Wielerella bovis gen. nov., sp. nov. a member of the family Neisseriaceae associated with bovine endocarditis.

Seven bacterial strains isolated from bovine endocarditis in six animals from different geographic regions were investigated in a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences placed all seven isolates on a distinct, monophyletic cluster in the family Neisseriaceae with closest similarity to type strains of Alysiella filiformis (97.06 %) and Kingella kingae (96.34 %). Whole genome sequence analysis of isolates confirmed their species status, with an average nucleotide identity >96 % between isolates and <80 % to other type species of genera of Neisseriaceae while digital DNA-DNA hybridization values were >80 % and<18 %, respectively. The DNA G+C content was 42.5-43.0 mol%. Whole genome sequence based phylogeny showed the isolates being monophyletic and separated from established genera, thereby forming a new genus within the family Neisseriaceae. Similarly, analysis of MALDI-TOF MS reference spectra clustered the isolates close together and clearly separated from other genera, making this the method of choice for identification. Biochemical markers based on classical as well as commercial identification schemes allowed separation from closely related Neisseriaceae genera, even though the new taxon is biochemically not very active. Major fatty acids are C12 : 0, C14 : 0 and C16 : 0. The major quinone is ubiquinone Q-8. In the polar lipid profile, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phospholipid were predominant. We propose the novel genus Wielerella with the type species Wielerella bovis gen. nov., sp. nov. The type strain is CCUG 44465T (=DSM 113289T=JF 2483T) isolated post mortem from a cow with endocarditis in Switzerland.

Novel HPAIV H5N8 Reassortant (Clade 2.3.4.4b) Detected in Germany.

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) was detected in a greater white-fronted goose in January 2020 in Brandenburg, Germany, and, in February 2020, in domestic chickens belonging to a smallholding in Baden-Wuerttemberg, Germany. Full-genome sequencing was conducted on the MinION platform, enabling further phylogenetic analyses. The virus of clade 2.3.4.4b holds six segments from a Eurasian/Asian/African HPAIV H5N8 reassortant and two segments from low pathogenic avian influenza H3N8 subtype viruses recently detected in wild birds in Central Russia. These new entries continue to show the reassortment potential of the clade 2.3.4.4 H5Nx viruses, underlining the necessity for full-genome sequencing and continuous surveillance.

Isolation and antimicrobial susceptibility of Streptococcus castoreus isolated from carcasses of European beavers (Castor fiber) in Germany.

Streptococcus (S.) castoreus was isolated from seven beavers (Castor fiber), which were found dead in the federal state of Brandenburg, Germany, and submitted to the Berlin-Brandenburg State laboratory for post mortem diagnostics. The isolates originated from various inflammatory processes where they were associated with other Gram negative and Gram positive aerobic and/or anaerobic bacteria (i. e. Actinobacillus sp., species of the Actinomycetaceae family, coliform bacteria, Fusobacterium sp., Prevotella sp.), but also from cloacal swabs. Testing their antimicrobial susceptibility, all S. castoreus isolates and the type strain S. castoreus (DSM 17536) were classified as being susceptible to penicillin G, ampicillin, amoxicillin-clavulanic acid, 1st generation cephalosporins, erythromycin, tetracycline, and chloramphenicole but intermediate to gentamicin. Considering both the polymicrobial flora isolated from the inflamed tissues and the fact that antimicrobials of a narrow spectrum (namely penicillins, 1st generation cephalosporins, macrolids and lincosamids) can be toxic to rodents, chloramphenicol might be a suitable drug for treatment of beavers suffering from S. castoreus (mixed)-bacterial infections.

Visibility of weak contrasts in subsurface scattering near-field microscopy.

We present a systematic study of the effect of the tip vibration amplitude in scattering-type scanning near-field optical microscopy (s-SNOM) on the visibility of buried structures in terms of image contrast, signal strength, and noise. When varying the tip vibration amplitude the visibility of structures and the image contrast change in opposite directions. We address the question how to optimize the tip vibration amplitude in practice and discuss the respective advantages of either high contrast or low noise. Besides for subsurface imaging, our findings can also be applied to the imaging of structures with a low material contrast on the surface.

