Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5–6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30 mg semi-engorged ticks fed more reliably, ticks as small as 15 mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10 mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

Methods to determine resistance to anthelmintics when continuing larval development occurs

The in vivo faecal egg count reduction test (FECRT) is the most commonly used test to detect anthelmintic resistance (AR) in gastrointestinal nematodes (GIN) of ruminants in pasture based systems. However, there are several variations on the method, some more appropriate than others in specific circumstances. While in some cases labour and time can be saved by just collecting post-drench faecal worm egg counts (FEC) of treatment groups with controls, or pre- and post-drench FEC of a treatment group with no controls, there are circumstances when pre- and post-drench FEC of an untreated control group as well as from the treatment groups are necessary. Computer simulation techniques were used to determine the most appropriate of several methods for calculating AR when there is continuing larval development during the testing period, as often occurs when anthelmintic treatments against genera of GIN with high biotic potential or high re-infection rates, such as Haemonchus contortus of sheep and Cooperia punctata of cattle, are less than 100% efficacious. Three field FECRT experimental designs were investigated: (I) post-drench FEC of treatment and controls groups, (II) pre- and post-drench FEC of a treatment group only and (III) pre- and post-drench FEC of treatment and control groups. To investigate the performance of methods of indicating AR for each of these designs, simulated animal FEC were generated from negative binominal distributions with subsequent sampling from the binomial distributions to account for drench effect, with varying parameters for worm burden, larval development and drench resistance. Calculations of percent reductions and confidence limits were based on those of the Standing Committee for Agriculture (SCA) guidelines. For the two field methods with pre-drench FEC, confidence limits were also determined from cumulative inverse Beta distributions of FEC, for eggs per gram (epg) and the number of eggs counted at detection levels of 50 and 25. Two rules for determining AR: (1) %reduction (%R) < 95% and lower confidence limit <90%; and (2) upper confidence limit <95%, were also assessed. For each combination of worm burden, larval development and drench resistance parameters, 1000 simulations were run to determine the number of times the theoretical percent reduction fell within the estimated confidence limits and the number of times resistance would have been declared. When continuing larval development occurs during the testing period of the FECRT, the simulations showed AR should be calculated from pre- and post-drench worm egg counts of an untreated control group as well as from the treatment group. If the widely used resistance rule 1 is used to assess resistance, rule 2 should also be applied, especially when %R is in the range 90 to 95% and resistance is suspected.

Computational methods for Bayesian model choice

In this note, we shortly survey some recent approaches on the approximation of the Bayes factor used in Bayesian hypothesis testing and in Bayesian model choice. In particular, we reassess importance sampling, harmonic mean sampling, and nested sampling from a unified perspective.

Stable Dispersions of Metal Oxide Nanowires and Nanoparticles in Water, Dimethylformamide and Toluene

In view of the important need to generate well-dispersed inorganic nanostructures in various solvents, we have explored the dispersion of nanostructures of metal oxides such as TiO2, Fe3O4 and ZnO in solvents of differing polarity in the presence of several surfactants. The solvents used are water, dimethylformamide (DMF) and toluene. The surfactant-solvent combinations yielding the best dispersions are reported alongwith some of the characteristics of the nanostructures in the dispersions. The surfactants which dispersed TiO2 nanowires in water were polyethylene oxide (PEO), Triton X-100 (TX-100), polyvinyl alcohol (PVA) and sodium bis(2-ethylhexyl) sulphosuccinate (AOT). TiO2 nanoparticles could also be dispersed with AOT and PEO in water, and with AOT in toluene. In DMF, PVA, PEO and TX-100 were found to be effective, while in toluene, only AOT gave good dispersions. Fe3O4 nanoparticles were held for long periods of time in water by PEO, AOT, PVA and polyethylene glycol (PEG), and by AOT in toluene. In the case of ZnO nanowires, the best surfactant-solvent combinations were found to be, PEO, sodium dodecyl sulphate (SIDS) and AOT in water and AOT, PEG, PVA, PEO and TX-100 in DMF In toluene, stable dispersions of ZnO nanowires were obtained with PEO. We have also been able to disperse oxide nanostructures in non-polar solvents by employing a hydrophobic silane coating on the surface.

Quantitative methods to measure pigmentation variation in farmed Giant Tiger Prawns, Penaeus monodon, and the effects of different harvest methods on cooked colour

Cooked prawn colour is known to be a driver of market price and a visual indicator of product quality for the consumer. Although there is a general understanding that colour variation exists in farmed prawns, there has been no attempt to quantify this variation or identify where this variation is most prevalent. The objectives of this study were threefold: firstly to compare three different quantitative methods to measure prawn colour or pigmentation, two different colorimeters and colour quantification from digital images. Secondly, to quantify the amount of pigmentation variation that exists in farmed prawns within ponds, across ponds and across farms. Lastly, to assess the effects of ice storage or freeze-thawing of raw product prior to cooking. Each method was able to detect quantitative differences in prawn colour, although conversion of image based quantification of prawn colour from RGB to Lab was unreliable. Considerable colour variation was observed between prawns from different ponds and different farms, and this variation potentially affects product value. Different post-harvest methods prior to cooking were also shown to have a profound detrimental effect on prawn colour. Both long periods of ice storage and freeze thawing of raw product were detrimental to prawn colour. However, ice storage immediately after cooking was shown to be beneficial to prawn colour. Results demonstrated that darker prawn colour was preserved by holding harvested prawns alive in chilled seawater, limiting the time between harvesting and cooking, and avoiding long periods of ice storage or freeze thawing of uncooked product.

