Lethal Dose Estimates Research Articles

Virulence of Burkholderia pseudomallei ATS2021 Unintentionally Imported to United States in Aromatherapy Spray.

In the United States in 2021, an outbreak of 4 cases of Burkholderia pseudomallei, the etiologic agent of melioidosis and a Tier One Select Agent (potential for deliberate misuse and subsequent harm), resulted in 2 deaths. The causative strain, B. pseudomallei ATS2021, was unintentionally imported into the United States in an aromatherapy spray manufactured in India. We established that ATS2021 represents a virulent strain of B. pseudomallei capable of robust formation of biofilm at physiologic temperatures that may contribute to virulence. By using mouse melioidosis models, we determined median lethal dose estimates and analyzed the bacteriologic and histopathologic characteristics of the organism, particularly the potential neurologic pathogenesis that is probably associated with the bimABm allele identified in B. pseudomallei strain ATS2021. Our data, combined with previous case reports and the identification of endemic B. pseudomallei strains in Mississippi, support the concept that melioidosis is emerging in the United States.

Quantifying resistance to very-long-chain fatty acid-inhibiting herbicides in Amaranthus tuberculatus using a soilless assay.

Resistance to preemergence (PRE) soil-applied herbicides, such as inhibitors of very-long-chain fatty acid (VLCFA) elongases, was documented in two waterhemp [Amaranthus tuberculatus (Moq.) J.D. Sauer] populations (SIR and CHR) from Illinois, USA. To limit the spread of resistant weed populations, rapid detection measures are necessary. Soil-based resistance assays are limited by edaphic factors, application timing, variable seeding depth and rainfall amount. Therefore, cost-effective techniques mitigating effects of edaphic factors that are appropriate for small- to large-scale assays are needed. Our research goal was to identify and quantify resistance to the VLCFA-inhibiting herbicides, S-metolachlor and pyroxasulfone, using a soilless greenhouse assay. Dose-response experiments were conducted under greenhouse conditions with pre-germinated waterhemp seeds planted on the vermiculite surface, which had been saturated with S-metolachlor (0.015-15 μM), pyroxasulfone (0.0005-1.5 μM), or S-metolachlor plus the cytochrome P450 (P450) inhibitor, malathion. Lethal dose estimates of 50% (LD50) and growth reduction of 50% (GR50) were calculated for S-metolachlor and pyroxasulfone PRE and used to determine resistance indices (RI) for resistant populations (CHR and SIR) relative to sensitive populations, SEN and ACR. RI values for S-metolachlor using LD50 values calculated relative to SEN and ACR were 17.2 and 15.2 (CHR) or 11.5 and 10.1 (SIR), while RI values for pyroxasulfone using LD50 values calculated relative to SEN and ACR were 3.8 and 3.1 (CHR) or 4.8 and 3.8 (SIR). Malathion decreased the GR50 of S-metolachlor to a greater degree in CHR compared to ACR, consistent with P450 involvement in S-metolachlor resistance in CHR. Results from these soilless assays are in accord with previous findings in soil-based systems that demonstrate CHR and SIR are resistant to S-metolachlor and pyroxasulfone. This method provides an effective, reproducible alternative to soil-based systems for studying suspected PRE herbicide-resistant populations and will potentially assist in identifying non-target-site resistance mechanisms.

Inheritance of resistance to S-metolachlor in a waterhemp (Amaranthus tuberculatus) population from central Illinois