Cryptosporidium baileyi--infection in Red-breasted Merganser (Mergus serrator) ducklings from a zoological garden.

Infection of the Bursa of Fabricius with Cryptosporidium baileyi was diagnosed in a group of hand reared Red-breasted Merganser (Mergus serrator) ducklings during one breeding season in a German zoological garden.The birds had died in an emaciated and anaemic state after problems with spontaneous feeding. The bursae were infected with moderate to high numbers of cryptosporidia, which were associated with hyperplasia, degeneration and sloughing of the affected epithelial cells and mild heterophilic bursitis, lesions typically seen in bursal cryptosporidiosis in other avian species. In addition, lymphatic tissue was nearly absent, which was probably caused by chronic stress and malnutrition related to the rearing of these highly stress-sensitive birds in an artificial environment. Companion ducklings from stress-resistant, spontaneously feeding species from the zoological collection were used to calm the Mergansers, but may have introduced the cryptosporidia into the rearing boxes. Another possible source for the introduction of C. baileyi were adult mallards (Anas platyrhynchos), which were used to increase the hatching rate by a phase of natural breeding in the middle third of the incubation period of the Merganser eggs.

Quasi-analytical model for scattering infrared near-field microscopy on layered systems.

We present a quantitative quasi-analytical model to predict and analyze signals on layered samples measured by infrared scattering-type scanning near-field optical microscopy. Our model predictions are compared to experimental data and to fully retarded calculations based on a point dipole approximation of the tip. The model is used to study the influence of the tip vibration amplitude and of the tip radius on the near-field contrasts of samples with particularly small variations in the layer thickness. Additionally the influence of a dielectric capping layer on the tip-substrate coupling is analyzed. When inversely applied, our calculation opens the possibility to extract the local layer thickness of thin films or the dielectric functions that allow one to draw conclusions on the material composition, conductivity or crystal structure on the nanoscale.

Tailored antiplatelet therapy can overcome clopidogrel and aspirin resistance--the BOchum CLopidogrel and Aspirin Plan (BOCLA-Plan) to improve antiplatelet therapy.

BACKGROUND: Dual antiplatelet therapy using acetylsalicylic acid (ASA, aspirin) and clopidogrel is of great importance following coronary stenting. However, the variable platelet inhibitory effectiveness compromises the antithrombotic advantages provided by dual antiplatelet therapy. The aim of this single-center prospective study was to reduce the low response incidence of dual antiplatelet therapy with ASA and clopidogrel according to a prespecified therapy algorithm. METHODS: Platelet function testing using whole blood aggregometry (Chronolog 590) was performed 48 hours following coronary stenting (for either acute coronary syndromes or stable coronary artery disease) on 504 patients. The antiplatelet therapy included a loading dose of 600 mg clopidogrel and 500 mg ASA, followed by 75 mg clopidogrel and 100 mg ASA once daily. Clopidogrel low responders (CLR: >5 ohm; adenosine diphosphate (ADP) 5 µM) and/or ASA low responders (ALR: >0 ohm; arachidonic acid 10 µM) were treated according to a structured therapy plan: in the case of CLR, the maintenance + dose was doubled (repeated loading dose followed by 150 mg daily), and when still ineffective ticlopidine or prasugrel, if available and not contraindicated, were used. ALR was treated by increasing the dose to 300 mg in a first step or to 500 mg ASA when the first modification did not take effect sufficiently. In addition, ADP receptor antagonist 2-methylthioadenosine 5'-monophosphate triethylammonium salt (MeSAMP) testing and ASA incubation were performed to rule out either a platelet ADP-receptor defect or an ASA pharmacokinetic resistance. RESULTS: Of the total cohort of 504 patients, we detected 30.8% clopidogrel low-responders and 19.4% aspirin low-responders. For ALR, with a dose adjustment of 300 mg ASA daily, 94.6% of ALR were effectively treated and the residual 5.4% by administration of daily dosages of 500 mg ASA. This means that after modification of the ASA maintenance dose, all initial ALRs had an adequate antiplatelet response. The results for clopidogrel revealed that 69% of the CLR were treated effectively by increasing the clopidogrel dose to 150 mg daily. When prasugrel was not available or contraindicated, 12.7% of the remaining low responders showed an adequate result after being switched to ticlopidine. Consequently, by applying the therapy algorithm, we were able to reduce the CLR prevalence by 86.6%. On including prasugrel in the therapy plan, we were finally able to eliminate thienopyridine low response. In addition, no ADP receptor defect was found in this study as a potential reason for CLR. We identified the following factors associated with both CLR and ALR status: acute coronary syndromes, positive troponin values as well as diabetes mellitus and elevated HbA1C values and a higher platelet count. Furthermore, our data revealed for CLR elevated C-reactive protein values and a high PREDICT-score (including an age >65 years, acute coronary syndrome, diabetes mellitus, renal failure, and reduced left ventricular function) as risk factors. The following factors correlated with the risk of ASA low response: patients with elevated hemoglobin, serum creatinine and C-reactive protein values. In addition, medication with nitrates reduced the risk of being CLR. As also holds true for CLR, we found the PREDICT-score to be correlated to the risk of being ALR. However, by far the strongest risk factor for CLR or ALR was the fact of dual resistance. CONCLUSION: Following a structured therapy plan based on a "test and treat" strategy, the prevalence of clopidogrel or aspirin low response can be significantly reduced and the risk of inadequate dual antiplatelet therapy minimized.

