IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology

BACKGROUND: Control of brucellosis in livestock, wildlife and humans depends on the reliability of the methods used for detection and identification of bacteria. In the present study, we describe the evaluation of the recently established real-time PCR assay based on the Brucella-specific insertion sequence IS711 with blood samples from 199 wild boars (first group of animals) and tissue samples from 53 wild boars (second group of animals) collected in Switzerland. Results from IS711 real-time PCR were compared to those obtained by bacterial isolation, Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and indirect ELISA (i-ELISA). RESULTS: In the first group of animals, IS711 real-time PCR detected infection in 11.1% (16/144) of wild boars that were serologically negative. Serological tests showed different sensitivities [RBT 15.6%, c-ELISA 7.5% and i-ELISA 5.5%] and only 2% of blood samples were positive with all three tests, which makes interpretation of the serological results very difficult. Regarding the second group of animals, the IS711 real-time PCR detected infection in 26% of animals, while Brucella spp. could be isolated from tissues of only 9.4% of the animals. CONCLUSION: The results presented here indicate that IS711 real-time PCR assay is a specific and sensitive tool for detection of Brucella spp. infections in wild boars. For this reason, we propose the employment of IS711 real-time PCR as a complementary tool in brucellosis screening programs and for confirmation of diagnosis in doubtful cases.

Real-time Human Motion Capture with Simple Marker Sets and Monocular Video

In this paper we present a hybrid method to track human motions in real-time. With simplified marker sets and monocular video input, the strength of both marker-based and marker-free motion capturing are utilized: A cumbersome marker calibration is avoided while the robustness of the marker-free tracking is enhanced by referencing the tracked marker positions. An improved inverse kinematics solver is employed for real-time pose estimation. A computer-visionbased approach is applied to refine the pose estimation and reduce the ambiguity of the inverse kinematics solutions. We use this hybrid method to capture typical table tennis upper body movements in a real-time virtual reality application.

[Field validation of a real-time PCR test for the detection of Mycoplasma hyopneumoniae in nasal swabs of live pigs]

Enzootic pneumonia (EP) of pigs, caused by Mycoplasma hyopneumoniae has been a notifiable disease in Switzerland since May 2003. The diagnosis of EP has been based on multiple methods, including clinical, bacteriological and epidemiological findings as well as pathological examination of lungs (mosaic diagnosis). With the recent development of a real-time PCR (rtPCR) assay with 2 target sequences a new detection method for M. hyopneumoniae became available. This assay was tested for its applicability to nasal swab material from live animals. Pigs from 74 herds (average 10 pigs per herd) were tested. Using the mosaic diagnosis, 22 herds were classified as EP positive and 52 as EP negative. From the 730 collected swab samples we were able to demonstrate that the rtPCR test was 100% specific. In cases of cough the sensitivity on herd level of the rtPCR is 100%. On single animal level and in herds without cough the sensitivity was lower. In such cases, only a positive result would be proof for an infection with M. hyopneumoniae. Our study shows that the rtPCR on nasal swabs from live pigs allows a fast and accurate diagnosis in cases of suspected EP.

Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

In order to improve the diagnosis of enzootic pneumonia (EP) in pigs two real-time polymerase chain reaction (rtPCR) assays for the detection of Mycoplasma hyopneumoniae in bronchial swabs from lung necropsies were established and validated in parallel. As a gold standard, the current "mosaic diagnosis" was taken, including epidemiological tracing, clinical signs, macro- and histopathological lesions of the lungs and immunofluorescence. One rtPCR is targeting a repeated DNA element of the M. hyopneumoniae genome (REP assay), the other a putative ABC transporter gene (ABC assay). Both assays were shown to be specific for M. hyopneumoniae and did not cross react with other bacteria and mollicutes from pig. With material from pigs of defined EP-negative farms the two assays showed to be 100% specific. When testing lungs from pig farms with EP, the REP assay detected 50% and the ABC assay 90% of the farms as positive. Both tests together detected all positive farms. Within a positive herd the two assays tested similarly with on average over 90% of the lung samples analysed from a single farm showing positive scores. A series of samples with suspicion of EP and samples from pigs with diseases other than respiratory taken from current routine diagnostic was assayed. None of the assays showed false positive results. The sensitivities in this sample group were 50% for the REP and 70% for the ABC assays and for both assays together 85%. The two assays run in parallel are therefore a valuable tool for the improvement of the current diagnosis of EP.

Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

Wearable schizophrenia treatment with real-time affective context awareness

The Personal Health Assistant Project (PHA) is a pilot system implementation sponsored by the Kozani Region Governors’ Association (KRGA) and installed in one of the two major public hospitals of the city of Kozani. PHA is intended to demonstrate how a secure, networked, multipurpose electronic health and food benefits digital signage system can transform common TV sets inside patient homes or hospital rooms into health care media players and facilitate information sharing and improve administrative efficiency among private doctors, public health care providers, informal caregivers, and nutrition program private companies, while placing individual patients firmly in control of the information at hand. This case evaluation of the PHA demonstration is intended to provide critical information to other decision makers considering implementing PHA or related digital signage technology at other institutions and public hospitals around the globe.

Application of real-time fluorescent PCR for quantitative assessment of Neospora caninum infections in organotypic slice cultures of rat central nervous system tissue

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection.

Real-time observation of the charge-transfer-to-solvent dynamics

Intermolecular electron-transfer reactions have a crucial role in biology, solution chemistry and electrochemistry. The first step of such reactions is the expulsion of the electron to the solvent, whose mechanism is determined by the structure and dynamical response of the latter. Here we visualize the electron transfer to water using ultrafast fluorescence spectroscopy with polychromatic detection from the ultraviolet to the visible region, upon photo-excitation of the so-called charge transfer to solvent states of aqueous iodide. The initial emission is short lived (~60 fs) and it relaxes to a broad distribution of lower-energy charge transfer to solvent states upon rearrangement of the solvent cage. This distribution reflects the inhomogeneous character of the solvent cage around iodide. Electron ejection occurs from the relaxed charge transfer to solvent states with lifetimes of 100–400 fs that increase with decreasing emission energy.

Simultaneous detection and discrimination of virulent and benign Dichelobacter nodosus in sheep of flocks affected by foot rot and in clinically healthy flocks by competitive real-time PCR.

Ovine foot rot caused by Dichelobacter nodosus is affecting sheep worldwide. The current diagnostic methods are difficult and cumbersome. Here, we present a competitive real-time PCR based on allelic discrimination of the protease genes aprV2 and aprB2. This method allows direct detection and differentiation of virulent and benign D. nodosus from interdigital skin swabs in a single test. Clinically affected sheep harbored high loads of only virulent strains, whereas healthy sheep had lower loads of predominantly benign strains.

A relation between screening masses and real-time rates

Thermal screening masses related to the conserved vector current are determined for the case that the current carries a non-zero Matsubara frequency, both in a weak-coupling approach and through lattice QCD. We point out that such screening masses are sensitive to the same infrared physics as light-cone real-time rates. In particular, on the perturbative side, the inhomogeneous Schrödinger equation determining screening correlators is shown to have the same general form as the equation implementing LPM resummation for the soft-dilepton and photon production rates from a hot QCD plasma. The static potential appearing in the equation is identical to that whose soft part has been determined up to NLO and on the lattice in the context of jet quenching. Numerical results based on this potential suggest that screening masses overshoot the free results (multiples of 2πT) more strongly than at zero Matsubara frequency. Four-dimensional lattice simulations in two-flavour QCD at temperatures of 250 and 340 MeV confirm the non-static screening masses at the 10% level. Overall our results lend support to studies of jet quenching based on the same potential at T ≳ 250 MeV.

Real-time simulation of large open quantum spin systems driven by dissipation

We consider a large quantum system with spins 12 whose dynamics is driven entirely by measurements of the total spin of spin pairs. This gives rise to a dissipative coupling to the environment. When one averages over the measurement results, the corresponding real-time path integral does not suffer from a sign problem. Using an efficient cluster algorithm, we study the real-time evolution from an initial antiferromagnetic state of the two-dimensional Heisenberg model, which is driven to a disordered phase, not by a Hamiltonian, but by sporadic measurements or by continuous Lindblad evolution.

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

BACKGROUND: Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP. RESULTS: By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%). CONCLUSIONS: There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease