Release kinetics of tumor necrosis factor-α and interleukin-1 receptor antagonist in the equine whole blood

Background: Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration dependently. Results: Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24–48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8–12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12–24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release. Conclusions: Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse

Comprehensive Assessment of the Virulence Factors sub 3, sub 6 and mcpA in the Zoonotic Dermatophyte Trichophyton benhamiae Using FISH and qPCR

Skin infections by keratinophilic fungi are commonly referred to as dermatophytosis and represent a major health burden worldwide. Although patient numbers are on the rise, data on virulence factors, their function and kinetics are scarce. We employed an ex vivo infection model based on guinea pig skin explants (GPSE) for the zoonotic dermatophyte Trichophyton (T.) benhamiae to investigate kinetics of the virulence factors subtilisin (sub) 3, sub 6, metallocarboxypeptidase A (mcpA) and isocitrate lyase (isol) at gene level for ten days. Fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) were used to detect and quantify the transcripts, respectively. Kingdom-spanning, species-specific and virulence factor-specific probes were successfully applied to isolated fungal elements showing inhomogeneous fluorescence signals along hyphae. Staining results for inoculated GPSE remained inconsistent despite thorough optimization. qPCR revealed a significant increase of sub 3- and mcpA-transcripts toward the end of culture, sub 6 and isol remained at a low level throughout the entire culture period. Sub 3 is tightly connected to the de novo formation of conidia during culture. Since sub 6 is considered an in vivo disease marker. However, the presented findings urgently call for further research on the role of certain virulence factors during infection and disease

The Influence of Glyphosate on the Microbiota and Production of Botulinum Neurotoxin During Ruminal Fermentation

Abstract The aim of the present study is to investigate the impact of glyphosate on the microbiota and on the botulinum neurotoxin (BoNT) expression during in vitro ruminal fermentation. This study was conducted using two DAISY II -incubators with four ventilated incubation vessels filled with rumen fluid of a 4-year-old non-lactating Holstein-Friesian cow. Two hundred milliliter rumen fluid and 800 ml buffer solution were used with six filter bags containing 500 mg concentrated feed or crude fiber-enriched diet. Final concentrations of 0, 1, 10, and 100 lg/ml of glyphosate in the diluted rumen fluids were added and incubated under CO 2 -aerated conditions for 48 h. The protozoal population was analyzed microscopically and the ruminal flora was characterized using the fluorescence in situ hybridization technique. Clostridium botulinum and BoNT were quantified using most probable number and ELISA, respectively. Results showed that glyphosate had an inhibitory effect on select groups of the ruminal microbiota, but increased the population of pathogenic species. The BoNT was produced during incubation when inoculum was treated with high doses of glyphosate. In conclusion, glyphosate causes dysbiosis which favors the production of BoNT in the rumen. The global regulations restrictions for the use of glyphosate should be re-evaluated

Identification of Zoophilic Dermatophytes Using MALDI-TOF Mass Spectrometry

Dermatophytoses represent a major health burden in animals and man. Zoophilic dermatophytes usually show a high specificity to their original animal host but a zoonotic transmission is increasingly recorded. In humans, these infections elicit highly inflammatory skin lesions requiring prolonged therapy even in the immunocompetent patient. The correct identification of the causative agent is often crucial to initiate a targeted and effective therapy. To that end, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents a promising tool. The objective of this study was to evaluate the reliability of species identification of zoophilic dermatophytes using MALDI-TOF MS. The investigation of isolates from veterinary clinical samples suspicious of dermatophytoses suggests a good MALDI-TOF MS based identification of the most common zoophilic dermatophyte Microsporum canis. Trichophyton (T.) spp. usually achieved scores only around the cutoff value for secure species identification because of a small number of reference spectra. Moreover, these results need to be interpreted with caution due to the close taxonomic relationship of dermatophytes being reflected in very similar spectra. In our study, the analysis of 50 clinical samples of hedgehogs revealed no correct identification using the provided databases, nor for zoophilic neither for geophilic causative agents. After DNA sequencing, adaptation of sample processing and an individual extension of the inhouse database, acceptable identification scores were achieved (T. erinacei and Arthroderma spp., respectively). A score-oriented distance dendrogram revealed clustering of geophilic isolates of four different species of the genus Arthroderma and underlined the close relationship of the important zoophilic agents T. erinacei, T. verrucosum and T. benhamiae by forming a subclade within a larger cluster including different dermatophytes. Taken together, MALDI-TOF MS proofed suitable for the identification of zoophilic dermatophytes provided fresh cultures are used and the reference library was previously extended with spectra of laboratory-relevant species. Performing independent molecular methods, such as sequencing, is strongly recommended to substantiate the findings from morphologic and MALDI-TOF MS analyses, especially for uncommon causative agents

Hypothesis and theory : a pathophysiological concept of stroke-induced acute phase response and increased intestinal permeability leading to secondary brain damage

