Search

The purpose of the study was to determine the histo-morphological effects on villous synovium after synovectomy using two different motorized synovial resectors and two different intensities ex-vivo. Thirty-three (n = 33) equine metacarpophalangeal/metatarsophalangeal joints were used. Synovectomy was performed along the dorsomedial/dorsolateral synovium (n = 66) using two motorized synovial resectors (aggressive full radius resector, AFRR, used at two intensities: single treatment, n = 24 vs. triple treatment, n = 21 vs. aggressive meniscus side cutter, AMSC, n = 21). Arthroscopic images were evaluated blindly for resector type and intensity. Histological images were evaluated descriptive for synovial morphology and the extent of tissue loss using a microscopic scale. Scanning electron microscopy described the synovial morphology. The synovectomized areas were specific for each resector used and distinguishable from arthroscopic images. The AFRR demonstrated a clear demarcation between treated and non-treated areas and removed the stratum synoviale completely including parts of the underlying stratum fibrosum. In contrast, the AMSC showed less clear demarcation, villous scaffolds and no involvement of the stratum fibrosum. Triple intense treated AFFR samples resulted in significantly deeper lesions compared to single treatments (p = 0.037) but could not be distinguished on arthroscopic images. The morphological effects on villous synovium differ according to the resector type used. The extent of synovial tissue loss cannot be estimated from arthroscopic images but histologically. The type and use of motorized synovial resector determines the morphological alterations of the treated synovium. Arthroscopic control is considered unsuitable to control synovectomy depth., Competing Interests: Declaration of Competing Interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective:  The aim of this study was to evaluate differences in paraspinal musculature between dogs with and without atlantoaxial instability (AAI) using computed tomography scans., Study Design:  Retrospective multicentre study evaluating transverse reconstructed computed tomography scans of 83 small breed dogs (34 with and 49 without AAI) for the cross-sectional paraspinal musculature area at three levels (Occiput/C1, mid-C1, mid-C2). Ratio of moments, dorsal-to-ventral muscle-area ratios (d-v-ratio) and ratios of the dorsal and ventral musculature to C2 height (d-C2-ratio and v-C2-ratio) were evaluated for differences between groups using multivariate analysis of variance ( p  < 0.05) taking the head-neck position into account., Results:  Dogs with AAI showed a significantly lower d-v-ratio at levels 2 and 3, d-C2-ratio at level 2 and ratio of moments at all levels. When head-neck positions were analysed separately, ratio of moments was significantly lower in affected dogs at level 1 and 2. Also lower was d-C2-ratio at level 2, but only in flexed positioning. The head-neck position had a significant influence on ratio of moments and d-v-ratio at all three levels and on d-C2-ratio at level 1., Conclusion:  Significant changes in muscle area were observed only for the hypaxial muscles at the C1 level, indicating a limited role of muscular adaption in AAI patients. Our results confirm an altered ratio of moments in dogs with AAI. The head-neck position has a significant impact and should be taken into account when evaluating spinal musculature., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

In the last decades, the scientific community spared no effort to elucidate the therapeutic potential of mesenchymal stromal cells (MSCs). Unfortunately, in vitro cellular senescence occurring along with a loss of proliferative capacity is a major drawback in view of future therapeutic applications of these cells in the field of regenerative medicine. Even though insight into the mechanisms of replicative senescence in human medicine has evolved dramatically, knowledge about replicative senescence of canine MSCs is still scarce. Thus, we developed a high-content analysis workflow to simultaneously investigate three important characteristics of senescence in canine adipose-derived MSCs (cAD-MSCs): morphological changes, activation of the cell cycle arrest machinery, and increased activity of the senescence-associated β-galactosidase. We took advantage of this tool to demonstrate that passaging of cAD-MSCs results in the appearance of a senescence phenotype and proliferation arrest. This was partially prevented upon immortalization of these cells using a newly designed PiggyBac™ Transposon System, which allows for the expression of the human polycomb ring finger proto-oncogene BMI1 and the human telomerase reverse transcriptase under the same promotor. Our results indicate that cAD-MSCs immortalized with this new vector maintain their proliferation capacity and differentiation potential for a longer time than untreated cAD-MSCs. This study not only offers a workflow to investigate replicative senescence in eukaryotic cells with a high-content analysis approach but also paves the way for a rapid and effective generation of immortalized MSC lines. This promotes a better understanding of these cells in view of future applications in regenerative medicine., (© 2021. The Author(s), under exclusive licence to American Aging Association.)

Translation of acute ischemic stroke research to the clinical setting remains limited over the last few decades with only one drug, recombinant tissue-type plasminogen activator, successfully completing the path from experimental study to clinical practice. To improve the selection of experimental treatments before testing in clinical studies, the use of large gyrencephalic animal models of acute ischemic stroke has been recommended. Currently, these models include, among others, dogs, swine, sheep, and nonhuman primates that closely emulate aspects of the human setting of brain ischemia and reperfusion. Species-specific characteristics, such as the cerebrovascular architecture or pathophysiology of thrombotic/ischemic processes, significantly influence the suitability of a model to address specific research questions. In this article, we review key characteristics of the main large animal models used in translational studies of acute ischemic stroke, regarding (1) anatomy and physiology of the cerebral vasculature, including brain morphology, coagulation characteristics, and immune function; (2) ischemic stroke modeling, including vessel occlusion approaches, reproducibility of infarct size, procedural complications, and functional outcome assessment; and (3) implementation aspects, including ethics, logistics, and costs. This review specifically aims to facilitate the selection of the appropriate large animal model for studies on acute ischemic stroke, based on specific research questions and large animal model characteristics.

Objective: Intravenous hydroxyethyl starch (HES) solutions are potentially nephrotoxic due to rapid renal tissue uptake, subsequent osmotic nephrosis, and long-lasting intracellular storage. This study aimed to investigate the severity of intracellular storage of HES in renal tissue samples from critically ill dogs receiving 6% HES 130/0.4. Materials and Methods: Fresh, post-mortem (<2 h after death) renal tissue samples were analyzed through histology, immunohistochemistry (HES 130/0.4-specific antibodies), and electron microscopy for the severity of renal tubular vacuolization (VAC), intravacuolar HES accumulation (ACC), and ultra-structure impairment. Moreover, we investigated the relationship between VAC or ACC grade and HES dose (mL/kg), duration of HES administration (h), and pre-HES plasma creatinine concentrations. Results: Histology revealed that 2/20 dogs (10%) had no, 11/20 dogs (55%) had mild, 5/20 dogs (25%) had moderate, and 2/20 dogs (10%) had severe VAC. Immunohistochemistry revealed that 5/20 dogs (25%) had no, 6/20 dogs (30%) had mild, 7/20 dogs (35%) had moderate, and 2/20 dogs (10%) had severe ACC. Both changes were predominantly found in the distal tubular epithelium of mild and moderate cases, and all tubular segments were affected in severe cases. Seven of 20 dogs (35%) had osmotic nephrosis (ON). On electron microscopy, large granules with an electron-dense content were repeatedly detected in individual cells, mainly in the distal tubules. No correlation was found between cumulative HES dose or duration of HES administration and VAC grade, ACC grade, or presence/absence of ON. Conclusion: A high percentage of dogs had renal tubular HES storage and one-third of dogs showed HES-induced ON. Short-term HES administration caused VAC and ACC, regardless of the dose or duration of administration. In contrast to previous studies, HES 130/0.4 deposits were mainly located in the renal distal tubule., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Adamik, Stoffel, Tangermann, de Breuyn Dietler and Stokar-Regenscheit.)