Responses of Super Rice (Oryza sativa L.) to Different Planting Methods for Grain Yield and Nitrogen-Use Efficiency in the Single Cropping Season

To break the yield ceiling of rice production, a super rice project was developed in 1996 to breed rice varieties with super high yield. A two-year experiment was conducted to evaluate yield and nitrogen (N)-use response of super rice to different planting methods in the single cropping season. A total of 17 rice varieties, including 13 super rice and four non-super checks (CK), were grown under three N levels [0 (N0), 150 (N150), and 225 (N225) kg ha−1] and two planting methods [transplanting (TP) and direct-seeding in wet conditions (WDS)]. Grain yield under WDS (7.69 t ha−1) was generally lower than TP (8.58 t ha−1). However, grain yield under different planting methods was affected by N rates as well as variety groups. In both years, there was no difference in grain yield between super and CK varieties at N150, irrespective of planting methods. However, grain yield difference was dramatic in japonica groups at N225, that is, there was an 11.3% and 14.1% average increase in super rice than in CK varieties in WDS and TP, respectively. This suggests that high N input contributes to narrowing the yield gap in super rice varieties, which also indicates that super rice was bred for high fertility conditions. In the japonica group, more N was accumulated in super rice than in CK at N225, but no difference was found between super and CK varieties at N0 and N150. Similar results were also found for N agronomic efficiency. The results suggest that super rice varieties have an advantage for N-use efficiency when high N is applied. The response of super rice was greater under TP than under WDS. The results suggest that the need to further improve agronomic and other management practices to achieve high yield and N-use efficiency for super rice varieties in WDS.

Development of analytical methods for the separation of plutonium, americium, curium and neptunium from environmental samples

In this work, separation methods have been developed for the analysis of anthropogenic transuranium elements plutonium, americium, curium and neptunium from environmental samples contaminated by global nuclear weapons testing and the Chernobyl accident. The analytical methods utilized in this study are based on extraction chromatography. Highly varying atmospheric plutonium isotope concentrations and activity ratios were found at both Kurchatov (Kazakhstan), near the former Semipalatinsk test site, and Sodankylä (Finland). The origin of plutonium is almost impossible to identify at Kurchatov, since hundreds of nuclear tests were performed at the Semipalatinsk test site. In Sodankylä, plutonium in the surface air originated from nuclear weapons testing, conducted mostly by USSR and USA before the sampling year 1963. The variation in americium, curium and neptunium concentrations was great as well in peat samples collected in southern and central Finland in 1986 immediately after the Chernobyl accident. The main source of transuranium contamination in peats was from global nuclear test fallout, although there are wide regional differences in the fraction of Chernobyl-originated activity (of the total activity) for americium, curium and neptunium.

Preparation of Industrially Important Hydroxy Acids and Diacids from 2,2-Disubstituted Propane-1,3-Diols and Linear Primary Diols by Green Chemistry Methods

Environmentally benign and economical methods for the preparation of industrially important hydroxy acids and diacids were developed. The carboxylic acids, used in polyesters, alkyd resins, and polyamides, were obtained by the oxidation of the corresponding alcohols with hydrogen peroxide or air catalyzed by sodium tungstate or supported noble metals. These oxidations were carried out using water as a solvent. The alcohols are also a useful alternative to the conventional reactants, hydroxyaldehydes and cycloalkanes. The oxidation of 2,2-disubstituted propane-1,3-diols with hydrogen peroxide catalyzed by sodium tungstate afforded 2,2-disubstituted 3-hydroxypropanoic acids and 1,1-disubstituted ethane-1,2-diols as products. A computational study of the Baeyer-Villiger rearrangement of the intermediate 2,2-disubstituted 3-hydroxypropanals gave in-depth data of the mechanism of the reaction. Linear primary diols having chain length of at least six carbons were easily oxidized with hydrogen peroxide to linear dicarboxylic acids catalyzed by sodium tungstate. The Pt/C catalyzed air oxidation of 2,2-disubstituted propane-1,3-diols and linear primary diols afforded the highest yield of the corresponding hydroxy acids, while the Pt, Bi/C catalyzed oxidation of the diols afforded the highest yield of the corresponding diacids. The mechanism of the promoted oxidation was best described by the ensemble effect, and by the formation of a complex of the hydroxy and the carboxy groups of the hydroxy acids with bismuth atoms. The Pt, Bi/C catalyzed air oxidation of 2-substituted 2-hydroxymethylpropane-1,3-diols gave 2-substituted malonic acids by the decarboxylation of the corresponding triacids. Activated carbon was the best support and bismuth the most efficient promoter in the air oxidation of 2,2-dialkylpropane-1,3-diols to diacids. In oxidations carried out in organic solvents barium sulfate could be a valuable alternative to activated carbon as a non-flammable support. In the Pt/C catalyzed air oxidation of 2,2-disubstituted propane-1,3-diols to 2,2-disubstituted 3-hydroxypropanoic acids the small size of the 2-substituents enhanced the rate of the oxidation. When the potential of platinum of the catalyst was not controlled, the highest yield of the diacids in the Pt, Bi/C catalyzed air oxidation of 2,2-dialkylpropane-1,3-diols was obtained in the regime of mass transfer. The most favorable pH of the reaction mixture of the promoted oxidation was 10. The reaction temperature of 40°C prevented the decarboxylation of the diacids.

