the role of TLR2 in the activation of the immune response by peste des petits ruminants virus (PPRV), a morbillivirus of the Paramixoviridae family that causes an acute, highly contagious disease in goats and sheep. Using human embryonic kidney (HEK) 293 cells stably expressing human (h)TLR2 but lacking any other TLR, we found that PPRV induces IL-8 production in a dose-dependent manner. That activation is only observed in cells expressing hTLR2 and is greatly reduced when the receptor is blocked by pretreatment with specific antibody. We identified hemagglutinin (H) as the viral protein responsible of TLR2 activation by performing the same assays with purified recombinant mammalian-expressed H protein. Exogenous addition of recombinant H protein to cell culture induces high levels of interleukin (IL)-8 only in TLR2-expressing cells. Moreover, H engagement on TLR2 in the monocytic cell line THP-1 activates extracellular-signal-regulated kinase (ERK) signaling. Stimulation of primary ovine dendritic cells with either inactivated PPRV or purified recombinant H protein results in transcription of pro-inflammatory cytokines and the secretion of the Th1-polarizing cytokine IL-12. The role of these host immune mechanisms in the control of PPR is discussed.This work was funded by grants AGL2015-64290R and RTI2018-094616-B-100 from the Ministerio de Ciencia (Spain); grant VetBioNet INFRAIA-731014 from the EU-H2020 and grant S2018/BAA-4370-PLATESA2 from Comunidad de Madrid (Fondo Europeo de Desarrollo Regional, FEDER). MA was funded by an FPI grant (BES-2013-066406). NS was the holder of a fellowship from OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems.Peer reviewed15 Pág

Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens

Ovine footrot is a complex multifactorial infectious disease, causing lameness in sheep with major welfare and economic consequences. Dichelobacter nodosus is the main causative bacterium; however, footrot is a polymicrobial disease with Fusobacterium necrophorum, Mycoplasma fermentans and Porphyromonas asaccharolytica also associated. There is limited understanding of the host response involved. The proinflammatory mediators, interleukin (IL)-1β and C-X-C Motif Chemokine Ligand 8 (CXCL8), have been shown to play a role in the early response to D. nodosus in dermal fibroblasts and interdigital skin explant models. To further understand the response of ovine skin to bacterial stimulation, and to build an understanding of the role of the cytokines and chemokines identified, primary ovine interdigital fibroblasts and keratinocytes were isolated, cultured and stimulated. The expression of mRNA and protein release of CXCL8 and IL-1β were measured after stimulation with LPS, D. nodosus or F. necrophorum, which resulted in increased transcript levels of IL-1β and CXCL8 in the M. fermentans-free cells. However, only an increase in the CXCL8 protein release was observed. No IL-1β protein release was detected, despite increases in IL-1β mRNA, suggesting the signal for intracellular pre-IL-1β processing may be lacking when culturing primary cells in isolation. The keratinocytes and fibroblasts naturally infected with M. fermentans showed little response to the LPS, a range of D. nodosus preparations or heat-inactivated F. necrophorum. Primary single cell culture models complement ex vivo organ culture models to study different aspects of the host response to D. nodosus. The ovine keratinocytes and fibroblasts infected with M. fermentans had a reduced response to the experimental bacterial stimulation. However, in the case of footrot where Mycoplasma spp. are associated with diseased feet, this natural infection gives important insights into the impact of multiple pathogens on the host response

Immunological homeostasis at the ovine placenta may reflect the degree of maternal foetal interaction