Pantoprazole does not influence the antiplatelet effect of clopidogrel-a whole blood aggregometry study after coronary stenting.

Recent attention has been drawn to a potential drug-drug interaction observed between clopidogrel and proton pump inhibitors (PPIs). However, this potential interaction may not be a class effect of PPIs. We investigated if pantoprazole, which has a different metabolism than omeprazole, diminishes the effectiveness of clopidogrel. Our study included 336 patients (mean age 64.6 years; 106 women) 48 hours after percutaneous coronary stent implantation with a loading dose of 600 mg clopidogrel hydrogensulfate and 500 mg aspirin, followed by 75 mg clopidogrel and 100 mg aspirin daily. Whereas 188 patients (59 women) were not given any PPI comedication, 122 patients received pantoprazole and 26 either omeprazole or esomeprazole. The platelet aggregation followed by impedance aggregometry (in Ohm) was induced by 5 mmol/L adenosine diphosphate. The percentage of clopidogrel low-response (CLR) was similar between the non-PPI group [2.75 Ohm (confidence interval, CI: 2.25-3.26); 21.9% CLR] and the pantoprazole group [2.33 Ohm (CI: 1.79-2.87); 16.4% CLR] but higher in patients treated with omeprazole/esomeprazole (3.00 Ohm (CI: 1.49-4.51); 30.8% CLR). Multivariate regression analysis reveals that the risk of CLR in the pantoprazole comedication group was not increased compared with the group without any PPI [odds ratio 0.59 (CI: 0.31-1.13) 0.11]. Our data suggest that pantoprazole does not diminish the antiplatelet effectiveness of clopidogrel early after coronary stenting. Therefore, the use of pantoprazole seems preferable in patients treated with clopidogrel when a concomitant medication with a PPI is indicated.

Comparing the antiplatelet effect of clopidogrel hydrogensulfate and clopidogrel besylate: a crossover study.

