Phaidra - University of Veterinary Medicine Vienna
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Little agreement among methodologies to determine fecal glucocorticoid metabolites in a mountain ungulate
No full textFaecal glucocorticoid metabolites (FGMs) have gained relevance in ecological studies and population monitoring, allowing non-invasive remote sampling without the need to capture and handle animals. Enzyme immunoassays (EIAs) and radioimmunoassays (RIAs) are commonly used for FGM determination. Although these methods should be validated for each species, non-validated tests are still widely used to determine FGMs in wildlife. Near-infrared reflectance spectroscopy (NIRS) is a predictive method requiring calibration against reference methods used to assess FGMs in wildlife. EIAs and RIAs have been utilized to determine FGMs in chamois (Rupicapra spp.), a medium-sized mountain ungulate.
This study aims to assess the potential of NIRS to determine FGMs and to evaluate the correlation among analytical methods used to determine FGMs in chamois. Faecal samples from 125 Alpine chamois (Rupicapra rupicapra rupicapra) and 125 Pyrenean chamois (R. pyrenaica pyrenaica) were collected from the field, frozen at − 20 °C, lyophilized, grounded, and scanned using a NIRSystems 5000 monochromator over a 1108–2492 nm wavelength. After this non-destructive NIRS analysis, FGMs were extracted and analysed using four immunoassays previously used in chamois studies: a 125-I-corticosterone RIA, a cortisol EIA, and two 11-oxoetiocholanolone EIAs (72a and 72T). Only the 11-oxoetiocholanolone 72T EIA has been validated for Alpine chamois. NIRS predictions were calibrated and cross-validated for each of the four immunoassays. The correlation among the four immunoassays was assessed using Spearman\u27s rank.
The coefficient of determination for NIRS calibration (R²) values ranged from 0.37 to 0.75, and the ratio of performance to deviation values from 1.2 to 1.6. Therefore, NIRS could not predict FGM concentration in chamois faeces. This could be due to the complexity and variability of the FGM detected by the immunoassays as reference methods, and by potential interference of other compounds in the faecal matrix. The correlation among the immunoassays was low overall.
As a conclusion, NIRS cannot be recommended for measuring FGMs in chamois. The low correlation among the immunoassays used for FGM determination raises concern about the reliability of previous studies using non-validated methods in chamois. Only biologically validated tests should be used to assess FGMs to avoid incorrect inferences about biological responses in physiological, conservational, and ecological studies or population monitoring. These conclusions are applicable beyond the species studied here
Documentation of antimicrobial resistance data in veterinary practices in Germany
No full textAntimikrobielle Resistenz stellt nach wie vor eine der größten Herausforderungen für die öffentliche Gesundheit dar. Der Einsatz von Antibiotika im Allgemeinen und der übermäßige Einsatz im Besonderen erzeugen einen Selektionsdruck, der das Auftreten und die Ausbreitung von antibiotikaresistenten Erregern in allen Bereichen (Human-, Veterinär- und Umweltbereich) begünstigt. Daher wurden zahlreiche (inter-)nationale Monitoringsysteme zur Überwachung des Einsatzes antimikrobieller Substanzen etabliert, jedoch ist die Erfassung von Antibiotikaresistenzen auf der Ebene landwirtschaftlicher Betriebe nach wie vor unzureichend.
In Deutschland ist die Dokumentation des Einsatzes antimikrobieller Mittel bei Tieren, die der Lebensmittelgewinnung dienen, seit 1975 vorgeschrieben. Seitdem hat sich ein standardisiertes Dokumentations- und Berichtsformat etabliert. Die Meldung der Ergebnisse von Empfindlichkeitstests auf Betriebsebene unterliegt jedoch keiner gesetzlichen Regelung. Obgleich einige rechtliche Rahmenbedingungen für die Dokumentation bestehen, lässt sich eine erhebliche Heterogenität hinsichtlich des Inhalts und der Form der dokumentierten Daten beobachten.
Ziel dieser Studie ist es, die bestehende Heterogenität der Dokumentation in den deutschen Tierarztpraxen ausführlich zu beschreiben, um eine Datenstruktur zu entwickeln, die für ein zukünftiges Antibiotikaresistenzmonitoring auf Betriebsebene, basierend auf den veterinärdiagnostischen Routinedaten, genutzt werden kann. Dafür werden der Datentransfer und die Datenaufbereitung beschrieben sowie die Dokumentation der einzelnen Komponenten der Empfindlichkeitsprüfung in teilnehmenden Praxen bzw. Laboren diskutiert. Darauf aufbauend wird aufgezeigt, welche der Komponenten zwingend in ein Monitoring aufgenommen werden sollten und warum. Abschließend werden zwei mögliche Wege für ein Monitoringsystem vorgestellt und Empfehlungen gegeben.Antimicrobial resistance remains one of the greatest public health challenges of our time. By creating a selection pressure, the use of antimicrobials in general and the inappropriate overuse especially, fuels the emergence and spread of antimicrobial resistant bacteria in all sectors (human, veterinary and environment). Therefore, numerous (inter-)national surveillance systems have been established to monitor the use of antimicrobial agents, yet the recording of antimicrobial resistance at the farm level remains scarce.