AbstractWaterhemp [Amaranthus tuberculatus (Moq.) Sauer] is a dioecious weed that has evolved resistance to very-long-chain fatty-acid elongase (VLCFAE)–inhibiting herbicides via rapid metabolism. Although detoxification enzyme activities are associated with S-metolachlor resistance in two multiple herbicide–resistant (MHR) A. tuberculatus populations from Illinois, the genetic basis of resistance is unknown. Therefore, our goal was to investigate inheritance of S-metolachlor resistance in the Stanford, Illinois–resistant (SIR) population. Specifically, our research objectives were to: (1) generate a uniformly resistant, full-sib near-inbred line (DK3-2) via three generations of recurrent selection for resistance using preemergence S-metolachlor; (2) develop A. tuberculatus populations segregating for S-metolachlor resistance via reciprocal single-plant (one male × one female) full-sib mating of DK3-2 and a VLCFAE-inhibiting herbicide-sensitive population, SEN; (3) quantify S-metolachlor resistance levels in parental lines and their F1 progenies via greenhouse dose–response analysis; and (4) evaluate inheritance of S-metolachlor resistance in F2 progenies. Dose–response analysis using six to eight S-metolachlor concentrations (0.015 to 15.0 μM, varying per population) generated lethal dose (LD) estimates of 50% (LD50) and 90% (LD90) for SIR, SEN, DK3-2, and F1 progenies. LD estimates indicated DK3-2 has a higher magnitude of S-metolachlor resistance than the SIR population, demonstrating single crosses significantly increased S-metolachlor resistance in DK3-2. Levels of S-metolachlor resistance in F1 populations were intermediate compared with DK3-2 and SEN. Segregation of S-metolachlor resistance in F2 families from the paternal-derived lines fit a single-gene model (R:S = 3:1), indicating a single, dominant gene confers S-metolachlor resistance in SIR. However, F2 segregation results from the maternal-derived lines fit a duplicate recessive epistasis model (R:S = 9:7), indicating a second recessive gene may also modify S-metolachlor resistance in SIR. Results and germplasm derived from this research can assist in identifying the gene(s) conferring resistance to S-metolachlor in A. tuberculatus.

Sensitivity of the stripe-faced dunnart, Sminthopsis macroura (Gould 1845), to the insecticide, fipronil; implications for pesticide risk assessments in Australia

A lack of toxicity data quantifying responses of Australian native mammals to agricultural pesticides prompted an investigation into the sensitivity of the stripe-faced dunnart, Sminthopsis macroura (Gould 1845) to the insecticide, fipronil (5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinyl pyrazole, CAS No. 120068-37-3). Using the Up-And-Down method for determining acute oral toxicity in mammals (OECD) median lethal dose estimates of 990 mg kg−1 (95% confidence interval (CI) = 580.7–4770.0 mg kg−1) and 270.4 mg kg−1 (95% CI = 0.0–>20,000.0 mg kg−1) were resolved for male and female S. macroura, respectively. The difference between median lethal dose estimates for males and females may have been influenced by the older ages of two female dunnarts. Consequently, further modelling of female responses to fipronil doses used the following assumptions: (a) death at 2000 mg kg−1, (b) survival at 500 mg kg−1 and (c) a differential response (both survival and death) at 990 mg kg−1. This modelling revealed median lethal dose estimates for female S. macroura of 669.1 mg kg−1 (95% CI = 550–990 mg kg−1; assuming death at 990 mg kg−1) and 990 mg kg−1 (95% CI = 544.7–1470 mg kg−1; assuming survival at 990 mg kg−1). These median lethal dose estimates are 3–10-fold higher than available LD50 values of 94 mg kg−1 for a similarly sized eutherian mammal, Mus musculus (L. 1758) and 97 mg kg−1 for Rattus norvegicus (Birkenhout 1769). Implications for pesticide risk assessments in Australia are discussed.

Increasing virulence, but not infectivity, associated with serially emergent virus strains of a fish rhabdovirus.

