A functional dual-coated (FDC) microtiter plate method to replace the botulinum toxin LD50 test

Conventional capture ("Sandwich") ELISAs equally detect denatured inactive and native active botulinum type A toxin. Light chain endoprotease activity assays also fail to distinguish between various inactive molecules including partially denatured and fragmented material still retaining this protease activity. By co-coating microtiter plates with SNAP25 substrate and a monoclonal antibody specific for a conformational epitope of the toxin's Hc domain, it was possible to develop a highly sensitive (130 aM LoD), precise (1.4% GCV) new assay specific for the biologically active toxin molecule. Capture was performed in phosphate buffer with a fixed optimal concentration of chaotropic agent (e.g., 1.2 M urea) to differentially isolate functional toxin molecules. Addition of enzymatically favorable buffer containing zinc and DTT reduced the interchain disulfide bond releasing and activating the captured L-chain with subsequent specific cleavage of the SNAP25(1-206) substrate. A neoepitope antibody specific for the newly exposed Q(197) epitope was used to quantify the cleaved SNAP25(1-197). The assay's requirement for the intact toxin molecule was demonstrated with pre-reduced toxin (heavy and light chains), recombinant LHn fragments, and stressed samples containing partially or fully denatured material. This is the first known immunobiochemical assay that correlates with in vivo potency and provides a realistic alternative.     

Why modification of the LD50 test will not be enough

During the last 10 years, the 'Three Rs' (reduction, refinement and replacement) concept of alternatives has come to be widely accepted, and new national and international laws require that non-animal procedures should replace animal experimentation wherever possible. Some reduction and refinement of animal use in toxicity testing has been achieved, and non-animal methods are becoming widely used as prescreens. However, even replacing the LD50 test by a modified and validated animal test, the Fixed Dose Procedure, will be a major achievement. In this paper it is argued that this is not good enough, and that more effort must be put into the development, validation, acceptance and use of genuine replacement alternative tests.     

Alternative methods for the median lethal dose (LD(50)) test: the up-and-down procedure for acute oral toxicity

The authors have developed an improved version of the up-and-down procedure (UDP) as one of the replacements for the traditional acute oral toxicity test formerly used by the Organisation for Economic Co-operation and Development member nations to characterize industrial chemicals, pesticides, and their mixtures. This method improves the performance of acute testing for applications that use the median lethal dose (classic LD50) test while achieving significant reductions in animal use. It uses sequential dosing, together with sophisticated computer-assisted computational methods during the execution and calculation phases of the test. Staircase design, a form of sequential test design, can be applied to acute toxicity testing with its binary experimental endpoints (yes/no outcomes). The improved UDP provides a point estimate of the LD50 and approximate confidence intervals in addition to observed toxic signs for the substance tested. It does not provide information about the dose-response curve. Computer simulation was used to test performance of the UDP without the need for additional laboratory validation.     

牛磺胆酸钠在大鼠急性胰腺炎模型中的半数致死量(LD50)研究

目的:通过使用不同浓度的牛磺胆酸钠(TC)建立急性胰腺炎(AP)模型,观察相应的死亡率,进而计算其半数致死量。方法:使用不同浓度的TC(0%(wt/vl)、1%、2%、3%、4%、5%、9%)观察AP造模72小时后各组的死亡率。同时分析TC浓度与组织病理评分、血清淀粉酶和外周血细胞变化的相关性。结果:TC的半数致死量为3.409%。并且,TC的浓度与组织病理评分、血清淀粉酶和外周血细胞变化密切相关。结论:TC所致AP造模的最佳剂量为3.409%。     

Potential of the insect growth regulator, fluazuron, in the control of Rhipicephalus sanguineus nymphs (Latreille, 1806) (Acari: Ixodidae): determination of the LD95 and LD50

Conventional pesticides have suffered two main drawbacks: (a) broad spectrum of action and (b) selection of target species resistant to the different active ingredients. Thus compounds that are less harmful to the environment and to human health, more specific and that do not induce resistance need to be developed. One alternative are insect growth regulators, such as fluazuron. The present study examined the efficacy of fluazuron (active ingredient of the acaricide Acatak®) and the sensitivity of Rhipicephalus sanguineus nymphs exposed to different doses of this chemical, and determined the lethal doses of fluazuron: 95% – LD₉₅ and 50% – LD₅₀. Different doses of fluazuron were applied in duplicates on the dorsal region of hosts (“pour on”). Distilled water was used in the control group. On the first day after the treatment with fluazuron, hosts were artificially infested with R. sanguineus nymphs. After engorgement, nymphs were removed, placed on Petri dishes, identified, and maintained in BOD incubator for 15 days. Dead R. sanguineus nymphs after the treatment with 13 different doses of fluazuron were quantified and the LD₉₅ was estimated to be 100 mg/kg and LD₅₀, 19.544 mg/kg (12.478–22.636), with a confidence interval of 95%. Nymphs of R. sanguineus were sensitive to fluazuron at various levels, indicating that this insect growth regulator (IGR) may be used to control this parasite in this stage of its biological cycle, reducing the significant damage it causes.     