In Germany, the documentation of the use of antimicrobial agents in food-producing animals has been mandatory since 1975, with a standardised documentation and reporting format in place today. However, the reporting of susceptibility test results at the farm level is not subject to legal regulation. While there are some regulatory frameworks regarding documentation, we observe considerable heterogeneity in terms of content and format of the documented data.
The objective of this investigation is to provide a detailed account of the existing heterogeneity of documentation in German veterinary practices, in order to develop a data structure that can be used for future AMR monitoring at the farm level, based on routine veterinary diagnostic data. To this end, the data transfer and processing are described, and the documentation of the individual components of the susceptibility tests in participating practices and laboratories is discussed. Based on this, it will be shown which components should be included in a monitoring system and why. Finally, two possible paths for a monitoring system will be presented, along with recommendations
Lead Contamination in Meat and Offal from Game (Ruminants), Destined for Raw Feeding of Dogs and Retailed in Austria
No full textBullet-derived lead (Pb) is a food safety hazard in meat from hunted wild game. Dogs can be exposed to alimentary Pb when fed with meat from wild game. We studied Pb contamination in 47 commercial “bones-and-raw-food” (BARF) packages from wild game meat and offal sold in Austria. Samples were first tested with a metal detector (calibrated for 2.5 mm diameter objects), then by X-ray, and Pb content was determined in sample areas free from metal-dense particles (as assessed in radiographs). Three samples tested positive by the metal detector (particle sizes ranged from 3 mm × 2 mm × 2 mm to 10 mm × 8 mm × 5 mm), with two particles from lead and one from tombac. Metal-dense objects were found in radiographs of 35/47 samples. In 13/47 samples, particles > 1 mm in diameter, and in 33/47 samples, smaller ones (median of five particles per sample) were noted. Lead content was above 1.5 mg/kg wet weight in 19/47 samples and exceeded the limit for general animal feed of 10 mg/kg wet weight in 5 of these 19 samples. The Pb contents as well as the presence of metal particles indicate that there is a feed safety issue in the samples analyzed. By a metal detector, the presence of large fragments was indicated in three samples, which casts doubt on whether all producers use a metal detector to scan the packages before delivery
Lactation-related dynamics of bacterial and fungal microbiomes in feces of sows and gut colonization in suckling and newly weaned piglets
No full textChanges in the gut microbial composition of the sow during lactation may influence the gut microbial colonization in their offspring, for which less information was available in the literature. This study aimed to assess: 1) the changes that occur in the bacterial and fungal communities in sow feces during the 28-d lactation period as well as in gastric and cecal digesta of piglets until one week after weaning, and 2) bacterial and fungal taxa in cecal digesta of the piglets postweaning that associate with fecal consistency. Aside from sow milk, piglets had access to creep feed from day of life (DoL) 3. Fecal samples from sows for microbial analysis were collected (n = 20) on days postpartum (DPP) 1, 6, 13, 20, and 27, as well as from weaned piglets for fecal scoring on DoL 30 and 34. Gastric and cecal digesta of piglets was collected on DoL3, 7, 14, 21, 28, 31, and 35 (n = 5/sex/DoL). Progressing lactation affected bacterial and fungal communities in sow feces, including 10.3- and 3.0-fold increases in the relative abundances of Lactobacillus from DPP1 to 6 and Kazachstania from DPP1 to 13, respectively (P < 0.001). Although time- and gut-site-related differences existed, bacterial and fungal taxa found in sow feces were also present in gastric and cecal digesta of piglets, which supports their role in gut colonization in neonatal piglets. In piglets, bacterial and fungal alpha-diversities showed certain fluctuations during the suckling period, whereby weaning affected the fungal than bacterial diversity at both gut sites (P < 0.05). At both gut sites, Lactobacillus largely increased from DoL3 to 7 and remained a dominating taxon until DoL35 (P < 0.05). Postweaning, plant-glycan fermenters (e.g., Prevotella-9) seemed to replace milk-glycan fermenting Fusobacterium and Bacteroides (P < 0.05). In gastric and cecal digesta, Kazachstania, Tausonia, Candida, and Blumeria were dominating fungi from DoL3 to 35, with Kazachstania becoming even more dominant postweaning (P < 0.001). Fecal consistency was softer on DoL34 than 30 (P < 0.05). Correlation analysis identified that softer feces were linked to the relative abundances of plant-glycan and proteolytic bacterial taxa including pathobionts (e.g., Clostridium sensu stricto) in the cecum on DoL34. However, the potential association between cecal mold and plant-pathogenic fungi Talaromyces, Mrakia, and Blumeria and softer feces are worth investigating in the future in relation to (gut) health of piglets