Surveillance and genetic typing of field isolates of a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), has identified four dominant viral genotypes that were involved in serial viral emergence and displacement events in steelhead trout (Oncorhynchus mykiss) in western North America. To investigate drivers of these landscape-scale events, IHNV isolates designated 007, 111, 110, and 139, representing the four relevant genotypes, were compared for virulence and infectivity in controlled laboratory challenge studies in five relevant steelhead trout populations. Viral virulence was assessed as mortality using lethal dose estimates (LD50), survival kinetics, and proportional hazards analysis. A pattern of increasing virulence for isolates 007, 111, and 110 was consistent in all five host populations tested, and correlated with serial emergence and displacements in the virus-endemic lower Columbia River source region during 1980–2013. The fourth isolate, 139, did not have higher virulence than the previous isolate 110. However, the mG139M genotype displayed a conditional displacement phenotype in that it displaced type mG110M in coastal Washington, but not in the lower Columbia River region, indicating that factors other than evolution of higher viral virulence were involved in some displacement events. Viral infectivity, measured as infectious dose (ID50), did not correlate consistently with virulence or with viral emergence, and showed a narrow range of variation relative to the variation observed in virulence. Comparison among the five steelhead trout populations confirmed variation in resistance to IHNV, but correlations with previous history of virus exposure or with sites of viral emergence varied between IHNV source and sink regions. Overall, this study indicated increasing viral virulence over time as a potential driver for emergence and displacement events in the endemic Lower Columbia River source region where these IHNV genotypes originated, but not in adjacent sink regions.

Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors.

BackgroundFrancisella tularensis is a select bio-threat agent and one of the most virulent intracellular pathogens known, requiring just a few organisms to establish an infection. Although several virulence factors are known, we lack an understanding of virulence factors that act through host-pathogen protein interactions to promote infection. To address these issues in the highly infectious F. tularensis subsp. tularensis Schu S4 strain, we deployed a combined in silico, in vitro, and in vivo analysis to identify virulence factors and their interactions with host proteins to characterize bacterial infection mechanisms.ResultsWe initially used comparative genomics and literature to identify and select a set of 49 putative and known virulence factors for analysis. Each protein was then subjected to proteome-scale yeast two-hybrid (Y2H) screens with human and murine cDNA libraries to identify potential host-pathogen protein-protein interactions. Based on the bacterial protein interaction profile with both hosts, we selected seven novel putative virulence factors for mutant construction and animal validation experiments. We were able to create five transposon insertion mutants and used them in an intranasal BALB/c mouse challenge model to establish 50 % lethal dose estimates. Three of these, ΔFTT0482c, ΔFTT1538c, and ΔFTT1597, showed attenuation in lethality and can thus be considered novel F. tularensis virulence factors. The analysis of the accompanying Y2H data identified intracellular protein trafficking between the early endosome to the late endosome as an important component in virulence attenuation for these virulence factors. Furthermore, we also used the Y2H data to investigate host protein binding of two known virulence factors, showing that direct protein binding was a component in the modulation of the inflammatory response via activation of mitogen-activated protein kinases and in the oxidative stress response.ConclusionsDirect interactions with specific host proteins and the ability to influence interactions among host proteins are important components for F. tularensis to avoid host-cell defense mechanisms and successfully establish an infection. Although direct host-pathogen protein-protein binding is only one aspect of Francisella virulence, it is a critical component in directly manipulating and interfering with cellular processes in the host cell.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2351-1) contains supplementary material, which is available to authorized users.

Clinical parameters, postmortem analysis and estimation of lethal dose in victims of a massive intoxication with diethylene glycol