Allergen-specific immunotherapy (AIT) constitutes the only curative approach for allergy treatment. There is need for improvement of AIT in veterinary medicine, such as in horses suffering from insect bite hypersensitivity, an IgE-mediated dermatitis to Culicoides. Dendritic cell (DC)-targeting represents an efficient method to increase antigen immunogenicity. It is studied primarily for its use in improvement of cancer therapy and vaccines, but may also be useful for improving AIT efficacy. Immunomodulators, like the Toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid-A (MPLA) has been shown to enhance the IL-10 response in horses, while CpG-rich oligonucleotides (CpG-ODN), acting as TLR-9 agonists, have been shown to induce Th1 or regulatory responses in horses with equine asthma. Our aim was to evaluate in vitro effects of antigen-targeting to equine DC with an antigen-fused peptide known to target human and mouse DC and investigate whether addition of MPLA or CpG-ODN would further improve the induced immune response with regard to finding optimal conditions for equine AIT. For this purpose, DC-binding peptides were fused to the model antigen ovalbumin (OVA) and to the recombinant Culicoides allergen Cul o3. Effects of DC-binding peptides on cellular antigen uptake and induction of T cell proliferation were assessed. Polarity of the immune response was analysed by quantifying IFN-γ, IL-4, IL-10, IL-17 and IFN-α in supernatants of antigen-stimulated peripheral blood mononuclear cells (PBMC) in presence or absence of adjuvants. Fusion of DC-binding peptides to OVA significantly enhanced antigen-uptake by equine DC. DC primed with DC-binding peptides coupled to OVA or Cul o3 induced a significantly higher T-cell proliferation compared to the corresponding control antigens. PBMC stimulation with DC-binding peptides coupled to Cul o3 elicited a significant increase in the pro-inflammatory cytokines IFN-γ, IL-4, IL-17, as well as the anti-inflammatory IL-10, but not of IFN-α. Adjuvant addition further enhanced the effect of the DC-binding peptides by significantly increasing the production of IFN-γ, IL-4, IL-10 and IFN-α (CpG-ODN) and IL-10 (MPLA), while simultaneously suppressing IFN-γ, IL-4 and IL-17 production (MPLA). Targeting equine DC with allergens fused to DC-binding peptides enhances antigen-uptake and T-cell activation and may be useful in increasing the equine immune response against recombinant antigens. Combination of DC-binding peptide protein fusions with adjuvants is necessary to appropriately skew the resulting immune response, depending on intended use. Combination with MPLA is a promising option for improvement of AIT efficacy in horses, while combination with CpG-ODN increases the effector immune response to recombinant antigens., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They encompass several important medical and veterinary pathogens that are often associated with a wide range of chronic diseases. The long persistence of mycoplasma cells in their hosts can exacerbate the spread of antimicrobial resistance observed for many species. However, the nature of the virulence factors driving this phenomenon in mycoplasmas is still unclear. Toxin-antitoxin systems (TA systems) are genetic elements widespread in many bacteria that were historically associated with bacterial persistence. Their presence on mycoplasma genomes has never been carefully assessed, especially for pathogenic species. Here we investigated three candidate TA systems in M. mycoides subsp. capri encoding a (i) novel AAA-ATPase/subtilisin-like serine protease module, (ii) a putative AbiEii/AbiEi pair and (iii) a putative Fic/RelB pair. We sequence analyzed fourteen genomes of M. mycoides subsp. capri and confirmed the presence of at least one TA module in each of them. Interestingly, horizontal gene transfer signatures were also found in several genomic loci containing TA systems for several mycoplasma species. Transcriptomic and proteomic data confirmed differential expression profiles of these TA systems during mycoplasma growth in vitro. While the use of heterologous expression systems based on E. coli and B. subtilis showed clear limitations, the functionality and neutralization capacities of all three candidate TA systems were successfully confirmed using M. capricolum subsp. capricolum as a host. Additionally, M. capricolum subsp. capricolum was used to confirm the presence of functional TA system homologs in mycoplasmas of the Hominis and Pneumoniae phylogenetic groups. Finally, we showed that several of these M. mycoides subsp. capri toxins tested in this study, and particularly the subtilisin-like serine protease, could be used to establish a kill switch in mycoplasmas for industrial applications., Competing Interests: The authors have declared that no competing interests exist.

Transiently increased teat wall thickness in response to machine milking has been documented by various methods, including ultrasound. However, correlative ultrasonography and histology to detect the origin of this phenomenon is lacking. The first goal of the present study was to evaluate and compare milking-related changes of the teat tissue in 2 breeds of dairy cows (11 Simmental and 3 Holstein) using B-mode ultrasonography. Additionally, the observed changes were compared with ultrasonographic findings in a Holstein cow with periparturient udder edema. Finally, corresponding histological sections of the Simmental teats were analyzed and compared with those from a lactating nonmilked Angus cow. We hypothesized that the mechanical load of both stretching by the vacuum during phases of open teat cup liner and compression by the closed liner during machine milking results in a transient congestion of blood vessels in the teat wall. The barrel of 1 front teat of each cow was scanned immediately before and after machine milking (system vacuum: 42 kPa; pulsation rate: 60 cycles/min; pulsation ratio: 65:35). Shortly after milking (33 ± 6 min), the Simmentals were slaughtered, and their scanned teat was immediately removed and processed for investigation by light microscopy. Ultrasonography after milking revealed anechoic tubular structures mainly in the inner half of the teat wall. Histological examination revealed these structures to be thick-walled veins. The left front and hind teats of the nonmilked lactating cow, collected and prepared identically to those from the Simmental cows, showed the same histological features. Ultrasonographic measurements showed that the diameter of these veins significantly increased after milking compared with matching images before milking. This effect was most pronounced in the Holstein cows. Similarly, these veins were very prominent in the periparturient cow. However, neither the milked cows, including the periparturient cow, nor the lactating nonmilked cow provided any evidence of edematous extravasation on ultrasonography or histology. These findings corroborated our hypothesis that the increase in size of thick-walled veins in the teat tissue is the main reason for the thickening of the teat walls in response to machine milking., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

A comparative study was carried out on common and agile frogs ( Rana temporaria and R. dalmatina ) naturally infected with ranid herpesvirus 3 (RaHV3) and common toads ( Bufo bufo ) naturally infected with bufonid herpesvirus 1 (BfHV1) to investigate common pathogenetic pathways and molecular mechanisms based on macroscopic, microscopic, and ultrastructural pathology as well as evaluation of gene expression. Careful examination of the tissue changes, supported by in situ hybridization, at different stages of development in 6 frogs and 14 toads revealed that the skin lesions are likely transient, and part of a tissue cycle necessary for viral replication in the infected hosts. Transcriptomic analysis, carried out on 2 naturally infected and 2 naïve common frogs ( Rana temporaria ) and 2 naturally infected and 2 naïve common toads ( Bufo bufo ), revealed altered expression of genes involved in signaling and cell remodeling in diseased animals. Finally, virus transcriptomics revealed that both RaHV3 and BfHV1 had relatively high expression of a putative immunomodulating gene predicted to encode a decoy receptor for tumor necrosis factor in the skin of the infected hosts. Thus, the comparable lesions in infected frogs and toads appear to reflect a concerted epidermal and viral cycle, with presumptive involvement of signaling and gene remodeling host and immunomodulatory viral genes.