Solving technical issues in flow cytometry to characterize porcine CD8α/β expressing lymphocytes
No full textThe CD8 molecule is a cell surface receptor and well described as co-receptor on T cells, binding directly to the major histocompatibility complex class I on antigen presenting cells. CD8 antigens are comprised of two distinct polypeptide chains, the α and the β chain. In the pig, the CD8 receptor is expressed by several lymphocyte subsets, including Natural Killer cells, γδ T cells and antigen experienced CD4+ αβ T cells. On these cell populations CD8 is expressed as αα homodimers. Porcine cytolytic T cells on the other hand exclusively express CD8 αβ heterodimers. Several monoclonal antibodies (mAbs) for either of the two chains are available and are frequently used in flow cytometry. We observed that distinct combinations of mAb clones for CD8α and CD8β chains can cause troubles in multi-color staining panels. Therefore, we aimed for an in-depth study of the usage of different CD8-specific mAb clones and optimizing co-staining strategies for flow cytometry. We tested mAb clones 11/295/33 and 76–2–11 for the detection of CD8α and mAb clones PPT23 and PG164A for the detection of CD8β. The results indicate that the CD8α clone 11/295/33 should not be used together with either of the two CD8β clones in the same incubation step, as co-staining led to a highly reduced ability of CD8β mAb binding and loss in signal in flow cytometry. This can lead to potential false results in detecting CD8αβ cytolytic T cells. In case of the CD8α mAb clone 76–2–11, no inhibition in binding of either CD8β mAb clones was observed, making it the preferred choice in multi-color staining panels. The obtained data will help in future panel designs for flow cytometry in the pig and therefore improving studies of porcine immune cells
Molecular basis of plastoquinone reduction in plant cytochrome b6f
No full textA multi-subunit enzyme, cytochrome b6f (cytb6f), provides the crucial link between photosystems I and II in the photosynthetic membranes of higher plants, transferring electrons between plastoquinone (PQ) and plastocyanin. The atomic structure of cytb6f is known, but its detailed catalytic mechanism remains elusive. Here we present cryogenic electron microscopy structures of spinach cytb6f at 1.9 Å and 2.2 Å resolution, revealing an unexpected orientation of the substrate PQ in the haem ligand niche that forms the PQ reduction site (Qn). PQ, unlike Qn inhibitors, is not in direct contact with the haem. Instead, a water molecule is coordinated by one of the carbonyl groups of PQ and can act as the immediate proton donor for PQ. In addition, we identify water channels that connect Qn with the aqueous exterior of the enzyme, suggesting that the binding of PQ in Qn displaces water through these channels. The structures confirm large movements of the head domain of the iron-sulfur protein (ISP-HD) towards and away from the plastoquinol oxidation site (Qp) and define the unique position of ISP-HD when a Qp inhibitor (2,5-dibromo-3-methyl-6-isopropylbenzoquinone) is bound. This work identifies key conformational states of cytb6f, highlights fundamental differences between substrates and inhibitors and proposes a quinone-water exchange mechanism
Information mismatch in PHH3-assisted mitosis annotation leads to interpretation shifts in H&E slide analysis
No full textThe count of mitotic figures (MFs) observed in hematoxylin and eosin (H&E)-stained slides is an important prognostic marker, as it is a measure for tumor cell proliferation. However, the identification of MFs has a known low inter-rater agreement. In a computer-aided setting, deep learning algorithms can help to mitigate this, but they require large amounts of annotated data for training and validation. Furthermore, label noise introduced during the annotation process may impede the algorithms\u27 performance. Unlike H&E, where identification of MFs is based mainly on morphological features, the mitosis-specific antibody phospho-histone H3 (PHH3) specifically highlights MFs. Counting MFs on slides stained against PHH3 leads to higher agreement among raters and has therefore recently been used as a ground truth for the annotation of MFs in H&E. However, as PHH3 facilitates the recognition of cells indistinguishable from H&E staining alone, the use of this ground truth could potentially introduce an interpretation shift and even label noise into the H&E-related dataset, impacting model performance. This study analyzes the impact of PHH3-assisted MF annotation on inter-rater reliability and object level agreement through an extensive multi-rater experiment. Subsequently, MF detectors, including a novel dual-stain detector, were evaluated on the resulting datasets to investigate the influence of PHH3-assisted labeling on the models\u27 performance. We found that the annotators\u27 object-level agreement significantly increased when using PHH3-assisted labeling (F1: 0.53 to 0.74). However, this enhancement in label consistency did not translate to improved performance for H&E-based detectors, neither during the training phase nor the evaluation phase. Conversely, the dual-stain detector was able to benefit from the higher consistency. This reveals an information mismatch between the H&E and PHH3-stained images as the cause of this effect, which renders PHH3-assisted annotations not well-aligned for use with H&E-based detectors. Based on our findings, we propose an improved PHH3-assisted labeling procedure
