Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses.

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Report of amoebic disease in a colony of Western honey bees (Apis mellifera).

The amoeba Malpighamoeba mellificae is the etiologic agent of amoebic (amoeba) disease of Western honey bees (Apis mellifera). M. mellificae damages the Malpighian tubules, which is believed to weaken and kill the host bee. Here, the authors describe the detection of this organism in a honey bee colony in the Yukon Territory, Canada. The Malpighian tubules of 14% (7/50) of the adult worker bees were discolored dark brown. Fifteen bees screened using conventional polymerase chain reaction for the 18S gene of M. mellificae were positive for the pathogen. Histologically, the lumens of Malpighian tubules were packed with amoebae, causing dilation of the tubules and attenuation and loss of the tubular epithelium. This phylogenetic analysis places M. mellificae in a new clade, a sister group to the Entamoebidae. This work provides a foundation for further investigation into the distribution, prevalence, and pathology associated with M. mellificae infection.

Divergent Serpentoviruses in Free-Ranging Invasive Pythons and Native Colubrids in Southern Florida, United States.

Burmese python (Python bivittatus) is an invasive snake that has significantly affected ecosystems in southern Florida, United States. Aside from direct predation and competition, invasive species can also introduce nonnative pathogens that can adversely affect native species. The subfamily Serpentovirinae (order Nidovirales) is composed of positive-sense RNA viruses primarily found in reptiles. Some serpentoviruses, such as shingleback nidovirus, are associated with mortalities in wild populations, while others, including ball python nidovirus and green tree python nidovirus can be a major cause of disease and mortality in captive animals. To determine if serpentoviruses were present in invasive Burmese pythons in southern Florida, oral swabs were collected from both free-ranging and long-term captive snakes. Swabs were screened for the presence of serpentovirus by reverse transcription PCR and sequenced. A total serpentovirus prevalence of 27.8% was detected in 318 python samples. Of the initial swabs from 172 free-ranging pythons, 42 (24.4%) were positive for multiple divergent viral sequences comprising four clades across the sampling range. Both sex and snout-vent length were statistically significant factors in virus prevalence, with larger male snakes having the highest prevalence. Sampling location was statistically significant in circulating virus sequence. Mild clinical signs and lesions consistent with serpentovirus infection were observed in a subset of sampled pythons. Testing of native snakes (n = 219, 18 species) in part of the python range found no evidence of python virus spillover; however, five individual native snakes (2.3%) representing three species were PCR positive for unique, divergent serpentoviruses. Calculated pairwise uncorrected distance analysis indicated the newly discovered virus sequences likely represent three novel genera in the subfamily Serpentovirinae. This study is the first to characterize serpentovirus in wild free-ranging pythons or in any free-ranging North America reptile. Though the risk these viruses pose to the invasive and native species is unknown, the potential for spillover to native herpetofauna warrants further investigation.

Multi-Institutional Retrospective Case-Control Study Evaluating Clinical Outcomes of Foals with Small Intestinal Strangulating Obstruction: 2000-2020.

Lower survival has been reported in foals than adults with small intestinal strangulating obstruction (SISO), but age-dependent outcomes have not been examined directly. Hospital records were collected from five US academic referral hospitals. It was hypothesized that foals would exhibit lower survival than case-matched adults. Foal cases 6-months-of-age or younger, and adult cases between 2- and 20-years-of-age were collected. Data revealed 24 of 25 (96.0%) foals and 66 of 75 (88.0%) adults that were recovered from surgery for SISO survived to hospital discharge. Sixteen of the total 41 (39.0%) foals studied were euthanized intraoperatively, whereas 30 of 105 (28.6%) adults were euthanized intraoperatively. Common lesions in foals that were recovered from surgery were volvulus (n = 13) and intussusception (n = 5), whereas common lesions in adults were volvulus (n = 25) and strangulating lipoma (n = 23). This study was limited by incomplete medical records, relatively small sample size, and lack of long-term follow-up. Unexpectedly, short-term survival tended to be higher in foals than adults and may have been partly driven by case selection prior to referral or surgery or decision-making intraoperatively. More optimism toward surgical treatment of foals with SISO may be warranted.

Measurement of Dark Ice-Ablator Mix in Inertial Confinement Fusion.