Successful mammalian pregnancies are a result of complex physiological, endocrinological and immunological processes that combine to create an environment where the mother is tolerant to the semi-allogeneic fetus. Our knowledge of the mechanisms thatcontribute to maternal tolerance is derived mainly from human and murine studies of haemochorial placentation. However, as thisis the most invasive type of placentation it cannot be assumed that identical mechanisms apply to the less invasive epitheliochorialplacentation found in other species such as ruminants. Here, we examine three features associated with reproductive immuneregulation in a transformed ovine trophoblast cell line and ex-vivo ovine reproductive tissues collected at term, namely: majorhistocompatibility complex (MHC) expression, Indoleamine 2, 3 dioxygenase-1 (IDO-1) expression and Natural Killer (NK) cellinfiltration. High levels of MHC class I protein expression were detected at the surface of the trophoblast cell line using a pan-MHCclass I specific monoclonal antibody. The majority of MHC class I transcripts isolated from the cell line clustered with classical MHCalleles. Transcriptional analysis of placental tissues identified only classical MHC class I transcripts. We found no evidence ofconstitutive transcription of IDO-1 in either the trophoblast cell line or placental tissues. Ex-vivo tissues collected from thematerno-fetal interface were negative for cells expressing NKp46/NCR1. Collectively, these observations suggest that the relatively non-invasive synepitheliochorial placentation found in sheep has a more limited requirement for local immunoregulation compared to the more invasive haemochorial placentation of primates and rodents

EU:n tietosuoja-asetus 2016/679 (GDPR) ohjelmistoyrityksessä

Insinöörityössä tavoitteena oli tutustua EU:n tietosuoja-asetukseen 2016/679 sekä selvittää, millaisia vaatimuksia se aiheuttaa työn tilanneessa ohjelmistoyrityksessä ja suunnitella ja toteuttaa osa muutostöistä. Työn tavoitteet suunniteltiin yhdessä tilaajayrityksen kanssa. Työn aikana selvitettiin tietosuoja-asetuksen sisältöä ja sitä, miltä osin se kohdistuu yritykseen ja kuinka asetuksen ehdot käytännössä toteutettaisiin. Selvityksen aikana pyrittiin muodostamaan selkeä kuva siitä, missä asetuksen määrittämissä rooleissa yritys toimii ja mitkä niistä muodostuvat vastuut ovat. Pyrittiin myös muodostamaan käsitys yrityksen asiakkaiden tulevista tarpeista, joiden perusteella yrityksen ohjelmistotuote voitaisiin valmistella vastaamaan asetuksen vaatimuksia. Selvityksen perusteella yritykselle tehtiin nykytila-analyysi, jolla pyrittiin dokumentoimaan yrityksen tietoturvakäytännöt ja määrittämään, mitä puutteita yrityksen hallinnollisissa ja teknisissä tietoturva ja -suojakäytännöissä on. Analyysin perusteella priorisoitiin kehitysprojekteja yrityksen toimintatapojen ja ohjelmistotuotteen parantamiseen. Työn tuloksena luotiin uusia ominaisuuksia työn tilaajan ohjelmistotuotteeseen. Näitä olivat rekisteröityjen suostumuksen pyyntö ja tiedotus heidän oikeuksistaan, oikeus tulla unohdetuksi ja tietojen siirto. Lisäksi tarkastettiin ja laajennettiin tilaajayrityksen tietoturvasuunnitelmaa, tietoturvaesitettä ja tietoturvaohjeistusta yrityksen henkilöstölle ja asiakkaille.Aim of this thesis was to get acquainted with EU data protection regulation 2016/679, find out which of its requirements are notable in a software company that ordered the thesis and implement a part of the changes needed. Goals of the thesis were planned together with the company. Contents of the Data protection regulation were studied and a viewed from the roles the company is in as a data controller and a data processor. Company’s clients were studied to predict what requirements they will have for the company’s main software product soon. Based on the theoretical study, a current state analysis was done to document company’s managerial and technical data protection practices and find their flaws. Based on the analysis a few development projects were formed and prioritized especially focusing on the company’s main software product. Thesis’ results were data protection documentation and guidelines for the company’s personnel and clients and software feature implementations which enable data subjects’ rights to be forgotten, to transfer their data and to gather their consent and inform them of their rights