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.

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.

Development of a constant pressure perfused ex vivo model of the equine larynx.

Distal axonopathy is seen in a broad range of species including equine patients. In horses, this degenerative disorder of the recurrent laryngeal nerve is described as recurrent laryngeal neuropathy (RLN). The dysfunctional innervation of the cricoarytenoideus dorsalis muscle (CAD) leads to a loss of performance in affected horses. In general, ex vivo models of the larynx are rare and for equine patients, just one short report is available. To allow for testing new therapy approaches in an isolated organ model, we examined equine larynges in a constant pressure perfused setup. In order to check the vitality and functionality of the isolated larynx, the vessels´ reaction to norepinephrine (NE) and sodium nitroprusside (NP) as vasoactive agents was tested. Additionally, the contractility of the CAD was checked via electrical stimulation. To determine the extent of hypoxic alterations, lactate dehydrogenase (LDH) and lactate were measured and an immunofluorescent analysis of hypoxia-inducible factor (HIF-1α), a key transcription factor in hypoxia, was performed. For this, a hypoxia-induced cell culture for HIF-1α was developed. The application of NE led to an expected vasoconstriction while NP caused the expected vasodilation. During a perfusion period of 352 ±20.78 min, LDH values were in the reference range and lactate values slightly exceeded the reference range at the end of the perfusion. HIF-1α nuclear translocation could reliably be detected in the hypoxia-induced cell cultures, but not in sections of the perfused CAD. With the approach presented here, a solid basis for perfusing equine larynges was established and may serve as a tool for further investigations of equine larynx disorders as well as a transferrable model for other species.

Detection of subtilisin 3 and 6 in skin biopsies of cattle with clinically manifested bovine ringworm.

Trichophyton (T.) verrucosum is a highly pathogenic dermatophyte causing zoonotic bovine ringworm that is transmissible to humans. The virulence factors subtilisin (Sub)3 and Sub6 are discussed to contribute to disease manifestation but no protein expression study is available for T. verrucosum. We used customized antibodies (against Trichophyton-species, Sub3 and Sub6) to examine skin biopsies of infected cattle via immunofluorescence stainings. Both virulence factors Sub3 and 6 were solely expressed by conidia and not only found in epidermal but also in dermal and hair structures. The anti-T-antibody reliably detected the fungus and proved more sensitive compared to histological stains. LAY SUMMARY: We examined the zoonotic dermatophyte Trichophyton (T.) verrucosum in bovine skin and studied two important virulence factors called subtilisin (Sub)3 and Sub6 that T. verrucosum produces and secretes using immunolabeling.

Horse hair follicles: A novel dermal stem cell source for equine regenerative medicine.

The easily accessible niche represented by skin and its appendages may serve as a promising source to complement modern regenerative medicine for horses. In humans and in animal models for human medicine, the hair follicle and its stem cell niches are well characterized. Since literature in this field of equine research is scarce, we sought to analyze cells of the dermal stem cell niche of the equine hair follicle morphologically and for a subset of markers useful for cell characterization via immunolabeling. We cultured equine forelock skin explants to obtain cultures with cells migrating from the hair follicles. Isolation of cells revealed typical fibroblast morphology with a strong tendency to aggregate and form spheroids. For immunofluorescent characterization of primary isolations, we tested an antibody panel consisting of lineage makers for the dermal compartment of the hair follicle, markers associated with an undifferentiated cell status and markers for epithelial cell types as negative controls. All antibodies used were also tested on equine skin sections. The isolated cells displayed clear profiles of dermal and undifferentiated cells. To substantiate our findings, we tested our primary isolations for established equine multipotent mesenchymal stromal cell antigen expression markers in flow cytometry experiments yielding strong convergence. The data presented here provide insights to a stem cell source in horses almost unnoticed to date. The basic investigations of the equine dermal hair follicle stem cell niche confirm the expression of standard markers used in other species and lay the foundation for future studies on this easily available adult stem cell source. © 2017 International Society for Advancement of Cytometry.