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 largest welfare problems. The complex etiology of footrot makes in situ or in vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved and how they may interact with the host, ultimately providing a way to identify targets for future hypothesis-driven investigative work. Here, we present the first combined global analysis of bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intratissue and surface bacterial populations and the most abundant bacterial transcriptomes were analyzed, demonstrating that footrot-affected skin has reduced diversity and increased abundances of not only the causative bacterium Dichelobacter nodosus but also other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica. Host transcriptomics reveals the suppression of biological processes related 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 compared 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.

Defining immune correlates during latent and active chlamydial infection in sheep.

Ovine enzootic abortion (OEA) caused by the obligate intracellular bacterial pathogen Chlamydia abortus (C. abortus), is an endemic disease in most sheep-rearing countries worldwide. Following infection, C. abortus establishes a complex host-pathogen interaction with a latent phase in non-pregnant sheep followed by an active disease phase in the placenta during pregnancy leading to OEA. Improved knowledge of the host-pathogen interactions at these different phases of disease will accelerate the development of new diagnostic tests and vaccines to control OEA. Current evidence indicates that cellular immunity is essential for controlling C. abortus infection. We have previously described a model of mucosal (intranasal) infection of non-pregnant sheep with C. abortus that replicates the latent and active phases of OEA. We have investigated antigen-specific recall responses of peripheral blood mononuclear cells (PBMC) in sheep infected with C. abortus via the intranasal route to determine how these change during the latent and active phases of disease. By analysing cytokines associated with the major CD4+ve Thelper (Th) cell subsets (Interferon-gamma (IFN-γ)/Th1; Interleukin (IL)-4/Th2; IL-17A/Th17; IL-10/Tregulatory), we show that there is selective activation of PBMC producing IFN-γ and/or IL-10 during the latent phase following infection. These cytokines are also elevated during the active disease phase and while they are produced by sheep that are protected from OEA, they are also produced by sheep that abort, highlighting the difficulties in finding specific cellular immunological correlates of protection for complex intracellular pathogens.

Enhancing the toolbox to study IL-17A in cattle and sheep.

The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.

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

Circulating 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(+)CD16(low/-)CD163(+) and CD14(-)CD16(++)CD163(low-) 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 CX3CR1(high)CCR2(low) whereas the major CD14(+) subset was CX3CR1(low)CCR2(high). 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)-1ß, 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.

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)-1ß 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-1ß were measured after stimulation with LPS, D. nodosus or F. necrophorum, which resulted in increased transcript levels of IL-1ß and CXCL8 in the M. fermentans-free cells. However, only an increase in the CXCL8 protein release was observed. No IL-1ß protein release was detected, despite increases in IL-1ß mRNA, suggesting the signal for intracellular pre-IL-1ß 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.

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

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.

The Veterinary Immunological Toolbox: Past, Present, and Future.

It is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. This creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. There have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. Various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. While short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. We review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the International Union of Immunological Societies (IUIS) Veterinary Immunology Committee (VIC) Immune Toolkit Workshop at the 12th International Veterinary Immunology Symposium (IVIS) in Seattle, USA, 16-19 August 2019. The future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for One Health initiatives.

Immunological Homeostasis at the Ovine Placenta May Reflect the Degree of Maternal Fetal 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 that contribute to maternal tolerance is derived mainly from human and murine studies of haemochorial placentation. However, as this is the most invasive type of placentation it cannot be assumed that identical mechanisms apply to the less invasive epitheliochorial placentation found in other species such as ruminants. Here, we examine three features associated with reproductive immune regulation in a transformed ovine trophoblast cell line and ex-vivo ovine reproductive tissues collected at term, namely: major histocompatibility complex (MHC) expression, Indoleamine 2,3 dioxygenase-1 (IDO-1) expression, and Natural Killer (NK) cell infiltration. High levels of MHC class I protein expression were detected at the surface of the trophoblast cell line using a pan-MHC class I specific monoclonal antibody. The majority of MHC class I transcripts isolated from the cell line clustered with classical MHC alleles. Transcriptional analysis of placental tissues identified only classical MHC class I transcripts. We found no evidence of constitutive transcription of IDO-1 in either the trophoblast cell line or placental tissues. Ex-vivo tissues collected from the materno-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.

Exploiting ovine immunology to improve the relevance of biomedical models.

Animal models of human disease are important tools in many areas of biomedicine; for example, in infectious disease research and in the development of novel drugs and medical devices. Most studies involving animals use rodents, in particular congenic mice, due to the availability of a wide number of strains and the ease with which they can be genetically manipulated. The use of mouse models has led to major advances in many fields of research, in particular in immunology but despite these advances, no animal model can exactly reproduce all the features of human disease. It is increasingly becoming recognised that in many circumstances mice do not provide the best model and that alternative species may be more appropriate. Here, we describe the relative merits of sheep as biomedical models for human physiology and disease in comparison to mice, with a particular focus on reproductive and respiratory pathogens.

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-γ), 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-γ) 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-γ-producing CD4+ve memory T cells.

Identification of CD4+CD25 high Foxp3+ T cells in ovine peripheral blood.

Regulatory T cells (Treg) are an important subset of T lymphocytes which play a key role in maintaining peripheral immunological tolerance. The most studied subpopulation of Treg in mice and humans are natural Treg, which differentiate in the thymus and are identified by expression of CD4, high levels of IL-2Rα (CD25), and forkhead box P3 (Foxp3), a transcription factor intimately associated with Treg function. We and others have previously identified Foxp3(+) T cells in ovine tissue, suggesting that Treg exist in this species. However, the existence of putative natural Treg in sheep, as identified by co-expression of CD4, CD25 and Foxp3, has yet to be determined. In this study we demonstrate that the anti-rat/mouse Foxp3 monoclonal antibody FJK-16s cross-reacts with ovine Foxp3. Using a transfected Chinese hamster ovary cell line that constitutively expresses recombinant ovine Foxp3 as a positive control, we have developed a sensitive triple-labelling flow cytometry protocol to simultaneously label CD4, CD25 and Foxp3. We demonstrate that Foxp3(+) T lymphocytes exist in ovine peripheral blood, and that the majority of Foxp3 expression occurs within the CD4(+)CD25(hi) population. These results are consistent with those seen in other mammalian species and indicate that putative natural Treg exist in sheep.