The common limitation of surgical revascularization procedures for severe tissue ischemia due to cardiovascular diseases is the need to interrupt blood flow during the intervention. We aim to introduce a new technique that allows a sutureless, non-occlusive revascularization. A 3-step technique was developed using rabbit's aorta to simulate a side-to-side anastomosis model. It enables the creation of a bypass circuit for revascularization. The first step was the soldering of 2 vessels in a side-to-side fashion based on the laser-assisted vascular anastomosis (LAVA) principle using a diode laser emitting irradiation at 810 nm with an albumin-based solder patch between them, followed by the creation of a channel within the patch using either a holmium-doped yttrium aluminum garnet laser (Ho:YAG) at λ = 2100 nm or a xenon-chloride excimer laser (XeCl) at λ = 308 nm. Thereby, a bypass circuit was created, thus allowing a non-ischemic revascularization. The system was deemed functional when a flow was observed across the anastomosis. The highest average tensile strength recorded after side-to-side LAVA using a diode laser power of 3.2 W for 60 s was 2278.6 ± 800 mN (n = 20). The Ho:YAG laser created the channels with less tension on the anastomosis than the excimer laser. Histological analysis showed limited thermal damage and good patch-tissue adaptation. The preliminary results of this feasibility study outline the foundations for an entirely sutureless laser-assisted revascularization procedure. The next studies will evaluate the rheological parameters across the bypass circuit to optimize the post-anastomotic flow.

Respiratory diseases negatively impact the global goat industry, but are understudied. There is a shortage of established and biological relevant in vitro or ex vivo assays to study caprine respiratory infections. Here, we describe the establishment of an in vitro system based on well-differentiated caprine airway epithelial cell (AEC) cultures grown under air liquid interface conditions as an experimental platform to study caprine respiratory pathogens. The functional differentiation of the AEC cultures was monitored and confirmed by light and immunofluorescence microscopy, scanning electron microscopy and examination of histological sections. We validated the functionality of the platform by studying Influenza D Virus (IDV) infection and Mycoplasma mycoides subsp. capri (Mmc) colonization over 5 days, including monitoring of infectious agents by titration and qPCR as well as colour changing units, respectively. The inoculation of caprine AEC cultures with IDV showed that efficient viral replication takes place, and revealed that IDV has a marked cell tropism for ciliated cells. Furthermore, AEC cultures were successfully infected with Mmc using a multiplicity of infection of 0.1 and colonization was monitored over several days. Altogether, these results demonstrate that our newly-established caprine AEC cultures can be used to investigate host-pathogen interactions of caprine respiratory pathogens., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The tracheobronchial tree is lined by a mucociliary epithelium containing millions of multiciliated cells. Their integrated oscillatory activity continuously propels an overlying pollution-protecting mucus layer in cranial direction, leading to mucociliary clearance - the primary defence mechanism of the airways. Mucociliary transport is commonly thought to co-emerge with the collective ciliary motion pattern under appropriate geometrical and rheological conditions. Proper ciliary alignment is therefore considered essential to establish mucociliary clearance in the respiratory system. Here, we used volume electron microscopy in combination with high-speed reflection contrast microscopy in order to examine ciliary orientation and its spatial organization, as well as to measure the propagation direction of metachronal waves and the direction of mucociliary transport on bovine tracheal epithelia with reference to the tracheal long axis (TLA). Ciliary orientation is measured in terms of the basal body orientation (BBO) and the axonemal orientation (AO), which are commonly considered to coincide, both equivalently indicating the effective stroke as well as the mucociliary transport direction. Our results, however, reveal that only the AO is in line with the mucociliary transport, which was found to run along a left-handed helical trajectory, whereas the BBO was found to be aligned with the TLA. Furthermore, we show that even if ciliary orientation remains consistent between adjacent cells, ciliary orientation exhibits a gradual shift within individual cells. Together with the symplectic beating geometry, this intracellular orientational pattern could provide for the propulsion of highly viscous mucus and likely constitutes a compromise between efficiency and robustness., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nanoparticle (NP)-assisted procedures including laser tissue soldering (LTS) offer advantages compared to conventional microsuturing, especially in the brain. In this study, effects of polymer-coated silica NPs used in LTS were investigated in human brain endothelial cells (ECs) and blood-brain barrier models. In the co-culture setting with ECs and pericytes, only the cell type directly exposed to NPs displayed a time-dependent internalization. No transfer of NPs between the two cell types was observed. Cell viability was decreased relatively to NP exposure duration and concentration. Protein expression of the nuclear factor ĸ-light-chain-enhancer of activated B cells and various endothelial adhesion molecules indicated no initiation of inflammation or activation of ECs after NP exposure. Differentiation of CD34+ ECs into brain-like ECs co-cultured with pericytes, blood-brain barrier (BBB) characteristics were obtained. The established endothelial layer reduced the passage of integrity tracer molecules. NP exposure did not result in alterations of junctional proteins, BBB formation or its integrity. In a 3-dimensional setup with an endothelial tube formation and tight junctions, barrier formation was not disrupted by the NPs and NPs do not seem to cross the blood-brain barrier. Our findings suggest that these polymer-coated silica NPs do not damage the BBB.

Objective: To localize vagal branches within the surgical field of laryngoplasty and identify potentially hazardous surgical steps., Study Design: Observational cadaveric study., Sample Population: Five equine head-neck specimens and four entire equine cadavers., Methods: Dissection of the pharyngeal region from a surgical perspective. Neuronal structures were considered at risk if touched or if the distance to instruments was less than 5 mm., Results: The branches of the pharyngeal plexus (PP) supplying the cricopharyngeal muscle (PPcr), the thyropharyngeal muscle (PPth), and the esophagus (PPes) were identified in the surgical field in nine of nine, five of nine, and one of nine specimens, respectively. The internal branch of the cranial laryngeal nerve (ibCLN) was identified within the carotid sheath in six of nine specimens. The external branch of the cranial laryngeal nerve (ebCLN) was identified close to the septum of the caudal constrictors in nine of nine specimens. The blade of the tissue retractor compressed the ibCLN in six of six, the ebCLN in four of six, the PPcr in six of six, the PPth in two of three, and the PPes in two of two specimens in which the respective nerves were identified after further dissection. Surgical exploration of the dorsolateral aspect of the pharynx and the incision of the septum of the caudal constrictors harmed the ebCLN in nine of nine, PPcr in seven of nine, and PPth in four of eight specimens., Conclusion: Several vagal branches were located in the surgical field and must be considered at risk because of their location., Clinical Significance: Use of the tissue retractor, dissection over the pharynx, and dissection of the septum of the caudal constrictors involve a risk to damage vagal branches., (© 2020 The American College of Veterinary Surgeons.)

Small extracellular vesicles (EVs) are among the most frequently investigated EVs and play major roles in intercellular communication by delivering various cargo molecules to target cells. They could potentially represent an alternative delivery strategy to treat ocular toxoplasmosis, a parasitosis affecting the retinal pigment epithelium (RPE). To date, the uptake of human small EVs by RPE cells has never been reported. In this study, we report on the intracellular uptake of fluorescently labelled human urine and fibroblast-derived small EVs by human RPE cells. In summary, both dye-labelled urinary small EVs and small EVs obtained from fibroblasts stably expressing membrane-bound green fluorescent protein were successfully internalized by RPE cells as revealed by immunohistochemistry. In recipient ARPE19 cells, BODIPY-labelled small EVs were found in close vicinity to the parasite Toxoplasma gondii. Additionally, an ultrastructural method was enabled to distinguish between labelled exogenous and endogenous small EVs within target cells.

