Fatty Acid Amide Hydrolase Deficiency Is Associated with Deleterious Cardiac Effects after Myocardial Ischemia and Reperfusion in Mice.

Ischemic cardiomyopathy leads to inflammation and left ventricular (LV) dysfunction. Animal studies provided evidence for cardioprotective effects of the endocannabinoid system, including cardiomyocyte adaptation, inflammation, and remodeling. Cannabinoid type-2 receptor (CB2) deficiency led to increased apoptosis and infarctions with worsened LV function in ischemic cardiomyopathy. The aim of our study was to investigate a possible cardioprotective effect of endocannabinoid anandamide (AEA) after ischemia and reperfusion (I/R). Therefore, fatty acid amide hydrolase deficient (FAAH)-/- mice were subjected to repetitive, daily, 15 min, left anterior descending artery (LAD) occlusion over 3 and 7 consecutive days. Interestingly, FAAH-/- mice showed stigmata such as enhanced inflammation, cardiomyocyte loss, stronger remodeling, and persistent scar with deteriorated LV function compared to wild-type (WT) littermates. As endocannabinoids also activate PPAR-α (peroxisome proliferator-activated receptor), PPAR-α mediated effects of AEA were eliminated with PPAR-α antagonist GW6471 i.v. in FAAH-/- mice. LV function was assessed using M-mode echocardiography. Immunohistochemical analysis revealed apoptosis, macrophage accumulation, collagen deposition, and remodeling. Hypertrophy was determined by cardiomyocyte area and heart weight/tibia length. Molecular analyses involved Taqman® RT-qPCR and immune cells were analyzed with fluorescence-activated cell sorting (FACS). Most importantly, collagen deposition was reduced to WT levels when FAAH-/- mice were treated with GW6471. Chemokine ligand-2 (CCL2) expression was significantly higher in FAAH-/- mice compared to WT, followed by higher macrophage infiltration in infarcted areas, both being reversed by GW6471 treatment. Besides restoring antioxidative properties and contractile elements, PPAR-α antagonism also reversed hypertrophy and remodeling in FAAH-/- mice. Finally, FAAH-/--mice showed more substantial downregulation of PPAR-α compared to WT, suggesting a compensatory mechanism as endocannabinoids are also ligands for PPAR-α, and its activation causes lipotoxicity leading to cardiomyocyte apoptosis. Our study gives novel insights into the role of endocannabinoids acting via PPAR-α. We hypothesize that the increase in endocannabinoids may have partially detrimental effects on cardiomyocyte survival due to PPAR-α activation.

Two alternative DNA extraction methods to improve the detection of Mycobacterium-tuberculosis-complex members in cattle and red deer tissue samples.

BACKGROUND: Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis and M. caprae, is a notifiable animal disease in Germany. Diagnostic procedure is based on a prescribed protocol that is published in the framework of German bTB legislation. In this protocol small sample volumes are used for DNA extraction followed by real-time PCR analyses. As mycobacteria tend to concentrate in granuloma and the infected tissue in early stages of infection does not necessarily show any visible lesions, it is likely that DNA extraction from only small tissue samples (20-40 mg) of a randomly chosen spot from the organ and following PCR testing may result in false negative results. In this study two DNA extraction methods were developed to process larger sample volumes to increase the detection sensitivity of mycobacterial DNA in animal tissue. The first extraction method is based on magnetic capture, in which specific capture oligonucleotides were utilized. These nucleotides are linked to magnetic particles and capture Mycobacterium-tuberculosis-complex (MTC) DNA released from 10 to 15 g of tissue material. In a second approach remaining sediments from the magnetic capture protocol were further processed with a less complex extraction protocol that can be used in daily routine diagnostics. A total number of 100 tissue samples from 34 cattle (n = 74) and 18 red deer (n = 26) were analyzed with the developed protocols and results were compared to the prescribed protocol. RESULTS: All three extraction methods yield reliable results by the real-time PCR analysis. The use of larger sample volume led to a sensitivity increase of DNA detection which was shown by the decrease of Ct-values. Furthermore five samples which were tested negative or questionable by the official extraction protocol were detected positive by real time PCR when the alternative extraction methods were used. By calculating the kappa index, the three extraction protocols resulted in a moderate (0.52; protocol 1 vs 3) to almost perfect agreement (1.00; red deer sample testing with all protocols). CONCLUSION: Both new methods yielded increased detection rates for MTC DNA detection in large sample volumes and consequently improve the official diagnostic protocol.

Molecular analysis of Coxiella burnetii in Germany reveals evolution of unique clonal clusters.