This work analyzes a massive intoxication that occurred in 1992 in Argentina as a result of the use of propolis syrup as a popular upper respiratory infection medicinal agent. The intoxicating agent was diethylene glycol (DEG), which caused metabolic acidosis, anuria, renal failure and death in 15 out of the 29 studied victims. DEG poisoning cases were classified in three groups according to survival time: Group 1—patients that survived up to 3 days; Group 2—patients that survived between 4 and 5 days; Group 3—patients that survived between 6 and 21 days. Patients from Group 1 showed the highest values of anion gap, the lowest measures of base excess (BE) and more severe clinical manifestations. Correlation between pH and BE was r 2 = 0.68, 0.99 and 0.55 for Groups 1, 2 and 3, respectively. A methanolic extraction was performed on the fatal victims’ viscera and blood, with subsequent concentration and purification. The semi-crystalline fraction obtained retained DEG by means of co-dissolution and adsorption as demonstrated by thin lay chromatography/flame ionisation detection (TLC/FID). In 3 out of the 15 fatal cases (from Group 1), DEG was isolated from viscera and blood (femoral venous), between 48 and 72 h post ingestion. The concentration relation (DEG) viscera/(DEG) blood ranged from 1.45 to 1.55 with a coefficient correlation r 2 = 0.96 ( n = 3). In the other victims, DEG could not be detected. The reason for this could be the long survival period of the victims after their ingestion of the syrup. Additionally, putrefying mechanisms could have been operating. Samples of the propolis syrup of each victim were studied by means of nuclear magnetic resonance (NMR) and quantified by gas chromatography/flame ionisation detection (GC/FID). Results showed that syrup samples contained 65.0% (w/v) of diethylene glycol (DEG) and 32.0% (w/v) of propylene glycol (PG). A good correlation between the amount of DEG ingested and the anion gap ( r 2 = 0.63) for the 15 victims studied could be observed. The lethal dose for human beings estimated in this work ranged from 0.014 to 0.170 mg DEG/kg body weight. This is a lower lethal dose than reported in a separate incident in Haiti. These results may contribute to the understanding of DEG's metabolic pathway and provides data from lethal doses in humans.

Use of a generalized linear mixed model to reduce excessive heterogeneity in petroleum spray oil bioassay data.

High heterogeneity (variance) is a consistent and significant problem in petroleum spray oil derived bioassay data. It can mask small statistical differences sought by researchers in relative toxicity or potency analysis. To compensate for excessive heterogeneity, researchers often use very large sample sizes to improve statistical accuracy. We present a statistical method of modeling heterogeneity extending the conventional probit model by adding random effects to it. We illustrate this by reanalyzing 26 of our own published experiments. Twelve of these had excessive heterogeneity that was significantly reduced in ten cases by including random replicate effects with or without random slopes. Five were further improved by allowing a nonlinear (spline) response. The result was tighter confidence intervals for the estimates of lethal dose.

Population-level effects of pesticides and other toxicants on arthropods.

New developments in ecotoxicology are changing the way pesticides and other toxicants are evaluated. An emphasis on life histories and population fitness through the use of demography, other measures of population growth rate, field studies, and modeling are being exploited to derive better estimates of pesticide impacts on both target and nontarget species than traditional lethal dose estimates. We review the state of the art in demographic toxicology, an approach to the evaluation of toxicity that uses life history parameters and other measures of population growth rate. A review of the literature revealed that 75 studies on the use of demography and similar measures of population growth rate in toxicology have been published since 1962. Of these 75 studies, the majority involved arthropods. Recent evaluations have indicated that ecotoxicological analysis based on population growth rate results in more accurate assessments of the impacts of pesticides and other toxicants because measures of population growth rate combine lethal and sublethal effects, which lethal dose/concentration estimates (LD/LC50) cannot do. We contend that to advance our knowledge of toxicant impacts on arthropods, the population growth rate approach should be widely adopted.

Dose-Response Tests on Red Flour Beetle and Confused Flour Beetle (Coleoptera: Tenebrionidae) Collected from Flour Mills in the United States

Adults of 14 field strains of the red flour beetle, Tribolium castaneum (Herbst), and 10 strains of confused flour beetle, Tribolium confusum Jacqueline DuVal, collected from flour mills were tested for resistance to malathion and dichlovos by topical application. Lethal dose estimates showed that all strains of T. castaneum were resistant to malathion and dichlorvos. Some strains showed the highest malathion resistance ever reported for this insect. Approximately half the strains of T. confusum tested were resistant to malathion and dichlorvos. T. castaneum was more resistant than T. confusum to both malathion and dichlorvos. There was a positive correlation of the lethal dose values for insecticide resistant strains of both species when compared with the survival of insects exposed to diagnostic doses. Therefore, survival of insects at a diagnostic dose may be used to predict field efficacy of malathion and dichlorvos in these 2 pest species if resistance levels are low.