PET imaging identifies anti-inflammatory effects of fluoxetine and a correlation of glucose metabolism during epileptogenesis with chronic seizure frequency
No full textThe serotonergic system has shown to be altered during epileptogenesis and in chronic epilepsy, making selective serotonin reuptake inhibitors interesting candidates for antiepileptogenic therapy. In this study, we aimed to evaluate disease-modifying effects of fluoxetine during experimental epileptogenesis. Status epilepticus (SE) was induced by lithium-pilocarpine, and female rats were treated either with vehicle or fluoxetine over 15 days. Animals were subjected to 18F-FDG (7 days post-SE), 18F-GE180 (15 days post-SE) and 18F-flumazenil positron emission tomography (PET, 21 days post-SE). Uptake (18F-FDG), volume of distribution (18F-GE180) and binding potential (18F-flumazenil) were calculated. In addition, hyperexcitability testing and video-EEG monitoring were performed. Fluoxetine treatment did not alter brain glucose metabolism. 18F-GE180 PET indicated lower neuroinflammation in the hippocampus of treated animals (-22.6%, p = 0.042), but no differences were found in GABAA receptor density. Video-EEG monitoring did not reveal a treatment effect on seizure frequency. However, independently of the treatment, hippocampal FDG uptake 7 days after SE correlated with seizure frequency during the chronic phase (r = -0.58; p = 0.015). Fluoxetine treatment exerted anti-inflammatory effects in rats during epileptogenesis. However, this effect did not alter disease outcome. Importantly, FDG-PET in early epileptogenesis showed biomarker potential as higher glucose metabolism correlated to lower seizure frequency in the chronic phase
Evaluation of \u27In-Parlour Scoring\u27 (IPS) to Detect Lameness in Dairy Cows during Milking
No full textThe objective of this study was to evaluate the potential of \u27In-Parlour Scoring\u27 (IPS) as an alternative to locomotion scoring in herringbone, side-by-side, and tandem milking parlours in Austria. Between January and May 2023, a total of 990 observations were conducted on 495 cows across eleven Austrian dairy farms by two investigators working simultaneously but independently of each other. The observation criteria included shifting weight, claw conformation, and obvious disorders of the distal limb. Locomotion scoring was conducted on all cows within 24 h of assessment in the milking parlour using a scale of 1 to 5 (LCS 1: not lame; LCS 5: severely lame). Functional hoof trimming was performed within ten days after IPS. The following indicators were identified as useful for predicting lameness (LCS ≥ 3): shifting weight, abnormal weight distribution, swollen heel, hock joint or interdigital space, skin lesion on the lateral hock, claw position score, digital dermatitis lesions, short dorsal claw wall, and hyperextension of one claw. The reliability of the individual indicators for intra- and inter-rater assessment exhibited considerable variation ((weighted) kappa values: -0.0020-0.9651 and -0.0037-1.0, respectively). The specificity and sensitivity for the prediction of lame cows were calculated to be ≥ 96% and ≤ 24%, respectively. It was demonstrated that a one-time IPS has limited suitability for lameness assessment on Austrian dairy farms with herringbone, side-by-side, and tandem milking parlours
Differential expression of the type III secretion system genes in Yersinia ruckeri: Preliminary investigations in different environmental conditions
No full textType III secretion system (T3SS) is an important virulence system in Gram-negative bacteria. In this investigation, different environmental conditions that regulate the expression of T3SS genes in Yersinia ruckeri were investigated aimed at obtaining a better understanding about its modulation after various environmental challenges. Four isolates of Y. ruckeri CSF007-82, ATCC29473, A7959-11 and YRNC10 were cultivated under the diverse in vitro challenges iron depletion, high salt, low pH and in the presence of fish serum or in the fish cell culture (Chinook Salmon Embryo - CHSE). The transcriptional modulation of the chromosomal genes ysaV, ysaC, ysaJ and prgH of ysa were investigated using quantitative real-time PCR. The expression of prgH, ysaV, ysaC and ysaJ was differentially expressed in all four strains under evaluation. The highest gene expression levels were observed for Y. ruckeri YRNC10 AN after addition of 0.3 M NaCl in Luria Bertani broth. The results obtained from this study provide initial insights into T3SS responses in Y. ruckeri, which pave the way for further studies aimed at expanding our knowledge on the functional roles of the T3SS genes in Y. ruckeri