We present measurements of ice-ablator mix at stagnation of inertially confined, cryogenically layered capsule implosions. An ice layer thickness scan with layers significantly thinner than used in ignition experiments enables us to investigate mix near the inner ablator interface. Our experiments reveal for the first time that the majority of atomically mixed ablator material is "dark" mix. It is seeded by the ice-ablator interface instability and located in the relatively cooler, denser region of the fuel assembly surrounding the fusion hot spot. The amount of dark mix is an important quantity as it is thought to affect both fusion fuel compression and burn propagation when it turns into hot mix as the burn wave propagates through the initially colder fuel region surrounding an igniting hot spot. We demonstrate a significant reduction in ice-ablator mix in the hot-spot boundary region when we increase the initial ice layer thickness.

Measurement of L-shell emission from mid-Z targets under non-LTE conditions using Transmission Grating Spectrometer and DANTE power diagnostics.

In this work, we present the measurement of L-band emission from buried Sc/V targets in experiments performed at the OMEGA laser facility. The goal of these experiments was to study non-local thermodynamic equilibrium plasmas and benchmark atomic physics codes. The L-band emission was measured simultaneously by the time resolved DANTE power diagnostic and the recently fielded time integrated Soreq-Transmission Grating Spectrometer (TGS) diagnostic. The TGS measurement was used to support the spectral reconstruction process needed for the unfolding of the DANTE data. The Soreq-TGS diagnostic allows for broadband spectral measurement in the 120 eV-2000 eV spectral band, covering L- and M-shell emission of mid- and high-Z elements, with spectral resolution λ/Δλ = 8-30 and accuracy better than 25%. The Soreq-TGS diagnostic is compatible with ten-inch-manipulator platforms and can be used for a wide variety of high energy density physics, laboratory astrophysics, and inertial confinement fusion experiments.

Demonstration of Geometric Effects and Resonant Scattering in the X-Ray Spectra of High-Energy-Density Plasmas.

In a plasma of sufficient size and density, photons emitted within the system have a probability of being reabsorbed and reemitted multiple times-a phenomenon known in astrophysics as resonant scattering. This effect alters the ratio of optically thick to optically thin lines, depending on the plasma geometry and viewing angle, and has significant implications for the spectra observed in a number of astrophysical scenarios, but has not previously been studied in a controlled laboratory plasma. We demonstrate the effect in the x-ray spectra emitted by cylindrical plasmas generated by high power laser irradiation, and the results confirm the geometrical interpretation of resonant scattering.

Establishment of transfusion-relevant bacteria reference strains for red blood cells.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCC) are susceptible to bacterial contamination despite cold storage. A reliable evaluation of strategies to minimize the risk of RBCC-associated bacterial transmission requires the use of suitable reference bacteria. Already existing Transfusion-Relevant Bacteria Reference Strains (TRBRS) for platelet concentrates fail to grow in RBCC. Consequently, the ISBT TTID, Working Party, Bacterial Subgroup, conducted an international study on TRBRS for RBCC. MATERIALS AND METHODS: Six bacterial strains (Listeria monocytogenes PEI-A-199, Serratia liquefaciens PEI-A-184, Serratia marcescens PEI-B-P-56, Pseudomonas fluorescens PEI-B-P-77, Yersinia enterocolitica PEI-A-105, Yersinia enterocolitica PEI-A-176) were distributed to 15 laboratories worldwide for enumeration, identification, and determination of growth kinetics in RBCC at days 7, 14, 21, 28, 35 and 42 of storage after low-count spiking (10-25 CFU/RBCC). RESULTS: Bacterial proliferation in RBCC was obtained for most strains, except for S. marcescens, which grew only at 4 of 15 laboratories. S. liquefaciens, S. marcescens, P. fluorescens and the two Y. enterocolitica strains reached the stationary phase between days 14 and 21 of RBCC storage with a bacterial concentration of approximately 109  CFU/ml. L. monocytogenes displayed slower growth kinetics reaching 106 -107  CFU/ml after 42 days. CONCLUSION: The results illustrate the importance of conducting comprehensive studies to establish well-characterized reference strains, which can be a tool to assess strategies and methods used to ameliorate blood safety. The WHO Expert Committee on Biological Standardization adopted the five successful strains as official RBCC reference strains. Our study also highlights the relevance of visual inspection to interdict contaminated RBC units.

Case Report: Invasive Pentastomes, Raillietiella orientalis (Sambon, 1922), in a Free-Ranging Banded Water Snake (Nerodia fasciata) in North Central Florida, USA.

Raillietiella orientalis is an obligate, crustacean parasite that resides in the respiratory tract of definitive snake hosts. Common throughout southeastern Asia and Australia, R. orientalis is believed to have been introduced into southern Florida, United States along with Burmese pythons (Python bivittatus) in the 1990s. While the invasive range of Burmese pythons is restricted to southern Florida, R. orientalis has advanced north in the state in native snake species. R. orientalis were recovered from the lungs, trachea, oral cavity, and esophagus of an emaciated adult female free-ranging banded water snake (Nerodia fasciata) in north central (Alachua County), Florida, USA. Concurrent findings included the recovery of Ochetosoma sp. trematodes from the oral cavity, and multifocal dermal lesions consistent with snake fungal disease (Ophidiomyces ophiodiicola). This is the first report of R. orientalis in north central Florida, well outside the invasive range of the Burmese python, documenting the substantial northward expansion of the known geographical range of this invasive pentastome in Florida.

Impact of Localized Radiative Loss on Inertial Confinement Fusion Implosions.

The impact to fusion energy production due to the radiative loss from a localized mix in inertial confinement implosions using high density carbon capsule targets has been quantified. The radiative loss from the localized mix and local cooling of the reacting plasma conditions was quantified using neutron and x-ray images to reconstruct the hot spot conditions during thermonuclear burn. Such localized features arise from ablator material that is injected into the hot spot from the Rayleigh-Taylor growth of capsule surface perturbations, particularly the tube used to fill the capsule with deuterium and tritium fuel. Observations, consistent with analytic estimates, show the degradation to fusion energy production to be linearly proportional to the fraction of the total emission that is associated with injected ablator material and that this radiative loss has been the primary source of variations, of up to 1.6 times, in observed fusion energy production. Reducing the fill tube diameter has increased the ignition metric χ_{no α} from 0.49 to 0.72, 92% of that required to achieve a burning hot spot.

Oral, Cloacal, and Hemipenal Actinomycosis in Captive Ball Pythons (Python regius).

Ball pythons (Python regius) are one of the most commonly kept and bred reptiles in captivity. In a large ball python breeding colony, a unique syndrome characterized by granulomatous inflammation of the cloaca and hemipenes (phalli) was observed in 140 of 481 (29.1%) breeding males, but only one of 1,446 breeding females. Lesions were absent in virgin males (n = 201) and virgin females (n = 293). On postmortem examination (n = 13, 12 males, 1 female), numerous well-defined mucosal and submucosal granulomas were present in the hemipenes (males) and cloaca (males and female). Extension into the coelomic cavity and liver was noted in a subset of these animals. An additional small subset of breeder animals (6/2027; 0.3%) presented with oral and mandibular swellings. Postmortem examination (n = 4, all female) showed oral lesions histologically indistinguishable from the cloacal/hemipenal lesions. Aerobic bacterial culture of a hepatic granuloma of one snake resulted in the isolation of filamentous, Gram-positive bacilli; amplification, and sequencing of the 16S rRNA gene and subsequent phylogenetic analysis of the isolate identified the bacterium as a novel species of Actinomyces. Screening of cloacal and oral granulomas using a specific, heminested 16S rRNA PCR assay confirmed the presence of the agent in all 17 snakes, as well as in cloacal swabs taken at the time of necropsy in 11/13 snakes. The Actinomyces sp. was also identified by PCR of cloacal swabs of unaffected snakes (n = 94) from the affected colony and two unrelated, grossly unaffected breeding colonies. In the affected colony, 65.5% of breeding animals (n = 23) but only 11.9% of virgin animals (n = 42) tested PCR positive, with breeding status being a significant predictor of bacterium presence (P < 0.00001). This study characterizes a granulomatous mucosal disease syndrome of breeding male ball pythons associated with a novel Actinomyces. In stark contrast to male snakes, the presence of the bacterium in both breeding and virgin females was very rarely associated with clinical disease. Though additional studies are necessary, these data suggest a role for the novel bacterium in the disease process, a predilection for clinical disease in male snakes, and the potential for sexual transmission of the disease.

Using L-shell x-ray spectra to determine conditions of non-local thermal dynamic equilibrium plasmas.