BACKGROUND: Clopidogrel hydrogensulfate is a thienopyridine acting as an important antiplatelet agent alone or in combination with acetyl salicylic acid to prevent cardiovascular complications. A different clopidogrel salt, clopidogrel besylate, was approved in Germany as a "new drug" in May 2008. Only one study with 46 healthy men compared the plasma concentrations of both clopidogrel formulas. In our crossover study we measured the antiplatelet effect of clopidogrel hydrogensulfate (CHS, clopidogrel bisulfate) and clopidogrel besylate (CB) using two techniques, whole blood impedance aggregometry and flow cytometry in healthy subjects. METHODS: Twenty-one healthy volunteers (14 male, 7 female, mean age 36.3 years) were treated either with CHS or CB (300 mg loading, followed by 75 mg/day) and after a wash-out period of at least 21 days, the participants were switched to the other clopidogrel salt in a crossover design. Blood samples were drawn before and 2, 4 and 48 h after the initial dose was taken. Flow cytometry measurements of CD62P (P-selectin) expression were done at baseline and 48 h thereafter with three different ADP concentrations (5, 15, 50 micromol/L ADP). Whole blood impedance aggregometry testing (Chrono-log Model 590) was performed at baseline and after 2, 4 and 48 h with two ADP concentrations (5 and 20 micromol/L ADP). RESULTS: Using flow cytometry, the mean inhibitory effect of clopidogrel on the CD62P expression was similar and no significant differences were noted in subjects treated with either of the clopidogrel formulas for hydrogensulfate or besylate salt (5 micromol/L ADP: 8.12 +/- 5.53 CHS vs. 6.48 +/- 5.01 CB; 15 micromol/L ADP: 9.33 +/- 6.44 CHS vs. 8.99 +/- 8.27 CB; 50 micromol/L ADP: 11.17 +/- 6.81 CHS vs. 9.52 +/- 6.17 CB). It is important to note that clopidogrel CB shows similar and conspicuously high interindividual variability as was reported earlier on CHS. We observed both possibilities, subjects responding less to the hydrogensulfate salt, but better to the besylate salt, and vice versa. Using aggregometry, both salt formulas achieved similar inhibitory effects regarding initial platelet function (2 h/5 micromol/L ADP: CHS 4.5 +/- 3.66 Omega; CB 3.89 +/- 3.81 Omega and 4 h/5 micromol/L ADP: CHS 5.78 +/- 3.51 Omega; CB 4.89 +/- 4.03 Omega) as well as during the maintenance phase (48 h/5 micromol/L ADP: CHS 2.86 +/- 2.92 Omega; CB 3.43 +/- 3.06 Omega). Once again the aggregometry results for CB showed a similarly high interindividual variability as also holds true for CHS. Some subjects had a better antiplatelet effect with clopidogrel besylate and vice versa with clopidogrel hydrogensulfate. CONCLUSION: The crossover study using whole blood aggregometry and flow cytometry shows no overall significant difference in the antiplatelet effect of clopidogrel hydrogensulfate as compared to clopidogrel besylate. However, it is important to note that besides high interindividual there is also high intraindividual variability between the two different clopidogrel formulas. We observed both: subjects responding less to besylate salt, but better to hydrogensulfate salt, and vice versa.

Highly pathogenic avian influenza virus (H5N1) in frozen duck carcasses, Germany, 2007.

We conducted phylogenetic and epidemiologic analyses to determine sources of outbreaks of highly pathogenic avian influenza virus (HPAIV), subtype H5N1, in poultry holdings in 2007 in Germany, and a suspected incursion of HPAIV into the food chain through contaminated deep-frozen duck carcasses. In summer 2007, HPAIV (H5N1) outbreaks in 3 poultry holdings in Germany were temporally, spatially, and phylogenetically linked to outbreaks in wild aquatic birds. Detection of HPAIV (H5N1) in frozen duck carcass samples of retained slaughter batches of 1 farm indicated that silent infection had occurred for some time before the incidental detection. Phylogenetic analysis established a direct epidemiologic link between HPAIV isolated from duck meat and strains isolated from 3 further outbreaks in December 2007 in backyard chickens that had access to uncooked offal from commercial deep-frozen duck carcasses. Measures that will prevent such undetected introduction of HPAIV (H5N1) into the food chain are urgently required.

Helcococcus ovis, an emerging pathogen in bovine valvular endocarditis.