Development of a novel bioassay for estimation of median lethal concentrations (LC50) and doses (LD50) of the entomopathogenic fungus Beauveria bassiana, against western flower thrips, Frankliniella occidentalis

To conduct laboratory experiments aimed at quantifying secondary acquisition of fungal conidia by western flower thrips (Frankliniella occidentalis), an efficient assay technique using Beauveria bassiana as the model fungus was developed. Various application protocols were tested and it was determined that the percent mortality did not vary among protocols. Peak mortality of second-instar nymphs, under constant exposure to conidia, occurred 5 days post-inoculation. Second-instar thrips that were exposed to conidia within 24 h of the molt to second instar were more susceptible to Beauveria bassiana than thrips exposed after times greater than 24 h post-molt. Conidia efficacy, which was monitored at 24 h intervals, did not differ significantly within 72 h. A test of the final bioassay system was conducted in a series of assays aimed at determining the LD50 of B. bassiana technical powder against second-instar western flower thrips. It was determined that B. bassiana (strain GHA) is highly effective at very low doses (LD50 of 33-66 conidia/insect).     

Genetic testing for late-onset diseases: effect of disease controllability, test predictivity, and gender on the decision to take the test

This study was designed to investigate the influence of the predictive power of the genetic test, the disease's controllability, and gender on the intention to take a predictive genetic test. Another aim was to examine the certainty of the intention to take the test. Nine scenarios were presented to a convenience sample of 121 men and women. Each scenario described a hypothetical and devastating late-onset disease and manipulated the level of disease controllability and test predictive value. The more predictivity the test offered and the more controllable the disease, the more the subjects were interested in being tested and the more certain their decision to take the test. Men's decisions were more positive than women's. There were also interactions between disease controllability and gender and between disease controllability and test predictivity. It seems that the factor that most influences the decision not to take the test is low disease controllability. This effect is more pronounced in women. Gender differences in decision making and information processing may be pertinent. The data show a marked gap between the decision itself and the certainty of that decision. The strength of the decision may be a better predictor of actual test uptake than the direction of the decision.     

LD50 and repellent effects of essential oils from Argentinian wild plant species on Varroa destructor

The repellent and acaricidal effects of some essential oils from the most typical wild plant species of northern Patagonia, Argentina, on Varroa destructor Anderson & Trueman were evaluated using a complete exposure test. Honey bees, Apis mellifera L., and mites (five specimens of each per dish) were introduced in petri dishes having different oil concentrations (from 0.1 to 25 micro per cage). Survival of bees and mites was registered after 24, 48, and 72 h. An attraction/repellence test was performed using a wax tube impregnated with essential oil and another tube containing wax only. The lowest LD50 values for mites were registered for Acantholippia seriphioides (A. Gray) Mold. (1.27 microl per cage) and Schinus molle L. (2.65 microl per cage) after 24 h, and for Wedelia glauca (Ortega) O. Hoffm. ex Hicken (0.59 microl per cage) and A. seriphioides (1.09 microl per cage) after 72 h of treatment. The oil with the highest selectivity ratio (A. mellifera LD50/V. destructor LD50) was the one extracted from S. molle (>16). Oils of Lippia junelliana (Mold.) Troncoso, Minthostachys mollis (HBK) Grieseb., and Lippia turbinata Grieseb. mixed with wax had repellent properties. None of the oils tested had attractive effects on Varroa mites.     

Lack of temperature-sensitivity of rat liver pyruvate kinase (Short Communication)

Rat liver pyruvate kinase, prepared by Sephadex treatment of a 10(5)g supernatant in phosphate buffer, is quite stable and gives reproducible results when a variety of parameters are altered in the enzyme assay. Incubation of this preparation at 25 degrees C or 0-2 degrees C has no effect on the activation by fructose 1,6-diphosphate or inhibition by ATP or alanine.