Hereditary nasal parakeratosis (HNPK) is an inherited disorder described in Labrador Retrievers and Greyhounds. It has been associated with breed-specific variants in the SUV39H2 gene encoding a histone 3 methyltransferase involved in epigenetic silencing. Formalin-fixed biopsies of the nasal planum of Labrador Retrievers were screened by immunofluorescence microscopy for the presence and distribution of epidermal proliferation and differentiation markers. Gene expression of these markers was further analysed using RNA sequencing (RNA-seq) and ultrastructural epidermal differences were investigated by electron microscopy. Differentiation of the nasal planum in the basal and suprabasal epidermal layers of HNPK-affected dogs (n = 6) was similar compared to control dogs (n = 6). In the upper epidermal layers, clear modifications were noticed. Loricrin protein was absent in HNPK-affected nasal planum sections in contrast to sections of the same location of control dogs. However, loricrin was present in the epidermis of paw pads and abdominal skin from HNPK dogs and healthy control dogs. The patterns of keratins K1, K10 and K14, were not markedly altered in the nasal planum of HNPK-affected dogs while the expression of the terminal differentiation marker involucrin appeared less regular. Based on RNA-seq, LOR and IVL expression levels were significantly decreased, while KRT1, KRT10 and KRT14 levels were up-regulated (log2fold-changes of 2.67, 3.19 and 1.71, respectively) in HNPK-affected nasal planum (n = 3) compared to control dogs (n = 3). Electron microscopical analysis revealed structural alterations in keratinocytes and stratum corneum, and disrupted keratinocyte adhesions and distended intercellular spaces in lesional samples (n = 3) compared to a sample of a healthy control dog (n = 1). Our findings demonstrate aberrant keratinocyte terminal differentiation of the nasal planum of HNPK-affected Labrador Retrievers and provide insights into biological consequences of this inactive SUV39H2 gene variant., Competing Interests: The authors have declared that no competing interests exist.

' Treponema phagedenis ' was originally described in 1912 by Noguchi but the name was not validly published and no type strain was designated. The taxon was not included in the Approved Lists of Bacterial Names and hence has no standing in nomenclature. Six Treponema strains positive in a ' T. phagedenis ' phylogroup-specific PCR test were isolated from digital dermatitis (DD) lesions of cattle and further characterized and compared with the human strain ' T. phagedenis ' ATCC 27087. Results of phenotypic and genotypic analyses including API ZYM, VITEK2, MALDI-TOF and electron microscopy, as well as whole genome sequence data, respectively, showed that they form a cluster of species identity. Moreover, this species identity was shared with ' T. phagedenis '-like strains reported in the literature to be regularly isolated from bovine DD. High average nucleotide identity values between the genomes of bovine and human ' T. phagedenis ' were observed. Slight genomic as well as phenotypic variations allowed us to differentiate bovine from human isolates, indicating host adaptation. Based on the fact that this species is regularly isolated from bovine DD and that the name is well dispersed in the literature, we propose the species Treponema phagedenis sp. nov., nom. rev. The species can phenotypically and genetically be identified and is clearly separated from other Treponema species. The valid species designation will allow to further explore its role in bovine DD. The type strain for Treponema phagedenis sp. nov., nom. rev. is B43.1 T (=DSM 110455 T =NCTC 14362 T ) isolated from a bovine DD lesion in Switzerland.

The adult anatomy and physiology of the bovine (Bos taurus) stomach have been investigated extensively. Despite the many studies, however, the early development of the stomach has not yet been fully elucidated. The goal of the present study, therefore, was to review the available literature, to visualize the embryonic and early foetal development of the bovine stomach and to shed light on unresolved issues. The stomachs of fifteen bovine embryos and eleven foetuses from 26 to 80 days of gestation were photographed both in situ and after exenteration and critical point drying. A series of photographs was obtained that yielded a contiguous and comprehensive view of all the developmental changes that occurred until the virtually final configuration of the stomach was attained. In addition, the serosal surface was studied by electron microscopy, thus revealing subtle regional differences in the lining of the peritoneal cavity. Our observations corroborate the contention that all the compartments evolve from the fusiform primordium and that no outgrowth at the level of the oesophagus occurs. The greater curvature as well as the attachment line of the dorsal mesogastrium shift to the left, which is similar to the process in monogastrians. The rumen and reticulum develop from separate protrusions, and further compartmentalization results from constrictions and bulges and not from folding. Between 55 and 60 days of gestation, the entire bovine stomach except for the abomasum eventually relocates to its final position. In summary, previously debated key issues were addressed and integrated with current findings., (© 2019 Blackwell Verlag GmbH.)

The atlantoaxial joint can be affected by instability, in most cases a congenital pathology in young small breed dogs. Causes of atlantoaxial instability (AAI) are variable but are usually attributed to a lack of ligamentous support. The purpose of the present study was to specify the role of the ligamentous structures in the stabilisation of the atlantoaxial joint and to find possible adaptations of the ligaments' internal structure to their specific function. Five Beagle cadavers were included in this study. Each dog was subjected to a computed tomography (CT) and a magnetic resonance imaging (MRI) examination of the upper cervical region. This region was then dissected and the ligamentous structures stabilising the atlantoaxial joint were measured and removed for histological analysis. A ligament to dens ratio (LDR) was established in order to provide a basis for comparison with the measurements taken in other dog breeds. MRI and gross anatomical measurements were very similar, confirming the validity of the results. MRI thus seems reliable for evaluating the ligamentous structures of the canine occipitoatlantoaxial region. The movement exerting the greatest stress on the atlantoaxial ligaments and inducing the greatest distension of the alar ligaments was a head flexion combined with a rotation. A clear adaptation of the ligamentous shape and internal structure to their specific function was observed. Histologically, alar ligaments consisted of wavy collagen fibres and a high proportion of elastic fibres, providing them with a remarkable elasticity compared to the transverse ligament structure which was much more rigid., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Laser tissue soldering is a novel treatment method for injuries of hollow organs such as cerebrovascular aneurysms. Nanomaterials contained in the solder are foreign to the body. Hence, it is indispensable to carefully examine possible adverse effects prior to introducing this technique. The aim of this study was to characterize the impact of different concentrations of polymer-coated silica nanoparticles (NPs) on mitochondrial function and integrity of brain endothelial cells using the rat brain capillary endothelial cell line rBCEC4. At maximal capacity, NP exposure resulted in a decrease in the oxygen consumption rate whereas glycolysis was not affected. In combination with a stressor, i.e. lack of glucose in the medium, NP exposure interfered primarily with glycolytic ATP generation rather than oxidative phosphorylation. Furthermore, NPs caused a metabolic shift towards a stressed phenotype, exhibiting increased levels of the oxygen consumption rate and the extracellular acidification rate compared to untreated controls. Overall, mitochondrial mass, distribution and morphology as well as intracellular ATP content were not altered. The mitochondrial membrane potential was increased after exposure to the highest NP concentration and the content of proteins involved in mitochondrial dynamics was changed slightly, indicating possible modifications of the fusion / fission balance. In conclusion, PCL-NP exposure changed mitochondrial respiration, especially under glucose deprivation, but did not affect mitochondrial morphology and distribution. Further studies are needed to investigate whether the functional effects are transient or long-term as this will be crucial for the use of these NPs in laser tissue soldering., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To investigate the feasibility and complications associated with ceratohyoidectomy (CHE) in standing sedated horses unaffected (experimental horses) and standing sedated horses affected (clinical cases) with temporohyoid osteoarthropathy (THO)., Study Design: Case series., Animals: Six experimental horses and four clinical cases., Methods: Standing CHE was performed in six experimental horses euthanized 30 minutes (n = 3) and 7 days (n = 3) postoperatively. The four clinical cases were presented because of central facial nerve paralysis (n = 3), vestibular ataxia (n = 3), auricular hemorrhage (n = 2), quidding (n = 1), and oesophageal impaction (n = 1). Evolution was assessed by clinical examination during hospitalization and later by telephone interviews for the clinical cases., Results: The procedure was successfully performed in all horses. Experimental horses did not show any short-term postoperative complications. Hemorrhage was experienced intraoperatively in one of the clinical cases and was successfully managed with placement of hemostatic forceps. Vestibular ataxia and other symptoms of THO improved within days, but facial nerve paralysis did not improve until 9 days to 6 months after surgery. Follow-up ranged from 9 to 24 months. All clinical cases returned to performance, and client satisfaction was excellent., Conclusion: Ceratohyoidectomy was consistently feasible in standing sedated horses. The method did not result in postoperative complications and led to resolution of clinical signs associated with THO., Clinical Significance: Standing CHE should be considered in horses affected with THO, especially when horses present with marked vestibular deficits and ataxia, to reduce risks associated with recovery from general anesthesia., (© 2019 The American College of Veterinary Surgeons.)