Gut integrity impairment leading to increased intestinal permeability (IP) is hypothesized to be a trigger of critically illness. Approximately 15–20% of human ischemic stroke (IS) victims require intensive care, including patients with impaired level of consciousness or a high risk for developing life-threatening cerebral edema. Local and systemic inflammatory reactions are a major component of the IS pathophysiology and can significantly aggravate brain tissue damage. Intracerebral inflammatory processes following IS have been well studied. Until now, less is known about systemic inflammatory responses and IS consequences apart from a frequently observed post-IS immunosuppression. Here, we provide a hypothesis of a crosstalk between systemic acute phase response (APR), IP and potential secondary brain damage during acute and subacute IS stages supported by preliminary experimental data. Alterations of the acute phase proteins (APPs) C-reactive protein and lipopolysaccharide-binding protein and serum level changes of antibodies directed against Escherichia coli-cell extract antigen (IgA-, IgM-, and IgG-anti-E. coli) were investigated at 1, 2, and 7 days following IS in ten male sheep. We found an increase of both APPs as well as a decrease of all anti-E. coli antibodies within 48 h following IS. This may indicate an early systemic APR and increased IP, and underlines the importance of the increasingly recognized gut-brain axis and of intestinal antigen release for systemic immune responses in acute and subacute stroke stages

Futtermittelhygiene

Im vorliegenden Heft der Schriftenreihe werden die Ergebnisse von zwei Projekten vorgestellt, die sich mit futtermittelhygienischen Aspekten befassten und von Wissenschaftlern der Universität Leipzig erarbeitet wurden. Untersuchungen zur Beeinflussung der Toxinexpression von Clostridium botulinum Typ B und C unter in-vitro-Bedingungen / Untersuchungen zu Vorkommen und Verbreitung von Clostridium botulinum in Rinderbeständen des Freistaates Sachsen / Mykotoxinscreening in Blut, Galle und Milch bei gesunden und kranken Kühe

Hypothesis and Theory: A Pathophysiological Concept of Stroke-Induced Acute Phase Response and Increased Intestinal Permeability Leading to Secondary Brain Damage

Gut integrity impairment leading to increased intestinal permeability (IP) is hypothesized to be a trigger of critically illness. Approximately 15–20% of human ischemic stroke (IS) victims require intensive care, including patients with impaired level of consciousness or a high risk for developing life-threatening cerebral edema. Local and systemic inflammatory reactions are a major component of the IS pathophysiology and can significantly aggravate brain tissue damage. Intracerebral inflammatory processes following IS have been well studied. Until now, less is known about systemic inflammatory responses and IS consequences apart from a frequently observed post- IS immunosuppression. Here, we provide a hypothesis of a crosstalk between systemic acute phase response (APR), IP and potential secondary brain damage during acute and subacute IS stages supported by preliminary experimental data. Alterations of the acute phase proteins (APPs) C-reactive protein and lipopolysaccharide-binding protein and serum level changes of antibodies directed against Escherichia coli-cell extract antigen (IgA-, IgM-, and IgG-anti-E. coli) were investigated at 1, 2, and 7 days following IS in ten male sheep. We found an increase of both APPs as well as a decrease of all anti- E. coli antibodies within 48 h following IS. This may indicate an early systemic APR and increased IP, and underlines the importance of the increasingly recognized gut-brain axis and of intestinal antigen release for systemic immune responses in acute and subacute stroke stages

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database - the quality controlled standard tool for routine identification of human and animal pathogenic fungi

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.This study was supported by an National Health and Medical Research Council of Australia (NH&MRC) grant [#APP1031952] to W Meyer, S Chen, V Robert, and D Ellis; CNPq [350338/2000-0] and FAPERJ [E-26/103.157/2011] grants to RM Zancope-Oliveira; CNPq [308011/2010-4] and FAPESP [2007/08575-1] Fundacao de Amparo Pesquisa do Estado de So Paulo (FAPESP) grants to AL Colombo; PEst-OE/BIA/UI4050/2014 from Fundacao para a Ciencia e Tecnologia (FCT) to C Pais; the Belgian Science Policy Office (Belspo) to BCCM/IHEM; the MEXBOL program of CONACyT-Mexico, [ref. number: 1228961 to ML Taylor and [122481] to C Toriello; the Institut Pasteur and Institut de Veil le Sanitaire to F Dromer and D Garcia-Hermoso; and the grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and the Fundacao de Amparo a Pesquisa do Estado de Goias (FAPEG) to CM de Almeida Soares and JA Parente Rocha. I Arthur would like to thank G Cherian, A Higgins and the staff of the Molecular Diagnostics Laboratory, Division of Microbiology and Infectious Diseases, Path West, QEII Medial Centre. Dromer would like to thank for the technical help of the sequencing facility and specifically that of I, Diancourt, A-S Delannoy-Vieillard, J-M Thiberge (Genotyping of Pathogens and Public Health, Institut Pasteur). RM Zancope-Oliveira would like to thank the Genomic/DNA Sequencing Platform at Fundacao Oswaldo Cruz-PDTIS/FIOCRUZ [RPT01A], Brazil for the sequencing. B Robbertse and CL Schoch acknowledge support from the Intramural Research Program of the NIH, National Library of Medicine. T Sorrell's work is funded by the NH&MRC of Australia; she is a Sydney Medical School Foundation Fellow.info:eu-repo/semantics/publishedVersio