The initial isolation of Helcococcus ovis from a valvular thrombus prompted us to investigate the prevalence of this bacterium in bovine valvular endocarditis. Specimens from 55 affected hearts were examined by culture using Columbia blood agar and cross streaking the inoculated plate with a Staphylococcus aureus strain. As confirmed by 16S rRNA gene sequencing, H. ovis was isolated with an unexpectedly high frequency of 33%, predominantly as heavy growth and pure culture. The majority of H. ovis isolates showed distinct satellitism around S. aureus and pyridoxal dependency, resembling "nutritionally variant streptococci" (now assigned to the genera Abiotrophia and Granulicatella). Using the API rapid ID 32 Strep, API ZYM, and Rosco Diatabs systems, incongruent results were obtained for alkaline phosphatase, beta-galactosidase, beta-glucuronidase, and leucine aminopeptidase activities. Based on the satellitism/pyridoxal dependency; hemolysis on blood agar; the API rapid ID 32 Strep results for arginine dihydrolase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, and pyroglutamic acid arylamidase activities; hippurate hydrolysis; and acidification of sucrose, a scheme for the identification of H. ovis and its differentiation from other members of the Helcococcus genus and the pyridoxal-dependent species Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans is proposed. By establishing specific fluorescence in situ hybridization, large H. ovis aggregates were specifically detected within the fibrinous exudate of the valvular thrombi. Our results demonstrate for the first time that H. ovis represents an emerging pathogen in bovine valvular endocarditis that is frequently isolated if appropriate culture conditions are used.

How to optimise clopidogrel therapy? Reducing the low-response incidence by aggregometry-guided therapy modification.

The inhibitory platelet effect of clopidogrel is insufficient in approximately 5 to 30% of patients. These low responders (LR) face a significantly higher risk of cardiovascular complications. The therapeutic management of LR is still undefined. In the present study, we evaluate a novel therapeutic algorithm to reduce the incidence of clopidogrel resistance. One hundred sixty-one patients on 100 mg of aspirin co-medication underwent elective coronary stenting and were given an initial dosage of 600 mg clopidogrel, followed by 75 mg clopidogrel daily. 48 h later, the platelet responsiveness was tested with ADP (5-20 microM) stimulation by impedance aggregometry (Chronolog 590). A significant rise in impedance (>5 Omega after 6 minutes, aggregation index >65%) was defined as LR. In this subgroup, platelets were stimulated with the selective P2Y(12)-ADP receptor antagonist 2-MeS-AMP. One hundred twenty-three patients were clopidogrel-responders (76.4%) and 38 patients were LR (23.6%). A defect of the ADP-receptor P2Y(12) was found in three out of 38 LR (7.9%). Inhibition of platelet aggregation indicating clopidogrel-responsiveness was achieved with either a clopidogrel high-dose regimen (22/38, 57.9%); a repeat loading dose, doubling the maintenance dose) or with an alternative therapy with ticlopidine (8/38 (21.1%); 250 mg twice daily). Thus the incidence of LR was reduced from 23.6% to 5.0%. Our aggregometer-guided therapeutic algorithm reduced the relative percentage of clopidogrel LR by 78.9%. This approach could prove to be helpful in achieving a further decrease in the incidence of clopidogrel resistance.

[Pancytopenia, arthralgia and myeloneuropathy due to copper deficiency]. / Kupfermangel als seltene Ursache von Panzytopenie, Arthralgien und Gangstörungen.

BACKGROUND: Copper deficiency leads to hematologic disorders like pancytopenia. In addition, myeloneuropathy was described in a few cases reports. CASE REPORT: A 71-year-old woman was hospitalized because of increasing pancytopenia and ataxic gait, that resulted in a near-complete inability to walk without assistance. Additional symptoms included arthralgia, reduced appetite and weight loss. Laboratory studies revealed a proteinuria of 3,700 mg/day. Magnetic resonance imaging of the cervical and thoracic spine revealed a wedge-shaped signal intensity in the dorsal part as a sign of damage in this area. A copper deficiency was then identified as the likely underlying cause for the low blood cell counts and neurologic deficits. In this patient, the copper deficiency may have resulted from a disturbance in absorption due to a partial gastrectomy (modified Billroth I) 10 years ago and due to urinary copper loss in view of mesangioproliferative glomerulonephritis. A therapy with copper gluconate 3 x 3 mg/day was initiated. Within 2 weeks, blood cell counts normalized and appetite became normal again; just so, arthralgia disappeared. The neurologic symptoms persisted, even though the copper substitution continued for 6 months. CONCLUSION: Copper deficiency may be a differential diagnosis for hematologic abnormalities like pancytopenia, even if a disorder of intestinal resorption or a proteinuria occurs. Myeloneuropathy is a rare complication of this deficiency. Hemograms may become normal after treatment with oral copper gluconate, but at least in the case presented here, neurologic symptomes did not show any improvement.