Disbudding of calves is a standard husbandry procedure to reduce the risk of injuries to other cattle and to workers. Whereas acute pain resulting from disbudding has been studied extensively, little is known about chronic pain as a potential long-term consequence. The goal of the present study was to investigate possible morphological changes in the cornual nerve as a function of disbudding. Samples were collected from 17 randomly selected bulls and from 21 calves from a prospective clinical study. Among the calves, 13 were disbudded and 8 were sham-disbudded. Out of the disbudded calves, 4 showed signs of chronic pain. In all the animals, the infraorbital nerve was used as a methodological check. Morphological analysis included measuring minimal diameters of the axons present in both the cornual and infraorbital nerves. Sympathetic fibers were identified as based on the presence of Tyroxine hydroxylase (TH). TH-negative fibers were considered afferents. Trigeminal ganglia from the calves were immunostained for glial fibrillary acidic protein (GFAP) and Activating transcription factor 3 (ATF3). R. cornualis and N. infraorbitalis differed in terms of axon diameters and proportion of TH-positive fibers. Weak evidence (p > .091) of a difference in axon diameters between control and disbudded calves was found in R. cornualis, but the proportion of TH-positive fibers was alike in both groups. Average glial envelope and the percentages of ATF3-positive neurons revealed no difference between calves with and without signs of pain. Thus, available evidence is insufficient to support neuropathic changes as a result of disbudding in calves., (Copyright © 2019. Published by Elsevier Ltd.)

Members of the " Mycoplasma mycoides cluster" are important animal pathogens causing diseases including contagious bovine pleuropneumonia and contagious caprine pleuropneumonia, which are of utmost importance in Africa or Asia. Even if all existing vaccines have shortcomings, vaccination of herds is still considered the best way to fight mycoplasma diseases, especially with the recent and dramatic increase of antimicrobial resistance observed in many mycoplasma species. A new generation of vaccines will benefit from a better understanding of the pathogenesis of mycoplasmas, which is very patchy up to now. In particular, surface-exposed virulence traits are likely to induce a protective immune response when formulated in a vaccine. The candidate virulence factor L-α-glycerophosphate oxidase (GlpO), shared by many mycoplasmas including Mycoplasma pneumoniae , was suggested to be a surface-exposed enzyme in Mycoplasma mycoides subsp. mycoides responsible for the production of hydrogen peroxide directly into the host cells. We produced a glpO isogenic mutant GM12::YCpMmyc1.1- ΔglpO using in-yeast synthetic genomics tools including the tandem-repeat endonuclease cleavage (TREC) technique followed by the back-transplantation of the engineered genome into a mycoplasma recipient cell. GlpO localization in the mutant and its parental strain was assessed using scanning electron microscopy (SEM). We obtained conflicting results and this led us to re-evaluate the localization of GlpO using a combination of in silico and in vitro techniques, such as Triton X-114 fractionation or tryptic shaving followed by immunoblotting. Our in vitro results unambiguously support the finding that GlpO is a cytoplasmic protein throughout the " Mycoplasma mycoides cluster." Thus, the use of GlpO as a candidate vaccine antigen is unlikely to induce a protective immune response.

Nanomedicine is a constantly expanding field, facilitating and improving diagnosis and treatment of diseases. As nanomaterials are foreign objects, careful evaluation of their toxicological and functional aspects prior to medical application is imperative. In this study, we aimed to determine the effects of gold and polymer-coated silica nanoparticles used in laser tissue soldering on brain endothelial cells and the blood-brain barrier using rat brain capillary endothelial cells (rBCEC4). All types of nanoparticles were taken up time-dependently by the rBCEC4 cells, albeit to a different extent, causing a time- and concentration-dependent decrease in cell viability. Nanoparticle exposure did not change cell proliferation, differentiation, nor did it induce inflammation. rBCEC4 cells showed blood-brain barrier characteristics including tight junctions. None of the nanoparticles altered the expression of tight junctions or impaired the blood-brain barrier permeability. The findings suggest that effects of these nanoparticles on the metabolic state of cells have to be further characterized before use for medical purposes.

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects, and plants. Here, we demonstrate that highly virulent Mycoplasma mycoides subspecies capri ( Mmc ) can be fully attenuated via targeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately 10% of the original Mmc genome, were successively deleted using Saccharomyces cerevisiae as an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually non-essential in the JCVI-Syn3.0 minimal cell, were excised from the genome. In vitro characterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though 10% of the genome content were removed. A novel in vivo challenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reached endpoint criteria within 6 days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of a Mycoplasma strain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequent in vitro or in vivo trials employing the Mycoplasma chassis will allow us to dissect the role of individual candidate Mycoplasma virulence factors and lead the way for the development of an attenuated designer vaccine.

Background: Silica-ε-polycaprolactone-nanoparticles (SiPCL-NPs) represent a promising tool for laser-tissue soldering in the brain. After release of the SiPCL-NPs in the brain, neuronal differentiation might be modulated. The present study was performed to determine effects of SiPCL-NP-exposure at different stages of neuronal differentiation in neuron-like SH-SY5Y cells. The resulting phenotypes were analyzed quantitatively and signaling pathways involved in neuronal differentiation and degeneration were studied. SH-SY5Y cells were differentiated with all-trans retinoic acid or staurosporine to obtain predominantly cholinergic or dopaminergic neurons. The resulting phenotype was analyzed at the end of differentiation with and without the SiPCL-NPs given at various times during differentiation., Results: Exposure to SiPCL-NPs before and during differentiation led to a decreased cell viability of SH-SY5Y cells depending on the differentiation protocol used. SiPCL-NPs co-localized with the neuronal marker β-3-tubulin but did not alter the morphology of these cells. A significant decrease in the number of tyrosine hydroxylase (TH) immunoreactive neurons was found in staurosporine-differentiated cells when SiPCL-NPs were added at the end of the differentiation. TH-protein expression was also significantly downregulated when SiPCL-NPs were applied in the middle of differentiation. Protein expression of the marker for the dopamine active transporter (DAT) was not affected by SiPCL-NPs. SiPCL-NP-exposure predominantly decreased the expression of the high-affinity choline transporter 1 (CHT1) when the NPs were given before the differentiation. Pathways involved in neuronal differentiation, namely Akt, MAP-K, MAP-2 and the neurodegeneration-related markers β-catenin and GSK-3β were not altered by NP-exposure., Conclusions: The decrease in the number of dopaminergic and cholinergic cells may implicate neuronal dysfunction, but the data do not provide evidence that pathways relevant for differentiation and related to neurodegeneration are impaired.

[This corrects the article DOI: 10.1038/s41541-018-0078-0.].