K-shell x-ray spectra of Li- to H-like ions have long been used to determine plasma conditions. The ratio of integrated line intensities is used to determine the temperature. At the density of non-local thermal dynamic equilibrium (NLTE) plasmas (n e ≈ 1021 cm-3), the K-shell spectrum is not very sensitive to density. We propose using the L-shell emission of open L-shell ions (C- to Li-like) as an alternative to determine both temperature and density of NLTE plasmas. First, the L-shell models of a mid-Z material need to be verified against the temperatures obtained using a K-shell spectrum of a low-Z material. A buried layer platform is being developed at the OMEGA laser to study the open L-shell spectra of NLTE plasmas of mid-Z materials. Studies have been done using a 250 µm diameter dot composed of a layer of 1200 Å thick Zn between two 600 Å thick layers of Ti, in the center of a 1000 µm diameter, 13 µm thick beryllium tamper. Lasers heat the target from both sides for up to 3 ns. The size of the emitting volume vs time was measured with x-ray imaging (face-on and side-on) to determine the density. The temperature was measured from the Ti K-shell spectra. The use of this platform for the verification of atomic L-shell models is discussed.

Determination of Total Biotin by Liquid Chromatography Coupled with Immunoaffinity Column Cleanup Extraction: Multilaboratory Testing, Final Action 2016.02.

Single- and multilaboratory testing data have provided systematic scientific evidence that a simple, selective, accurate, and precise method can be used as a potential candidate reference method for dispute resolution in determining total biotin in all forms of infant, adult, and/or pediatric formula. Using LC coupled with immunoaffinity column cleanup extraction, the method fully meets the intended purpose and applicability statement in AOAC Standard Method Performance Requirement 2014.005. The method was applied to a cross-section of infant formula and adult nutritional matrixes, and acceptable precision and accuracy were established. The analytical platform is inexpensive, and the method can be used in almost any laboratory worldwide with basic facilities. The immunoaffinity column cleanup extraction is the key step to successful analysis.

Disruption of dopamine homeostasis has sexually dimorphic effects on senescence characteristics of Drosophila melanogaster.

The neurotransmitter dopamine (DA) is known to be involved in a multitude of physiological processes. We investigated sexually dimorphic effects of disruptions in DA homeostasis and its relationship to senescence using three different Drosophila melanogaster mutants namely Catsup (Catsup26 ) with elevated DA levels, and pale (ple2 ), Punch (PuZ22 ) with depleted DA levels. In all genotypes including controls, DA levels were significantly lower in old (45-50-day-old) flies compared with young (3-5-day-old) in both sexes. Interestingly, females had lower DA content than males at young age whereas this difference was not observed in old age, suggesting that males had a larger decline in DA levels with age. Females, in general, were longer lived compared with males in all genotypes except ple2 mutants with depleted DA levels. This phenotype was abolished in the ple2 rescue flies. Interestingly, females also demonstrated marked age-related decline in circadian locomotor activity compared with males. Old Catsup26 males with elevated DA levels accumulated significantly lower levels of lipid peroxidation product 4-hydroxy 2-nonenal (4-HNE) compared with age-matched wild type, ple2 and PuZ22 mutant males. In Catsup26 revertant lines this phenomenon was absent. We also observed a sexually dimorphic response in the expression levels of key stress and aging associated and/or related transcription factor genes across genotypes with elevated or depleted DA levels which was reverted to wild type levels in specific rescue lines. Taken together, our results reveal a novel sexually dimorphic involvement of DA in senescence characteristics of D. melanogaster.

Perturbations in dopamine synthesis lead to discrete physiological effects and impact oxidative stress response in Drosophila.

The impact of mutations in four essential genes involved in dopamine (DA) synthesis and transport on longevity, motor behavior, and resistance to oxidative stress was monitored in Drosophila melanogaster. The fly lines used for this study were: (i) a loss of function mutation in Catecholamines up (Catsup(26)), which is a negative regulator of the rate limiting enzyme for DA synthesis, (ii) a mutant for the gene pale (ple(2)) that encodes for the rate limiting enzyme tyrosine hydroxylase (TH), (iii) a mutant for the gene Punch (Pu(Z22)) that encodes guanosine triphosphate cyclohydrolase, required for TH activity, and (iv) a mutant in the vesicular monoamine transporter (VMAT(Δ14)), which is required for packaging of DA as vesicles inside DA neurons. Median lifespans of ple(2), Pu(Z22) and VMAT(Δ14) mutants were significantly decreased compared to Catsup(26) and wild type controls that did not significantly differ between each other. Catsup(26) flies survived longer when exposed to hydrogen peroxide (80 µM) or paraquat (10mM) compared to ple(2), Pu(Z22) or VMAT(Δ14) and controls. These flies also exhibited significantly higher negative geotaxis activity compared to ple(2), Pu(Z22), VMAT(Δ14) and controls. All mutant flies demonstrated rhythmic circadian locomotor activity in general, albeit Catsup(26) and VMAT(Δ14) flies had slightly weaker rhythms. Expression analysis of some key antioxidant genes revealed that glutathione S-transferase Omega-1 (GSTO1) expression was significantly up-regulated in all DA synthesis pathway mutants and especially in Catsup(26) and VMAT(Δ14) flies at both mRNA and protein levels. Taken together, we hypothesize that DA could directly influence GSTO1 transcription and thus play a significant role in the regulation of response to oxidative stress. Additionally, perturbations in DA synthesis do not appear to have a significant impact on circadian locomotor activity rhythms per se, but do have an influence on general locomotor activity levels.