Toxicity of Activated Cryl Proteins from Bacillus thuringiensis to Six Forest Lepidoptera and Bombyx mori

The insecticidal activity of the CryIB, CryIC, CryID, and CryIE δ-endotoxin proteins from Bacillus thuringiensis was investigated in force-feeding experiments with larvae of Choristoneura fumiferana, Orgyia leucostigma, Lymantria dispar, Malacosoma disstria, Lambdina fiscellaria fiscellaria, Actebia fennica, and Bombyx mori. The toxins were prepared by proteolytic activation of protoxins expressed in Escherichia coli and quantified by scanning densitometry after separation on sodium dodecyl sulfate-polyacrylamide gels. All toxins retained full insecticidal activity during 7 months of refrigerator storage at pH 10.5. The most sensitive response was recorded for B. mori, the only species that exhibited sufficient mortality at generally <10 ng of toxin per larva to permit estimation of lethal dose (LD 50) requirements. In all forest insect species, low and variable mortality necessitated the use of a feeding inhibition response (frass failure dose, FFD 50). C. fumiferana and M. disstria were susceptible to all four toxins. O. leucostigma and L. dispar were not susceptible to CryIB and CryIE. L. fiscellaria and B. mori showed no response to CryIB but were highly susceptible to CryIE. None of the toxins elicited a response in A. fennica . Activity of the toxins was compared to previously published activity of the CryIA toxins by calculating potencies relative to the activated toxin mixture derived from native HD-1 crystals. The four proteins were in most cases less active than the individual CryIA proteins and were at best equally but usually less active than the HD-1 toxin mixture. A notable exception was CryID, which was about as potent as the most active CryIA toxin against M. disstria and B. mori. Published binassay data for seven CryI proteins against 16 target insects are summarized.

Estimation of median human lethal radiation dose computed from data on occupants of reinforced concrete structures in Nagasaki, Japan.

This paper presents an estimate of the median lethal dose for humans exposed to total-body irradiation and not subsequently treated for radiation sickness. The median lethal dose was estimated from calculated doses to young adults who were inside two reinforced concrete buildings that remained standing in Nagasaki after the atomic detonation. The individuals in this study, none of whom have previously had calculated doses, were identified from a detailed survey done previously. Radiation dose to the bone marrow, which was taken as the critical radiation site, was calculated for each individual by the Engineering Physics and Mathematics Division of the Oak Ridge National Laboratory using a new three-dimensional discrete-ordinates radiation transport code that was developed and validated for this study using the latest site geometry, radiation yield, and spectra data. The study cohort consisted of 75 individuals who either survived > 60 d or died between the second and 60th d postirradiation due to radiation injury, without burns or other serious injury. Median lethal dose estimates were calculated using both logarithmic (2.9 Gy) and linear (3.4 Gy) dose scales. Both calculations, which met statistical validity tests, support previous estimates of the median lethal dose based solely on human data, which cluster around 3 Gy.

Evaluation of the Influence of Droplet Size and Density of Bacillus thuringiensis Against Gypsy Moth Larvae (Lepidoptera: Lymantriidae)

A study was conducted to determine the effect of spray droplet size and density on the efficacy of a commercial preparation of Bacillus thuringiensis against the gypsy moth ( Lymantria dispar L.). A concentration of 5,795 International Units (IU)/µl was used. In the laboratory, various size droplets and densities were tested against larvae using 12-cm2 red oak leaflets. Results show that producing relatively high densities of small (50-150 µm) droplets of B. thuringiensis will increase the efficacy compared with larger droplets (> 150 µm) present at low densities at the same dose. Median lethal dose estimates are made for droplets in three size classes (50-150, 150-250, and 250-350 µm). LD95’s for the generalized size classes of 100, 200, and 300 µm were 10.8, 2.2, and 0.9 drops/cm2, respectively. Implications of these results are discussed.