Stereotaxic systems and automatic tissue segmentation routines enable neuronavigation as well as reproducible processing of neuroimage datasets. Such systems have been developed for humans, non-human-primates, sheep, and rodents, but not for dogs. Although dogs share important neurofunctional and -anatomical features with humans, and in spite of their importance in translational neuroscience, little is known about the variability of the canine brain morphology and, possibly related, function. Moreover, we lack templates, tissue probability maps (TPM), and stereotaxic brain labels for implementation in standard software utilities such as Statistical Parametric Mapping (SPM). Hence, objective and reproducible, image-based investigations are currently impeded in dogs. We have created a detailed stereotaxic reference frame for dogs including TPM and tissue labels, enabling inter-individual and cross-study neuroimage analysis. T2w datasets were acquired from 16 neurologically inconspicuous dogs of different breeds by 3T MRI. The datasets were averaged after initial preprocessing using linear and nonlinear registration algorithms as implemented in SPM8. TPM for gray (GM) and white matter (WM) as well as cerebrospinal fluid (CSF) were created. Different cortical, subcortical, medullary, and CSF regions were manually labeled to create a spatial binary atlas being aligned with the template. A proof-of-concept for automatic determination of morphological and volumetrical characteristics was performed using additional canine datasets (n = 64) including a subgroup of laboratory beagles (n = 24). Overall, 21 brain regions were labeled using the segmented tissue classes of the brain template. The proof-of-concept trial revealed excellent suitability of the created tools for image processing and subsequent analysis. There was high intra-breed variability in frontal lobe and hippocampus volumes, and noticeable inter-breed corpus callosum volume variation. The T2w brain template provides important, breed-averaged canine brain anatomy features in a spatial standard coordinate system. TPM allows automatic tissue segmentation using SPM and enables unbiased automatic image processing or morphological characterization in different canine breeds. The reported volumetric and morphometric results may serve as a starting point for further research aimed at in vivo analysis of canine brain anatomy and function., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae is a severe disease widespread in Africa and Asia. Limited knowledge is available on the pathogenesis of this organism, mainly due to the lack of a robust in vivo challenge model and the means to do site-directed mutagenesis. This work describes the establishment of a novel caprine challenge model for CCPP that resulted in 100% morbidity using a combination of repeated intranasal spray infection followed by a single transtracheal infection employing the recent Kenyan outbreak strain ILRI181. Diseased animals displayed CCPP-related pathology and the bacteria could subsequently be isolated from pleural exudates and lung tissues in concentrations of up to 10 9 bacteria per mL as well as in the trachea using immunohistochemistry. Reannotation of the genome sequence of ILRI181 and F38 T revealed the existence of genes encoding the complete glycerol uptake and metabolic pathways involved in hydrogen peroxide (H 2 O 2 ) production in the phylogenetically related pathogen M. mycoides subsp. mycoides. Furthermore, the expression of L-α-glycerophosphate oxidase (GlpO) in vivo was confirmed. In addition, the function of the glycerol metabolism was verified by measurement of production of H 2 O 2 in medium containing physiological serum concentrations of glycerol. Peroxide production could be inhibited with serum from convalescent animals. These results will pave the way for a better understanding of host-pathogen interactions during CCPP and subsequent vaccine development.

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention., Competing Interests: PV, MG, JK, SP, NE, SB, CS, JP, HS, VG, RB, MS, MH, RD, VT No competing interests declared, (© 2019, V'kovski et al.)

Here we report the discovery and partial characterization of a novel herpesvirus tentatively named Bufonid herpesvirus 1 (BfHV1) from severe dermatitis in free ranging common toads (Bufo bufo) in Switzerland. The disease has been observed in toads every year since 2014, in spring, during the mating season, at different and distant locations. The virus is found in the skin and occasionally in the brain of infected toads. The genome of the virus is at least 158 Kb long and contains at least 152 open reading frames with a minimal length of 270 nt. The genome of BfHV1 contains all the signature genes that are present in alloherpesviruses. Phylogenetic analysis based on the amino acid sequence of the DNA polymerase and terminase proteins positions the novel virus among the members of the genus Batrachovirus, family Alloherpesviridae. This is the first herpesvirus ever characterized in common toads.

Collagen is the main structural element of connective tissues, and its favorable properties make it an ideal biomaterial for regenerative medicine. In dental medicine, collagen barrier membranes fabricated from naturally occurring tissues are used for guided bone regeneration. Since the morphological characteristics of collagen membranes play a crucial role in their mechanical properties and affect the cellular behavior at the defect site, in-depth knowledge of the structure is key. As a base for the development of novel collagen membranes, an extensive morphological analysis of four porcine membranes, including centrum tendineum, pericardium, plica venae cavae and small intestinal submucosa, was performed. Native membranes were analyzed in terms of their thickness. Second harmonic generation and two-photon excitation microscopy of the native membranes showed the 3D architecture of the collagen and elastic fibers, as well as a volumetric index of these two membrane components. The surface morphology, fiber arrangement, collagen fibril diameter and D-periodicity of decellularized membranes were investigated by scanning electron microscopy. All the membrane types showed significant differences in thickness. In general, undulating collagen fibers were arranged in stacked layers, which were parallel to the membrane surface. Multiphoton microscopy revealed a conspicuous superficial elastic fiber network, while the elastin content in deeper layers varied. The elastin/collagen volumetric index was very similar in the investigated membranes and indicated that the collagen content was clearly higher than the elastin content. The surface of both the pericardium and plica venae cavae and the cranial surface of the centrum tendineum revealed a smooth, tightly arranged and crumpled morphology. On the caudal face of the centrum tendineum, a compact collagen arrangement was interrupted by clusters of circular discontinuities. In contrast, both surfaces of the small intestinal submucosa were fibrous, fuzzy and irregular. All the membranes consisted of largely uniform fibrils displaying the characteristic D-banding. This study reveals similarities and relevant differences among the investigated porcine membranes, suggesting that each membrane represents a unique biomaterial suitable for specific applications., Competing Interests: Geistlich Pharma AG provided the salary of TM and covered the costs related to 2-photonmicroscopy. Geistlich (BS, NS) was further involved in the experimental design, data analysis, decision to publish and in the preparation of the manuscript. Internal institutional resources (government funding) covered personnel costs (JB, MHS, HM, BV, SK) as well as the expenses for laboratory consumables and scanning electron microscopy. There was no additional external funding received for this study. The authors declare no competing interests. None of the institutions involved has filed a patent application or is considering to do so. The University of Bern (Veterinary Anatomy) and Geistlich Pharma AG adopted a Research Collaboration Agreement. The commercial affiliation does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Inactivated vaccines lack immunogenicity and therefore require potent adjuvants. To understand the in vivo effects of adjuvants, we used a system immunology-based analysis of ovine blood transcriptional modules (BTMs) to dissect innate immune responses relating to either antibody or haptoglobin levels. Using inactivated foot-and-mouth disease virus as an antigen, we compared non-adjuvanted to liposomal-formulated vaccines complemented or not with TLR4 and TLR7 ligands. Early after vaccination, BTM relating to myeloid cells, innate immune responses, dendritic cells, and antigen presentation correlated positively, whereas BTM relating to T and natural killer cells, as well as cell cycle correlated negatively with antibody responses. Interestingly, similar BTM also correlated with haptoglobin, but in a reversed manner, indicating that acute systemic inflammation is not beneficial for early antibody responses. Analysis of vaccine-dependent BTM modulation showed that liposomal formulations induced similar responses to those correlating to antibody levels. Surprisingly, the addition of the TLR ligands appeared to reduce early immunological perturbations and mediated anti-inflammatory effects, despite promoting antibody responses. When pre-vaccination BTM were analyzed, we found that high vaccine responders expressed higher levels of many BTM relating to cell cycle, antigen-presenting cells, and innate responses as compared with low responders. In conclusion, we have transferred human BTM to sheep and identified early vaccine-induced responses associated with antibody levels or unwanted inflammation in a heterogeneous and small group of animals. Such readouts are applicable to other veterinary species and very useful to identify efficient vaccine adjuvants, their mechanism of action, and factors related to low responders., Competing Interests: The authors declare no competing interests.