The causative agent of Q fever, Coxiella burnetii, is a query agent occurring naturally all over the world. We studied 104 German Coxiella burnetii strains/DNA samples obtained between 1969 and 2011 using a 14 microsatellite marker Multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA) technique. We were able to divide our collection into 32 different genotypes clustered into four major groups (A-D). Two of these (A and C) formed predominant clonal complexes that covered 97% of all studied samples. Group C consisted exclusively of cattle-associated isolates/DNA specimens, while group A comprised all other affected species including all sheep-derived strains/DNA samples. Within this second cluster, two major genotypes (A1, A2) were identified. Genotype A2 occurred in strains isolated from ewes in northern and central Germany, whereas genotype A1 was found in most areas of Germany. MLVA analysis of C. burnetii strains from neighbouring countries revealed a close relationship to German strains. We thus hypothesize that there is a western and central European cluster of C. burnetii. We identified predominant genotypes related to relevant host species and geographic regions which is in line with findings of the Dutch Q fever outbreak (2007-2010). Furthermore three of our analyzed German strains are closely related to the Dutch outbreak clone. These findings support the theory of predominant genotypes in the context of regional outbreaks. Our results show that a combination of 8 MLVA markers provides the highest discriminatory power for attributing C. burnetii isolates to genotypes. For future epidemiological studies we propose the use of three MLVA markers for easy and rapid classification of C. burnetii into 4 main clusters.

Effects of odors on sleep quality in 139 healthy participants.

The present study aimed to systematically examine whether laurinal, orange odor, and a specifically designed "perfume" influence sleep quality. During sleep, healthy participants (n = 139) were presented with odor or no odor through nose clips for fourteen consecutive nights (phase one). We collected physiological parameters together with subjective reports. Later on, longer lasting effects of this manipulation were examined for the following fourteen nights (phase two) without exposition to odors. Additionally, olfactory, cognitive and non-cognitive measures were conducted before phase one, between both phases and after phase two. One-way analyses of variance for repeated measures with nights and condition (1 vs 2) as the within-subject factor and odor condition (0, 1, 2 or 3) together with odor pleasantness rating as between-subject factor, was employed to analyse data. Overall, the present results demonstrated that the odor condition in comparison to control had no consistent effect on sleep in healthy participants which can be possibly explained by exposure to odors via nose clips. However, the analyses indicated that the individual pleasantness of odors enhanced the positive assessment of sleep quality. Altogether, the present results indicate that the subjective perception of an odor's hedonic value appears to be crucial for sleep quality, not the odors themselves.

Mismatch repair deficiency occurs very rarely in seminomas.

BACKGROUND: Dense tumor-associated lymphocyte infiltration is linked to mismatch repair (MMR) deficiency in colorectal and endometrial cancer. MMR deficiency is of high clinical importance as MMR deficient cancers tend to react favorably to treatment with immune checkpoint inhibitors. Strong lymphocytic infiltration is a morphological hallmark of seminomas. We thus asked whether seminomas may exhibit MMR deficiency at relevant frequency. METHODS: To screen for tumors with MMR deficiency, protein expression of MLH1, PMS2, MSH2, and MSH6 was analyzed by immunohistochemistry (IHC) on a tissue microarray (TMA) containing 574 seminomas. RESULTS: In total, 536 cases were evaluable resulting in 481 seminomas with unequivocally intact MMR protein expression. In 55 cancers, one or several IHC stains were equivocal and lacked detectable MMR protein in both tumor and stromal cells. Large section IHC analysis of all 55 equivocal cases demonstrated substantial staining issues due to improper fixation in 54 cases and identified one tumor with clear-cut MLH1 and PMS2 protein loss. This seminoma showed homogeneous loss of MLH1 and PMS2 in the entire tumor mass whereas minor adjacent foci of associated germ cell neoplasia in situ (GCNIS) were MMR intact. Polymerase chain reaction (PCR) analysis using the 5 microsatellite loci of the "Bethesda Panel" revealed instability in 1 of 4 interpretable loci ("MSI-low") and additional instability of the complex tetra-penta repeat locus MYCL1 in this tumor. CONCLUSIONS: In summary, one single seminoma with MMR deficiency, characterized by protein loss of MLH1 and PMS2, was identified among 536 interpretable seminomas (0.19%). MMR deficiency is not a relevant determinant of lymphocyte influx in seminoma.

Genome Sequence Analysis of Clostridium chauvoei Strains of European Origin and Evaluation of Typing Options for Outbreak Investigations.