Effects of Dose Selection and Sample Size on the Precision of Lethal Dose Estimates in Dose–Mortality Regression

As a guide to experimental design for bioassays, the effects of total sample size and dose selection on the precision of LD50 and LD90 estimates were determined by Monte Carlo simulations with the logit model. A sample size of 120 appears to be the minimum necessary for reliable estimation. For increased precision, sample sizes of 240 or more are required. A computer procedure was used to search for the percentage of mortality resulting in minimum predicted 95% confidence interval lengths for the LD50 and LD90 in dose-response experiments with three to eight doses, equal numbers of test subjects at each dose, and total sample sizes of 120, 240, 480, and 720. Regardless of the number of doses, the most precise LD50 estimates were obtained when responses when evenly distributed between 25 and 75%. Precise LD90 estimates, however, required one or two responses ≤10% and the majority between 75 and 95%.

Effect of Body Weight on Lethal Dose Estimates for the Western Spruce Budworm12

Procedures for calculating lethal dose and confidence interval estimates for larvae of Choristoneura occidentalis Freeman of a given body weight based on multiple probit or logit regression techniques are described. Results of experiments with mexaearbate, pyrethrins, and DDT were similar for both probit and logit methods. Estimated LDs and their 95% confidence limits increased with increasing body weight. When the hypothesis of proportionality is rejected, use of the proportional model may produce estimates of LDs which are too low and 95% confidence limits which are too narrow, particularly in higher weight classes. These results suggest that topical application experiments should be done with a uniform application volume, and the hypothesis of porportional response should be tested. Since grouping tends to mask departures from proportionality in the data, the use of the basic model as a standard procedure for data analysis is recommended.

Acute toxicity of ochratoxins A and B in chicks.

Ochratoxins A and B were given to 1-day-old Babcock B-300 cockerels to evaluate acute toxic effects. Two trials with ochratoxin A gave 7-day oral median lethal dose estimates of 116 mug (3.3 mg/kg) and 135 mug (3.9 mg/kg) per chick. Chicks given daily oral doses of 100 mug of ochratoxin A died on the second day. Single subcutaneous doses of 400 mug of ochratoxin A were also lethal. The 7-day oral median lethal dose of B was estimated at 1,890 mug (54 mg/kg) per chick. Chicks given oral doses of 100 mug of ochratoxin B daily for 10 days survived. Sublethal doses of both ochratoxins A and B resulted in growth suppression which was proportional to the amount of ochratoxin given. Visceral gout was the principal gross finding. Microscopic examinations revealed acute nephrosis, hepatic degeneration or focal necrosis, and enteritis. Suppression of hematopoiesis in the bone marrow and depletion of lymphoid elements from the spleen and bursa of Fabricius were frequently seen. Both ochratoxins appeared to have similar pathological effects. This is the first report on the toxicity of ochratoxin B.

Acute Toxicity of Ochratoxins A and B in Chicks 1

Ochratoxins A and B were given to 1-day-old Babcock B-300 cockerels to evaluate acute toxic effects. Two trials with ochratoxin A gave 7-day oral median lethal dose estimates of 116 mug (3.3 mg/kg) and 135 mug (3.9 mg/kg) per chick. Chicks given daily oral doses of 100 mug of ochratoxin A died on the second day. Single subcutaneous doses of 400 mug of ochratoxin A were also lethal. The 7-day oral median lethal dose of B was estimated at 1,890 mug (54 mg/kg) per chick. Chicks given oral doses of 100 mug of ochratoxin B daily for 10 days survived. Sublethal doses of both ochratoxins A and B resulted in growth suppression which was proportional to the amount of ochratoxin given. Visceral gout was the principal gross finding. Microscopic examinations revealed acute nephrosis, hepatic degeneration or focal necrosis, and enteritis. Suppression of hematopoiesis in the bone marrow and depletion of lymphoid elements from the spleen and bursa of Fabricius were frequently seen. Both ochratoxins appeared to have similar pathological effects. This is the first report on the toxicity of ochratoxin B.