Evaluating and validating abundance monitoring methods in the absence of populations of known size: review and application to a passive tracking index

Rarely is it possible to obtain absolute numbers in free-ranging populations and although various direct and indirect methods are used to estimate abundance, few are validated against populations of known size. In this paper, we apply grounding, calibration and verification methods, used to validate mathematical models, to methods of estimating relative abundance. To illustrate how this might be done, we consider and evaluate the widely applied passive tracking index (PTI) methodology. Using published data, we examine the rationality of PTI methodology, how conceptually animal activity and abundance are related and how alternative methods are subject to similar biases or produce similar abundance estimates and trends. We then attune the method against populations representing a range of densities likely to be encountered in the field. Finally, we compare PTI trends against a prediction that adjacent populations of the same species will have similar abundance values and trends in activity. We show that while PTI abundance estimates are subject to environmental and behavioural stochasticity peculiar to each species, the PTI method and associated variance estimate showed high probability of detection, high precision of abundance values and, generally, low variability between surveys, and suggest that the PTI method applied using this procedure and for these species provides a sensitive and credible index of abundance. This same or similar validation approach can and should be applied to alternative relative abundance methods in order to demonstrate their credibility and justify their use.

Critical review on the stability of illicit drugs in sewers and wastewater samples

Wastewater-based epidemiology (WBE) applies advanced analytical methods to quantify drug residues in wastewater with the aim to estimate illicit drug use at the population level. Transformation processes during transport in sewers (chemical and biological reactors) and storage of wastewater samples before analysis are expected to change concentrations of different drugs to varying degrees. Ignoring transformation for drugs with low to medium stability will lead to an unknown degree of systematic under- or overestimation of drug use, which should be avoided. This review aims to summarize the current knowledge related to the stability of commonly investigated drugs and, furthermore, suggest a more effective approach to future experiments. From over 100 WBE studies, around 50 mentioned the importance of stability and 24 included tests in wastewater. Most focused on in-sample stability (i.e., sample preparation, preservation and storage) and some extrapolated to in-sewer stability (i.e., during transport in real sewers). While consistent results were reported for rather stable compounds (e.g., MDMA and methamphetamine), a varying range of stability under different or similar conditions was observed for other compounds (e.g., cocaine, amphetamine and morphine). Wastewater composition can vary considerably over time, and different conditions prevail in different sewer systems. In summary, this indicates that more systematic studies are needed to: i) cover the range of possible conditions in sewers and ii) compare results more objectively. To facilitate the latter, we propose a set of parameters that should be reported for in-sewer stability experiments. Finally, a best practice of sample collection, preservation, and preparation before analysis is suggested in order to minimize transformation during these steps.

Forest inventory-based large-scale forest biomass and carbon budget assessment: new enhanced methods and use of remote sensing for verification

In recent years, concern has arisen over the effects of increasing carbon dioxide (CO2) in the earth's atmosphere due to the burning of fossil fuels. One way to mitigate increase in atmospheric CO2 concentration and climate change is carbon sequestration to forest vegeta-tion through photosynthesis. Comparable regional scale estimates for the carbon balance of forests are therefore needed for scientific and political purposes. The aim of the present dissertation was to improve methods for quantifying and verifying inventory-based carbon pool estimates of the boreal forests in the mineral soils. Ongoing forest inventories provide a data based on statistically sounded sampling for estimating the level of carbon stocks and stock changes, but improved modelling tools and comparison of methods are still needed. In this dissertation, the entire inventory-based large-scale forest carbon stock assessment method was presented together with some separate methods for enhancing and comparing it. The enhancement methods presented here include ways to quantify the biomass of understorey vegetation as well as to estimate the litter production of needles and branches. In addition, the optical remote sensing method illustrated in this dis-sertation can be used to compare with independent data. The forest inventory-based large-scale carbon stock assessment method demonstrated here provided reliable carbon estimates when compared with independent data. Future ac-tivity to improve the accuracy of this method could consist of reducing the uncertainties regarding belowground biomass and litter production as well as the soil compartment. The methods developed will serve the needs for UNFCCC reporting and the reporting under the Kyoto Protocol. This method is principally intended for analysts or planners interested in quantifying carbon over extensive forest areas.