Mycoplasma bovis causes bovine mycoplasmosis. The major clinical manifestations are pneumonia and mastitis. Recently an increase in the severity of mastitis cases was reported in Switzerland. At the molecular level, there is limited understanding of the mechanisms of pathogenicity of M. bovis . Host-pathogen interactions were primarily studied using primary bovine blood cells. Therefore, little is known about the impact of M. bovis on other cell types present in infected tissues. Clear in vitro phenotypes linked to the virulence of M. bovis strains or tissue predilection of specific M. bovis strains have not yet been described. We adapted bovine in vitro systems to investigate infection of epithelial cells with M. bovis using a cell line (MDBK: Madin-Darby bovine kidney cells) and two primary cells (PECT: bovine embryonic turbinate cells and bMec: bovine mammary gland epithelial cells). Two strains isolated before and after the emergence of severe mastitis cases were selected. Strain JF4278 isolated from a cow with mastitis and pneumonia in 2008 and strain L22/93 isolated in 1993 were used to assess the virulence of M. bovis genotypes toward epithelial cells with particular emphasis on mammary gland cells. Our findings indicate that M. bovis is able to adhere to and invade different epithelial cell types. Higher titers of JF4278 than L22/93 were observed in co-cultures with cells. The differences in titers reached between the two strains was more prominent for bMec cells than for MDBK and PECT cells. Moreover, M. bovis strain L22/93 induced apoptosis in MDBK cells and cytotoxicity in PECT cells but not in bMec cells. Dose-dependent variations in proliferation of primary epithelial cells were observed after M. bovis infection. Nevertheless, an indisputable phenotype that could be related to the increased virulence toward mammary gland cells is not obvious.

T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b -/- CD8 + T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b -/- CD8 + T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b -/- CD8 + T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8 + T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue-resident T cell populations., (© 2018 Moalli et al.)

This study reports the occurrence of the lysosomal storage disease GM2 gangliosidosis (Sandhoff disease) in two 11-mo-old captive-bred, male and female mongoose siblings ( Mungos mungo). The clinical signs and the pathological findings reported here were similar to those reported in other mammalian species. Light microscopy revealed an accumulation of stored material in neurons and macrophages accompanied by a significant neuronal degeneration (swelling of neuronal soma, loss of Nissl substance, and neuronal loss) and gliosis. Electron microscopy of brain tissue identified the stored material as membrane-bound multilamellar bodies. An almost complete lack of total hexosaminidase activity in serum suggested a defect in the HEXB gene (Sandhoff disease in humans). High-performance thin-layer chromatography and mass spectrometry confirmed the accumulation of GM2 ganglioside in brain and kidney tissue, and the lectin staining pattern of the brain tissue further corroborated the diagnosis of a Sandhoff-type lysosomal storage disease.

Stock management of the Swiss water buffalo livestock results in the slaughtering of about 350 animals per year. As the stunning of water buffaloes still is an unresolved issue, we investigated the terminal ballistics of currently used perforating stunning devices. Cartridge fired captive bolt devices, handguns and a bullet casing gun were tested in a shooting steep by firing on bisected heads, forehead plates and soap blocks. Energy loss of captive bolts confirmed their inadequacy when used for heavy water buffaloes, notably adult males. As for the free projectiles, ballistics revealed that beyond the impact energy, bullet deformation has a strong impact on the outcome. Light 9mm Luger or .38 Spl bullets as well as large deformable .44 Rem. Magnum bullets should be avoided in favor of heavier .357 Magnum deformation ammunition. These data have been translated into the development of a new stunning device for water buffaloes meeting both animal welfare and occupational safety requirements., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Water buffaloes and cattle differ considerably with respect to the anatomy of the head. As a result, captive bolt stunners often fail to reliably produce adequate loss of consciousness in water buffaloes and, thus, do not fulfill animal welfare requirements. The goal of the present study was to assess and validate a new stunning device for water buffaloes meeting animal welfare and occupational safety requirements. The newly designed bullet casing gun uses .357Mag/10.2g hollow point bullets and has additional safety features. Its effectiveness and usability were assessed under practical conditions in an abattoir as based on widely accepted criteria. Stunning resulted in deep unconsciousness in 19 out of 20 water buffaloes. One 9-year old male did not immediately collapse. Except for very old bulls, the device presented herewith provides a means to stun water buffaloes of both sexes effectively and reliably while keeping occupational hazards to a minimum., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background and Objective: The clinical application of laser-assisted vascular anastomosis is afflicted by unreliable and low bonding strengths as well as tedious handling during microvascular surgery. The challenge to be met arises from the flow-off of the chromophore during soldering that changes the absorption and stains the surrounding tissue, leading to an uncontrollable thermal damage zone. In this study, we investigated the feasibility to produce an indocyanine green (ICG)-loaded patch by electrospinning and tested its applicability to both in vitro and in vivo microvascular laser soldering., Materials and Methods: A blend of polycaprolactone and ICG was electrospun to produce a pliable patch. Prior to soldering, the patch was soaked in 40% wt. bovine serum albumin solution. The solder patch was wrapped in vitro around blood vessel stumps of rabbit aortas. An intraluminal balloon catheter enabled an easy alignment and held the setup in place. The soldering energy was delivered via a diffusor fiber from the vessel lumen using a diode laser at 810 nm. During the procedure, the surface temperature was observed with an infrared camera. Afterward, samples were embedded in methylmethacrylate and epon to study thermal damage. The quality of the fusion was assessed by measuring the tensile strength. After in vitro tests with rabbit aortas, eight large white pigs were subjected to an acute in vivo experiment, and the artery of the latissimus dorsi flap was anastomosed to the distal femoral artery., Results: The ICG-loaded patch, produced by electrospinning, has a thickness of 279 ± 62 μm, a fiber diameter of 1.20 ± 0.19 μm, and an attenuation coefficient of 1,119 ± 183 cm -1 at a wavelength of 790 nm. The patch was pliable and easy to handle during surgery. No leakage of the chromophore was observed. Thermal damage was restricted to the Tunica adventitia and Tunica media and the area of the vessel wall that was covered with the patch. Six pigs were successfully treated, without any bleeding and with a continuous blood flow. The in vivo flap model yielded a similar tensile strength compared to in vitro laser-assisted vascular anastomoses (138 ± 52 vs. 117 ± 30 mN/mm 2 )., Conclusion: Our study demonstrated the applicability of the ICG-loaded patch for laser-assisted vascular anastomosis. By using electrospinning, ICG could be bound to polymer fibers, avoiding its flow-off and the staining of the surrounding tissue. This patch demonstrated several advantages over liquid solder as it was easier to apply, ensured a high and reliable bonding strength while maintaining a constant concentration of ICG concentration during the surgery. Lasers Surg. Med. 49:928-939, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Clostridium perfringens β -toxin (CPB) is the major virulence factor of C. perfringens type C causing a hemorrhagic enteritis in animals and humans. In experimentally infected pigs, endothelial binding of CPB was shown to be associated with early vascular lesions and hemorrhage but without obvious thrombosis of affected vessels, suggesting altered hemostasis in the early phase of the disease. The objective of the present study was to investigate the effect of CPB on platelets, with respect to primary hemostasis. Our results demonstrate that CPB binds to porcine and human platelets and forms oligomers resulting in a time- and dose-dependent cell death. Platelets showed rapid ultrastructural changes, significantly decreased aggregation and could no longer be activated by thrombin. This indicates that CPB affects the physiological function of platelets and counteracts primary hemostasis. Our results add platelets to the list of target cells of CPB and extend the current hypothesis of its role in the pathogenesis of C. perfringens type C enteritis., Competing Interests: The authors declare no conflict of interest.