Development of a ten inch manipulators-based, flexible, broadband two-crystal spectrometer.

We have developed and implemented a broadband X-ray spectrometer with a variable energy range for use at the Atomic Weapons Establishment's Orion Laser. The spectrometer covers an energy bandwidth of ∼1-2 keV using two independently mounted, movable Bragg diffraction crystals. Using combinations of cesium hydrogen pthlate, ammonium dihydrogen phosphate, and pentaerythritol crystals, spectra covering the 1.4-2.5, 1.85-3.15, or 3.55-5.1 keV energy bands have been measured. Image plate is used for detection owing to its high dynamic range. Background signals caused by high energy X-rays and particles commonly produced in high energy laser experiments are reduced by a series of tantalum baffles and filters installed between the source and crystal and also between the crystals and detector.

Observations of the effect of ionization-potential depression in hot dense plasma.

The newly commissioned Orion laser system has been used to study dense plasmas created by a combination of short pulse laser heating and compression by laser driven shocks. Thus the plasma density was systematically varied between 1 and 10 g/cc by using aluminum samples buried in plastic foils or diamond sheets. The aluminum was heated to electron temperatures between 500 and 700 eV allowing the plasma conditions to be diagnosed by K-shell emission spectroscopy. The K-shell spectra show the effect of the ionization potential depression as a function of density. The data are compared to simulated spectra which account for the change in the ionization potential by the commonly used Stewart and Pyatt prescription and an alternative due to Ecker and Kröll suggested by recent x-ray free-electron laser experiments. The experimental data are in closer agreement with simulations using the model of Stewart and Pyatt.

Shorter bevacizumab infusions do not increase the incidence of proteinuria and hypertension.

BACKGROUND: A previous study has shown that shorter bevacizumab infusions (0.5 mg/kg/min) can be safely administered without increasing the risk of infusion-related hypersensitivity reactions (HSRs). However, the risk of proteinuria and hypertension in patients receiving shorter infusions of bevacizumab is undetermined. PATIENTS AND METHODS: This was a multicenter, prospective, observational study in patients receiving <10 mg/kg of bevacizumab infused over 0.5 mg/kg/min. Patients were observed until discontinuation of bevacizumab for progression of cancer or toxicity. The incidence of hypertension and proteinuria was compared with a prior cohort of patients who had received standard duration infusions of bevacizumab. RESULTS: Sixty-three patients received a total of 392 doses of shorter bevacizumab infusions. Nineteen (30.2%) patients experienced proteinuria while receiving bevacizumab. Out of 19 patients, 13 had grade 1 and 6 had grade 2 proteinuria. None of the patients experienced grade 3 or 4 proteinuria. Hypertension was reported in 32 (50.8%) patients receiving bevacizumab. Twelve (19%) patients developed grade 3 or greater hypertension on bevacizumab. The incidence of proteinuria and hypertension was 38.3% and 56.6%, respectively, in patients (N = 120, 1347 infusions) receiving standard duration infusions of bevacizumab. CONCLUSIONS: Shorter bevacizumab infusions (0.5 mg/kg/min) do not increase the risk of proteinuria and hypertension.

Ultra fast x-ray streak camera for ten inch manipulator based platforms.

Ultra fast x-ray streak cameras are a staple for time resolved x-ray measurements. There is a need for a ten inch manipulator (TIM) based streak camera that can be fielded in a newer large scale laser facility. The Lawrence Livermore National Laboratory ultra fast streak camera's drive electronics have been upgraded and redesigned to fit inside a TIM tube. The camera also has a new user interface that allows for remote control and data acquisition. The system has been outfitted with a new sensor package that gives the user more operational awareness and control.