Black quarter caused by Clostridium (C.) chauvoei is an important bacterial disease that affects cattle and sheep with high mortality. A comparative genomics analysis of 64 C. chauvoei strains, most of European origin and a few of non-European and unknown origin, was performed. The pangenome analysis showed limited new gene acquisition for the species. The accessory genome involved prophages and genomic islands, with variations in gene composition observed in a few strains. This limited accessory genome may indicate that the species replicates only in the host or that an active CRISPR/Cas system provides immunity to foreign genetic elements. All strains contained a CRISPR type I-B system and it was confirmed that the unique spacer sequences therein can be used to differentiate strains. Homologous recombination events, which may have contributed to the evolution of this pathogen, were less frequent compared to other related species from the genus. Pangenome single nucleotide polymorphism (SNP) based phylogeny and clustering indicate diverse clusters related to geographical origin. Interestingly the identified SNPs were mostly non-synonymous. The study demonstrates the possibility of the existence of polymorphic populations in one host, based on strain variability observed for strains from the same animal and strains from different animals of one outbreak. The study also demonstrates that new outbreak strains are mostly related to earlier outbreak strains from the same farm/region. This indicates the last common ancestor strain from one farm can be crucial to understand the genetic changes and epidemiology occurring at farm level. Known virulence factors for the species were highly conserved among the strains. Genetic elements involved in Nicotinamide adenine dinucleotide (NAD) precursor synthesis (via nadA, nadB, and nadC metabolic pathway) which are known as potential anti-virulence loci are completely absent in C. chauvoei compared to the partial inactivation in C. septicum. A novel core-genome MLST based typing method was compared to sequence typing based on CRISPR spacers to evaluate the usefulness of the methods for outbreak investigations.

ChromID® CARBA Agar Fails to Detect Carbapenem-Resistant Enterobacteriaceae With Slightly Reduced Susceptibility to Carbapenems.

After first detections of carbapenemase-producing Enterobacteriaceae (CPE) in animals, the European Union Reference Laboratory for Antimicrobial Resistance has provided a protocol for the isolation of carbapenemase-producing Escherichia (E.) coli from cecum content and meat. Up to now, only few isolates were recovered using this procedure. In our experience, the choice of the selective agar is important for the efficacy of the method. Currently, the use of the prevailing method fails to detect CPE that exhibit a low resistance against carbapenems. Thus, this study aims to evaluate the suitability of selective media with antibiotic supplements and commercial ChromID® CARBA agar for a reliable CPE detection. For comparative investigations, detection of freeze-dried carbapenemase-resistant bacteria was studied on different batches of the ChromID® CARBA agar as well as on MacConkey agar supplemented with 1 mg/L cefotaxime and 0.125 mg/L meropenem (McC+CTX+MEM). The suitability of the different media was assessed within a time of 25 weeks, starting at least six weeks before expiration of the media. Carbapenem-resistant isolates exhibiting a serine-based hydrolytic resistance mechanism (e.g., bla KPC genes) were consistently detected over 25 weeks on the different media. In contrast, carbapenemase producers with only slightly reduced susceptibility and exhibiting a zinc-catalyzed activity (e.g., bla VIM, bla NDM, and bla IMP) could only be cultivated on long-time expired ChromID® CARBA, but within the whole test period on McC+CTX+MEM. Thus, ChromID® CARBA agar appears to be not suitable for the detection of CPE with slightly increased minimum inhibitory concentrations (MIC) against carbapenems, which have been detected in German livestock and thus, are of main interest in the national monitoring programs. Our data are in concordance with the results of eleven state laboratories that had participated in this study with their ChromID® CARBA batches routinely used for the German CPE monitoring. Based on the determined CPE detection rate, we recommend the use of McC+CTX+MEM for monitoring purposes. This study indicates that the use of ChromID® CARBA agar might lead to an underestimation of the current CPE occurrence in food and livestock samples.

Cowpox virus transmission from pet rats to humans, Germany.

We describe a cluster of cowpox virus (CPXV) infections in humans that occurred near Munich, Germany, around the beginning of 2009. Previously, only sporadic reports of CPXV infections in humans after direct contact with various animals had been published. This outbreak involved pet rats from the same litter.

Evaluating diagnostic tests for bovine tuberculosis in the southern part of Germany: A latent class analysis.

Germany has been officially free of bovine tuberculosis since 1996. However, in the last years there has been an increase of bovine tuberculosis cases, particularly in the southern part of Germany, in the Allgäu region. As a consequence a one-time tuberculosis surveillance program was revisited with different premortal and postmortal tests. The aim of this paper was to estimate diagnostic sensitivities and specificities of the different tests used within this surveillance program. In the absence of a perfect test with 100% sensitivity and 100% specificity, thus in the absence of a gold standard, a Bayesian latent class approach with two different datasets was performed. The first dataset included 389 animals, tested with single intra-dermal comparative cervical tuberculin (SICCT) test, PCR and pathology; the second dataset contained 175 animals, tested with single intra-dermal cervical tuberculin (SICT) test, Bovigam® assay, pathology and culture. Two-way conditional dependencies were considered within the models. Additionally, inter-laboratory agreement (five officially approved laboratories) of the Bovigam® assay was assessed with Cohen's kappa test (21 blood samples). The results are given in posterior means and 95% credibility intervals. The specificities of the SICT test, SICCT test, PCR and pathology ranged between 75.8% [68.8-82.2%] and 99.0% [96.8-100%]. The Bovigam® assay stood out with a very low specificity (6.9% [3.6-11.1%]), though it had the highest sensitivity (95.7% [91.3-99.2%]). The sensitivities of the SICCT test, PCR, SICT test, pathology and culture varied from 57.8% [48.0-67.6%] to 88.9% [65.5-99.7%]. The prevalences were 19.8% [14.6-26.5%] (three-test dataset) and 7.7% [4.2-12.3%] (four-test dataset). Among all pairwise comparisons the highest agreement was 0.62 [0.15-1]). In conclusion, the specificity of the Bovigam® assay and the inter-laboratory agreement were lower than expected.

Real-time PCR protocol for the detection of porcine parvovirus in field samples.

This report describes a real-time polymerase chain reaction assay with SYBR Green for detection of a broad range of porcine parvoviruses (PPV) and accurate virus quantification in porcine tissues. The assay targets the VP2 gene of PPV and the porcine genomic c-myc gene for normalization. The detection limit of the SYBR Green reaction was shown to be equivalent to 6 x 10(0) to 6 x 10(1) PPV copies/reaction and the overall detection limit equivalent to 0.1 TCID(50)/100 microl. The assay was linear over a 10(7) dilution range of template concentrations. Other porcine pathogens involved in reproductive disorders such as porcine circovirus 2 (PCV-2), porcine reproductive and respiratory virus (PRRSV), Aujeszky's disease virus (PRV) and other parvoviruses such as feline parvovirus (FPV), canine parvovirus (CPV), minute virus of canines (MVC) and a human parvovirus (B19) were not detected by this assay.

Real-time PCR assay for the detection of tanapox virus and yaba-like disease virus.

The yatapoxvirus genus contains three members: tanapox virus (TPV), yaba-like disease virus (YLDV) and yaba monkey tumor virus (YMTV), two of which (TPV and YLDV) may infect humans. However, only a very small number of patients have been diagnosed with TPV outside Africa. Given the increased international travel and the similarity of clinical signs during the early stages of a TPV/YLDV infection as compared to diseases caused by agents of potential biological warfare, such as smallpox, monkeypox, tularemia and anthrax, the rapid and reliable recognition of a TPV/YLDV infection is crucial. A real-time PCR assay using TaqManchemistry was developed in order to identify unambiguously TPV/YLDV. Primers and probe targeting a 101bp region of the PstI L fragment of TPV, initial optimisations steps were carried out with YLDV DNA as template. Using probit regression analysis, the lower limit of detection was calculated to be ca. 8 copies per assay. A total of five TPV strains, one YDLV strain and scab-derived DNA from a patient with a TPV infection yielded specific amplification, whereas the DNA of YMTV was not amplified. Various viral and bacterial pathogens (n=29) associated with rash-causing illnesses were not detected using this assay.

[Severe purulent and necrotizing glossitis in a fallow deer (Dama dama) due to an infection with the involvement of Mannheimia granulomatis]. / Hochgradige eitrig-nekrotisierende Glossitis bei einem Damwild (Dama dama) nach Infektion unter Beteiligung von Mannheimia granulomatis.

Post mortem examination of a young fallow deer (Dama dama) revealed a severe purulent and necrotizing glossitis as well as a multifocal necrotizing and ulcerative rumenitis and typhlitis. The animal was cachectic. Mannheimia (M.) sp. was isolated from the tongue lesions and identified as M. granulomatis by MALDI-TOF MS and 16S rRNA sequencing. Mycosis and BVDV infection were excluded. Few publications are dealing with similar macroscopic findings associated with the isolation of M. granulomatis in cattle and roe deer. Therefore, M. granulomatis should also be taken into consideration when such lesions occur in other ruminants. Based on our findings in case of gross pathological lesions of the tongue of ruminants a Mannheimia granulomatis-infection should be investigated as well as the possible role of Fusobacterium necrophorum, Actinobacillus lignieresii or Actinomyces bovis.