Keel bone damage is a wide-spread welfare problem in laying hens. It is unclear so far whether bone quality relates to keel bone damage. The goal of the present study was to detect possible associations between keel bone damage and bone properties of intact and damaged keel bones and of tibias in end-of-lay hens raised in loose housing systems. Bones were palpated and examined by peripheral quantitative computer tomography (PQCT), a three-point bending test, and analyses of bone ash. Contrary to our expectations, PQCT revealed higher cortical and trabecular contents in fractured than in intact keel bones. This might be due to structural bone repair after fractures. Density measurements of cortical and trabecular tissues of keel bones did not differ between individuals with and without fractures. In the three-point bending test of the tibias, ultimate shear strength was significantly higher in birds with intact vs. fractured keel bones. Likewise, birds with intact or slightly deviated keel bones had higher mineral and calcium contents of the keel bone than birds with fractured keel bones. Calcium content in keel bones was correlated with calcium content in tibias. Although there were some associations between bone traits related to bone strength and keel bone damage, other factors such as stochastic events related to housing such as falls and collisions seem to be at least as important for the prevalence of keel bone damage.

Amphibian pathogens are of current interest as contributors to the global decline of amphibians. However, compared with chytrid fungi and ranaviruses, herpesviruses have received relatively little attention. Two ranid herpesviruses have been described: namely, Ranid herpesvirus 1 (RHV1) and Ranid herpesvirus 2 (RHV2). This article describes the discovery and partial characterization of a novel virus tentatively named Ranid herpesvirus 3 (RHV3), a candidate member of the genus Batrachovirus in the family Alloherpesviridae. RHV3 infection in wild common frogs (Rana temporaria) was associated with severe multifocal epidermal hyperplasia, dermal edema, a minor inflammatory response, and variable mucous gland degeneration. Intranuclear inclusions were numerous in the affected epidermis together with unique extracellular aggregates of herpesvirus-like particles. The RHV3-associated skin disease has features similar to those of a condition recognized in European frogs for the last 20 years and whose cause has remained elusive. The genome of RHV3 shares most of the features of the Alloherpesviruses. The characterization of this presumptive pathogen may be of value for amphibian conservation and for a better understanding of the biology of Alloherpesviruses.

Objectives: Atlantoaxial instability has been reported in humans, dogs, equids and ruminants. The functional role of the atlantoaxial ligaments has only been described rudimentarily in equids and ruminants. The goal of the present cadaveric study was to compare the anatomy between the different species and to comparatively assess the role of the stabilizing ligaments of the atlantoaxial joint under sagittal shear loading in canine, equine, and bovine cervical spines., Methods: Three equine, bovine, and canine cadaveric specimens were investigated. Biomechanical testing was performed using a purpose built shear-testing device driven by a uniaxial servo-hydraulic testing machine. Three cycles in a dorsoventral direction with a constant quasi-static velocity of 0.2 mm/s up to a limiting force of 50 N (canine) or 250 N (bovine, equine), respectively, were performed for each specimen tested. Load and linear displacement were measured by the displacement sensor and load cell of the testing system at a sampling rate of 20 Hz. Tests were performed and the range of motion determined with both intact and transected atlantoaxial ligaments., Results: The range of motion was significantly increased after transection of the ligaments only in the canine specimens. The bovine atlantoaxial joint was biomechanically more stable than in equids., Clinical Significance: Species-specific anatomical and biomechanical differences of the atlantoaxial ligaments in canines, equids, and bovines were detected. The significance of these differences and their impact on the pathogenesis of atlantoaxial subluxations and subsequent treatment remain open questions.

Nanomedicine offers a promising tool for therapies of brain diseases, but they may be associated with potential adverse effects. The aim of this study was to investigate the uptake of silica-nanoparticles engineered for laser-tissue soldering in the brain using SH-SY5Y cells, dissociated and organotypic slice cultures from rat hippocampus. Nanoparticles were predominantly taken up by microglial cells in the hippocampal cultures but nanoparticles were also found in differentiated SH-SY5Y cells. The uptake was time- and concentration-dependent in primary hippocampal cells. Transmission electron microscopy experiments demonstrated nanoparticle aggregates and single particles in the cytoplasm. Nanoparticles were found in the endoplasmic reticulum, but not in other cellular compartments. Nanoparticle exposure did not impair cell viability and neuroinflammation in primary hippocampal cultures at all times investigated. Neurite outgrowth was not significantly altered in SH-SY5Y cells, but the neuronal differentiation markers indicated a reduction in neuronal differentiation induction after nanoparticle exposure., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sarcoids are the most frequently observed skin tumours in equids and consist of cutaneous accumulations of transformed fibroblasts. Their aetiopathogenesis is closely linked to a presumably abortive infection by bovine papillomavirus (BPV) types 1 and 2. In cattle, dermal fibropapillomas induced by BPV1/2 usually regress spontaneously due to a local, cell-mediated, immune response; however, equids appear to lack an effective immune response to BPV1/2 and mechanisms of immune evasion have been postulated. As a consequence, equine sarcoids tend to persist and are prone to recur. In this study, cryosections were analysed by immunofluorescent staining and a high content analysis system to determine the presence and distribution of CD4(+), CD8(+), FoxP3(+), RORγt(-), CD206(+) and CD14(+) cells, along with expression of the BPV1 early regulatory protein E2. A higher density of cells was positive for BPV1 E2(+) within the transformed tissue than in perilesional tissue or normal skin of horses with sarcoids and control horses. The proportion of CD8(+) cytotoxic T cells was significantly increased in perilesional and lesional tissues, whereas CD4(+) T helper cells were present in higher density only in lesional tissue compared to normal skin from horses with and without sarcoids. The proportion of pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells was decreased in sarcoid tissue compared to perilesional, distant and control tissue. There were no significant differences in densities of CD4(+)FoxP3(+) RORγt(-) regulatory T cells between sarcoids and control tissues. Equine sarcoids are characterised by infiltrations of CD8(+) and CD4(+) T cells, with decreased representation by pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study aims to enhance the anatomical knowledge of the ear of the adult quail (Coturnix coturnix) through the creation of a scaled 3D model utilizing data from micro-CT images. In addition, 17 annotated histological sections of the quail's ear are aligned to their 3D position in the model. The resulting anatomical atlas provides an intuitive insight into the 3D anatomy and can be used for medical education. The model also allows measuring anatomical structures and can thus serve as reference for the quail's auricular anatomy and as a basis to evaluate clinical diagnostic imaging results., (© 2015 Blackwell Verlag GmbH.)

Owing to their ubiquitous distribution, expected beneficial effects and suspected adverse effects, nanoparticles are viewed as a double-edged sword, necessitating a better understanding of their interactions with tissues and organisms. Thus, the goals of the present study were to develop and present a method to generate quantitative data on nanoparticle entry into cells in culture and to exemplarily demonstrate the usefulness of this approach by analyzing the impact of size, charge and various proteinaceous coatings on particle internalization. N9 microglial cells and both undifferentiated and differentiated SH-SY5Y neuroblastoma cells were exposed to customized gold nanoparticles. After silver enhancement, the particles were visualized by epipolarization microscopy and analysed by high-content analysis. The value of this approach was substantiated by assessing the impact of various parameters on nanoparticle uptake. Uptake was higher in microglial cells than in neuronal cells. Only microglial cells showed a distinct size preference, preferring particles with a diameter of 80 nm. Positive surface charge had the greatest impact on particle uptake. Coating with bovine serum albumin, fetuin or protein G significantly increased particle internalization in microglial cells but not in neuronal cells. Coating with wheat germ agglutinin increased particle uptake in both N9 and differentiated SH-SY5Y cells but not in undifferentiated SH-SY5Y cells. Furthermore, internalization was shown to be an active process and indicators of caspase-dependent apoptosis revealed that gold nanoparticles did not have any cytotoxic effects. The present study thus demonstrates the suitability of gold nanoparticles and high-content analysis for assessing numerous variables in a stringently quantitative and statistically significant manner. Furthermore, the results presented herein showcase the feasibility of specifically targeting nanoparticles to distinct cell types.