Identification of bacteria isolated from veterinary clinical specimens using MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged as a rapid and accurate identification method for bacterial species. Although it has been successfully applied for the identification of human pathogens, it has so far not been well evaluated for routine identification of veterinary bacterial isolates. This study was performed to compare and evaluate the performance of MALDI-TOF MS based identification of veterinary bacterial isolates with commercially available conventional test systems. Discrepancies of both methods were resolved by sequencing 16S rDNA and, if necessary, the infB gene for Actinobacillus isolates. A total of 375 consecutively isolated veterinary samples were collected. Among the 357 isolates (95.2%) correctly identified at the genus level by MALDI-TOF MS, 338 of them (90.1% of the total isolates) were also correctly identified at the species level. Conventional methods offered correct species identification for 319 isolates (85.1%). MALDI-TOF identification therefore offered more accurate identification of veterinary bacterial isolates. An update of the in-house mass spectra database with additional reference spectra clearly improved the identification results. In conclusion, the presented data suggest that MALDI-TOF MS is an appropriate platform for classification and identification of veterinary bacterial isolates.

Draft Genome Sequence of Strain BF-4, a Lysinibacillus-Like Bacillus Isolated during an Anthrax Outbreak in Bavaria.

We report the draft genome sequence of Lysinibacillus sp. strain BF-4. Strain BF-4 has a notably small genome for a free-living bacillus, with a size of 2.63 Mbp. In agreement with phenotypic observations, the genome lacks genes essential for endospore formation.

Correlates of diagnostic accuracy in patients with nonspecific complaints.

OBJECTIVE: To investigate diagnostic accuracy in patient histories involving nonspecific complaints and the extent to which characteristics of physicians and structural properties of patient histories are associated with accuracy. METHODS: Six histories of patients presenting to the emergency department (ED) with nonspecific complaints were provided to 112 physicians: 36 ED physicians, 50 internists, and 26 family practitioners. Physicians listed the 3 most likely diagnoses for each history and indicated which cue(s) they considered crucial. Four weeks later, a subset of 20 physicians diagnosed the same 6 histories again. For each history, experts had previously determined the correct diagnoses and the diagnostic cues. RESULTS: Accuracy ranged from 14% to 64% correct diagnoses (correct diagnosis listed as the most likely) and from 29% to 87% correct differential diagnoses (correct diagnosis listed in the differential). Acute care physicians (ED physicians and internists) included the correct diagnosis in the differential in, on average, 3.4 histories, relative to 2.6 for the family practitioners (P = 0.001, d = .75). Diagnostic performance was fairly reliable (r = .61, P < 0.001). Clinical experience was negatively correlated with diagnostic accuracy (r = -.25, P = 0.008). Two structural properties of patient histories-cue consensus and cue substitutability-were significantly associated with diagnostic accuracy, whereas case difficulty was not. Finally, prevalence of diagnosis also proved significantly correlated with accuracy. CONCLUSIONS: Average diagnostic accuracy in cases with nonspecific complaints far exceeds chance performance, and accuracy varies with medical specialty. Analyzing cue properties in patient histories can help shed light on determinants of diagnostic performance and thus suggest ways to enhance physicians' ability to accurately diagnose cases with nonspecific complaints.

Evaluation of six commercial DNA extraction kits for recovery of Burkholderia pseudomallei DNA.

Six commercially available DNA extraction kits, as well as thermal lysis and proteinase K DNA extraction were evaluated regarding bacterial inactivation, DNA yield and purity, and their use in a Burkholderia pseudomallei real-time PCR. While all methods successfully inactivated the bacteria, by measuring DNA purity and the level of detection by real-time PCR, the proteinase K method was the most sensitive.

Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis.

Specific identification of Bacillus anthracis and differentiation from closely related Bacillus cereus and Bacillus thuringiensis strains is still a major diagnostic problem. Commercially available diagnostic kits targeting plasmid-markers cannot differentiate between B. anthracis, non-anthracis Bacillus species harbouring anthrax-specific virulence plasmids, and plasmidless B. anthracis strains. A TaqMan PCR assay was designed targeting sequences of gene locus BA_5345 of the B. anthracis strain Ames. Specificity was determined by using a panel of 328 Bacillus strains; sensitivity was determined by probit analysis. All B. anthracis isolates (n=92) were specifically detected by using the genomic TaqMan PCR assay whereas 236 strains belonging to 19 Bacillus species other than B. anthracis were PCR negative. The detection limit was determined to be 12.7 copies per reaction (95% confidence interval 10.2-17.5 copies). Here we present an extensively evaluated and - to our current knowledge - specific TaqMan PCR assay for the detection of B. anthracis based on a chromosomal marker.